Mutation Research, 258 (1991) 3-49
© 1991 Elsevier Science Publishers B.V. 0165-1110/91/$03.50 ADONIS 016511109100061D MUTREV 07297
Ionizing radiation and genetic risks I. Epidemiological, population genetic, biochemical and molecular aspects of Mendelian diseases K. Sankaranarayanan MGC Department of Radiation Genetics and Chemical Mutagenesis, Sylvius Laboratories, State University of Leiden, Leiden (The Netherlands)
(Received 31 July 1990) (Revision received 28 November 1990) (Accepted 3 December 1990)
Keywords: Mendelian diseases; Epidemiology; Population Genetics; Biochemicalaspects; Molecular aspects
Summa~ This paper reviews the currently available information on naturally occurring Mendelian diseases in man; it is aimed at providing a background and framework for discussion of experimental data on radiation-induced mutations (papers II and III) and for the estimation of the risk of Mendelian disease in human populations exposed to ionizing radiation (paper IV). Current consensus estimates indicate that a total of about 125 per 104 livebirths are directly affected by one or another naturally occurring Mendelian disease (autosomal dominants, 95/104; X-linked ones, 5 / 1 0 4, and autosomal recessives, 25/104). These estimates are conservative and take into account conditions which are very rare and for which prevalence estimates are unavailable. Most, although not all, of the recognized " c o m m o n " dominants have onset in adult ages while most sex-linked and autosomal recessives have onset at birth or in childhood. Autosomal dominant and X-linked diseases (i.e., the responsible mutant alleles) presumed to be maintained in the population due to a balance between mutation and selection are the ones which m a y be expected to increase in frequency as a result of radiation exposures. Viewed from this standpoint, the above assumption seems safe only for a small proportion of such diseases; for the remainder, there is no easy way to discriminate between different mechanisms that may be responsible or to rigorously exclude some in favor of some others. Mutations in genes that code for enzymic proteins are more often recessive in contrast to those that code for non-enzymic proteins, which are more often dominant. At the molecular level, with recessives, a wide variety of changes is possible and these include specific types of point mutations, small and large intragenic deletions, multilocus deletions and rearrangements. In the case of dominants, however, the kinds of recoverable point mutations and deletion-type changes are less extensive because of functional constraints. The mutational potential of genes varies, depending on the gene, its size, sequence content and arrangement, location and its normal functions, and can be grouped into three groups: those in which only
Correspondence: Prof. K. Sankaranarayanan, Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands).
point mutations have been found to occur, those in which only deletions or other gross changes have been recovered and those in which both kinds of changes are known. Molecular data are available for about 75 Mendelian conditions and these suggest that in approximately 50% of them, the changes categorized to date are point mutations and in the remainder, intragenic deletions or other gross changes; there does not seem to be any fundamental difference between dominants and recessives with respect to the underlying molecular defect. Onset age for Mendelian diseases appears related to the normal function disrupted or abolished by the mutation and not necessarily to the underlying molecular change. Point mutations do not appear to be distributed at random throughout the gene; C p G dinucleotide sequences, when present in the gene, provide "hot-spots" for transition-type mutations, but not all transitions occur at CpG sequences. The breakpoints involved in intragenic deletions also are not distributed at random within the gene. In cases analyzed, the length of the deletion per se is not correlated with the severity of the clinical effect. The data from a number of well-analyzed gene deletions are consistent with mechanisms that assume base mispairing between repeat sequences and slippage during replication, homologous unequal recombination between evolutionarily related genes, homologous unequal recombination between repetitive sequences such as Alu and non-homologous recombination. Not all deletions involve Alu sequences. There is circumstantial evidence supporting the hypothesis that repetitive sequences may play an important role in chromosome pairing; if true, the deletions and duplications that have been found to be associated with some diseases may represent the inevitable by-products of occasional mispairing.
In all biological systems, mutations arise "spontaneously" and can be induced by exposure to ionizing radiation. Irrespective of whether they are spontaneous or induced, mutations are recognized because they lead to altered phenotypes and a large number of spontaneously arising mutations which cause disease states in man are known. The principal reasons for the continuing efforts to estimate the genetic risks of ionizing radiation in man are: the fact that ionizing radiation is capable of inducing mutations (and, if they are induced in germ cells, they can be transmitted to the progeny), the concern that such induced mutations can cause genetic disease and the need to protect our genetic heritage. A careful examination of the progress achieved in this field over the past three decades (reviewed in Sankaranarayanan, 1988a; UNSCEAR, 1988) will reveal that (i) the estimates of risk of genetic disease in humans, attributable to radiation-induced mutations, are based on data on mutation rates obtained in studies with the mouse; for various reasons, the emphasis has been on induced mutations having dominant phenotypic effects in the
progeny of the first (and subsequent) post-irradiation generations; (ii) so far, there have been no known instances of radiation-induced heritable mutations in human germ cells or of transmissible genetic disease as we perceive it; and (iii) the extensive genetic data collected in the Hiroshima and Nagasaki studies continue to suggest that the magnitude of genetic risks to man may be lower than that extrapolated from animal data; this discrepancy has not been satisfactorily resolved. During the past several decades, considerable advances have been made in the identification of biochemical defects in human diseases and, in the last 10-15 years, the methods of, and research in, molecular biology have contributed greatly to our understanding of the nature of molecular defects underlying many of the Mendelian diseases. On the radiation front, although only a handful of mutant genes have thus far been subjected to molecular scrutiny (in the mouse in vivo system and in mammalian in vitro systems), it is already clear that there are similarities as well as differences between the spectra of spontaneous and radiation-induced mutations. However, until now,
findings in biochemical and molecular studies have had no perceptible impact on the thinking of "genetic risk estimators". As we enter the 1990s, it seems useful to take stock of the important advances in fields relevant for genetic risk estimation; reflect on whether and to what extent the concepts and assumptions hitherto used in this endeavor can still be justified; identify the changes that seem warranted; and define areas of research which are likely to contribute to a "refinement" of genetic risk estimates in the coming years. These then represent the overall aims of this series of papers of which the first four appear in this issue. These four papers together are intended to catalyze discussion on the risk of Mendelian disease in man, as a result of exposure to radiation. Work along similar lines on multifactorial and chromosomal diseases is underway and will be published in due course. The present paper, the first in this series, focuses on epidemiological, population genetic, biochemical and molecular aspects of Mendelian diseases. (Just before the submission of this paper for publication, a relevant one by Mohrenweiser and Jones (1990) appeared in Vol. 231 of Mutation Research; while it covers some of the same ground as that in my paper, its scope is rather different.) The second and third papers (Sankaranarayanan, 1991a,b) will summarize data on spontaneously arising and radiation-induced mutations in mammalian experimental systems. The fourth paper (Sankaranarayanan, 1991c) will review the "state of the art" in the field of genetic risk estimation (methods, assumptions, experimental and human databases and risk estimates) and examine the relevance of advances in our knowledge of the nature of spontaneous and radiation-induced mutations and the underlying mechanisms, for the estimation of the risk of Mendelian diseases in human populations exposed to ionizing radiation. More specifically, it will present arguments based on the above and other lines of reasoning to support the view that current estimates of risk for this group of diseases may be conservative. What are genetic diseases? Nearly all diseases are to some extent genetic and to some extent environmental and, with re-
gard to the relative importance of genetic and environmental factors in pathogenesis, it is convenient to think of them as covering a spectrum. Towards one end of the spectrum, genetic factors dominate and towards the other, environmental factors. Close to (and not necessarily at) the "genetic end" are conditions which are relatively simple in their formal genetics and which tend to be rare (i.e., Mendelian diseases and constitutional chromosomal anomalies). Towards the "environmental end" are other conditions such as infectious diseases. At different "distances" between these two ends are conditions which are common, which do not follow simple Mendelian patterns of inheritance but which tend to cluster in families (and thus have a genetic component in their etiology). These are termed "multifactorial" diseases. The term "genetic disease" thus includes not only Mendelian and chromosomal diseases, but also multifactorial ones. Furthermore, although not considered in this review, diseases which are due to mutations in the mitochondrial genome (see Grossman, 1990) such as Leber optic atrophy, mitochondrial myopathies, etc. (e.g., Novotny et al., 1986; Nakase et al., 1990; Holt et al., 1988; Wallace et al., 1988), also qualify for inclusion under "genetic disease". Knowledge of naturally occurring genetic diseases, apart from providing a perspective of illhealth in man attributable to genetic causes, also provides a convenient frame of reference to judge whether the projected increases in inherited disease as a result of radiation (or other mutagenic exposures) are trivial, small or substantial. Mendelian diseases General considerations In his 1988 compendium, McKusick has listed 2208 phenotypes (not all of these are pathological states) determined at distinct gene loci, that are well documented as being inherited in a Mendelian manner (1443 autosomal dominants, 139 X-linked ones and 626 autosomal recessives). In addition, a further 2136 conditions are included (1114 autosomal dominants, 171 X-linked and 851 autosomal recessives) for which the evidence for
T A B L E IA ESTIMATES O F LIVEBIRTH P R E V A L E N C E S (X104), FITNESS ( f ) , A P P R O X I M A T E A G E A T O N S E T (years) A N D M U T A T I O N RATES F O R SOME A U T O S O M A L D O M I N A N T DISEASES IN M A N ( A D A P T E D F R O M C A R T E R , 1982; CHILDS, 1981; BEIR, 1990; U N S C E A R , 1982) System/organ affected
Prevalence ( x 10 4) a
Fitness
Onset/clini-
Mutation rate
Carter
Childs
(Childs)
cal detection (years)
( × 10 6) per gamete per generation
4.0 5.0 0.3 0.1 2.0 0.5 0.5
3.50 3.00 1.00 0.25 2.20 --
0.5 0.8 b 0.8 0.2 0.7 ? ?
20 + 45 30--40 5 37 Adult 20-30
2.0
-
?
12
Diaphyseal aclasia (multiple exotoses) Aehondroplasia Osteogenesis imperfecta Osteopetrosis tarda Marfan syndrome Ehlers-Danlos syndrome
5.0 0.2 0.4 0.1 0.4 0.1
0.50 0.30 0.40 0.10 0.30 -
0.7 0.2 0.6 0.8 0.7 ?
Childhood At birth At birth At birth At birth Childhood
Intestines
Multiple polyposis coil
1.0
0.71
0.8
10-70
Kidney
Polycystic kidney disease
8.0
8.60
0.8
20 & upwards
Nervous
Skeletal
Disease
Neurofibromatosis Huntington disease Primary basilar impression Tuberous sclerosis Myotonic dystrophy Cerebellar ataxia (dominant) Spastic paraplegia Dominant peroneal muscular atrophy (Charcot-MarieTooth disease)
93 5 10 10 28 ? ?
?
8 12 9 1 5 ? 7 76
Dominant forms of early childhood onset deafness Dominant otosclerosis
Monogenic hypercholesterolemia
Congenital spherocytosis
Dentinogenesis imperfecta Amelogenesis imperfecta
Acute intermittent porphyria Variegate porphyria
Cleft lip + cleft palate with mucous pits of lip
Ear
Circulatory
Blood
Teeth
Metabolism
Face
95.0
(29.6) d
0.1
0.1 0.1
1.0 0.2
2.0
58.40
1.30
0.11
0.15 0.15
1.25 0.60
2.20
20.00
10.0
10.0 20.0
0.69
0.30 0.24 0.15 0.40
1.0
1.0 0.3 -
0.5
0.8
0.9 1.0
1.0 1.0
0.8
1.0
1.0
0.3
0.3 0.5 0.9 b 0.7
At birth
Early childhood onwards Early childhood onwards
Childhood Childhood
Childhood
Adult
Adult
Early childhood
At birth Early childhood At birth Early childhood
30
1
--1 c 1¢
< 1¢ -- 1 c
22
< 20 c
< 20 ¢
24
10 6 3 6
a The figures are those published by the respective authors, except that Carter presented them on a per-1000 basis and Childs on a per-million basis; the presence of two decimal places for Childs' figures, does not represent greater accuracy! b Apparent reproductive fitness does not agree with estimated mutation rate (Childs, 1981). c Mutation rate estimates subject to considerable uncertainty (Childs, 1981). d Implied in the overall estimate of total prevalence of 95/104.
Total
Rare diseases of early onset
Dominant forms of blindness Retinoblastoma Aniridia Cataracts with early onset
Eye
8
TABLE IB ESTIMATED LIVEBIRTH PREVALENCES ( x 104) OF X-LINKED AND AUTOSOMAL RECESSIVE DISEASES IN MAN (AFTER CARTER, 1982; CHILDS, 1981) System/organ affected
Prevalence ( × 104) a
Disease
Carter
Childs
X-linked conditions
Neuromuscular Blood clotting Skin Mental retardation (severe)
Duchenne muscular dystrophy. Hemophilia A (factor VIII) Hemophilia B (factor IX) Ichthyosis Non-specific X-linked Rare conditions b Very rare conditions c Total X-linked Rounded
2 1 -1 1 3 8 10
1.80 1.45
0.25 0.60 1.33
0.10 5.53
Common autosomal recessive conditions
Metabolism Nervous system Red blood cells Endocrine glands Ear Eye Severe mental retardation
Cystic fibrosis of pancreas Classical phenylketonuria Neurogenic muscle atrophies Sickle cell anemia Adrenal hyperplasias Severe congenital deafness Recessive forms of blindness Non-specific recessive forms
5 1 1 1 1 2 1 5
Rare autosomal recessive conditions
Metabolism
Tay-Sachs disease Mucopolysaccharidoses I Mucopolysaccharidoses II Metachromatic leukodystrophy Galactokinase deficiency Galactosemia Homocystinuria Cystinuria Total recessives Rounded
0.4 0.2 0.1 0.2 0.1 0.2 0.1 0.6 18.9 25.0
a For X-linked conditions, the estimates pertain to 104 male livebirths. b Those with birth prevalences in the range of 0.1-1/104 males (hemophilia B, X-linked deafness, ocular albinism, X-linked nystagmus, the Burton type of hypogammaglobulinemia, hypophosphatemic rickets, anhidrotic ectodermal dysplasia, and X-linked amelogenesis imperfecta) in Carter's compilation; in those in Childs' compilation, those with birth prevalences in the range of 0.01 0.15/104 males (11 conditions). c Those with birth prevalences of less than 1 per million males.
M e n d e l i a n i n h e r i t a n c e is i n c o m p l e t e . In using the terms "dominant" and "recessive", firstly, it s h o u l d b e r e a l i z e d t h a t t h e c l a s s i c a l c o n cepts of dominance and recessivity have been changing over the years and these designations (which connote attributes of the phenotype), while still q u i t e u s e f u l i n t h e b i o c h e m i c a l o r m o l e c u l a r s e a r c h f o r b a s i c d e f e c t s , h a v e m u c h less s i g n i f i -
c a n c e a t t h e level o f p r i m a r y g e n e a c t i o n . A s M c K u s i c k ( 1 9 8 8 ) p o i n t s o u t , m a n y p h e n o t y p e s in h i s c a t a l o g u e c l a s s i f i e d as d o m i n a n t s a r e i n c o m p l e t e l y so (i.e., t h e m u t a n t h o m o z y g o u s s t a t e l e a d s to a more severe and somewhat different phenot y p e ) a n d l i k e w i s e m a n y o f t h o s e c l a s s i f i e d as r e c e s s i v e s a r e i n c o m p l e t e l y r e c e s s i v e (i.e., m i l d m a n i f e s t a t i o n s a r e d i s c e r n i b l e i n h e t e r o z y g o t e s if
appropriate methods are used). Further, the concept that the genotype determines the phenotype precisely (i.e., if a condition is dominant, it is always manifested in the heterozygote and if it is recessive, all appropriate homozygotes exhibit the condition) is not always true. The human genetic literature is replete with examples of Mendelian conditions which have reduced penetrance a n d / o r variable expressivity. Secondly, there is the phenomenon of codominance (both alleles are expressed in the heterozygote) which is characteristic of many enzyme variants and which can be detected by electrophoretic methods. For instance, the erythrocyte enzyme adenosine deaminase (autosomal dominant; McKusick No. 10270) has three genetically determined electrophoretic phenotypes and shows polymorphism. Adenosine deaminase deficiency however is a recessive disease. Thirdly, there are conditions which, although formally classified under "recessives", have been found to be caused by mutations in different codons of the gene in question (i.e., the phenotype of the affected individual is not due to homozygosity for mutation at the same site); in other words, they are "genetic compounds". Most flthalassemia clinical "homozygotes" for instance are genetic compounds. Fourthly, there are situations exemplified by conditions such as retinoblastoma: susceptibility to retinoblastoma can be transmitted as a dominant; however, for a tumor to develop, a mutation must also occur at the same site on the other normal chromosome 13 - - a somatic mutation. Thus, in a strict sense, retinoblastoma is recessive since homozygosity is required for its expression. Finally, there are mosaics. Mosaicism for constitutional chromosomal abnormalities such as sex-chromosomal anomalies, Down syndrome, etc. (detected in lymphocyte cultures) has long been known. Only in recent Years, however has the fact that mosaics for single gene mutations can occur begun to be appreciated, especially through the use of D N A techniques. While any condition appearing in two or more affected children of unaffected parents may be reasonably assumed to be a recessive, it cannot be readily distinguished from the effects of germ-line mosaicism for a dominant mutation in that generation. It is also possible that
with some dominant mutations, because of the severity of the effect (if present in all cells of the body), only somatic mosaicism m a y be compatible with viability. Examples of conditions where germ-line mosaicism has actually been demonstrated or strongly inferred include Duchenne muscular dystrophy, hemophilia A, ornithine t r a n s c a r b a m y l a s e deficiency, achondroplasia, pseudochondroplasia, Apert syndrome, Crouzon syndrome, osteogenesis imperfecta type II, tuberous sclerosis, aniridia, etc. (reviewed in Hall, 1988; see also Edwards, 1989). In view of these and other reasons, some arbitrariness had to be exercised in classification in McKusick's catalogue: many (although not all) enzymes have been listed under dominants because of codominant polymorphism or under recessives because of deficiency.
Epidemiological aspects Overall prevalences The current consensus estimates of livebirth prevalences (UNSCEAR, 1988) are about 95/104 for autosomal dominants, 5 / 1 0 4 for X-linked ones and 25/104 for autosomal recessives (Table IA,B). The figure for autosomal dominantsis derived from several ad hoc studies (see Carter, ]977, 1982 for reviews) and is based on conditions with individual prevalences in the range of 1 20 per 104 livebirths ( " c o m m o n dominants"; about a dozen conditions) and of 1 - - 5 per 105 livebirths ("less common dominants"; also about a dozen conditions). The resulting total of 65.4/104 has been rounded upwards to about 95/104 to take into account the much rarer conditions. The estimates of Childs (1981) for basically the same autosomal dominant conditions (with some modifications) yield a slightly lower total prevalence figure of 58.4/104 livebirths (see Table IA). For X-linked recessives, Carter's estimate of 5/104 livebirths (Table IB) is based on conditions whose individual prevalences are equal to or greater than 1/104 male livebirths (four conditions; total prevalence, 5/104 male livebirths) and on those for a number of rarer conditions (estimated total of the order of 3,/104 male livebirths). The resulting total of 8 / 1 0 4 male livebirths was divided by 2 and rounded upwards to 5 / ] 0 4. The estimates of Childs for the total prevalence of
10 TABLE II A COMPARISON OF THE PREVALENCE ESTIMATES FOR SOME SELECTED AUTOSOMAL DOMINANT AND X-LINKED CONDITIONS MANIFEST AT BIRTH BETWEEN HUNGARIAN STUDIES AND THOSE PUBLISHED IN THE LITERATURE a MIM No. b
Conditions
Prevalence per 104 livebriths c Hungary
Other countries
A utosomal dominants
10080 10120 10620 11620 11930 12105 12350 12990 14290 15450 16620 17470 18020 18360 18760 19407
Achondroplasia Apert syndome Aniridia, bilateral Cataract, congenital bilateral Van der Woude syndrome Contractural arachnodactyly Crouzon syndrome EEC syndrome Holt-Oram syndrome Treacber-Collins syndrome Osteogenesisimperfecta, type I Polysyndactyly,preaxial, type IV Retinoblastoma Split hand and/or foot Thanotophoric dwarfism Wilmstumor Total
0.56 0.09 0.03 0.38
0.29-0.83 0.06-0.29 0.18 0.4
0.01
0.11
0.02
d
0.10 0.03 0.14 0.06
d 0.04 0.07 0.14
0.26
0.40
0.91
d
0.32 0.14 0.08 0.40 3.53
0.24-0.33 0.15 0.58 0.40
Hemophilia A Hemophilia B Incontinentiapigmenti Martin-Bell syndrome Duchenne muscular dystrophy Gorlin-Psaume syndrome Total
2.73 0.25 1.13 5.40 2.50
1-2 0.2-0.3 1.22 4.40 2.50
0.04
0.02
X-linked conditions
30670 30690 30890 30955 31020 31120
12.05
a From Czeizel (1989) and Sankaranarayanan and Czeizel (1990); these articles also provide references to the Hungarian and international studies; the autosomal dominant conditions have been ascertained through the program of the Hungarian Surveillance of Sentinel Anomalies (1980-1986) and involve 1,061,879 livebirths. For X-linked conditions, the sources are the following: 30670, 30690 and 30955: ad hoc epidemiological studies; 30830 and 31120: Hungarian Surveillance of Sentinel Anomalies; 31020: regional screening. b Mendelian Inheritance in Man (McKusick, 1988) numbers. c In the case of X-linked conditions, per 1 0 4 male livebirths. d Not available.
X-linked c o n d i t i o n s ( 5 . 5 3 / 1 0 4 male births or 2 . 8 / 1 0 4 total births) are lower. F o r a u t o s o m a l recessive diseases (Table IB), Carter's figure of 2 5 / 1 0 4 livebirths is based o n eight c o n d i t i o n s with i n d i v i d u a l prevalences equal to or greater t h a n 1 / 1 0 4 (total 1 7 / 1 0 4 ) a n d a n o t h e r eight with lower i n d i v i d u a l prevalences (total 1.9/104). The total for all a u t o s o m a l recessives is thus 1 8 . 9 / 1 0 4 a n d this figure has been adjusted u p w a r d s (to take i n t o a c c o u n t the very rare conditions) to 2 5 / 1 0 4 . A l t h o u g h there a p p e a r to be n o reasons to seriously q u e s t i o n the overall livebirth prevalence estimate of 2 5 / 1 0 4 for these diseases, a r g u m e n t s / s u g g e s t i o n s have b e e n advanced to s u p p o r t the view that the frequency of homozygotes for recessives at conception could be m u c h higher t h a n this figure (Neel, 1978; Vogel a n d Motulsky, 1986; Searle a n d Edwards, 1986). O n e aspect of M e n d e l i a n diseases hitherto not m e n t i o n e d pertains to the l o n g - s t a n d i n g evidence for a n increase in s p o n t a n e o u s m u t a t i o n frequencies with increasing p a t e r n a l age (e.g., a c h o n d r o plasia, A p e r t syndrome, M a r f a n s y n d r o m e , osteogenesis imperfecta, etc.) (Carothers et al., 1986; Evans, 1988; see also C h a p t e r 5 in Vogel a n d Motulsky, 1986). The rates of increase however differ b e t w e e n genes. The causes for these increases r e m a i n unresolved. It must be realized that the entries in T a b l e IA,B - although they p r o v i d e us with some insights into the load of M e n d e l i a n diseases in the h u m a n species - represent a synthesis of i n f o r m a tion from different p o p u l a t i o n s . Therefore, they do n o t reflect either the profile or the aggregate b u r d e n of such diseases in a n y specific h u m a n p o p u l a t i o n ; ideally, however, it is this which is required to project risks in context.
Clinical onset
As indicated in T a b l e IA, m a n y of the recognized " c o m m o n " d o m i n a n t s i n c l u d e d in these listings first a p p e a r in adult life (e.g., H u n t i n g t o n disease (prevalence: 5 / 1 0 4 ) , familial hypercholesterolemia ( 2 0 / 1 0 4 ) , polycystic k i d n e y disease ( 8 / 1 0 4 ) , d o m i n a n t a d u l t o n s e t otosclerosis ( 1 0 / 1 0 4 ) ) . I n contrast, m a n y of the "less comm o n " a n d " r a r e " d o m i n a n t c o n d i t i o n s are m a n i fest at b i r t h or in early childhood. Estimates of b i r t h frequencies for some of these latter conditions, from the o n g o i n g p r o g r a m m e of H u n g a r i a n
11 Surveillance of Sentinel Anomalies, have been summarized by Czeizel (1989) and Sankaranarayanan and Czeizel (1990) and are shown in Table 2. The total livebirth prevalence for these rare conditions listed in this table is only 3.53/104 which is less than 4% of the estimated total of 95/104 . The livebirth prevalence figure of Baird et al. (1988) for dominant conditions in British Columbia diagnosed through the first 25 years of life is about 14/104 , and this is about 15% of the estimated total of 95/104 . In contrast to autosomal dominants, most Xlinked and autosomal recessive diseases have onset in infancy or childhood and are generally clinically more severe. As will be discussed later, these differences in onset age seem related to the function impaired as a result of the mutation and the "natural history" of the disease process.
Some population genetic aspects As stated earlier, naturally occurring mutations constitute the basis for Mendelian diseases. Although new mutations arise spontaneously in each g e n e r a t i o n , the p o p u l a t i o n frequencies of Mendelian diseases remain stable. Therefore, there must be mechanisms that operate to maintain this stability. Exposure of a population to radiation results in the introduction of additional mutant genes in its gene pool (over and above those that arise spontaneously) and their rates of persistence and elimination will determine how m a n y additional cases of Mendelian diseases will result in any given generation. An understanding of the mechanism(s)/factors t h a t m a i n t a i n gene frequency equilibria is thus of paramount importance both for explaining the natural prevalence of Mendelian diseases and for estimating expected increases as a result of exposure to radiation. At a more general level, these questions relate to mechanisms of maintenance of population variability of which the maintenance of stable frequencies of Mendelian diseases is just one aspect. All the mechanisms postulated to explain the maintenance of natural variability in a population depend on some kind of balance between opposing forces and are adequately dealt with in population genetics text books (see for instance, Li, 1955; Crow and Kimura, 1970). Crow and Kimura
(1970) list eight possible mechanisms for maintaining population variability noting that "these are neither mutually exclusive nor collectively exhaustive". Of these, four are very briefly considered here: (i) balance between mutation and selection; (ii) selection favoring heterozygotes; (iii) neutral polymorphism; and (iv) transient polymorphism.
Mutation-selection balance Balance between mutation and selection represents one, perhaps a major, mechanism in maintaining population variability. The theory is that in each generation, new mutations arise and most of them are harmful (although to different degrees) to their carriers (i.e., they affect their "fitness") and are eliminated by natural selection sooner or later. At equilibirum, a balance is achieved between mutation rate and the rate of selective elimination. The term "fitness" as used here refers to reproductive fitness and is not always synonymous with disease. Consider a mutation that leads to a rare autosomal dominant disease which is strongly selected against. The birth frequency (rn) of the disease is directly proportional to the mutation rate (u) and to the number of generations that the mutant gene persists in the population; the latter is inversely proportional to the decrease in fitness ( f ) . The relationship b e t w e e n m u t a t i o n rate, birth frequency and fitness for these is given by the well-known equation u=m(1
-f)/2
(1)
This formula has been used to estimate most of the mutation rates summarized in Table 1A. For an X-linked condition maintained by m u t a t i o n selection balance, the principle is the same: if the fitness of the mutant males is 1 - f relative to normal males and heterozygous females are not impaired, then, u = m(1 - f ) / 3
(2)
where m represents the birth frequency in males. For autosomal recessives, the situation is complicated (see Morton, 1981; Crow and Kimura, 1970; Crow and Denniston, 1985 for detailed discussions) and will not be gone into here. It suffices
12 to note that (i) the phenotypic incidence of recessives is not directly related to mutation rates; (ii) estimates of mutation rates for recessive mutations depend on the assumption that at some time an equilibrium was reached between spontaneous mutation and loss by selection (although reduction in inbreeding in recent generations makes current equilibrium untenable); and (iii) in calculating mutation rates, one should take into account elimination of mutant alleles through their effects in heterozygotes, through consanguinity and through the mutant gene's meeting a preexising, non-complementing mutant allele by chance.
Selection favoring heterozygotes Another mechanism that can maintain population variability relies on the concept of superiority of fitness of heterozygotes, relative to that of the homozygotes. In a simple two-allele situation (say, A 1 and A 2 ) if the heterozygote (AIA2) has higher fitness relative to both homozygotes (A1A a and A z A 2 ) , selection will result in an equilibrium and both A x and A 2 will continue to occur in the population indefinitely. The population frequencies of the alleles are entirely determined by the selection coefficients against the homozygotes. Letting s~ and s 2 denote the selection coefficents for the two homozygotes (fitness being (1 s~) for the A~A 1, (1 - s2) for the A 2 A 2 and 1 for the A~A2), it can be shown that equilibrium will be attained when the frequencies in the gene pool are equal to s2/(s 1 + s 2) for the A 1 allele and s~/(sa + s2) for the A 2 allele. This situation is referred to as balanced polymorphism. There is a vast amount of literature on the existence and importance of balanced p o l y m o r phism (due to chromosomal inversions) in natural populations of Drosophila and these studies date back to the 1950s (e.g., Dobzhansky, 1951). In man, evidence strongly suggestive of balanced polymorphism is provided by the sickle-cell hemoglobin heterozygote which has neither the anemia of one homozygote nor the susceptibility to malaria of the other (Allison, 1954a,b, 1955). Associated geographically with malaria are two other polymorphic genetical defects, fl-thalassemia and glucose-6-phosphate dehydrogenase deficiency, and it seems probable that similar mechanisms of hetero-
zygous advantage to the malarial challenge have played a role in their high frequencies in endemically malarial areas (e.g., Luzzato et al., 1969; Undevia, 1973; Motulsky, 1975; Miller et al., 1976).
Neutral polymorphism " I f a series of alleles are nearly equivalent in their effect on fitness (near enough that the differences among them are of a lower order than the mutation rates or the reciprocal of the effective population number), there may be neutral polymorphism in which the frequencies of the different types are determined largely by their mutation rates. In such a situation, the effects of a finite population become especially important and the number of alleles actually maintained will be determined between mutation and random extinction through gene frequency drift" (Crow and Kimura, 1970). Evidence for the existence of potentially neutral polymorphisms in man, originally provided by protein studies, has now been extended at the D N A level through the demonstration of restriction fragment length polymorphisms (RFLPs). At their simplest, the R F L P s reflect base-pair changes which create or destroy a cleavage site for a specific restriction enzyme, causing a change in the length of a D N A fragment (e.g., W y m a n and White, 1980; Jeffreys et al., 1985a,b, 1988; Kovacs et al., 1990). Several hyperpolymorphic V N T R (Variable N u m b e r of Tandem Repeat) loci are known in the human genome. The mutation rates to new length alleles vary substantially from locus to locus; for some of these, these estimates rates are quite high (e.g., 1 × 1 0 - 2 / g a m e t e (Kovacs et al., 1990); 5 × 1 0 - 2 / g a m e t e (Jeffreys et al., 1988)).
Transient polymorphism The mechanisms thus far mentioned are all stabilizing ones. However, a population may be polymorphic because it is in the process of change and a transient polymorphism may persist for a long time and in nature this may be very difficult to distinguish from a stable polymorphism (Crow and Kimura, 1970).
13 What mechanisms can be invoked to explain the prevalence of Mendelian diseases in human populations? In inquiring about mechanisms for the maintenance of Mendelian diseases in the population, one should bear in mind the following facts. Firstly, the "stability" of the frequencies is a convenient assumption and in fact, there is no irrefutable epidemiological evidence on this point nor would one expect such evidence to be easily obtained. Secondly, it is not a simple matter to apply the concept of reproductive fitness in most present-day human populations; this is because family sizes are dictated by social and cultural considerations (in which individual choices play a dominant role) and not by purely biological ones and reproductive compensation is not uncommon; these realities do not however detract from the possibility that differences in reproductive fitness could have existed in early periods of human history. Thirdly, the dynamics of mutant genes in the past determines their present prevalences and provides a basis for future predictions; during the last 3 - 4 generations or about 100 years, human populations have experienced dramatic changes in living conditions. It would thus be an oversimplification to treat selection as constant over long periods of time. Fourthly, with conditions such as Huntington disease, a positive family history has traditionally been one of the criteria by which the clinician decides as to whether a case in question is likely to represent a given disease entity or not; it is not surprising, therefore, that one does not find " n e w " mutations. The point is that the ascertainment of a specific heritable disease is rarely as random as one would like it to be. Fifthly, the data can appear to be consistent with more than one mechanism, and in such cases, statements about the most probable mechanism are essentially matters of informed judgement. Finally, population genetic considerations are relatively straightforward if a disease can be considered as a single, homogeneous entity; however, as data on molecular heterogeneities of mutations underlying Mendelian diseases continue to accumulate, another dimension of complexity is introduced; for instance, the molecular nature of the
mutation and the bodily s y s t e m / f u n c t i o n affected may have an important bearing on onset age (the latter could be very early for some large deletions so that they are eliminated through early embryonic losses a n d / o r spontaneous abortions) and thus, for the determination of selection coefficients. As stated earlier, the relevance of the question of mechanisms in the context of the present series of papers stems from the theory that the prevalence of diseases (i.e., the respective mutant genes) maintained by mutation-selection balance would, in principle, be expected to increase as a result of exposure to radiation. Since the prevalence of autosomal recessive diseases is only very indirectly related to mutation rate, in what follows, only autosomal dominants and X-linked recessives will be considered. It is safe to assume that most of the rare autosomal dominant and X-linked conditions listed in Table 2 (and some additional ones in Table 1A,B such as neurofibromatosis, Marfan syndrome and autosomal dominant forms of osteogenesis imperfecta) are likely to be maintained by mutation-selection balance because of onset at birth or in childhood and severity of effect which preclude reproduction in most (if not all) cases. The total prevalence of these " b i r t h or childhood onset" conditions is only 3.53/104 in the population of Hungary (Table 2) and for those with onset through the first 25 years of life, 14/104 in British Columbia (Baird et al., 1988). These values represent less than 5 and 15% respectively of the estimated total prevalence of autosomal dominant diseases (95/104). With the relatively common dominants such as familial hypercholesterolemia, adult form of polycystic kidney disease, Huntington disease, myotonic dystrophy and dominant otosclerosis (which together account for nearly one-half of the total birth prevalence of 95/104 ) the situation is not simple; these conditions are compatible with a substantial degree of reproductive fitness because they are of relatively late onset. Searle and Edwards (1986) speculated that " s o m e severe dominants, including Huntington's chorea and myotonic dystrophy, so rarely appear as new mutants in well-conducted surveys (Harper, 1979; Reed et al., 1958; Shaw and Caro, 1982)
14 that their population frequency may well be maintained by some sort of heterozygous advantage during the reproductive period rather than by mutation .... The dominant hyperlipidemias and hypercholesterolemias are also so common that they are unlikely to be maintained by mutation." The problem, however, is our current total ignorance of the nature of this advantage and of the biological mechanisms involved. Differences of opinion exist on the mechanisms of maintenance of Huntington disease (HD). H D was inferred to be selectively deleterious in a Michigan population (Reed and Neel, 1959), selectively neutral in a Queensland population (Wallace and Parker, 1973) and selectively advantageous in both a Minnesota (Marx, 1973) and a Welsh population (Walker et al., 1983). In a more recent paper on H D in the Afrikaners of South Africa, Stine and Smith (1990) concluded that the current H D gene frequency estimate of 4.2 x 10 -5 in this population (based on 210 H D cases who were descendants of an original settler who was a presumed heterozygote, identified among 2.5 x 10 6 Afrikaners) is consistent with a selection coefficient of about 0.34 (i.e., the H D mutation is deleterious). It is worth noting that attempts to explain the relatively high prevalences of conditions such as familial hypercholesterolemia (FH) on models based on neutral or transient polymorphisms is also not free of problems. Such an explanation would require that thr allele frequencies are largely determined by a balance between mutation and random extinction through gene frequency drift. The concepts of "founder effect" and gene frequency drift have in fact been used to explain the high frequencies of homo- and the heterozygous cases of F H in Afrikaners and French Canadians (Seftel et al., 1980; Brink et al., 1987; Hobbs et al., 1987). However, although these mechanism cannot be excluded, they do not seem compatible with the widespread nature of F H in different populations. For FH, the birth frequency estimate (i.e., that of heterozygotes) is about 20/104, but less than 1% of F H cases are presumed to be due to new mutations. (This appears to be true for H D and adult polycystic kidney disease as well; see chapter 5, page 419 in Vogel and Motulsky, 1986.) The
frequency of F H homozygotes is generally of the order of about 10 6. If the F H mutations are selectively neutral or near-neutral in heterozygotes, one can formally estimate that the mutation rate is about one-half of that of new mutants (i.e., one half of 1% of 20/104); this is about 10 5 which is not unduly high. However, this figure depends critically on the estimated proportion of sporadic cases; if the latter is much lower than assumed, then the mutation rate estimate will be correspondingly lower. It is conceivable that, in the absence of selection against heterozygotes, the condition is maintained in the population through selection against homozygotes for the dominant mutation (these individuals die in their teens or early 20s). If this assumption is correct, in a hypothetically large population in which the "gain" of mutant alleles is balanced by their "loss" in homozygotes (and if there is no inbreeding), it can be shown that the mutation rate will be equal to the frequency of the homozygotes which is 10 6, i.e., an order of magnitude lower than that estimated in the preceding paragraph. If F H and H D are illustrative of the relatively common dominant conditions, the principal message from this discussion is that the available data may be compatible with more than one mechanism of maintenance of mutant genes involved. This in turn means that the assumption implicit in the use of one principal method of risk estimation (to be dealt with in paper IV), namely that all autosomal dominant and X-linked diseases are maintained in the population through a balance between mutation and selection, may need to be carefully qualified. Biochemical basis of Mendelian diseases General considerations A great variety of different enzymes and other non-enzymic proteins are synthesized in the cells of every multicellular eukaryote. On general biochemical grounds, one would expect that mutations in genes that affect the nature, synthesis, stability or quantity of enzymic proteins are likely to be reflected in the phenotype only in the homozygote (or in hemizygotes in the case of mutations in X-linked genes), i.e., formally, they are "reces-
15 sives". This is so b e c a u s e o f the facts that (i) in vivo, the enzymes d o not act in isolation, b u t are k i n e t i c a l l y linked to o t h e r enzymes via their substrates a n d p r o d u c t s a n d (ii) few enzymes are so r a t e - l i m i t i n g as to cause a serious r e d u c t i o n in the f u n c t i o n i n g of a m e t a b o l i c p a t h w a y when the enz y m e has 4 0 - 6 0 % n o r m a l activity, which is generally the case in heterozygotes. M o s t i n b o r n errors of m e t a b o l i s m are recessives and, as K a c s e r a n d Burns (1981) stated, " t h e w i d e s p r e a d o c c u r r e n c e of recessives is... an inevitable c o n s e q u e n c e of the kinetic structure of e n z y m e n e t w o r k s . " I n contrast, m u t a t i o n s in genes that c o d e for non-enzymic p r o t e i n s or for enzymes that effect changes in the structure of n o n - e n z y m i c p r o t e i n s (e.g., collagen, several p r o t e i n s involved in b l o o d c o a g u l a t i o n , c o m p l e m e n t system, t r a n s p o r t p r o teins, b i n d i n g proteins, etc.), in general, w o u l d be e x p e c t e d to result in " d o m i n a n t " p h e n o t y p e s . This is b e c a u s e the " b u i l t - i n safety f a c t o r " c o m m o n to m o s t e n z y m e systems does not exist with the result that the p r o d u c t of a n o r m a l allele is likely to be insufficient to m a i n t a i n h e a l t h y function a n d / o r the m u t a n t p r o d u c t interferes in some w a y with that of the n o r m a l allele a n d thus with the develo p m e n t of the n o r m a l p h e n o t y p e . However, instances are k n o w n where e n z y m e deficiencies result in " d o m i n a n t " p h e n o t y p e s (e.g., the five different forms of p o r p h y r i a ) . This k i n d of situation m a y occur when the e n z y m e in q u e s t i o n h a p p e n s to b e r a t e - l i m i t i n g in the m e t a b o l i c p a t h w a y in which it takes part, b e c a u s e the level of activity of such enzymes in the n o r m a l situation will in general b e closer to the m i n i m u m r e q u i r e d to m a i n tain n o r m a l function (Harris, 1970) or as a result of c h a n g e in the specificity of the enzyme. Likewise, deficiencies or a b n o r m a l i t i e s in n o n - e n z y m i c p r o t e i n s l e a d i n g to " r e c e s s i v e " p h e n o t y p e s are also known, a n d can arise as a result of m u t a t i o n s in genes whose p r o d u c t s are involved in r a t e - c o n trolled processes (see M c K u s i c k , 1986, 1988 for examples).
Actual observations T a b l e III, e x t r a c t e d f r o m A p p e n d i x A of McK u s i c k (1988), s u m m a r i z e s our c u r r e n t k n o w l e d g e on a b o u t 400 M e n d e l i a n c o n d i t i o n s with d e f i n e d b i o c h e m i c a l a b n o r m a l i t i e s . As can b e seen, defects in enzymic proteins more often result in recessives
TABLE III GROUPING OF MENDELIAN DISORDERS ACCORDING TO THE NATURE OF THE IDENTIFIED BIOCHEMICAL DEFECT a Defect in
Enzymic protein Non-enzymic protein
Autosomal Autosomal X-linked Total dominant recessive n % n n % n % 34 14.9 179 78.5 15 101 56.7
6.6 228 100
59 33.1 18 10.1 178 100
a Based on McKusick (1988). In this table, horizontal comparisons (percentages of the different Mendelian classes) within each of the two groups are unlikely to be biased (see text); however, vertical comparisons ('enzymic' versus 'non-enzymic' among the three Mendelian groups) will be biased since, until now, more abnormalities have been detected in enzymes (or deficiencies) than in non-enzymic proteins.
than dominants (179 a u t o s o m a l recessives to 34 d o m i n a n t s , o r a ratio of 5.3 to 1) while those in non-enzymic structural proteins are more likely to result in dominants than recessives (101 a u t o s o m a l d o m i n a n t s to 59 recessives or a r a t i o of 1.7 to 1). These d a t a are thus c o n s i s t e n t with theoretical expectations, c o n s i d e r i n g the fact that there is s o m e a r b i t r a r i n e s s in M c K u s i c k ' s classification discussed earlier (see also H o o k , 1987). It is also w o r t h n o t i n g that M e n d e l i a n c o n d i t i o n s for which the a c t u a l b i o c h e m i c a l basis is k n o w n r e p r e s e n t o n l y a small subset of the over 4000 c o n f i r m e d plus p r o b a b l e ones. Molecular basis of Mendelian diseases General considerations In c o n s i d e r i n g the m o l e c u l a r basis of M e n d e l i a n diseases, o n e should recognize three i m p o r t a n t facts. First, n o t all m u t a t i o n a l changes result in disease a n d those that do, r e p r e s e n t b u t a small p r o p o r t i o n of changes p o t e n t i a l l y possible. O n e a l r e a d y k n e w this to b e true from, a m o n g others, o b s e r v a t i o n s on the w i d e s p r e a d o c c u r r e n c e of p r o tein p o l y m o r p h i s m s a n d the fact t h a t o n l y s o m e of these are a s s o c i a t e d with clinical disease. N o w , research in m o l e c u l a r b i o l o g y h a s s h o w n t h a t varia t i o n s in D N A sequence ( c a u s e d b y n u c l e o t i d e substitutions, d e l e t i o n s a n d d u p l i c a t i o n s ) w i t h i n
13006 13050
12310
11845 11930
Hereditary persistence of fetal hemoglobin, deletion type Methemoglobinemia, hemoglobinopathic, of Hb M (beta type) Sickle cell anemia Hereditary persistence of fetal hemoglobin, non-deletion type A Hereditary persistence of fetal hemoglobin, non-deletion type G
Adenine phosphoribosyltransferase deficiency Severe combined immunodeficiency Amyloidosis, neuropathic, type I Amyloidosis, neuropathic, type II Amyloidosis, Icelandic, cerebrovascular type Thrombophilia, familial, due to antithrombin III deficiency Thrombophilia, familial, due to defect in protein C Alpha-l-antitrypsin deficiency Hypoalphalipoproteinemia Alagille syndrome Van der Woude syndome, mental retardation Craniosynostosis, mental retardation Ehlers-Danlos syndrome, type VIIAI Elliptocytosis, Rh-linked Methemoglobinemia, hemoglobinopathic, of Hb M (alpha type) a-Tlialassemia fl-Thalassemia
10260
10480 10490 10515
Disorder
MIM
17686 10740 10768
Protein C Alpha-l-antitrypsin Apolipoprotein A-I
14190 14190 14220 14225
fl-Globin yA-Globin TG-Globin
PMp
PMm PMp
PMm
PMm PMm, PMn, PMp, PMrp, PMs, PMt
14180 14190
fl-Globin
PMm
14180
14190
PMs?
PMm PMm
PMm, PMn
PMm
PMm PMm PMm PMm
LMd, LMr
LMd
LMd LMr
LMd
LMd LMd
LMd
LMd
2,4
2,4
1
1
2,4
1
1
1
1,2 1
2
2,3 3 2 1,2
3
1,2
1 3 3 3
3
detection
PMm
Onset/
PM
LM
Type(s) of
12016
fl-Globin
/3-Globin
a-Globin
Procollagen, type I Protein 4.1 et-Globin
10730
Antithrombin III
11930
10270 17630 17630
MIM
Adenosine deaminase Prealbumin (transthyretin) Prealbumin (transthyretin) Gamma-trace (cystatin C)
A utosomal dominants
Defective gene product
The first MIM number (McKusick, 1988), on the left, refers to an entry for the disorder; the second MIM number, on the right, refers to an entry for a particular molecule (gene product). Sometimes, when it is not certain that the given molecule is the site of the defect, two entries exist in McKusick (1988) and both are given.
INTRAGENIC LESIONS IDENTIFIED IN MENDELIAN DISORDERS IN MAN (BASED ON MCKUSICK, 1988; HARPER et al., 1989 WITH SOME ADDITIONS)
TABLE IV
20775 21410 22402 22960 23080 23205 23440 24665
20332
20191
19340 19407
19035
18020 18050 18290 18840 18840
17570 17610 17627
17280 17510
15023 16621
14389
Albinism, oculocutaneous (tyrosinase deficient) Apolipoprotein C-II deficiency Zellweger syndrome Hyperlipoproteinemia, type III Fructose intolerance, hereditary Gaucher disease Propionicacidemia, type II Angelman syndrome Lipoprotein lipase deficiency
Pyruvate dehydrogenase E-I deficiency Adrenal hyperplasia
Immunodeficiency, T cell, with neurological disorder Piebald trait, mental retardation Adenomatods polyposis, mental retardation Greig cephalopolysyndactyly Porphyria cutanea tarda Prader-Willi syndrome Diabetes mellitus, rare forms Retinoblastoma Rieger syndrome Spherocytosis I, mental retardation DiGeorge sequence DiGeorge sequence, mental retardation Hemolytic anemia due to triosephosphate isomerase deficiency Trichorhinophalangeal syndrome, type I, mental retardation yon Willebrand disease WAGR syndrome
Giedion-Langer syndrome Osteogenesis imperfecta congenita, type II
Kappa light chain deficiency
Hypercholesterolemia, familial
Lipoprotein lipase
Apotipoprotein E Fructose-l-phosphate aldolase Acid fl-glucosidase Propionyl-CoA-carboylase
Apolipoprotein C-II
Pyruvate dehydrogenase E-I a subunit Steroid cytochrome P450-21hydroxylase Tyrosinase
A utosomal recessives
von Willebrand factor
Triosephosphate isomerase
19045
PMm, PMs PMm PMm
PMm
PMm
PMm
Insulin Retina-specific protein
PMm
PMm
PMm
PMm 17673
16405
12015 12016
14720
PMm, PMn
Uroporphyrinogen decarboxylase
al-Chain of type I collagen; a2-chain of type II collagen Purine nucleoside phosphorylase
Low defisity lipoprotein receptor Immmunoglobulin kappa constant chain
LMd LMd LMd
3 2 3
1,2,3
2,3 1 3 2,3
1
LMd, LMr
LMd LMd
3
2,3 3
LMd LMd LMd
1
3 1,2 3 2 2 2 1 1
1
2,3 3
1,2
1,2 1
4
3
LMd
LMd LMd LMd LMd LMd
LMd
LMd
LMd LMd
LMd LMd
LMd, LMr
30150 30310
30020 30050
27280 27660
26160 26240 26880
Adrenal hypoplasia (Addison disease) Ocular albinism (Nettleship-Falls type OA1) and ichthyosis Fabry disease Choroideremia
Miller-Dieker lissencephaly syndrome Omithinemia with gyrate atrophy of choroid and retina Phenylketonuria Pituitary dwarfism I Sandhoff disease (GMs-gangliosidosis, variant 0) Tay-Sachs disease Tyrosinemia II (Richner-Hanhart syndrome)
24720
25887
Disorder
MIM
TABLE IV (continued)
a-Galactosidase A
X-linked
Phenylalanine hydroxylase Growth hormone /3-Hexosaminidase A & B, fl-chain B-Hexosaminidase A, a-chain Tyrosine transaminase
Ornithine ketoacidaminotrans ferase
Autosomal recessioes
Defective gene product
13925
MIM
PMm, PMs
PMm, PMi
PM
Type(s) of
LMd, LMdu LMd LMd
LMd LMd
LMd LMd
LMd LMd
LMd
LMd
LM
2 2
1,2 2
1
2
1
Onset/ detection
Microphthalmia, iridoschisis, goiter, labium synechia, craniotabes Muscular dystrophy, Becket & Duchenne Norrie disease Ornithine transcarbamylase deftciency Pelzaeus-Merzbacher disease
Choroideremia, deafness, mental retardation Color blindness Granulomatous disease, X-linked Hemophilia A Hemophilia B Glycerol kinase deficiency, adrenal hypoplasia, hypogonadotropic hypogonadism, mental retardation Ichthyosis, chondrodysplasia punctata, Kallman syndrome, mental retardation Lesch-Nyhan syndrome
Omithine transcarbamylase
Dystrophin
PMm
PMm
LMd, LMr, LMdu LMd LMd
LMd, LMdr, LMdu LMd
PMm
Hypoxanthine guanine phosphoribosyltransferase
30800
LMd
PMm, PMn PMm, PMn
LMd, LMdu LMd LMd, LMi LMd, LMi LMd
Steroid sulfatase
Cytochrome b (-245), fl-chain Factor VIII Factor IX
1
2,3 1
2
3
1
1
2 2 1 1 2
PM, point mutations; PMi, PM in initiator codon; PMm, PM, missense type; PMn, PM, nonsense type; PMp, PM in promoter, PMrp, PM affecting RNA processing (other than splicing); PMs, PM affecting RNA splicing; PMt, PM in terminator codon; LM, length mutation; LMd, deletion; LMi, insertion; LMr, rearrangement; LMdu, duplication. Onset/detection: 1, at birth; 2, childhood; 3, adults; 4, can be asymptomatic.
31208
31060 31125
31020
30970
30810 30295
30390 30640 30670 30680 30703, 30020
30310
20 human populations, as revealed through the use of restriction enzymes, are common (e.g., Botstein et al., 1980; Cooper and Schmidtke, 1984; Jeffreys et al., 1986; N a k a m u r a et al., 1987; Mohrenweiser et al., 1989, 1990). Most of these R F L P variants do not seem to have functional significance or association with any discernible clinical phenotypes. A few of course represent mutations known to be associated with diseases (e.g., sickle-cell mutation; Kan and Dozy, 1978). Second, most Mendelian diseases are heterogeneous, phenotypically and at the molecular genetic level: genetic heterogeneity in terms of more than one locus for a single clinical phenotype as well as multiple disease phenotypes determined by a single locus have been recognized for many years. Molecular biology has taught us that heterogeneity in terms of the underlying molecular changes is also common. The latter is due to the fact that a wide variety of molecular changes is theoretically possible - and does occur, as revealed by D N A analysis although the number of clinical and biochemical phenotypes that result is often limited, as was first extensively documented in the case of globin gene mutations. Third, the kind of changes that one sees in a mutant would be expected to be dependent on the D N A sequence organization and function of the normal gene in the organism, the kind of changes it can tolerate and whether the loss of the gene or alteration of its function is compatible with viability and thus recoverable for study. When a recessive mutation first arises, it is "sheltered" by the wild-type allele in the heterozygote; therefore, for these mutations, a wide array of molecular changes is possible. These changes can be specific point mutations (base-pair changes) in the gene or more drastic events such as intragenic deletions, multilocus deletions (provided such multilocus loss is not incompatible with viability), rearrangements, etc. In contrast, for most dominant mutations, the kinds of recoverable molecular changes are likely to be less since any major disturbance in normal function due to total gene deletions or rearrangements will be incompatible with viability, i.e., there are functional constraints.
Actual obseroations As of 1989, information on gene localization
(to specific c h r o m o s o m e s / c h r o m o s o m a l regions) is available for 464 clinical disorders (Harper et al., 1989) and the molecular changes underlying 76 Mendelian conditions have been ascertained, although to different levels of precision and completeness (McKusick, 1988; Harper et al., 1989 and other published results). Following McKusick (1988), one can classify these changes into two principal groups, namely point mutations (PMs) to include base pair changes anywhere in the gene and length mutations (LMs) to include D N A deletions (small and large), insertions, rearrangements and duplications; some of the latter are fundamentally the same as microscopically detectable chromosomal aberrations, e.g., deletions, rearrangements, insertions and duplications. Table IV, based on McKusick (1988) and Harper et al. (1989) with some additions, presents the currently available information on molecular changes in Mendelian conditions, together with an indication of the age at onset (see also Mohrenweiser and Jones, 1990). It should be mentioned here that: (i) although enzyme deficiencies have been demonstrated in well over 200 recessives (autosomal and X-linked), only those for which data from molecular studies exist are included in the table; and (ii) the table does not provide information on the proportion of PM or LM type of events at any given locus but only some indication of the " m u t a t i o n a l potential" of the genes studied. Table IV permits two general conclusions. First, both PMs (of different kinds) as well as LMs have been found; however, with some genes, a considerable variety of changes has been recorded whereas with some others, PMs alone or LMs alone have been found. There are several reasons for this: clinical relevance and the amount of effort expended, rate of occurrence of mutants and availability for analysis, the specificity of the type of mutational event (i.e., whether it can occur anywhere in the gene and still give a detectable phenotype or whether it is restricted to specific codons), sequence organization, size and function of the gene. Second, most of the autosomal recessive and X-linked conditions have onset at birth or in childhood and this seems unrelated to the nature of the underlying molecular event; with autosomal dominants, however, the situation appears to be
21 TABLE V CLASSIFICATION OF MENDELIAN DISEASES ACCORDING TO THE NATURE OF THE UNDERLYING MOLECULAR DEFECT INTO THOSE WHICH ARE DUE TO POINT MUTATIONS (PM), POINT OR LENGTH MUTATIONS (PM OR LM) OR LENGTH MUTATIONS (LM) ONLY Disease classification Autosomal dominants X-linked Autosomal recessives
PM only 18 1 4 Total 23 (30.2%)
22 3 sub-total 25 (43.1%) Autosomal recessives 8 Grand total 33 (43.4%)
Autosomal dominants X-linked
PM or LM a
LM only
Total
6 4 4 14 (18.4%)
18 11 10 39 (51.3%)
42 16 18 76 (100%)
2b 2c 4 (6.9%) 0 4 (5.2%)
18 11 29 (50.0%) 10 39 (51.3%)
42 16 58 18 76 (100%)
For the top half of the table, the criterion for inclusion in this group is that at least one LM has been identified; for the bottom half, the criterion is that the frequency of LM should be higher than 5%. See text for details. b Alpha-thalassemia and familial hypercholesterolemia. c Lesch-Nyhan syndrome and ornithine transcarbamylase deficiency. a
different: those d u e to P M s m a n i f e s t themselves at birth, in c h i l d h o o d or in a d u l t s while those d u e to L M s m a n i f e s t themselves p r e d o m i n a n t l y at b i r t h or in childhood. It is easy to u n d e r s t a n d the s i t u a t i o n with respect to a u t o s o m a l a n d X - l i n k e d recessives: m o s t ( a l t h o u g h n o t all) are d u e to e n z y m e deficiencies which d i s r u p t n o r m a l m e t a b o lism a n d so are d e t e c t e d early. H o w e v e r , the m e c h a n i s m or m e c h a n i s m s t h a t p e r m i t s o m e a u t o s o m a l d o m i n a n t P M s to express themselves at a n y early stage in life while for s o m e o t h e r s onset is d e l a y e d until a d u l t h o o d , s o m e t i m e s late a d u l t h o o d , are n o t fully delineated. O n e w o u l d a p r i o r i expect that onset will b e d e p e n d e n t on the b o d i l y f u n c t i o n affected o r c o m p r o m i s e d as a result of the m u t a t i o n : for i n s t a n c e if it is a vital b o d y f u n c t i o n (e.g., the i m m u n e function) that is affected, then this is likely to c o m e to the attention of the clinician early on in the life of the patient. I n contrast, if the " n a t u r a l h i s t o r y " of the disease is such that an overt clinical m a n i f e s t a t i o n takes m a n y years (e.g., familial h y p e r c h o l e s t e r o lemia, familial a m y l o i d p o l y n e u r o p a t h y ) , clinical d e t e c t i o n m a y be d e l a y e d until later. T h e essence of the r e a s o n i n g then is that, with a u t o s o m a l d o m i n a n t PMs, onset age is m o s t p r o b a b l y r e l a t e d to the n a t u r e of the i m p a i r e d f u n c t i o n a n d is n o t necessarily d e p e n d e n t on the k i n d of u n d e r l y i n g
m o l e c u l a r defect ( P M or LM). S t a t e d differently, with respect to onset age, there m a y b e no f u n d a m e n t a l m o l e c u l a r - a l t e r a t i o n - r e l a t e d differences between a u t o s o m a l d o m i n a n t s o n the one h a n d a n d a u t o s o m a l recessive a n d X - l i n k e d ones on the other. T h e rare a u t o s o m a l d o m i n a n t c o n d i t i o n s for which o n l y L M s have b e e n r e c o v e r e d so far ( T a b l e 4) merit s o m e c o m m e n t . M o s t of these are m a n i fest at b i r t h or c h i l d h o o d . M a n y of these c o n d i tions are m i c r o d e l e t i o n ( c o n t i g u o u s gene deletion) s y n d r o m e s (Schinzel, 1988); in these, a p a r t f r o m the p u t a t i v e gene in question, a d d i t i o n a l D N A sequences that are vital for s o m e essential b o d i l y functions ( a n d not necessarily for survival) m a y also be deleted. T h e m o l e c u l a r b i o l o g y of these c o n d i t i o n s has n o t yet b e e n w o r k e d o u t in detail.
Mutational potential of different genes A n overview of the relative i m p o r t a n c e of P M s a n d L M s in M e n d e l i a n diseases s t u d i e d thus far c a n be o b t a i n e d b y r e g r o u p i n g the i n f o r m a t i o n c o n t a i n e d in T a b l e IV u n d e r the h e a d i n g s of " P M s o n l y " , " P M s or L M s " a n d " L M s " only. T h e resuits are given in the top half of T a b l e V. It c a n b e seen that a m o n g the 76 genes s t u d i e d (i) o n l y P M s have thus far b e e n f o u n d in 23 (30%), P M s or L M s in 14 (18%) a n d o n l y L M s in 39 (51%) a n d
22 (ii) there seems to be a preponderance of LMs among X-linked diseases. Of interest in the present context, however, are the relative proportions of PMs versus LMs in the different Mendelian diseases. The situation is straightforward with respect to genes at which only PMs or only LMs have actually been observed; however, those in which either PMs or LMs have been found (n = 14) need further scrutiny for ascertaining the nature of the predominant type of change. To do this, an arbitrary criterion was adopted: genes at which the recorded LMs are 5% or less were included in the " P M only" group and those at which this proportion was exceeded, in the " P M or L M " group. When the " P M or L M " group was reanalyzed with the above criterion, only four conditions "qualified" for inclusion in the above group. These are: c~-thalassemia (frequency of LMs over 80%; Antonarakis, 1985; Orkin, 1987), familial hypercholesterolemia (frequency of LMs in the range of 6-63%; see McKusick, 1988 for details and references); ornithine transcarbamylase deficiency (frequency of LMs about 7%; Rozen et al., 1985) and Lesch-Nyhan syndrome (frequency of LMs about 18%; Yang et al., 1984). For the remaining 10 entries, LMs are infrequent (around 5% or less of the events, when recorded), and therefore were included in the " P M only" group. The results of the above regrouping are given in the bottom half of Table V. Of the total of 76, 33 (43.4%) are now classified under PMs, four (5.2%) under " P M s or LMs" and 39 (51.3%) under LMs. If only the autosomal dominant and X-linked diseases are included, these percentages are nearly the same (43.1%, 6.9% and 50%, respectively). One tentative conclusion from this analysis is that in roughly one half of the genes at which spontaneous mutational events (leading to Mendelian disease) have been studied, P M is the underlying event and in the remainder, the events are DNA deletions or other gross changes.
Origin of spontaneous mutations General considerations
The question of why mutations arise "spontaneously" still cannot be answered and the term
"spontaneous" remains a thinly veiled admission of our ignorance of the phenomenon. Already early in the history of radiation genetics, Muller and Mott-Smith (1930) showed that natural background radiation can account for only a negligible fraction of spontaneous mutations in Drosophila and the same is likely to be true of humans as well. The question of whether and to what extent the very low levels of chemical mutagens present in our environment (in the food we eat, the water we drink and the air we breathe) may together contribute to spontaneous mutations has not yet been answered but it seems unlikely that a significant fraction of "spontaneous" mutations is due to these. During the last decade, there has been much discussion, although in the context of aging and cancer, on the possibility that endogenous mutagens produced by normal aerobic metabolism can contribute to naturally occurring D N A damage at least to some extent (e.g., Adelman et al., 1988; Cutler, 1986; Ames, 1988a,b, 1989; Ames and Saul, 1988; Richter et al., 1988; Simic et al., 1989). Four important endogenous processes leading to significant D N A damage are likely to be oxidation, methylation, deamination and depurination. The existence of specific D N A - r e p a i r glycosylases for oxidative, methylated and deaminated adducts and a repair system for apurinic sites produced by spontaneous depurination provide support for this premise (Ames, 1989). Perhaps, as Sargentini and Smith (1985) state " w i t h continued study, the term "spontaneous mutagenesis" will be replaced by more specific terms such as 5-methylcytosine deamination mutagenesis, fatty acid oxidation mutagenesis, phenylalanine mutagenesis and imprecise-recombination mutagenesis." On the question of how spontaneous mutations arise, there is a considerable wealth of data from diverse biological systems and these show that spontanous mutations can occur as a result of errors during D N A replication, repair and recombination. Additionally, there is now substantial evidence f r o m bacteria, yeast, maize and Drosophila which demonstrates that a significant proportion of spontaneous mutations originates from insertion of mobile genetic elements into genes (see Lambert et al., 1988). In humans, at present, the evidence for this is limited.
23 Mutational mechanisms
Table 6 (which is an expanded version of Table 4 discussed earlier) provides details on the kinds of molecular changes observed in various Mendelian diseases; they also enable one to gain some insight into the mechanisms involved in spontaneously occurring PM and LM events. These are discussed in the following paragraphs. A r e P M events distributed throughout the gene or are there specific exons / codons in which they arise?
To answer this question, genes at which almost every PM change has been ascertained and studied would represent the ideal situation; the only genes that come anywhere near this "ideal" situation are the globin genes. The less-than-ideal situation, which would permit at least some insights into major trends, would be instances of conditions due to several independent point mutations at the same gene. There are many such conditions as will be discussed below. Examples of conditions in which the PM events appear restricted to specific c o d o n s / e x o n s (Table 6) are (i) familial amyloid polyneuropathy, type I (a single PM, in exon 2 of the transthyretin gene in Portugese, Japanese, Swedish and Greek patients); (ii) familial hypercholesterolemia (the three characterized PMs in the low density lipoprotein receptor gene are in exons 14 and 17); (iii) Gaucher disease (PMs in exons 5, 9 and 10 of the glucocerebrosidase gene); (iv) phenylketonuria (PMs in exons 7, 9 and 12); (v) hemophilia A (PMs in exons 7, 18, 22-24 and 26 of the factor VIII gene); (vi) hemophilia B (PMs in exons 2, 3, 6, 8 and in IVS-3 and IVS-6 of the factor IX gene); and (vii) Lesch-Nyhan syndrome (in exons 3, 4 and 8). In fi-thalassemia, mutations in the /~-globin gene are found over the entire gene, but there seems to be a clustering of the events in the 5' part of the gene, exon 1 in particular. In severe combined immunodeficiency, the PM events appear to be distributed throughout the entire coding sequence. In osteogenesis imperfecta (OI), the PMs in the COL1A1 and COL1A2 genes are distributed over the entire gene, but the phenotypic effects vary depending on which of the procollagen chains contains the mutation, its location,
etc.; most of the PM events identified in the COL1A1 gene result in the lethal OI (type II) phenotype. Finally, PM events in the globin genes affecting practically each one of the amino acids in the a, /~, ~, and 8 chains have been discovered, but only a small proportion of these is associated with clinical effects. It would thus seem that (i) for most genes considered, PM events do not appear to be distributed at random throughout the gene and (ii) when these events appear to be distributed throughout the entire gene, either there is some clustering or only a limited number of the changes are associated with clinical effects. To what extent can one explain the spontaneous occurrence o f P M events on the basis o f the structure o f the gene?
This question can now be answered to some extent for mutations of the transition type. It has long been known (Vogel, 1972; Vogel and Kopun, 1977; Li et al., 1985) that among point mutations, transitions (A to G or G to A and C to T and T to C) occur more frequently than transversions in the genes of higher organisms. However, it was only relatively recently that the potential significance of the so-called C p G dinucleotide sequences to spontaneous mutagenesis (among others) began to attract attention. Vertebrate D N A is highly methylated at the cytosine residue and about 90% of 5-methylcytosine occurs within C p G sequences; the latter occurs, however, at only 0.25-0.2 of the frequency expected from the base composition (see Gardiner-Garden and Frommer, 1987 for a detailed discussion and citation of relevant references). At the level of the gene, C to T transitions and the corresponding G to A transitions in the complementary D N A strand occur at a high frequency within methylated gene regions (Seleker and Stevens, 1985; Savatier et al., 1985; Cooper et al., 1987; Bird et al., 1987; Long, 1987; Koeberl et al., 1989, 1990). This is thought to be due to the propensity of 5-methylcytosine to undergo spontaneous deamination to form thymine (Coulondre et al., 1978). Cooper and Youssoufian (1988) made a systematic analysis of published results to examine the role of C p G sequences in spontaneous muta-
LMd: Microdeletion in 7p21.2-.3 LMd: Microdeletion in 20pll.2-p12.1 P M s / L M d : In-frame deletion of exon 6 which codes for the amino acid residues 54-70 which includes the site at which N-proteinase normally cleaves the protein (pro-alpha-2 of type I collagen) or resulting from an RNA splicing defect LMr: A D N A rearrangement upstream from the initiator codon for translation in the gene coding for protein 4.1 PMm." His to Tyr at 58 or 87 in the alpha globin
9. Van der Woude syndrome, mental retardation
10. Craniosynostosis, mental retardation
11. Alagille syndrome, mental retardation 12. Ehlers-Danlos syndrome, type VIIA1
14. Methemoglobinemia, hemoglobinopathic, of Hb M (a-type)
13. Elliptocytosis, Rh-linked
PMm." At the cleavage site of apoA1 LMr: Inversion between 4th exon of ApoA1 and first intron of ApoC3 LMd." Microdeletion in 1q32-41; 2q34-36
8. Hypoalphalipoproteinemia
PMn: In null Bellingham allele: A to T (Lys to Stop at 217)
P M m : G to C (Trp to Cys at 402) P M n : C to T (Arg to Stop at 306) PM m : In P I * Z allele: G to A in exon 5 (Gly to Lys at 342); T to C in exon 3 (Val to Ala at 213); In PI*P: A to T in exon 3, Asp to Val at 256
PMm." T to G (Ile to Ser at 84); Thr to Ala at 60; Ser to Tyr at 77 of transthyretin P M m : T to A (Leu to Gin at 68) in cystatin C PM m : C to T (arg to Cys at 47) in antithrombin
3. Amyloidosis, neuropathic, type If
4. Amyloidosis, Icelandic, cerebrovascular type 5. Thrombophilia, familial, due to antithrombin-llI deficiency * 6. Thrombophilia, familial, due to defect in protein C* 7. al-Antitrypsin deficiency *
PMm: G to A in exon 2 (Val to Met at 30) of transthyretin gene
LMd: 3.2-kb deletion spanning the ADA promoter and the first exon
A utosomal dominants PM m : In exons 4, 7 10 and 11 and thus appear to be scattered over the coding sequence; the changes include C to T at position 396, G to A at 397, G to A at 727, C to T at 1081 and others at positions 334 and 1006 (the position numbers refer to the cDNA sequence of Wiginton et al., 1986); those at 396, 397, 727 and 1081 are at C p G dinucleotides, known to be hot spots for mutation (Cooper and Youssoufian, 1988); thus a high proportion of mutations is associated with a specific PM change; evidence for genetic compounds
Comments on mutational changes
2. Amyloidosis, neuropathic, type I *
1. Severe combined immunodeficiency (SCID) *
Disorder
See McKusick (1988)
Conboy et al. (1986)
Antonarakis (1988) Karathanasis et al. (1987) Bocian and Walker (1987); Hughes et al. (1988) Gong et al. (1976); see Harper et al. (1989) Schnittger et al. (1989) Wirtz et al. (1987); Byers (1989); Prockop et al. (1989)
Kidd et al., (1983); Nukiwa et al. (1986); Satoh et al. (1988); Fraizer et al. (1989); Farber et al. (1989)
Tawara et al. (1983); Dwulet and Benson (1984); Yoshioka et al. (1986); Saraiva et al. (1986); Maeda et al. (1986) Dwulet and Benson (1986); Wallace et al. (1986) Abrahamson et al. (1987) Duchange et al. (1986, 1987); Koide et al. (1984) Romeo et al. (1987)
Bonthron et al. (1985); Valerio et al. (1986); Akeson et al. (1987); Berkvens (1988); Berkvens et al. (1987); Markert et al. (1989)
References
DETAILS OF THE N A T U R E OF MUTATIONS IN M E N D E L I A N DISORDERS (GENERAL REFERENCES: McKUSICK, 1988; HARPER et al., 1989)
TABLE VI
17. Hereditary persistence of fetal hemoglobin, deletion type (HPFH)
16. /3-Thalassemia
15. a-Thalassemia
HPFH-3: < 70 kb, similar to the ones above; the 5' end is slightly 5' to that of HPFH-2, but the 3' end is more proximal and located about 30 kb 3' to the/3-globin gene
HPFH-2: > 70 kb, similar to the one above; the 5' and 3' end points are displaced by about 5 - 6 kb to the 5' side of the corresponding end points of HPFH-1
Deletions of /3-globin gene leading to /3-thalassemia are rare; a 619-bp deletion which removed the distal (3') third of the IVS-2 and the entire exon 3 of the gene plus 209 bp of adjacent 3' flanking D N A accounts for 1 / 3 of the /3-thalassemic genes in Asiatic Indians; a 1.3-kb deletion involving the 5' flanking DNA, exons 1 and 2 and the 5' end of IVS-2, found in Blacks; a 10-kb deletion involving the entire ,8-globin gene but sparing the 8-globin gene has been described in a Dutch family; all of these are B ° thalassemic patients HPFH-I: > 70 kb, involving deletion of both the 8- and /3-globin genes with the 5' end of the deletion located 3.75 kb to the 5' side of the ~ gene while the 3' end is located at a site greater than 60 kb to the 3' side of the /3-globin gene
4. Evidence for genetic c o m p o u n d s
3. More mutations in exon 1 than in 2 and 3.
2. B°: (a) nonsense orframeshifts (in codons 6, 8, 8 / 9 , 15, 16, 17, 39, 41/42, 71/72); (b) RNA processing defects: IVS-1, position 1, G to A, G to T; position 116, T to G; position 108, 25 bp deletion; position 117, 17 bp deletion; IVS-2, position 654, C to T; position 705, T to G
extend rightward and some left; c o m m o n deletion is 2.7 kb, rightward 1. B + : (a) transcription defects (C to G at - 87 from cap site; C to T at - 8 8 ; A to G at - 2 8 ; A to G at - 2 9 ) ; (b) RNA processing defects (codon 27, G to T; codon 26, G to A; IVS-1, position 5, G to T, G to C; position 6, T to C; position 110, G to A; IVS-2, position 745, C to G; polyadenylation signal (1000 bp from exon 3), T to C
LMd: Partial or total deletion of al a n d / o r a2 genes; some of these
PMi: A U G to ACG, alpha-2-globin gene; frameshift in codon 14 ( T G G to GG, alpha-l); PMrp: A A T A A A to A A T A A G (alpha-2); a 5-bp deletion at 5'-IVS-1 splice junction (alpha-2); PMt: T A A to C A A (Hb Constant Spring); T A A to A A A (Hb Icaria); T A A to T C A (Hb Koya Dora); T A A to G A A (Hb Seal Rock) (all the PMts are in alpha-2); unstable variant: codon 125, C T G to C G G (alpha-2); the PMs are generally rare among alpha-thalassemias
See Weatherall (1986)
See Antonarakis et al. (1985) and Weatherall (1986) and references cited therein
hemoglobin,
21. Hereditary persistence of fetal non-deletion type G 22. Hypercholesterolemia, familial *
25. Osteogenesis imperfecta (OI) *
24. Giedion-Langer syndrome
23. lmmunoglobulin kappa light chain deficiency *
hemoglobin,
fetal
20. Hereditary persistence of non-deletion type A
18. Methemoglobinemia, hemoglobinopathic, of Hb M (fl-type) 19. Sickle cell anemia
Disorder
TABLE VI (continued)
Russell et al. (1986); Lehrman et al. (1987); Langlois et at. (1988) Zannis and Breslow (1985)
PMm: G to A in exon 17; PMn: in exons 14, 17; m a n y mutations remain to be characterized LM: 2-kb insertion in IVS-1, 4-bp insertion in exon 17; 12- and 27-bp deletions in exon 4; other deletions (at least 16 known) are of various sizes ranging from 0.8 kb (in exon 5) to 11 kb (encompassing exons 4 - 8 ; evidence for genetic compounds LMdu: 14-kb duplication involving exons 2-8. PMm: T to G and C to T leading to the loss of the invariant tryptophan at position 148 on one chromosome and of the invariant cysteine at position 94 on the other; no effect on health Microdeletion in 8q23.3-q24.13
Pro-al mutations 1. Splice exon 6 ( - 24 aa) 2. Gly to Cys at 175 3. Insertion 50-70 aa 4. Gly to Val at 256 5. Gly to Arg at 391 6. Deletion exons 24-26 ( - 8 4 a a ) 7. Gly to Arg at 664 8. Gly to Cys at 748 9. Gly to Cys at 904 10. Gly to Cys at 988 11. Gly to Cys at CT3 (C-terminal telopeptide) 12. Frameshift deletion, 5 bp
OI
Consequence EDS VII OI Lethal OI Lethal OI Lethal Ol Letal OI Lethal Ol Lethal Ol Lethal OI Lethal OI OI
Most mutations involve the pro-al (COL1A1) or pro-or2 (COL1A2) gene, lead to alterations of the structure of the pro-0d (I) chain of type I procollagen and cause either osteogenesis imperfecta (OI) or EhlersDanlos syndrome (EDS)
See Weatherall (1986)
See Harper et al. (1989) for references Prockop et al. (1989); Byers (1989)
Stavnezer-Nordgren et al. (1985)
See Weatherall (1986)
Ingram (1956, 1957, 1986)
See McKusick (1988)
,4 utosomal dominants PMm: His to Tyr at 63 or 92 and Val to Glu at 67 in the fl-globin gene PMm: At the sixth codon from the 5' end of the fl-globin gene ( G A G to G T G ; Glu to Val) Rare; PMp: G to A at - 1 1 7 from the cap site of the A-T-globin gene (i.e., in close proximity to the C C A A T box); C to T at - 196 from the cap site of the A-T-globin gene Rare; PMp: C to G at - 202 from the cap site of the G-y-globin gene
References
C o m m e n t s on mutational changes
Microdeletion in 10p13 Microdeletion in 4q25-27 PM m : G to C (Glu to Asp at 104)
35. DiGeorge sequence 36. Rieger syndrome, mental retardation
37. Hemolytic anemia due to triosephosphate isomerase deficiency * 38. DiGeorge sequence, mental retardation
39. Trichorhinophalangeal syndrome, type 1, mental retardation 40. WAGR syndrome (Wilms tumor, Aniridia, Gonadoblastoma, mental retardation)
Microdeletion in 11p13
Microdeletion in 8q23.3-q24.13
Microdeletion in 22qll
Microdeletion in 8p21.1-p11.22
34. Spherocytosis, I, mental retardation
33. Retinoblastoma
Microdeletion in 7p13 Microdeletion in 15qll-q13; paternally inherited P M m : His to Asp at 10 (beta chain) Phe to Leu at 25 (beta chain) PHE to Ser at 24 Arg to His at 65 Microdeletion in 13q14
Lethal O1 Ol OI at 89) in the gene for purine
Lethal OI
OI Lethal OI Lethal Ol Lethal OI
Consequence EDS VII EDS/OI
30. Greig cephalopolysyndactyly 31. Prader-Willi syndrome 32. Diabetes mellitus, rare forms *
26. Immunodeficiency, T cell, with neurological disorder * 27. Porphyria cutanea tarda * 28. Piebald trait, mental retardation 29. Adenomatous polyposis, mental retardation
Pro-a2 mutations 1. Splice exon 6 ( - 18 aa) 2. Splice/deletion exon 11) ( - 18 aa) 3. Deletion - 20 aa 4. Splice exon 28 ( - 1 8 aa) 5. Gly to Asp at 547 6. Splice/deletion exon 33 ( - 18 aa) 7. Deletion exons 33-40 ( - 1 8 0 aa) 8. Gly to Asp at 907 9. Gly to Arg at 1012 10. Frameshift deletion of 4 bp PM m : G to A in exon 3 (Glu to Lys nucleoside phosphorylase PM m : G to A (Gly to Glu at 281) Microdeletion in 4q11-q21 Microdeletion in 5q21
de la Chapelle et al. (1981); Greenberg et al. (1988); Mascarello et al. (1989); Whitlock et al. (1989) Goldblatt and Smart (1986); Yamamoto et al. (1989) Francke et al. (1979); Porteous et al. (1989); Rose et al. (1989); Glaser et al. (1989)
Francke (1976); Cavenee et al. (1985); Lee et al. (1987) Chilcote et al. (1987); Kitatami et al. (1988) Greenberg et al. (1988) Ligutic et al. (1981); Motegi et al. (1988) Daar et al. (1986)
Verneuil et al. (1988) See Harper et al. (1989) Herrera et al. (1986); Hockey et al. (1989) Rosenkranz et al. (1989) See Harper et al. (1989) Chan et al. (1987); Shoelson et al. (1983a,b); Haneda et al. (1983); Shibasaki et al. (1985)
Williams et al. (1987)
C-II
deficiency
53. Zellweger syndrome 54. Phenylketonuria *
P M m : G to A (Val to Met at 332); C to A (Asp to Lys at 54)
52. Ornithinemia with gyrate atrophy of choroid and retina
Evidence for genetic compounds Microdeletion in 7ql 1 PMm." C to T (Arg to Try at 408) in exon 12; G to A in exon 5 (Arg to Gin at 158); T to C (Leu to Pro at 311) in exon 9, G to A in exon 7 (Ghi
PMi." G to A (Met to lie) LMd." partial deletion of the ornithine amino-transferase gene
Microdeletion in 17p13.3
51. Miller-Dieker lissencephaly syndrome
CII
L M d : a 6-kb deletion; L M d u : a 2-kb direct tandem duplication extending from within exon 6 to the 3' end of an A l u within intron 6
(Apo
termination codon Originally interpreted as a resulL of deletion of the functional gene (P450c21B; steroid cytochrome P450 21-hydroxylase) (at least 25% of cases), the current view is that these apparent deletions represent gene conversions, unequal cross-overs or polymorphism, but not physical loss of the gene; some gene deletions and duplications do occur LMd." A deletion of one base of the ACT codon for Thr at position 68 causing a frameshift and a premature termination at amino acid 68 instead of at the normal amino acid 79 P M m : ApoE2, Arg to Cys at 158 P M m : G to C in exon 5 (Ala to Pro at 149) of the fructose-l-phosphate aldolase gene P M m : T to C in exon 10 (Leu to Pro at 444); C to G in exon 9 (Pro to Arg at 415); A to G in exon 9 (Asp to Ser at 370); G to A in exon 5 (Arg to Gin at 120); C to T (Pro to Leu at 470); A to G (His to Arg at 495); evidence for genetic compounds Microdeletion in 15qll-q13 (as in Prader-Willi), but maternally inherited
A utosomal recessives L M d : A 4-bp deletion at the second codon (cDNA) upstream from the
50. Lipoprotein lipase deficiency
49. Angleman syndrome
48. Gaucher disease, types 1 to Ill *
46. Hyperlipoproteinemia, type IIl 47. Fructose intolerance, hereditary
45. Apolipoprotein Toronto)
44. Adrenal hyperplasia
43. Pyruvate dehydrogenase El deficiency
42. Adenine phosphoribosyl transferase deficiency
1988); Theo-
Naritomi et al. (1988) DiLella et al. (1986, 1987); Avigad et al. (1987); Lichter-Konecki et al.
Knoll et al. (1989); Pembrey et al. (1989) Langlois et al. (1989); Devlin et al. (1990) van Tuinen et al. (1988); Schwartz et al. (1988) Ramesh et al. (1988); Mitchell et al. (1988); Hotta et al. (1989)
Tsuji et al. (1987, phihis et al. (1989);
Smit (1987, 1989) Dazzo et al. (1989)
Cox et al. (1988)
White et al. (1987, 1988); Miller (1988); Matteson et al. (1987); Higashi et al. (1988); Rumsby et al. (1988)
Endo et al. (1989)
Kamatani et al. (1989) Hikada et al. (1987)
Shelton-lnloes et al. (1987)
A utosomal dominants L M d : 2 unrelated patients were shown to have large deletions within the
41. von Willebrand disease gene (12pter-12pl2), corresponding to at least 7.4 kb at the 3' end of cDNA; estimated size of deletion; at least 110 kb P M m : Met to Thr at 136 L M d : IN a patient with complete deficiency, in one allele, a trinucleotide deletion corresponding to phenylalanine in the deduced amino acid sequence, and in the other, a single nucleotide insertion immediately adjacent to the splice site at the 5' end of IVS-4
References
Comments on mutational changes
Disorder
TABLE VI (continued)
65. Granulomatous disease, X-linked
64. Choroideremia, mental retardation
61. Adrenal hypoplasia (Addison disease), mental retardation 62. Ocular albinism (Nettleship-Falls type OA1) and ichthyosis 63. Fabry disease
60. Propionicacidemia, type 11
59. Albinism (tyrosinase-deficient, oculocutaneous)
58. Tyrosinemia, II (tyrosinase transammase deficiency: TAT)
57. Tay-Sachs disease
56. Sandhoff disease (GM2-gangliosidosis
55. Pituitary dwarfism
LMd: in Xp21,1 and abnormal transcript
Merry et al. (1989); Cremers et al. (1989) Royer-Pokora et al. (1986); Orkin (1987)
Kornreich et al. (1988)
Schnur et al. (1989)
Microdeletion in Xp22.3 LMd." Deletions range in size from 0.4 kb to 5.5 kb; four of these had a breakpoint in IVS-2 and three of these families had breakpoints in an Atu-rich region LMdu: A partial duplication of about 8 kb encompassing exons 2 - 6 Microdeletion in Xq21
Yates et al. (1987)
Tahara et al. (1989)
Spritz et al. (1989)
Natt et al. (1987)
Tanaka et al. (1990) Myerowitz and Costigan (1988); Myerowitz and Hogikyan (1986, 1987); McDowell et al. (1989); Triggs-Raine et al. (1989)
O'Dowd et al. (1986)
Goosens et al. (1986); Braga et al. (1986)
(1988); Lyonett et al. (1989); Wang et al. (1989); John et al. (1989); Levy (1989); Okano et al. (1990); Scriver et al. (1988)
Microdeletion in Xp21
X-linked
extending 2 kb upstream past the putative promoter region of H E X - A gene encoding the alpha subunit (in French Canadians) Evidence for genetic c o m p o u n d s LMd: In one genetic compound, both T A T alleles deleted; the deletion from the mother at least 27 kb long and included the complete T A T gene; the other, inherited from the father had a de novo interstitial deletion in chromosome 16 (pter-q22 q22.3-qter) PM m : Thr to Lys (classical tyrosinase-negative OCA) which abolishes one of six putative N-linked glycosylation sites; Pro to Leu ('yellow' OCA) may interfere with normal folding of the tyrosinase polypeptide LMd." A 2-kb deletion in the gene for beta subunit of propinyl CoA carboxylase; 10 out of 30 m u t a n t alleles analyzed have this change
LMi: A 4-bp insertion in exon 11 (common in Ashkenazi Jewish patients) LMd: A 7.6-kb deletion which includes part of intron 1, all of exon 1 and
flanking the chorionic s o m a t o m a m m o t r o p i n gene; homozygosity for a 7.6-kb deletion within the growth hormone gene cluster LMd." Partial gene deletion located to the 5' end of the hexosaminidase B gene P M m : G to A (Arg to His at 178); C to T (Arg to Cys at 178) PMs: A splice junction defect in IVS-12 P M m : In exon 7 (rare)
LMd." A total of about 40 kb of D N A absent due to 2 separate deletions
Evidence for genetic c o m p o u n d s
PMi: A to G (Met to Val) PMs: G to A in intron 12 L M d. deletion of exon 3
to Lys at 280); C to T in exon 3 (Arg to Ter at 111)
70. Lesch-Nyhan syndrome
68. Glycerol kinase deficiency, adrenal hypoplasia, hypogonadotropic hypogonadism, mental retardation 69. Ichthyosis, chondrodysplasia punctata, Kallman syndrome, mental retardation
67. Hemophilia B (factor IX) *
66. Hemophilia A (factor VIII gene) *
Disorder
TABLE Vl (continued)
Severe Severe Severe Severe
PMm:C T G T G T
to A (Phe to Leu at 73) to A (Val to Asp at 129) to T (Ala to Set at 160) to G (Phe to Val at 198) to A (Cys to Tyr at 205) to C (Leu to Ser at 130)
LMd." Most patients examined have complete or partial deletions of the gene for steroid sulfatase located at Xp22.3
PMn." C to T, exon 8 (Arg to Stop at 252) C to T, exon 8 (Arg to Stop at 238) PMs: GT to T r , IVS-6, nt 1 GT to GG, IVS-3, nt2 LMd: Over 10 deletions ranging in size from about 1.5 kb up to about 115 kb LMi: Full length LINE-1 near the 5' end of Factor IX gene; A 6-kb insertion within the factor IX gene A Microdeletion in Xp21-pll.2
X-linked Change Effect P M m : A to G, exon 7 (Glu to Gly at 272) Moderately severe G to A, exon 26 (Arg to Gln at 2326) Moderately severe PMn: C to T, exon 18 (Arg to Stop at 1960) Severe C to T, exon 26 (Arg to Stop at 2326) Severe C to T, exon 22 (Arg to Stop at 2135) Severe C to T, exon 24 (Arg to Stop at 2228) Severe C to T, exon 23 (Arg to Stop at 2166) Severe All these mutations affect CpG nucleotides LMd: At least 12 deletions are known, ranging in size from about 2.5 kb to 210 kb; all except the 6-kb deletion of exon 22 (which is moderately severe) are severe LMi: Two patients with insertions of L IN E-1 sequences (3.8 kb and 2.3 kb) in exon 14 PMm.'G to A, exon 6 (Arg to His at 145) Mild A to G, exon 3 (Asp to Gly at 47) Mild G to A, exon 2 (Arg to Gin) Mild
Comments on mutational changes
Youssoufian
Shapiro et al. (1987); Bonifas et al. (1987); Ballabio et al. (1988); Bick et al. (1989) Stout and Caskey (1985, 1988); Wilson et al. (1983a,b); Wilson and Kelly (1984); Davidson et al. (1988a,b,c, 1989a,b); Cariello et al. (1988, 1989); Gibbs et al. (1989); Yang et al. (1984)
See Harper et al. (1989) for references
Antonarakis (1988); Chen et al. (1988, 1989); Ludwig et al. (1989) Scott et al. (1987); Yoshitake et al. (1985)
Kazazian et al. (1988)
Antonarakis (1988); et al. (1988a, b)
References
* Asterisks denote entries included in the analysis of Cooper and Youssoufian (1988). The references are given as a separate section in the Reference list.
L M d, LMdu: unequal exchanges between the red pigment gene and the green pigment genes
75. Pelizaeus-Merzbacher disease
76. Color blindness
LMd: Partial deletions of the OTC gene PM m : C to T (Pro to Ser at 225)
73. Norrie disease 74. Ornithine transcarbamylase deficiency *
LMd: A large number of intragenic deletions of various sizes, but these do not occur uniformly over the gene; large deletions are typically found in the first third of the gene and a large number of smaller deletions cluster in the middle of the gene; deletions appear to occur with approximately equal frequency in the D M D and the milder BMD and deletion size per se does not correlate with the severity of the disease; D M D largely results from frameshift deletions while BMD usually results from deletions that maintain the translational reading frame LMdu: Duplication of one or few exons LMr: Involves translocation of the D M D gene and the ribosomal RNA genes on 21p A microdeletion in X p l l . 3 PM m : G t o A , C t o T
71. Microphthalmia, iridoschisis, goiter, labium synechia, craniotabes 72. Muscular dystrophy, Duchenne and Becker (DMD, BMD)
C to G (His to Asp at 203) G to C (Gly to Arg at 71) C to T (Ser to Leu at 110) T to C (Leu to Pro at 41) G to A (Gly to Glu at 70) A to T (Asp to Val at 80) G to T (Ala to Ser at 161) T to G (Phe to Val at 199) T to A (Val to Asp at 130) A to G (Asp to Gly at 201) T to G (Iso to Met at 132) C to A (Ser to Arg at 103) C to U (Ser to Leu at 109) PMn: C to T (Arg to Stop at 169) LMd: A variety of very small (a few bps) to medium (a few kbs) to whole-gene deletions LMi: Insertion of one nucleotide at base 56, 57 or 58; potential insertion (GM 2227) in the region between exons 4-6. LMdu: Involves exons 2 and 3 (GM 1662) Microdeletion in Xp22
Gencic et al. (1989); Abuelo et al. (1989) Nathans et al. (1986a,b)
Donnai et al. (1988) Nussbaum et al. (1986); Rozen et al. (1985)
Monaco and Kunkel (1987, 1988); Monaco et al. (1988); Worton and Thompson (1988) and references cited in these reviews; Lindloff et al. (1989); Koenig et al., (1989); Gillard et al. (1989); den Dunnen et al. (1989); Bakker (1989); Worton et al. (1984); Hu et al. (1988, 1989) See Hu et al. (1988, 1989)
Thies et al. (1989)
32 genesis. The considerations which prompted this analysis are: (i) the now well established general observation that there is a very high frequency of RFLPs in human D N A when restiction enzymes are used whose recognition sequences contain a C p G dinucleotide; (ii) their findings and those of others that recurrent mutations occurred exclusively in CpG dinucleotides in the factor VIII gene (Youssofian et al., 1986: Antonarakis et al., 1987) and that these mutations were CG to T G or CG to CA transitions; and (iii) the strong inference from all these findings, that the presence of D N A methylation could be one cause of point mutations within human genes. Seventeen autosomal dominant and three Xlinked conditions (in which precise changes have been identified by D N A sequencing or by oligonucleotide hybridization) were included (these conditions are marked with an asterisk in Table 6 of this paper; the latter includes all but three conditions listed in Tables 1 and 2 of Cooper and Youssoufian, 1988). Excluded from their compilation were mutations causing thalassemia, because many of these remain in the population for a long time and therefore may be found in many individuals (and additionally, because of the very large number of reports) and those causing neutral polymorphisms. With these exclusions, a total of 45 independent mutations at different genes were found. For the purpose of their inquiry, they also excluded from analysis all the 11 factor VIII mutations since these mutations were not detected in an unbiased fashion (i.e., use of restriction enzymes such as TaqI which contain CpG in their recognition sequence and thus detect single basepair changes more frequently than other enzymes). The main finding was that 12 out of the remaining 34 (35%) occurred in C p G dinucleotides and of these 11 were either C to T or G to A mutations indicating the important role of naturally occurring deamination of 5-methylcytosine in the genesis of spontaneous transition mutations. Cooper and Youssoufian (1988) estimate that the rate of occurrence is about 42-fold higher than that predicted from random mutation. From a recent analysis of mutations in the factor IX gene (hemophilia B), Koeberl et al. (1990) have estimated that transitions at the CpG sites are elevated 24-fold over transitions at other sites and that the
former account for 44% of all the transitions and for 37% of all the mutations. These findings are entirely in line with the conclusion that CpG sites may be " h o t spots" for spontaneous mutations.
Mechanisms underlying naturally occurring DNA deletions and duplications The mechanisms that have so far been proposed to explain the origin of deletions and duplications in Mendelian diseases - replication slippage and recombination are related to the nucleotide sequence organization of the genome. Relevant in this context is the existence of genes in the human genome that are evolutionarily related (i.e., those that show significant sequence homologies) and of various families of repetitive D N A sequences (e.g., Alu, LINE-1 and VNTRs) within and between gene sequences.
Replication slippage In 1966, on the basis of their work with bacteriophage T4, Streisinger and colleagues proposed that frameshift mutations are most likely to occur in regions of repeats as a consequence of base mispairing (between two repeats) and slippage during D N A replication. Subsequent research in E. coli has provided ample support for this hypothesis (Farabaugh et al., 1978; Glickman et al., 1985; Schaaper et al., 1986). In this organism, a large proportion of spontaneous mutations in the lacI gene is a consequence of gain or loss of one copy of the 4-bp sequence T G G C which is tandemly repeated three times at nucleotides 621632 which also represents a hotspot for mutations of this type. Slippage during replication is one of the mechanisms that has been proposed to explain insertions and deletions in tandem repeat sequences mentioned earlier (e.g., Jeffreys et al., 1988; Wolff et al., 1989; Kovacs et al., 1990 and references cited therein). Examples of Mendelian diseases in which small deletions have been found and which have been interpreted on the basis of the replication slippage model include some fl-globin structural variants and some deletions in the retinoblastoma gene. The fl-globin structural variants were originally identified by amino acid sequence analyis (Bunn et al., 1977) and were found to lack one to eight amino acids. Comparison of the nucleotide
33 sequences in normal DNA within and adjacent to the codons for amino acids deleted in the variants revealed the presence of small 2-8-bp direct repeat sequences (Marotta et al., 1977). Efstratiadis et al. (1980) have argued that these small deletions are unlikely to be a consequence of unequal crossing-over between repeat sequences for two reasons: (i) most of the repeat sequences are very short and (ii) an unequal crossing-over event would produce one daughter molecule with three intact copies of the repeat sequence and another molecule with one intact copy; only one example of the former and very few of the latter were observed in their analysis. In studies involving deletions in the retinoblastoma gene (Canning and Dryja, 1989), sequence analysis of regions surrounding breakpoints of four deletions revealed that three deletions had termini within pairs of short, direct repeats ranging in size from four to seven base pairs. The authors have interpreted this finding as indicating that the replication slippage mechanism may predominate in the generation of deletions at this locus. Recombinational mechanisms By genetic methods, Sturtevant (1925) and Sturtevant and Morgan (1923) first inferred the occurrence of unequal recombination at the Bar locus in Drosophila. The repeat element in this case turned out to be the Bar locus itself, as was subsequently shown cytologically by Muller et al. (1936) in salivary gland chromosome preparations. These studies thus provided much of the conceptual framework for envisaging the origin of deletions and duplications in other organisms as well as in the human genome. Gene duplications have played an important role in the evolution of multigene families (e.g., haptoglobins, hemoglobins, L D L receptor, ubiquitin, etc.; see reviews by Maeda and Smithies, 1986; Fodde, 1990). Common to all multigene families are (i) the initial, largely unpredictable events that enabled single-copy genes to become ancestral to families of genes and (ii) the more predictable events (unequal recombination and gene conversion) that increased or decreased the number of copies within a family and transfer of information between its individual members. These
events, which have occurred in the course of evolution of the multigene families, still occur spontaneously and sometimes result in pathological phenotypes. For convenience, in what follows, the examples are considered under four headings: (i) homologous, unequal recombination between evolutionarily related genes; (ii) homologous, unequal recombination between DNA repeat sequences, the latter present within a n d / o r between genes; (iii) non-homologous recombination; and (iv) possible mechanisms underlying deletions/duplications in the D M D / B M D gene. Homologous unequal recombination between evolutionarily related genes. Most of the a-thalassemias result from gross D N A deletions within the a-globin gene complex and have been interpreted to arise as a result of homologous, unequal recombination between the two closely spaced a-globin genes (Higgs and Weatherall, 1983). This interpretation also holds for some complex thalassemias (unequal crossing-over between 8- and fl-globin genes or between A-T- and fl-globin genes resulting in 8-/3 (hemoglobin-Lepore), /3-8 (anti-Lepore) and 3,-/3 (hemoglobin-Kenya) fusion genes (reviewed in Weatherall, 1986)). These findings and the interpretation can be easily understood in the context of what is known about the globin genes: (i) the al- and a2-globin genes are located next to each other (in a stretch of about 30 kb), have almost identical nucleotide sequences and the sequence homologies extend beyond the coding regions (Lauer et al., 1980); (ii) the G-3,-, A-T- (fetal genes), 8- and/3-(adult) globin genes are located in a 50-kb region; (iii) the 8- and /3-globin genes are highly homologous in their coding region (Lawn et al., 1978; Efstratiadis et al., 1980); (iv) in the two fetal globin genes, the coding, intervening and flanking sequences are virtually identical (Slightom et al., 1980); and (v) there is an abundance of A lu repeats in both the a- and/3-globin gene clusters (Fritsch et al., 1980; Nicholls et al., 1987). In one type of color blindness, anomalous trichromacy, the individuals have either a 5' red-3' green or a 5' green-3' red photopigment gene; in another form of color blindness, dichromacy, the red or green photopigment is missing as a conse-
34 quence of either deletion of one of the genes or red-green gene fusion (Nathans et al., 1986a,b). The occurrence of these conditions is most easily explained by homologous but unequal recombination during meiosis. The red and green pigment genes are 96% identical, the green pigment genes vary in number (1, 2 or 3) and together with the single red pigment gene, reside in a head-to-tail tandem array among color-normal individuals (Nathans et al., 1986a,b). Homologous, unequal recombination between repeat sequences. In the a-globin cluster, one deletion has been found to be due to homologous unequal recombination between Alu repeats (Nicholls et al., 1987). Of four deletions and one duplication occurring within the low density lipoprotein receptor gene (causal in familial hypercholesterolemia) that were analyzed, three were attributed to homologous unequal recombination between Alu repeats oriented in the same direction and one from intrastrand recombination between Alu oriented in opposite directions (Lehrman et al., 1985, 1987a,b; Russell et al., 1986; Langlois et al., 1988). The remaining deletion has one breakpoint within an Alu repeat; thus nine of the 10 breakpoints are within Alu repeats. In Fabry disease, the breakpoints of four intragenic deletions were in an Alu-rich region (Kornreich et al., 1988). Other examples of Alu involvement include Tay-Sachs disease in French Canadians (Myerovitz and Hogikyan, 1987) and one reported case of A D A deficiency (Berkvens et al., 1987). A sex-chromosome rearrangement in an XX male was found to be due to homologous unequal exchange between Alu repeats present in the terminal parts of Xp and Yp (Rouyer et al., 1987). Yen et al. (1987) have suggested that the frequent occurrence of deletions of the steroid sulfatase gene (McKusick No. 30810) might be related to unequal exchanges between Alu repeats in this region. Other examples of unequal recombination between repeat sequences (although not necessarily resulting in disease phenotypes) are those at hypervariable loci. It has been suggested that the core sequences shared by many hypervariable minisatellites might serve as a recombination signal (e.g., Jeffreys et al., 1985; Chandley and
Mitchell, 1988). This view is supported by the presence of a core-related sequence at or near a meiotic recombination hot spot in the mouse major histocompatibility complex (Steinmetz et al., 1987). Non-homologous recombination. Examples of deletions arising as a result of non-homologous recombination are provided by some a-thalassemias (recombination between an al-globin gene and an Alu repeat resulting in a partially deleted a-globin gene (Orkin and Michelson, 1980; Nicholls et al., 1985)), some fl-thalassemias (Anand et al., 1988; Henthorn et al., 1990), and some complex (3'-6-fl) thalasssemia and H P F H cases (Jagadeeswaran et al., 1982; Ottolenghi and Giglioni, 1982; Vanin et al., 1983; Henthorn et al., 1986). In all these deletions (except in those studied by Henthorn et al., 1990), the 5' breakpoints mapped either in or close to Alu sequences. The interpretation is that these deletions presumably arose during D N A replication (when sequences widely separated in the linear D N A molecule might be physically close to each other as a result of anchorage to the nuclear matrix and chromatin loop formation) as a consequence of non-homologous intrachromosomal breakage and reunion events (Anand et al., 1988; Vanin et al., 1983). The models proposed by Anand et al. and Vanin et al. differ in some details. In the work of Henthorn et al. (1990) with four independent fl-globin gene deletions, none of the eight breakpoints occurred within Alu sequences. Their work and analysis of other published data permitted them to conclude that "deletion break points are more likely to occur within transcriptional units of the fl-globin gene region than would be expected by chance.., hence it is the preponderance of break points within genes, rather than in repetitive elements, that is of note in this region of the genome." However, their examination of the data for c~-globin gene deletions interpreted as having arisen as a result of non-homologous recombination suggests that four of the six breakpoints are within Alu repeats. In a case of lipoprotein lipase deficiency, Devlin et al. (1990) found a tandem duplication of approximately 2 kb in the LPE gene (from within exon 6 to the 3' end of an Alu element located within intron 6) and suggested that this probably
35 arose as a result of a non-homologous exchange event.
Duch'enne and Becker muscular dystrophies ( D M D / B M D ) . Intragenic partial deletions (well over 300 are now known) appear to be the most c o m m o n defect (accounting for about 50-70% of mutations at the D M D / B M D locus) leading to B M D or D M D (Forrest et al., 1987; den Dunnen et al., 1989; Koenig et al., 1987, 1989; Gillard et al., 1989). Much less common are intragenic duplications which appear to duplicate one or a few exons by tandem duplication of a portion of the gene, presumably by unequal crossing-over between repeat elements (Hu et al., 1988; Koenig et al., 1989). More recently, Hu et al. (1989) have published data suggesting that duplications can arise as an intrachromosomal event through unequal sister-chromatid exchange. The deletions appear to occur with approximately equal frequencies in D M D and in the milder BMD patients and are of different sizes; sequences deleted in D M D patients may also be deleted in B M D patients and there does not seem to be any correlation between the size of deletions and the severity of the condition (Hart et al., 1987; Davies et al., 1988; Darras et al., 1988; Lindlof et al., 1988). There is a striking non-rand o m distribution of deletions and the spectrum is such that large deletions are typically found within the first third of the gene and a large number of smaller deletions are clustered in the middle of the gene; the clustered deletions begin in one of the three introns in this region, suggesting that these introns may contain sequences that predispose to deletions (Koenig et al., 1987). As Koenig et al. speculated, " t h e multiple breaks could be due a sequence-sepcific rearrangement hot spot, or an extraordinarily large genomic distance spanned by this particular exon." Monaco et al. (1988) advanced the hypothesis that deletions resulting in the clinically less severe B M D bring together exons that maintain the translational reading frame of the m R N A and that such deletions should allow the production of an abnormal, but partially functional protein product; in D M D patients, however, the deletions bring together exons that disrupt the reading frame and would lead to the production of a severely trun-
cated product. Detailed analysis of deletion breakpoints published recently by Koenig et al. (1989), Gillard et al. (1989), den Dunnen et al. (1989) and Hodgson et al. (1989) lend strong support to the above "reading frame hypothesis" although there are exceptions. The mechanisms involved in the genesis of deletions in D M D / B M D are not fully understood, but they do not seem to be related to the deletions of integral copies of the repeat structural units identified in the D M D gene (Koenig et al., 1988) since a large majority of breakpoints do not involve the exons at all (den Dunnen et al., 1989). Gillard et al. (1989) suggest that the non-random clustering of breakpoints might be due to either sequence-specific (homologous) or structurespecific (non-homologous) recombination. As they stated, "non-homologous mechanisms are attractive for the relative ease with which they can account for the diversity of deletions .... By comparison, one might predict that homologous recombination mechanisms would result in a smaller number of (deletion) endpoints.., given the high frequency of deletions and the considerable heterogeneity of deletion endpoints, it seems probable that both types of mechanisms might contribute to the generation of deletions."
Molecular
requirements for
recombination.
Bearing on the general theme of recombination is the obvious requirement of sequence homology and its minimal extent, at the molecular level. Studies in bacteria have shown that as few as 20 bp of homologous sequence resulted in recombination (Watt et al., 1985). In work with a series of mutants that contained variable-sized deletions of the neomycin phosphotransferase gene in an eukaryotic-prokaryotic shuttle vector system, Ayares et al. (1986) showed that for mammalian cells, 25 bp of homologous sequence was sufficient to yield recombination products. Since the length of the homologous sequence required for recombination is much less than that of most of the repeat sequences present in the human genome, one inference is that both homologous and non-homologous recombination must be relatively c o m m o n and there must exist safeguards to prevent these from becoming excessive.
36 In experiments similar to those of Ayares et al. (1986) but using a synthetic hypervariable minisatellite sequence ((SAT)6.5), Wahtis et al. (1990) found that it stimulated homologous recombination up to 13.5-fold in the human EJ bladder carcinoma cell line. Other mutational mechanisms Gene conversions The term gene conversion was introduced by fungal geneticists to describe the non-reciprocal transfer of genetic markers from one chromosome to another (Radding, 1978; see also Whitehouse, 1982). Molecular geneticists working with D N A of higher eukaryotes use this term to describe the local transfer of D N A sequences from one gene to a related gene elsewhere in the genome in an event that resembles a double cross-over (Maeda and Smithies, 1986). Gene conversions and expansion/contraction of gene number by homologous, but unequal, crossing over have been proposed as mechanisms for concerted evolution (Hood et al., 1975) and a number of ideas have been published as to how these events could occur at the molecular level (see Maeda and Smithies, 1986 for references). Gene conversion events leading to disease phenotypes are now well-documented in the case of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (Donohoue et al., 1986; Harada et al., 1987; Higashi et al., 1988; Miller, 1988; Urabe et al., 1990). 21-Hydroxylase is mediated by a specific type of cytochrome P450 termed P450c21. In man, there are two 21-hydroxylase genes CYP21A and CYP21B, the former being a pseudogene and the latter the functional gene. Both are located in tandem immediately downstream (3') from one of the C4 genes encoding for the fourth component of serum complement on the short arm of chromosome 6 between HLA-B and H L A - D R (Carroll et al., 1985; White et al., 1985). The pseudogene contains an 8-bp deletion in the third exon, a 1-bp insertion in the seventh exon and a C to T transition in the eighth exon. These mutations generate frameshifts and a nonsense codon, respectively, which would prevent synthesis of functional protein. Therefore, if any of the mutations found in the CYP21A gene is intro-
duced into the CYP21B gene by gene conversion, it will result in deficiency for the enzyme. Such cases have in fact been reported and confirmed by molecular analysis (Higashi et al., 1988; Urabe et al., 1990). It is worth noting here that in human somatic cells in vitro, there are now data documenting a significant role of gene conversion events in the origin of spontaneous mutations at the H L A - A and thymidine kinase (TK) loci (reviewed in Sankaranarayanan, 1991b). Mutations due to insertion of mobile genetic elements There is good evidence in bacteria, yeast, maize and Drosophila and some limited evidence in the mouse indicating that insertion of mobile D N A sequences play an important role in the genesis of spontaneous mutations (Lambert et al., 1988). In man, the evidence for the occurrence of spontaneous mutations due to mobility of such elements (e.g., Alu, L I N E - l ) is quite limited at present (although that for their involvement in spontaneously occurring deletions is extensive, as reviewed in the preceding section). The situation with respect to the former has not changed much (with one exception) since the publication of the earlier review (Sankaranarayanan, 1988). Kazazian et al. (1987) found insertions of LINE-1 elements into exon 14 of the factor VIII gene in two of 240 unrelated patients with hemophilia A; in both cases the parents did not have the insertion and therefore the event must have occurred de novo. Other relevant information The fact that the overall organization of the human genome is complex and consists of different sequence classes (unique, moderately repetitive and highly repetitive sequences) has been known for a long time (Britten and Kohne, 1968; Jelinek and Schmid, 1982; Evans, 1984; Jeppsen and Bower, 1987). Considering just two classes of moderately repetitive sequences, the Alu and the LINE-1 elements, it is clear that they number around 500,000 (Alu) or 200,000-500,000 copies ( L I N E - I ; Singer, 1982; Skowronski and Singer, 1986). Likewise, the V N T R sequences (belonging to the highly repetitive sequence class) are quite abundant and appear to be dispersed throughout
37 the genome (Jeffreys et al., 1985a,b, 1986; N a k a m u r a et al., 1987). Alu and V N T R sequences are GC-rich and LINE-1 AT-rich. At the chromosomal level, using a combination of high resolution in situ hybridization with quantitative solid state imaging, Korenberg and Rykowski (1988) have shown that the Alu repeat family dominates in R (reverse bands) and the LINE-1 in G and Q (Giemsa and Quinacrine) bands. In in situ hybridization studies, Royle et al. (1988) found that six human minisatellite loci are not dispersed at random but show preferential (though not exclusive) localization to terminal Gbands of human autosomes. The functional significance of these and other repetitive elements has not been fully unravelled. F r o m circumstantial evidence, Chandley (1989; see also Chandley and Mitchell, 1988) has hypothesized that in meiosis, G-rich sequences (Alu and V N T R ) may be powerful primary sites for synaptic initiation within terminal and interstitial R-bands while the AT-rich sequences may play a more passive role in pairing being brought into alignment later, when homology is established. If essentially correct, these repeat sequences have an important functional role in meiotic pairing. (Royle et al. (1988), however, suggest that the proterminal minisatellites may arise as a by-product of generally enhanced recombination near chromosomal termini rather than being directly involved in recombination a n d / o r synapsis in these regions.) It is tempting to hypothesize that the deletions and duplications without clinical effects (e.g., those arising as a result of recombination between V N T R sequences) and those with clinical effects (e.g., the deletions and duplications in L D L gene) are the inevitable by-products of occasional mispairing. A similar line of reasoning may be applicable to "chromosomal variants" such as those involving centromeric heterochromatic regions in h u m a n chromosomes 1, 9 and 16 (the c-band polymorphisms) observed by cytogeneticists. Most of them may be innocuous; however, a small proportion may be associated with pathological effects of one kind or another (e.g., Shabtai and Halbrecht, 1979; Genest, 1979; Sudek and Sroka, 1979) but unlikely to be classified as Mendelian disease.
Conclusions The material discussed thus far with respect to mutational mechanisms that underlie Mendelian disease permits the following conclusions. (1) A variety of molecular changes (different types of point mutations, small and large intragenic deletions, multigene deletions, insertions, duplications, etc.) underlie Mendelian diseases; the lesions have been found in the coding or non-coding regions as well as in flanking sequences. (2) Point mutational events are not distributed at random throughout the gene; there is good evidence to suggest that transition mutations (C to T and the corresponding G to A transitions in the complementary D N A strand) occur at a high frequency within methylated gene regions. However, not all transition mutations are associated with C p G dinucleotides. (3) The occurrence of D N A deletions and duplications of genes is non-random and, in cases analyzed, the evidence is consistent with the view that there are at least four principal mechanisms: base mispairing (between two repeats) and slippage during D N A replication; homologous unequal recombination between genes that are related, homologous unequal recombination between Alu sequences (located within or between genes) and non-homologous intrachromosomal recombination which may involve Alu sequences; these findings suggest that Alu sequences may be deletion/duplication hotspots. However, not all deletions studied at the molecular level involve Alu sequences. (4) The repetitive sequences present in the human D N A may have a functional role in chromosome pairing and the deletions and duplications of genetic material that arise and lead to Mendelian disease may be construed as by-products of this natural process.
Acknowledgements I am grateful to my colleagues, Prof. L. Bernini and Dr. E. Bakker both at the D e p a r t m e n t of H u m a n Genetics, University of Leiden for their valuable comments on an earlier draft of this paper. I had the benefit of discussing a number of
38 ideas with Prof. W.J. Schull ( H o u s t o n ) over the past 2 - 3 years. He a n d Prof. H.J. Evans (Edinburgh) made a number of perceptive comments on the manuscript. I thank Prof. F.H. Sobels for a n u m b e r o f p r o f i t a b l e d i s c u s s i o n s , f o r his e n c o u r a g e m e n t to p r e p a r e this a n d the other p a p e r s f o r t h e s p e c i a l i s s u e a n d f o r his c r i t i c a l c o m m e n t s . I am
also i n d e b t e d
t o Dr.
N.
Yasuda
(Chiba,
J a p a n ) a n d to P r o f P . H . M . L o h m a n (Leiden) for their useful c o m m e n t s . T h e writing of this p a p e r was supported by EURATOM 6-0226-NL
C o n t r a c t N o . BI-
with the University of Leiden.
References N o t e that references to the text a n d references to T a b l e 6 a r e g i v e n s e p a r a t e l y .
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References to Table VI A utosomal dominants
Severe combined immunodeficiency Akeson, A.L., D.A. Wiginton, J.C. States, C.M. Perme et al. (1987) Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing. Proc. Natl. Acad. Sci. (U.S.A.) 84, 5947-5951. Berkvens, T.M. (1988) Normal and abnormal expression of the human adenosine deaminase gene. Ph.D. Thesis, Leiden University. Berkvens, T.M., E.J.A. Gerritsen, M. Oldenburg et al. (1987) Severe combined immunodeficiency due to a homozygous 3.2 kb deletion spanning the promoter and first exon of the adenosine deaminase gene. Nucleic Acids Res. 15, 93659378. Bonthron, D.T., A.F. Markham, D. Ginsburg, S.H. Orkin (1985) Identification of point mutation in the adenosine deaminase gene responsible for immunodeficiency. J. Clin. Invest. 76, 894-897. Cooper, D.N., H. Youssofian (1988) See References to the text. Markert, M.L., C. Norby-Slycord, F.E. Ward (1989) A high proportion of ADA point mutations associated with a specific alanine to valine substitution. Am J. Hum. Genet. 45, 354-361 (1989). Valerio, D., B.M.M. Dekker, M.G.C. Duyvesteyn et al. (1987) One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity. EMBO J. 5, 113-119. Wiginton, D.A., D.J. Kaplan, J.C. States et al. (1986) Complete sequence and structure of the gene for human adenosine deaminase. Biochemistry 25, 8234-8244. Amyloidosis, neuropathic, type I Dwulet, F.E., M.D. Benson (1984) Primary structure of an amyloid prealbumin and its plasma precursor in heredofamilial polyneuropathy of Swedish origin. Proc. Natl. Acad. Sci. (U.S.A.) 81,694-698 Maeda, S., S. Mira, S. Araki, K. Shimada (1986) Structure and expression of the mutant prealbumin gene associated with familial amyloidotic polyneuropathy. Mol. Biol. Med. 3, 329-338. Tawara, S., M. Nakazato, K. Kanagawa, H. Matsuo, S. Araki (1983) Identification of amyloid prealbumin variant in familial amyloidotic polyneuropathy (Japanese type). Biochem. Biophys. Res. Commun. 116, 880-888 (1983) Saraiva, M.J.M, P.P. Costa, D.S. Goodman (1986) Genetic expression of a transthyretin mutation in typical and late onset Portugese families with familial amyloidotic polyneuropathy. Neurology 36, 1413-1417.
Yoshioka, K., H. Sasaki, N. Yoshioka et al. (1986) Structure of the mutant prealbumin gene responsible for familial amyloidotic polyneuropathy. Mol. Biol. Med. 3, 319-328. Amyloidosis, neuropathic, type II Dwulet, F.E., M.D. Benson (1986) Characterization of a transthyretin (prealbumin) variant associated with familial amyloidotic polyneuropathy type II (Indiana/Swiss). J. Clin. Invest. 78, 880-886. Wallace, M.R., F.E. Dwulet, P.M. Conneally, M.D. Benson (1986) Biochemical and molecular genetic characterization of a new variant prealbumin associated with hereditary amyloidosis. J. Clin. Invest. 78, 6-12 (1986). Amyloidosis, Icelandic Abrahamson, M., A. Grubb, I. Olafsson, A. Lundwall (1987) Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C. FEBS Lett. 216, 229-233. Thrombophilia, familial Duchange, N., J.F. Chasse, G.N. Cohen, M.M. Zakin (1986) Antithrombin III Tours gene: identification of a point mutation leading to an arginine to cysteine replacement in a silent deficiency. Nucleic Acids Res. 14, 2408. Duchange, N., J.F. Chasse, G.N. Cohen, M.M. Zakin (1987) Molecular characterization of the antithrombin Tours deficiency. Thromb. Res. 45, 115-121. Koide, T., S. Odani, K. Takahashi, T. Ono, N. Sakuragawa (1984) Antithrombin III Toyama: replacement of arginine47 by cysteine in hereditary abnormal antithrombin III than lacks heparin binding ability. Proc. Natl. Acad. Sci. (U.S.A.) 81,289-293. Thrombophilia, familial, protein C Romeo, G., H.J. Hassan, S. Staempfli et al. (1987) Hereditary thrombophilia: identification of nonsense and missense mutations in the protein C gene. Proc. Natl. Acad. Sci. (U.S.A.) 84, 2829-2832. Alpha-l-antitrypsin deficiency Farber, J.P., S. Weidinger, H.W. Goedde, K. Olek (1989) The deficient a-l-antitrypsin phenotype is associated with an A to T transversion in exon III of the gene. Am. J. Hum. Genet. 45, 161-163. Fraizer, G.C., T.R. Harrold, M.M. Hofker, D.W. Cox (1989) In-frame single codon deletion in the Mmalton deficiency allele of a-l-antitrypsin. Am. J. Hum. Genet. 44, 894-902. Kidd, V.J., R.B. Wallace, K. Itakura, S.L.C. Woo (1983) AIpha-l-antitrypsin deficiency detection by direct analysis of the mutation in the gene. Nature 304, 230-234. Nukiwa, T., K. Satoh, M.L. Brantly, F. Ogushi et al. (1986) Identification of a second mutation in the protein-coding sequence ofthe Z type a-l-antitrypsin gene. J. Biol. Chem. 261, 15989-15994. Satoh, K., T. Nukiwa, M. Brantly, R.I. Garver et al. (1988) Emphysema associated with complete absence of a-l-antitrypsin of a stop codon in an a-l-antitrypsin coding exon. Am.J. Hum. Genet. 42, 77-83.
44 Hypocdipoproteinemia Antonarakis, S.E., Personal communication to McKusick (1988). Karathanasis, S.K., E. Ferris, I.A. Haddad (1987) D N A inversion within the apolipoproteins A I / C I I I / A I V encoding gene cluster of certain patients with p r e m a t u r e atherosclerosis. Proc. Natl. Acad. Sci. (U.S.A.) 84, 71987202. Van der Woude syndrome, M R Bocian, M., A.P. Walker (1987) Lip pits Am. J. Med. Genet. 26, 437-443. Hughes, M., F. Greenberg, E. McCabe, Does van der Woude syndrome map 36? Proc. Greenwood Genet. Center
and deletion lq32-41. D.H. Ledbetter (1988) to chromosome 2q347, 176-177.
Craniosynostosis, M R Gong, B.T., T.H. Norwood, H. Hoehn et al. (1986) Chromosome 7 short arm deletion and craniosynostosis. A 7psyndrome. Hum. Genet. 29, 117-123. Alagille syndrome, MR Schnittger, S., C. Hoefers, F. Beermann, P. Heidemann, I. H a n s m a n n (1989) Alagille-Watson syndrome is assigned to 20pll.2-p12.1 and provisionally to the region pll.23-p12.1. H G M 10, 1074. Ehlers-Danlos syndrome, type VIIA2 Byers, P.H. (1989) Inherited disorders of collagen gene structure and expression. Am. J. Med. Genet. 34, 72-80. Prockop, D.J., C.D. Constantinou, K.E. Dombrowski et al. (1989) Type 1 procollagen: the gene-protein system that harbors most of the mutations causing osteogenesis imperfecta and probably more c o m m o n heritable disorders of connective tissue. Am. J. Med. Genet. 34, 60-67. Wirtz, M.K., R.W. Glanville, B. Steinmann et al. (1987) Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-a2(1) chain. J. Biol. Chem. 262, 16376-16385. Elliptocytosis, Rh-linked Conboy, J., N. Mohandas, G. Tchernia, S.B. Shobet, Y.W. K a n (1986) Molecular basis of hereditary elliptocytosis due to protein 4.1 deficiency. New Engl. J. Med. 315, 680-685. Methemoglobinemia, of HbM, a type McKusick, V.A. (1988) See References to the text. Alpha-thalassemia, beta-thalassemia, H P F H Antonarakis, S.E., H.H. Kazazian, S.H. Orkin (1985) See References to the text. Weatherall, D.J. (1986) See References to the text. Sickle cell anemia Ingram, V.M. (1956) A specific chemical difference between the globins of normal and sickle cell anemia hemoglobin. Nature 178, 792. Ingram, V.M. (1957) Gene mutations in h u m a n hemoglobin:
the chemical difference between normal and sickle cell hemoglobin. Nature 180, 326. lngram, V.M. (1986) Sickle cell disease: molecular and cellular pathogenesis, Chapter 11. In: Hemoglobin: Molecular, Genetic and Clinical Aspects (H.F. Bunn, B.G. Forget, Eds.), W.B. Saunders, Philadelphia, PA, pp. 453-501. Hypercholesterolemia, familial Langlois, S., J.J. Kastelein, M.R. Hayden (1988) See References to the text. Lehrman, M.A., J.L. Goldstein, D.W. Russell, M.S. Brown (1987) See References to the text. Russell, D.W., M.A. Lehrman, T.C. Sudhof, T. Yamamoto, C.G. Davis, H.H. Hobbs, M.S. Brown, J.L. Goldstein (1986) See References to the text. Zannis, V.I., J.L. Breslow (1985) Genetic mutations affectinglipoprotein metabolism. Chapter 3. In: Advances in H u m a n Genetics (H. Harris, K. Hirschhorn, Eds.), Vol. 14, Plenum Press, New York, pp. 125-215 Immunoglobulin kappa light chain deficiency Stavnezer-Nordgren, J., O. Kekish, B.J.M. Zegers (1985) Molecular defects in a h u m a n immunoglobulin kappa chain deficiency. Science 230, 458-461. Giedion Langer syndrome Harper, P.S., J. Frezal, M.A. Ferguson-smith, A. Schinzel (1989) See References to the text. Osteogenesis imperfecta Byer, P.H. (1989) See Ehlers-Danlos syndrome, type VIIA2. Prockop, D.J., C.D. Constantinou, K.E. Dombrowski et al. (1989) See Ehlers-Danlos syndrome, type VIIA2. Immunodeficiency, T cell, with neurological disorder Williams, S.R., V. Gekeler, R.S. Mclvor, D.W. Martin Jr. (1987) A h u m a n purine nucleoside phosphorylase deficiency caused by a single base change. J. Biol. Chem. 263, 2332-2338. Porphyria cutanea Verneuil, H. de, J. the 281 (gly porphyria and 101-102.
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Piebald trait, M R Harper, P.S., J. Frezal, M.A. Ferguson-Smith, A. Schinzel (1989) See References to the text. Adenomatous polyposis, M R Herrera, L., S. Kakati, L. Gibas, E. Pietrzak, A.A. Sandberg (1986) Brief clinical report: Gardner syndrome in a m a n with an interstitial deletion of 5q. Am. J. Med. Genet. 25, 473-476. Hockey, K.A., M.T.Mulcahy, P. Montgomery, S. Levitt (1989) Deletion of chromosome 5q and familial adenomatous polyposis. J. Med. Genet. 26, 61-68.
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© 1991 Elsevier Science Publishers B.V. 0165-1110/91/$03.50 ADONIS 016511109100061D MUTREV 07297
Ionizing radiation and genetic risks I. Epidemiological, population genetic, biochemical and molecular aspects of Mendelian diseases K. Sankaranarayanan MGC Department of Radiation Genetics and Chemical Mutagenesis, Sylvius Laboratories, State University of Leiden, Leiden (The Netherlands)
(Received 31 July 1990) (Revision received 28 November 1990) (Accepted 3 December 1990)
Keywords: Mendelian diseases; Epidemiology; Population Genetics; Biochemicalaspects; Molecular aspects
Summa~ This paper reviews the currently available information on naturally occurring Mendelian diseases in man; it is aimed at providing a background and framework for discussion of experimental data on radiation-induced mutations (papers II and III) and for the estimation of the risk of Mendelian disease in human populations exposed to ionizing radiation (paper IV). Current consensus estimates indicate that a total of about 125 per 104 livebirths are directly affected by one or another naturally occurring Mendelian disease (autosomal dominants, 95/104; X-linked ones, 5 / 1 0 4, and autosomal recessives, 25/104). These estimates are conservative and take into account conditions which are very rare and for which prevalence estimates are unavailable. Most, although not all, of the recognized " c o m m o n " dominants have onset in adult ages while most sex-linked and autosomal recessives have onset at birth or in childhood. Autosomal dominant and X-linked diseases (i.e., the responsible mutant alleles) presumed to be maintained in the population due to a balance between mutation and selection are the ones which m a y be expected to increase in frequency as a result of radiation exposures. Viewed from this standpoint, the above assumption seems safe only for a small proportion of such diseases; for the remainder, there is no easy way to discriminate between different mechanisms that may be responsible or to rigorously exclude some in favor of some others. Mutations in genes that code for enzymic proteins are more often recessive in contrast to those that code for non-enzymic proteins, which are more often dominant. At the molecular level, with recessives, a wide variety of changes is possible and these include specific types of point mutations, small and large intragenic deletions, multilocus deletions and rearrangements. In the case of dominants, however, the kinds of recoverable point mutations and deletion-type changes are less extensive because of functional constraints. The mutational potential of genes varies, depending on the gene, its size, sequence content and arrangement, location and its normal functions, and can be grouped into three groups: those in which only
Correspondence: Prof. K. Sankaranarayanan, Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands).
point mutations have been found to occur, those in which only deletions or other gross changes have been recovered and those in which both kinds of changes are known. Molecular data are available for about 75 Mendelian conditions and these suggest that in approximately 50% of them, the changes categorized to date are point mutations and in the remainder, intragenic deletions or other gross changes; there does not seem to be any fundamental difference between dominants and recessives with respect to the underlying molecular defect. Onset age for Mendelian diseases appears related to the normal function disrupted or abolished by the mutation and not necessarily to the underlying molecular change. Point mutations do not appear to be distributed at random throughout the gene; C p G dinucleotide sequences, when present in the gene, provide "hot-spots" for transition-type mutations, but not all transitions occur at CpG sequences. The breakpoints involved in intragenic deletions also are not distributed at random within the gene. In cases analyzed, the length of the deletion per se is not correlated with the severity of the clinical effect. The data from a number of well-analyzed gene deletions are consistent with mechanisms that assume base mispairing between repeat sequences and slippage during replication, homologous unequal recombination between evolutionarily related genes, homologous unequal recombination between repetitive sequences such as Alu and non-homologous recombination. Not all deletions involve Alu sequences. There is circumstantial evidence supporting the hypothesis that repetitive sequences may play an important role in chromosome pairing; if true, the deletions and duplications that have been found to be associated with some diseases may represent the inevitable by-products of occasional mispairing.
In all biological systems, mutations arise "spontaneously" and can be induced by exposure to ionizing radiation. Irrespective of whether they are spontaneous or induced, mutations are recognized because they lead to altered phenotypes and a large number of spontaneously arising mutations which cause disease states in man are known. The principal reasons for the continuing efforts to estimate the genetic risks of ionizing radiation in man are: the fact that ionizing radiation is capable of inducing mutations (and, if they are induced in germ cells, they can be transmitted to the progeny), the concern that such induced mutations can cause genetic disease and the need to protect our genetic heritage. A careful examination of the progress achieved in this field over the past three decades (reviewed in Sankaranarayanan, 1988a; UNSCEAR, 1988) will reveal that (i) the estimates of risk of genetic disease in humans, attributable to radiation-induced mutations, are based on data on mutation rates obtained in studies with the mouse; for various reasons, the emphasis has been on induced mutations having dominant phenotypic effects in the
progeny of the first (and subsequent) post-irradiation generations; (ii) so far, there have been no known instances of radiation-induced heritable mutations in human germ cells or of transmissible genetic disease as we perceive it; and (iii) the extensive genetic data collected in the Hiroshima and Nagasaki studies continue to suggest that the magnitude of genetic risks to man may be lower than that extrapolated from animal data; this discrepancy has not been satisfactorily resolved. During the past several decades, considerable advances have been made in the identification of biochemical defects in human diseases and, in the last 10-15 years, the methods of, and research in, molecular biology have contributed greatly to our understanding of the nature of molecular defects underlying many of the Mendelian diseases. On the radiation front, although only a handful of mutant genes have thus far been subjected to molecular scrutiny (in the mouse in vivo system and in mammalian in vitro systems), it is already clear that there are similarities as well as differences between the spectra of spontaneous and radiation-induced mutations. However, until now,
findings in biochemical and molecular studies have had no perceptible impact on the thinking of "genetic risk estimators". As we enter the 1990s, it seems useful to take stock of the important advances in fields relevant for genetic risk estimation; reflect on whether and to what extent the concepts and assumptions hitherto used in this endeavor can still be justified; identify the changes that seem warranted; and define areas of research which are likely to contribute to a "refinement" of genetic risk estimates in the coming years. These then represent the overall aims of this series of papers of which the first four appear in this issue. These four papers together are intended to catalyze discussion on the risk of Mendelian disease in man, as a result of exposure to radiation. Work along similar lines on multifactorial and chromosomal diseases is underway and will be published in due course. The present paper, the first in this series, focuses on epidemiological, population genetic, biochemical and molecular aspects of Mendelian diseases. (Just before the submission of this paper for publication, a relevant one by Mohrenweiser and Jones (1990) appeared in Vol. 231 of Mutation Research; while it covers some of the same ground as that in my paper, its scope is rather different.) The second and third papers (Sankaranarayanan, 1991a,b) will summarize data on spontaneously arising and radiation-induced mutations in mammalian experimental systems. The fourth paper (Sankaranarayanan, 1991c) will review the "state of the art" in the field of genetic risk estimation (methods, assumptions, experimental and human databases and risk estimates) and examine the relevance of advances in our knowledge of the nature of spontaneous and radiation-induced mutations and the underlying mechanisms, for the estimation of the risk of Mendelian diseases in human populations exposed to ionizing radiation. More specifically, it will present arguments based on the above and other lines of reasoning to support the view that current estimates of risk for this group of diseases may be conservative. What are genetic diseases? Nearly all diseases are to some extent genetic and to some extent environmental and, with re-
gard to the relative importance of genetic and environmental factors in pathogenesis, it is convenient to think of them as covering a spectrum. Towards one end of the spectrum, genetic factors dominate and towards the other, environmental factors. Close to (and not necessarily at) the "genetic end" are conditions which are relatively simple in their formal genetics and which tend to be rare (i.e., Mendelian diseases and constitutional chromosomal anomalies). Towards the "environmental end" are other conditions such as infectious diseases. At different "distances" between these two ends are conditions which are common, which do not follow simple Mendelian patterns of inheritance but which tend to cluster in families (and thus have a genetic component in their etiology). These are termed "multifactorial" diseases. The term "genetic disease" thus includes not only Mendelian and chromosomal diseases, but also multifactorial ones. Furthermore, although not considered in this review, diseases which are due to mutations in the mitochondrial genome (see Grossman, 1990) such as Leber optic atrophy, mitochondrial myopathies, etc. (e.g., Novotny et al., 1986; Nakase et al., 1990; Holt et al., 1988; Wallace et al., 1988), also qualify for inclusion under "genetic disease". Knowledge of naturally occurring genetic diseases, apart from providing a perspective of illhealth in man attributable to genetic causes, also provides a convenient frame of reference to judge whether the projected increases in inherited disease as a result of radiation (or other mutagenic exposures) are trivial, small or substantial. Mendelian diseases General considerations In his 1988 compendium, McKusick has listed 2208 phenotypes (not all of these are pathological states) determined at distinct gene loci, that are well documented as being inherited in a Mendelian manner (1443 autosomal dominants, 139 X-linked ones and 626 autosomal recessives). In addition, a further 2136 conditions are included (1114 autosomal dominants, 171 X-linked and 851 autosomal recessives) for which the evidence for
T A B L E IA ESTIMATES O F LIVEBIRTH P R E V A L E N C E S (X104), FITNESS ( f ) , A P P R O X I M A T E A G E A T O N S E T (years) A N D M U T A T I O N RATES F O R SOME A U T O S O M A L D O M I N A N T DISEASES IN M A N ( A D A P T E D F R O M C A R T E R , 1982; CHILDS, 1981; BEIR, 1990; U N S C E A R , 1982) System/organ affected
Prevalence ( x 10 4) a
Fitness
Onset/clini-
Mutation rate
Carter
Childs
(Childs)
cal detection (years)
( × 10 6) per gamete per generation
4.0 5.0 0.3 0.1 2.0 0.5 0.5
3.50 3.00 1.00 0.25 2.20 --
0.5 0.8 b 0.8 0.2 0.7 ? ?
20 + 45 30--40 5 37 Adult 20-30
2.0
-
?
12
Diaphyseal aclasia (multiple exotoses) Aehondroplasia Osteogenesis imperfecta Osteopetrosis tarda Marfan syndrome Ehlers-Danlos syndrome
5.0 0.2 0.4 0.1 0.4 0.1
0.50 0.30 0.40 0.10 0.30 -
0.7 0.2 0.6 0.8 0.7 ?
Childhood At birth At birth At birth At birth Childhood
Intestines
Multiple polyposis coil
1.0
0.71
0.8
10-70
Kidney
Polycystic kidney disease
8.0
8.60
0.8
20 & upwards
Nervous
Skeletal
Disease
Neurofibromatosis Huntington disease Primary basilar impression Tuberous sclerosis Myotonic dystrophy Cerebellar ataxia (dominant) Spastic paraplegia Dominant peroneal muscular atrophy (Charcot-MarieTooth disease)
93 5 10 10 28 ? ?
?
8 12 9 1 5 ? 7 76
Dominant forms of early childhood onset deafness Dominant otosclerosis
Monogenic hypercholesterolemia
Congenital spherocytosis
Dentinogenesis imperfecta Amelogenesis imperfecta
Acute intermittent porphyria Variegate porphyria
Cleft lip + cleft palate with mucous pits of lip
Ear
Circulatory
Blood
Teeth
Metabolism
Face
95.0
(29.6) d
0.1
0.1 0.1
1.0 0.2
2.0
58.40
1.30
0.11
0.15 0.15
1.25 0.60
2.20
20.00
10.0
10.0 20.0
0.69
0.30 0.24 0.15 0.40
1.0
1.0 0.3 -
0.5
0.8
0.9 1.0
1.0 1.0
0.8
1.0
1.0
0.3
0.3 0.5 0.9 b 0.7
At birth
Early childhood onwards Early childhood onwards
Childhood Childhood
Childhood
Adult
Adult
Early childhood
At birth Early childhood At birth Early childhood
30
1
--1 c 1¢
< 1¢ -- 1 c
22
< 20 c
< 20 ¢
24
10 6 3 6
a The figures are those published by the respective authors, except that Carter presented them on a per-1000 basis and Childs on a per-million basis; the presence of two decimal places for Childs' figures, does not represent greater accuracy! b Apparent reproductive fitness does not agree with estimated mutation rate (Childs, 1981). c Mutation rate estimates subject to considerable uncertainty (Childs, 1981). d Implied in the overall estimate of total prevalence of 95/104.
Total
Rare diseases of early onset
Dominant forms of blindness Retinoblastoma Aniridia Cataracts with early onset
Eye
8
TABLE IB ESTIMATED LIVEBIRTH PREVALENCES ( x 104) OF X-LINKED AND AUTOSOMAL RECESSIVE DISEASES IN MAN (AFTER CARTER, 1982; CHILDS, 1981) System/organ affected
Prevalence ( × 104) a
Disease
Carter
Childs
X-linked conditions
Neuromuscular Blood clotting Skin Mental retardation (severe)
Duchenne muscular dystrophy. Hemophilia A (factor VIII) Hemophilia B (factor IX) Ichthyosis Non-specific X-linked Rare conditions b Very rare conditions c Total X-linked Rounded
2 1 -1 1 3 8 10
1.80 1.45
0.25 0.60 1.33
0.10 5.53
Common autosomal recessive conditions
Metabolism Nervous system Red blood cells Endocrine glands Ear Eye Severe mental retardation
Cystic fibrosis of pancreas Classical phenylketonuria Neurogenic muscle atrophies Sickle cell anemia Adrenal hyperplasias Severe congenital deafness Recessive forms of blindness Non-specific recessive forms
5 1 1 1 1 2 1 5
Rare autosomal recessive conditions
Metabolism
Tay-Sachs disease Mucopolysaccharidoses I Mucopolysaccharidoses II Metachromatic leukodystrophy Galactokinase deficiency Galactosemia Homocystinuria Cystinuria Total recessives Rounded
0.4 0.2 0.1 0.2 0.1 0.2 0.1 0.6 18.9 25.0
a For X-linked conditions, the estimates pertain to 104 male livebirths. b Those with birth prevalences in the range of 0.1-1/104 males (hemophilia B, X-linked deafness, ocular albinism, X-linked nystagmus, the Burton type of hypogammaglobulinemia, hypophosphatemic rickets, anhidrotic ectodermal dysplasia, and X-linked amelogenesis imperfecta) in Carter's compilation; in those in Childs' compilation, those with birth prevalences in the range of 0.01 0.15/104 males (11 conditions). c Those with birth prevalences of less than 1 per million males.
M e n d e l i a n i n h e r i t a n c e is i n c o m p l e t e . In using the terms "dominant" and "recessive", firstly, it s h o u l d b e r e a l i z e d t h a t t h e c l a s s i c a l c o n cepts of dominance and recessivity have been changing over the years and these designations (which connote attributes of the phenotype), while still q u i t e u s e f u l i n t h e b i o c h e m i c a l o r m o l e c u l a r s e a r c h f o r b a s i c d e f e c t s , h a v e m u c h less s i g n i f i -
c a n c e a t t h e level o f p r i m a r y g e n e a c t i o n . A s M c K u s i c k ( 1 9 8 8 ) p o i n t s o u t , m a n y p h e n o t y p e s in h i s c a t a l o g u e c l a s s i f i e d as d o m i n a n t s a r e i n c o m p l e t e l y so (i.e., t h e m u t a n t h o m o z y g o u s s t a t e l e a d s to a more severe and somewhat different phenot y p e ) a n d l i k e w i s e m a n y o f t h o s e c l a s s i f i e d as r e c e s s i v e s a r e i n c o m p l e t e l y r e c e s s i v e (i.e., m i l d m a n i f e s t a t i o n s a r e d i s c e r n i b l e i n h e t e r o z y g o t e s if
appropriate methods are used). Further, the concept that the genotype determines the phenotype precisely (i.e., if a condition is dominant, it is always manifested in the heterozygote and if it is recessive, all appropriate homozygotes exhibit the condition) is not always true. The human genetic literature is replete with examples of Mendelian conditions which have reduced penetrance a n d / o r variable expressivity. Secondly, there is the phenomenon of codominance (both alleles are expressed in the heterozygote) which is characteristic of many enzyme variants and which can be detected by electrophoretic methods. For instance, the erythrocyte enzyme adenosine deaminase (autosomal dominant; McKusick No. 10270) has three genetically determined electrophoretic phenotypes and shows polymorphism. Adenosine deaminase deficiency however is a recessive disease. Thirdly, there are conditions which, although formally classified under "recessives", have been found to be caused by mutations in different codons of the gene in question (i.e., the phenotype of the affected individual is not due to homozygosity for mutation at the same site); in other words, they are "genetic compounds". Most flthalassemia clinical "homozygotes" for instance are genetic compounds. Fourthly, there are situations exemplified by conditions such as retinoblastoma: susceptibility to retinoblastoma can be transmitted as a dominant; however, for a tumor to develop, a mutation must also occur at the same site on the other normal chromosome 13 - - a somatic mutation. Thus, in a strict sense, retinoblastoma is recessive since homozygosity is required for its expression. Finally, there are mosaics. Mosaicism for constitutional chromosomal abnormalities such as sex-chromosomal anomalies, Down syndrome, etc. (detected in lymphocyte cultures) has long been known. Only in recent Years, however has the fact that mosaics for single gene mutations can occur begun to be appreciated, especially through the use of D N A techniques. While any condition appearing in two or more affected children of unaffected parents may be reasonably assumed to be a recessive, it cannot be readily distinguished from the effects of germ-line mosaicism for a dominant mutation in that generation. It is also possible that
with some dominant mutations, because of the severity of the effect (if present in all cells of the body), only somatic mosaicism m a y be compatible with viability. Examples of conditions where germ-line mosaicism has actually been demonstrated or strongly inferred include Duchenne muscular dystrophy, hemophilia A, ornithine t r a n s c a r b a m y l a s e deficiency, achondroplasia, pseudochondroplasia, Apert syndrome, Crouzon syndrome, osteogenesis imperfecta type II, tuberous sclerosis, aniridia, etc. (reviewed in Hall, 1988; see also Edwards, 1989). In view of these and other reasons, some arbitrariness had to be exercised in classification in McKusick's catalogue: many (although not all) enzymes have been listed under dominants because of codominant polymorphism or under recessives because of deficiency.
Epidemiological aspects Overall prevalences The current consensus estimates of livebirth prevalences (UNSCEAR, 1988) are about 95/104 for autosomal dominants, 5 / 1 0 4 for X-linked ones and 25/104 for autosomal recessives (Table IA,B). The figure for autosomal dominantsis derived from several ad hoc studies (see Carter, ]977, 1982 for reviews) and is based on conditions with individual prevalences in the range of 1 20 per 104 livebirths ( " c o m m o n dominants"; about a dozen conditions) and of 1 - - 5 per 105 livebirths ("less common dominants"; also about a dozen conditions). The resulting total of 65.4/104 has been rounded upwards to about 95/104 to take into account the much rarer conditions. The estimates of Childs (1981) for basically the same autosomal dominant conditions (with some modifications) yield a slightly lower total prevalence figure of 58.4/104 livebirths (see Table IA). For X-linked recessives, Carter's estimate of 5/104 livebirths (Table IB) is based on conditions whose individual prevalences are equal to or greater than 1/104 male livebirths (four conditions; total prevalence, 5/104 male livebirths) and on those for a number of rarer conditions (estimated total of the order of 3,/104 male livebirths). The resulting total of 8 / 1 0 4 male livebirths was divided by 2 and rounded upwards to 5 / ] 0 4. The estimates of Childs for the total prevalence of
10 TABLE II A COMPARISON OF THE PREVALENCE ESTIMATES FOR SOME SELECTED AUTOSOMAL DOMINANT AND X-LINKED CONDITIONS MANIFEST AT BIRTH BETWEEN HUNGARIAN STUDIES AND THOSE PUBLISHED IN THE LITERATURE a MIM No. b
Conditions
Prevalence per 104 livebriths c Hungary
Other countries
A utosomal dominants
10080 10120 10620 11620 11930 12105 12350 12990 14290 15450 16620 17470 18020 18360 18760 19407
Achondroplasia Apert syndome Aniridia, bilateral Cataract, congenital bilateral Van der Woude syndrome Contractural arachnodactyly Crouzon syndrome EEC syndrome Holt-Oram syndrome Treacber-Collins syndrome Osteogenesisimperfecta, type I Polysyndactyly,preaxial, type IV Retinoblastoma Split hand and/or foot Thanotophoric dwarfism Wilmstumor Total
0.56 0.09 0.03 0.38
0.29-0.83 0.06-0.29 0.18 0.4
0.01
0.11
0.02
d
0.10 0.03 0.14 0.06
d 0.04 0.07 0.14
0.26
0.40
0.91
d
0.32 0.14 0.08 0.40 3.53
0.24-0.33 0.15 0.58 0.40
Hemophilia A Hemophilia B Incontinentiapigmenti Martin-Bell syndrome Duchenne muscular dystrophy Gorlin-Psaume syndrome Total
2.73 0.25 1.13 5.40 2.50
1-2 0.2-0.3 1.22 4.40 2.50
0.04
0.02
X-linked conditions
30670 30690 30890 30955 31020 31120
12.05
a From Czeizel (1989) and Sankaranarayanan and Czeizel (1990); these articles also provide references to the Hungarian and international studies; the autosomal dominant conditions have been ascertained through the program of the Hungarian Surveillance of Sentinel Anomalies (1980-1986) and involve 1,061,879 livebirths. For X-linked conditions, the sources are the following: 30670, 30690 and 30955: ad hoc epidemiological studies; 30830 and 31120: Hungarian Surveillance of Sentinel Anomalies; 31020: regional screening. b Mendelian Inheritance in Man (McKusick, 1988) numbers. c In the case of X-linked conditions, per 1 0 4 male livebirths. d Not available.
X-linked c o n d i t i o n s ( 5 . 5 3 / 1 0 4 male births or 2 . 8 / 1 0 4 total births) are lower. F o r a u t o s o m a l recessive diseases (Table IB), Carter's figure of 2 5 / 1 0 4 livebirths is based o n eight c o n d i t i o n s with i n d i v i d u a l prevalences equal to or greater t h a n 1 / 1 0 4 (total 1 7 / 1 0 4 ) a n d a n o t h e r eight with lower i n d i v i d u a l prevalences (total 1.9/104). The total for all a u t o s o m a l recessives is thus 1 8 . 9 / 1 0 4 a n d this figure has been adjusted u p w a r d s (to take i n t o a c c o u n t the very rare conditions) to 2 5 / 1 0 4 . A l t h o u g h there a p p e a r to be n o reasons to seriously q u e s t i o n the overall livebirth prevalence estimate of 2 5 / 1 0 4 for these diseases, a r g u m e n t s / s u g g e s t i o n s have b e e n advanced to s u p p o r t the view that the frequency of homozygotes for recessives at conception could be m u c h higher t h a n this figure (Neel, 1978; Vogel a n d Motulsky, 1986; Searle a n d Edwards, 1986). O n e aspect of M e n d e l i a n diseases hitherto not m e n t i o n e d pertains to the l o n g - s t a n d i n g evidence for a n increase in s p o n t a n e o u s m u t a t i o n frequencies with increasing p a t e r n a l age (e.g., a c h o n d r o plasia, A p e r t syndrome, M a r f a n s y n d r o m e , osteogenesis imperfecta, etc.) (Carothers et al., 1986; Evans, 1988; see also C h a p t e r 5 in Vogel a n d Motulsky, 1986). The rates of increase however differ b e t w e e n genes. The causes for these increases r e m a i n unresolved. It must be realized that the entries in T a b l e IA,B - although they p r o v i d e us with some insights into the load of M e n d e l i a n diseases in the h u m a n species - represent a synthesis of i n f o r m a tion from different p o p u l a t i o n s . Therefore, they do n o t reflect either the profile or the aggregate b u r d e n of such diseases in a n y specific h u m a n p o p u l a t i o n ; ideally, however, it is this which is required to project risks in context.
Clinical onset
As indicated in T a b l e IA, m a n y of the recognized " c o m m o n " d o m i n a n t s i n c l u d e d in these listings first a p p e a r in adult life (e.g., H u n t i n g t o n disease (prevalence: 5 / 1 0 4 ) , familial hypercholesterolemia ( 2 0 / 1 0 4 ) , polycystic k i d n e y disease ( 8 / 1 0 4 ) , d o m i n a n t a d u l t o n s e t otosclerosis ( 1 0 / 1 0 4 ) ) . I n contrast, m a n y of the "less comm o n " a n d " r a r e " d o m i n a n t c o n d i t i o n s are m a n i fest at b i r t h or in early childhood. Estimates of b i r t h frequencies for some of these latter conditions, from the o n g o i n g p r o g r a m m e of H u n g a r i a n
11 Surveillance of Sentinel Anomalies, have been summarized by Czeizel (1989) and Sankaranarayanan and Czeizel (1990) and are shown in Table 2. The total livebirth prevalence for these rare conditions listed in this table is only 3.53/104 which is less than 4% of the estimated total of 95/104 . The livebirth prevalence figure of Baird et al. (1988) for dominant conditions in British Columbia diagnosed through the first 25 years of life is about 14/104 , and this is about 15% of the estimated total of 95/104 . In contrast to autosomal dominants, most Xlinked and autosomal recessive diseases have onset in infancy or childhood and are generally clinically more severe. As will be discussed later, these differences in onset age seem related to the function impaired as a result of the mutation and the "natural history" of the disease process.
Some population genetic aspects As stated earlier, naturally occurring mutations constitute the basis for Mendelian diseases. Although new mutations arise spontaneously in each g e n e r a t i o n , the p o p u l a t i o n frequencies of Mendelian diseases remain stable. Therefore, there must be mechanisms that operate to maintain this stability. Exposure of a population to radiation results in the introduction of additional mutant genes in its gene pool (over and above those that arise spontaneously) and their rates of persistence and elimination will determine how m a n y additional cases of Mendelian diseases will result in any given generation. An understanding of the mechanism(s)/factors t h a t m a i n t a i n gene frequency equilibria is thus of paramount importance both for explaining the natural prevalence of Mendelian diseases and for estimating expected increases as a result of exposure to radiation. At a more general level, these questions relate to mechanisms of maintenance of population variability of which the maintenance of stable frequencies of Mendelian diseases is just one aspect. All the mechanisms postulated to explain the maintenance of natural variability in a population depend on some kind of balance between opposing forces and are adequately dealt with in population genetics text books (see for instance, Li, 1955; Crow and Kimura, 1970). Crow and Kimura
(1970) list eight possible mechanisms for maintaining population variability noting that "these are neither mutually exclusive nor collectively exhaustive". Of these, four are very briefly considered here: (i) balance between mutation and selection; (ii) selection favoring heterozygotes; (iii) neutral polymorphism; and (iv) transient polymorphism.
Mutation-selection balance Balance between mutation and selection represents one, perhaps a major, mechanism in maintaining population variability. The theory is that in each generation, new mutations arise and most of them are harmful (although to different degrees) to their carriers (i.e., they affect their "fitness") and are eliminated by natural selection sooner or later. At equilibirum, a balance is achieved between mutation rate and the rate of selective elimination. The term "fitness" as used here refers to reproductive fitness and is not always synonymous with disease. Consider a mutation that leads to a rare autosomal dominant disease which is strongly selected against. The birth frequency (rn) of the disease is directly proportional to the mutation rate (u) and to the number of generations that the mutant gene persists in the population; the latter is inversely proportional to the decrease in fitness ( f ) . The relationship b e t w e e n m u t a t i o n rate, birth frequency and fitness for these is given by the well-known equation u=m(1
-f)/2
(1)
This formula has been used to estimate most of the mutation rates summarized in Table 1A. For an X-linked condition maintained by m u t a t i o n selection balance, the principle is the same: if the fitness of the mutant males is 1 - f relative to normal males and heterozygous females are not impaired, then, u = m(1 - f ) / 3
(2)
where m represents the birth frequency in males. For autosomal recessives, the situation is complicated (see Morton, 1981; Crow and Kimura, 1970; Crow and Denniston, 1985 for detailed discussions) and will not be gone into here. It suffices
12 to note that (i) the phenotypic incidence of recessives is not directly related to mutation rates; (ii) estimates of mutation rates for recessive mutations depend on the assumption that at some time an equilibrium was reached between spontaneous mutation and loss by selection (although reduction in inbreeding in recent generations makes current equilibrium untenable); and (iii) in calculating mutation rates, one should take into account elimination of mutant alleles through their effects in heterozygotes, through consanguinity and through the mutant gene's meeting a preexising, non-complementing mutant allele by chance.
Selection favoring heterozygotes Another mechanism that can maintain population variability relies on the concept of superiority of fitness of heterozygotes, relative to that of the homozygotes. In a simple two-allele situation (say, A 1 and A 2 ) if the heterozygote (AIA2) has higher fitness relative to both homozygotes (A1A a and A z A 2 ) , selection will result in an equilibrium and both A x and A 2 will continue to occur in the population indefinitely. The population frequencies of the alleles are entirely determined by the selection coefficients against the homozygotes. Letting s~ and s 2 denote the selection coefficents for the two homozygotes (fitness being (1 s~) for the A~A 1, (1 - s2) for the A 2 A 2 and 1 for the A~A2), it can be shown that equilibrium will be attained when the frequencies in the gene pool are equal to s2/(s 1 + s 2) for the A 1 allele and s~/(sa + s2) for the A 2 allele. This situation is referred to as balanced polymorphism. There is a vast amount of literature on the existence and importance of balanced p o l y m o r phism (due to chromosomal inversions) in natural populations of Drosophila and these studies date back to the 1950s (e.g., Dobzhansky, 1951). In man, evidence strongly suggestive of balanced polymorphism is provided by the sickle-cell hemoglobin heterozygote which has neither the anemia of one homozygote nor the susceptibility to malaria of the other (Allison, 1954a,b, 1955). Associated geographically with malaria are two other polymorphic genetical defects, fl-thalassemia and glucose-6-phosphate dehydrogenase deficiency, and it seems probable that similar mechanisms of hetero-
zygous advantage to the malarial challenge have played a role in their high frequencies in endemically malarial areas (e.g., Luzzato et al., 1969; Undevia, 1973; Motulsky, 1975; Miller et al., 1976).
Neutral polymorphism " I f a series of alleles are nearly equivalent in their effect on fitness (near enough that the differences among them are of a lower order than the mutation rates or the reciprocal of the effective population number), there may be neutral polymorphism in which the frequencies of the different types are determined largely by their mutation rates. In such a situation, the effects of a finite population become especially important and the number of alleles actually maintained will be determined between mutation and random extinction through gene frequency drift" (Crow and Kimura, 1970). Evidence for the existence of potentially neutral polymorphisms in man, originally provided by protein studies, has now been extended at the D N A level through the demonstration of restriction fragment length polymorphisms (RFLPs). At their simplest, the R F L P s reflect base-pair changes which create or destroy a cleavage site for a specific restriction enzyme, causing a change in the length of a D N A fragment (e.g., W y m a n and White, 1980; Jeffreys et al., 1985a,b, 1988; Kovacs et al., 1990). Several hyperpolymorphic V N T R (Variable N u m b e r of Tandem Repeat) loci are known in the human genome. The mutation rates to new length alleles vary substantially from locus to locus; for some of these, these estimates rates are quite high (e.g., 1 × 1 0 - 2 / g a m e t e (Kovacs et al., 1990); 5 × 1 0 - 2 / g a m e t e (Jeffreys et al., 1988)).
Transient polymorphism The mechanisms thus far mentioned are all stabilizing ones. However, a population may be polymorphic because it is in the process of change and a transient polymorphism may persist for a long time and in nature this may be very difficult to distinguish from a stable polymorphism (Crow and Kimura, 1970).
13 What mechanisms can be invoked to explain the prevalence of Mendelian diseases in human populations? In inquiring about mechanisms for the maintenance of Mendelian diseases in the population, one should bear in mind the following facts. Firstly, the "stability" of the frequencies is a convenient assumption and in fact, there is no irrefutable epidemiological evidence on this point nor would one expect such evidence to be easily obtained. Secondly, it is not a simple matter to apply the concept of reproductive fitness in most present-day human populations; this is because family sizes are dictated by social and cultural considerations (in which individual choices play a dominant role) and not by purely biological ones and reproductive compensation is not uncommon; these realities do not however detract from the possibility that differences in reproductive fitness could have existed in early periods of human history. Thirdly, the dynamics of mutant genes in the past determines their present prevalences and provides a basis for future predictions; during the last 3 - 4 generations or about 100 years, human populations have experienced dramatic changes in living conditions. It would thus be an oversimplification to treat selection as constant over long periods of time. Fourthly, with conditions such as Huntington disease, a positive family history has traditionally been one of the criteria by which the clinician decides as to whether a case in question is likely to represent a given disease entity or not; it is not surprising, therefore, that one does not find " n e w " mutations. The point is that the ascertainment of a specific heritable disease is rarely as random as one would like it to be. Fifthly, the data can appear to be consistent with more than one mechanism, and in such cases, statements about the most probable mechanism are essentially matters of informed judgement. Finally, population genetic considerations are relatively straightforward if a disease can be considered as a single, homogeneous entity; however, as data on molecular heterogeneities of mutations underlying Mendelian diseases continue to accumulate, another dimension of complexity is introduced; for instance, the molecular nature of the
mutation and the bodily s y s t e m / f u n c t i o n affected may have an important bearing on onset age (the latter could be very early for some large deletions so that they are eliminated through early embryonic losses a n d / o r spontaneous abortions) and thus, for the determination of selection coefficients. As stated earlier, the relevance of the question of mechanisms in the context of the present series of papers stems from the theory that the prevalence of diseases (i.e., the respective mutant genes) maintained by mutation-selection balance would, in principle, be expected to increase as a result of exposure to radiation. Since the prevalence of autosomal recessive diseases is only very indirectly related to mutation rate, in what follows, only autosomal dominants and X-linked recessives will be considered. It is safe to assume that most of the rare autosomal dominant and X-linked conditions listed in Table 2 (and some additional ones in Table 1A,B such as neurofibromatosis, Marfan syndrome and autosomal dominant forms of osteogenesis imperfecta) are likely to be maintained by mutation-selection balance because of onset at birth or in childhood and severity of effect which preclude reproduction in most (if not all) cases. The total prevalence of these " b i r t h or childhood onset" conditions is only 3.53/104 in the population of Hungary (Table 2) and for those with onset through the first 25 years of life, 14/104 in British Columbia (Baird et al., 1988). These values represent less than 5 and 15% respectively of the estimated total prevalence of autosomal dominant diseases (95/104). With the relatively common dominants such as familial hypercholesterolemia, adult form of polycystic kidney disease, Huntington disease, myotonic dystrophy and dominant otosclerosis (which together account for nearly one-half of the total birth prevalence of 95/104 ) the situation is not simple; these conditions are compatible with a substantial degree of reproductive fitness because they are of relatively late onset. Searle and Edwards (1986) speculated that " s o m e severe dominants, including Huntington's chorea and myotonic dystrophy, so rarely appear as new mutants in well-conducted surveys (Harper, 1979; Reed et al., 1958; Shaw and Caro, 1982)
14 that their population frequency may well be maintained by some sort of heterozygous advantage during the reproductive period rather than by mutation .... The dominant hyperlipidemias and hypercholesterolemias are also so common that they are unlikely to be maintained by mutation." The problem, however, is our current total ignorance of the nature of this advantage and of the biological mechanisms involved. Differences of opinion exist on the mechanisms of maintenance of Huntington disease (HD). H D was inferred to be selectively deleterious in a Michigan population (Reed and Neel, 1959), selectively neutral in a Queensland population (Wallace and Parker, 1973) and selectively advantageous in both a Minnesota (Marx, 1973) and a Welsh population (Walker et al., 1983). In a more recent paper on H D in the Afrikaners of South Africa, Stine and Smith (1990) concluded that the current H D gene frequency estimate of 4.2 x 10 -5 in this population (based on 210 H D cases who were descendants of an original settler who was a presumed heterozygote, identified among 2.5 x 10 6 Afrikaners) is consistent with a selection coefficient of about 0.34 (i.e., the H D mutation is deleterious). It is worth noting that attempts to explain the relatively high prevalences of conditions such as familial hypercholesterolemia (FH) on models based on neutral or transient polymorphisms is also not free of problems. Such an explanation would require that thr allele frequencies are largely determined by a balance between mutation and random extinction through gene frequency drift. The concepts of "founder effect" and gene frequency drift have in fact been used to explain the high frequencies of homo- and the heterozygous cases of F H in Afrikaners and French Canadians (Seftel et al., 1980; Brink et al., 1987; Hobbs et al., 1987). However, although these mechanism cannot be excluded, they do not seem compatible with the widespread nature of F H in different populations. For FH, the birth frequency estimate (i.e., that of heterozygotes) is about 20/104, but less than 1% of F H cases are presumed to be due to new mutations. (This appears to be true for H D and adult polycystic kidney disease as well; see chapter 5, page 419 in Vogel and Motulsky, 1986.) The
frequency of F H homozygotes is generally of the order of about 10 6. If the F H mutations are selectively neutral or near-neutral in heterozygotes, one can formally estimate that the mutation rate is about one-half of that of new mutants (i.e., one half of 1% of 20/104); this is about 10 5 which is not unduly high. However, this figure depends critically on the estimated proportion of sporadic cases; if the latter is much lower than assumed, then the mutation rate estimate will be correspondingly lower. It is conceivable that, in the absence of selection against heterozygotes, the condition is maintained in the population through selection against homozygotes for the dominant mutation (these individuals die in their teens or early 20s). If this assumption is correct, in a hypothetically large population in which the "gain" of mutant alleles is balanced by their "loss" in homozygotes (and if there is no inbreeding), it can be shown that the mutation rate will be equal to the frequency of the homozygotes which is 10 6, i.e., an order of magnitude lower than that estimated in the preceding paragraph. If F H and H D are illustrative of the relatively common dominant conditions, the principal message from this discussion is that the available data may be compatible with more than one mechanism of maintenance of mutant genes involved. This in turn means that the assumption implicit in the use of one principal method of risk estimation (to be dealt with in paper IV), namely that all autosomal dominant and X-linked diseases are maintained in the population through a balance between mutation and selection, may need to be carefully qualified. Biochemical basis of Mendelian diseases General considerations A great variety of different enzymes and other non-enzymic proteins are synthesized in the cells of every multicellular eukaryote. On general biochemical grounds, one would expect that mutations in genes that affect the nature, synthesis, stability or quantity of enzymic proteins are likely to be reflected in the phenotype only in the homozygote (or in hemizygotes in the case of mutations in X-linked genes), i.e., formally, they are "reces-
15 sives". This is so b e c a u s e o f the facts that (i) in vivo, the enzymes d o not act in isolation, b u t are k i n e t i c a l l y linked to o t h e r enzymes via their substrates a n d p r o d u c t s a n d (ii) few enzymes are so r a t e - l i m i t i n g as to cause a serious r e d u c t i o n in the f u n c t i o n i n g of a m e t a b o l i c p a t h w a y when the enz y m e has 4 0 - 6 0 % n o r m a l activity, which is generally the case in heterozygotes. M o s t i n b o r n errors of m e t a b o l i s m are recessives and, as K a c s e r a n d Burns (1981) stated, " t h e w i d e s p r e a d o c c u r r e n c e of recessives is... an inevitable c o n s e q u e n c e of the kinetic structure of e n z y m e n e t w o r k s . " I n contrast, m u t a t i o n s in genes that c o d e for non-enzymic p r o t e i n s or for enzymes that effect changes in the structure of n o n - e n z y m i c p r o t e i n s (e.g., collagen, several p r o t e i n s involved in b l o o d c o a g u l a t i o n , c o m p l e m e n t system, t r a n s p o r t p r o teins, b i n d i n g proteins, etc.), in general, w o u l d be e x p e c t e d to result in " d o m i n a n t " p h e n o t y p e s . This is b e c a u s e the " b u i l t - i n safety f a c t o r " c o m m o n to m o s t e n z y m e systems does not exist with the result that the p r o d u c t of a n o r m a l allele is likely to be insufficient to m a i n t a i n h e a l t h y function a n d / o r the m u t a n t p r o d u c t interferes in some w a y with that of the n o r m a l allele a n d thus with the develo p m e n t of the n o r m a l p h e n o t y p e . However, instances are k n o w n where e n z y m e deficiencies result in " d o m i n a n t " p h e n o t y p e s (e.g., the five different forms of p o r p h y r i a ) . This k i n d of situation m a y occur when the e n z y m e in q u e s t i o n h a p p e n s to b e r a t e - l i m i t i n g in the m e t a b o l i c p a t h w a y in which it takes part, b e c a u s e the level of activity of such enzymes in the n o r m a l situation will in general b e closer to the m i n i m u m r e q u i r e d to m a i n tain n o r m a l function (Harris, 1970) or as a result of c h a n g e in the specificity of the enzyme. Likewise, deficiencies or a b n o r m a l i t i e s in n o n - e n z y m i c p r o t e i n s l e a d i n g to " r e c e s s i v e " p h e n o t y p e s are also known, a n d can arise as a result of m u t a t i o n s in genes whose p r o d u c t s are involved in r a t e - c o n trolled processes (see M c K u s i c k , 1986, 1988 for examples).
Actual observations T a b l e III, e x t r a c t e d f r o m A p p e n d i x A of McK u s i c k (1988), s u m m a r i z e s our c u r r e n t k n o w l e d g e on a b o u t 400 M e n d e l i a n c o n d i t i o n s with d e f i n e d b i o c h e m i c a l a b n o r m a l i t i e s . As can b e seen, defects in enzymic proteins more often result in recessives
TABLE III GROUPING OF MENDELIAN DISORDERS ACCORDING TO THE NATURE OF THE IDENTIFIED BIOCHEMICAL DEFECT a Defect in
Enzymic protein Non-enzymic protein
Autosomal Autosomal X-linked Total dominant recessive n % n n % n % 34 14.9 179 78.5 15 101 56.7
6.6 228 100
59 33.1 18 10.1 178 100
a Based on McKusick (1988). In this table, horizontal comparisons (percentages of the different Mendelian classes) within each of the two groups are unlikely to be biased (see text); however, vertical comparisons ('enzymic' versus 'non-enzymic' among the three Mendelian groups) will be biased since, until now, more abnormalities have been detected in enzymes (or deficiencies) than in non-enzymic proteins.
than dominants (179 a u t o s o m a l recessives to 34 d o m i n a n t s , o r a ratio of 5.3 to 1) while those in non-enzymic structural proteins are more likely to result in dominants than recessives (101 a u t o s o m a l d o m i n a n t s to 59 recessives or a r a t i o of 1.7 to 1). These d a t a are thus c o n s i s t e n t with theoretical expectations, c o n s i d e r i n g the fact that there is s o m e a r b i t r a r i n e s s in M c K u s i c k ' s classification discussed earlier (see also H o o k , 1987). It is also w o r t h n o t i n g that M e n d e l i a n c o n d i t i o n s for which the a c t u a l b i o c h e m i c a l basis is k n o w n r e p r e s e n t o n l y a small subset of the over 4000 c o n f i r m e d plus p r o b a b l e ones. Molecular basis of Mendelian diseases General considerations In c o n s i d e r i n g the m o l e c u l a r basis of M e n d e l i a n diseases, o n e should recognize three i m p o r t a n t facts. First, n o t all m u t a t i o n a l changes result in disease a n d those that do, r e p r e s e n t b u t a small p r o p o r t i o n of changes p o t e n t i a l l y possible. O n e a l r e a d y k n e w this to b e true from, a m o n g others, o b s e r v a t i o n s on the w i d e s p r e a d o c c u r r e n c e of p r o tein p o l y m o r p h i s m s a n d the fact t h a t o n l y s o m e of these are a s s o c i a t e d with clinical disease. N o w , research in m o l e c u l a r b i o l o g y h a s s h o w n t h a t varia t i o n s in D N A sequence ( c a u s e d b y n u c l e o t i d e substitutions, d e l e t i o n s a n d d u p l i c a t i o n s ) w i t h i n
13006 13050
12310
11845 11930
Hereditary persistence of fetal hemoglobin, deletion type Methemoglobinemia, hemoglobinopathic, of Hb M (beta type) Sickle cell anemia Hereditary persistence of fetal hemoglobin, non-deletion type A Hereditary persistence of fetal hemoglobin, non-deletion type G
Adenine phosphoribosyltransferase deficiency Severe combined immunodeficiency Amyloidosis, neuropathic, type I Amyloidosis, neuropathic, type II Amyloidosis, Icelandic, cerebrovascular type Thrombophilia, familial, due to antithrombin III deficiency Thrombophilia, familial, due to defect in protein C Alpha-l-antitrypsin deficiency Hypoalphalipoproteinemia Alagille syndrome Van der Woude syndome, mental retardation Craniosynostosis, mental retardation Ehlers-Danlos syndrome, type VIIAI Elliptocytosis, Rh-linked Methemoglobinemia, hemoglobinopathic, of Hb M (alpha type) a-Tlialassemia fl-Thalassemia
10260
10480 10490 10515
Disorder
MIM
17686 10740 10768
Protein C Alpha-l-antitrypsin Apolipoprotein A-I
14190 14190 14220 14225
fl-Globin yA-Globin TG-Globin
PMp
PMm PMp
PMm
PMm PMm, PMn, PMp, PMrp, PMs, PMt
14180 14190
fl-Globin
PMm
14180
14190
PMs?
PMm PMm
PMm, PMn
PMm
PMm PMm PMm PMm
LMd, LMr
LMd
LMd LMr
LMd
LMd LMd
LMd
LMd
2,4
2,4
1
1
2,4
1
1
1
1,2 1
2
2,3 3 2 1,2
3
1,2
1 3 3 3
3
detection
PMm
Onset/
PM
LM
Type(s) of
12016
fl-Globin
/3-Globin
a-Globin
Procollagen, type I Protein 4.1 et-Globin
10730
Antithrombin III
11930
10270 17630 17630
MIM
Adenosine deaminase Prealbumin (transthyretin) Prealbumin (transthyretin) Gamma-trace (cystatin C)
A utosomal dominants
Defective gene product
The first MIM number (McKusick, 1988), on the left, refers to an entry for the disorder; the second MIM number, on the right, refers to an entry for a particular molecule (gene product). Sometimes, when it is not certain that the given molecule is the site of the defect, two entries exist in McKusick (1988) and both are given.
INTRAGENIC LESIONS IDENTIFIED IN MENDELIAN DISORDERS IN MAN (BASED ON MCKUSICK, 1988; HARPER et al., 1989 WITH SOME ADDITIONS)
TABLE IV
20775 21410 22402 22960 23080 23205 23440 24665
20332
20191
19340 19407
19035
18020 18050 18290 18840 18840
17570 17610 17627
17280 17510
15023 16621
14389
Albinism, oculocutaneous (tyrosinase deficient) Apolipoprotein C-II deficiency Zellweger syndrome Hyperlipoproteinemia, type III Fructose intolerance, hereditary Gaucher disease Propionicacidemia, type II Angelman syndrome Lipoprotein lipase deficiency
Pyruvate dehydrogenase E-I deficiency Adrenal hyperplasia
Immunodeficiency, T cell, with neurological disorder Piebald trait, mental retardation Adenomatods polyposis, mental retardation Greig cephalopolysyndactyly Porphyria cutanea tarda Prader-Willi syndrome Diabetes mellitus, rare forms Retinoblastoma Rieger syndrome Spherocytosis I, mental retardation DiGeorge sequence DiGeorge sequence, mental retardation Hemolytic anemia due to triosephosphate isomerase deficiency Trichorhinophalangeal syndrome, type I, mental retardation yon Willebrand disease WAGR syndrome
Giedion-Langer syndrome Osteogenesis imperfecta congenita, type II
Kappa light chain deficiency
Hypercholesterolemia, familial
Lipoprotein lipase
Apotipoprotein E Fructose-l-phosphate aldolase Acid fl-glucosidase Propionyl-CoA-carboylase
Apolipoprotein C-II
Pyruvate dehydrogenase E-I a subunit Steroid cytochrome P450-21hydroxylase Tyrosinase
A utosomal recessives
von Willebrand factor
Triosephosphate isomerase
19045
PMm, PMs PMm PMm
PMm
PMm
PMm
Insulin Retina-specific protein
PMm
PMm
PMm
PMm 17673
16405
12015 12016
14720
PMm, PMn
Uroporphyrinogen decarboxylase
al-Chain of type I collagen; a2-chain of type II collagen Purine nucleoside phosphorylase
Low defisity lipoprotein receptor Immmunoglobulin kappa constant chain
LMd LMd LMd
3 2 3
1,2,3
2,3 1 3 2,3
1
LMd, LMr
LMd LMd
3
2,3 3
LMd LMd LMd
1
3 1,2 3 2 2 2 1 1
1
2,3 3
1,2
1,2 1
4
3
LMd
LMd LMd LMd LMd LMd
LMd
LMd
LMd LMd
LMd LMd
LMd, LMr
30150 30310
30020 30050
27280 27660
26160 26240 26880
Adrenal hypoplasia (Addison disease) Ocular albinism (Nettleship-Falls type OA1) and ichthyosis Fabry disease Choroideremia
Miller-Dieker lissencephaly syndrome Omithinemia with gyrate atrophy of choroid and retina Phenylketonuria Pituitary dwarfism I Sandhoff disease (GMs-gangliosidosis, variant 0) Tay-Sachs disease Tyrosinemia II (Richner-Hanhart syndrome)
24720
25887
Disorder
MIM
TABLE IV (continued)
a-Galactosidase A
X-linked
Phenylalanine hydroxylase Growth hormone /3-Hexosaminidase A & B, fl-chain B-Hexosaminidase A, a-chain Tyrosine transaminase
Ornithine ketoacidaminotrans ferase
Autosomal recessioes
Defective gene product
13925
MIM
PMm, PMs
PMm, PMi
PM
Type(s) of
LMd, LMdu LMd LMd
LMd LMd
LMd LMd
LMd LMd
LMd
LMd
LM
2 2
1,2 2
1
2
1
Onset/ detection
Microphthalmia, iridoschisis, goiter, labium synechia, craniotabes Muscular dystrophy, Becket & Duchenne Norrie disease Ornithine transcarbamylase deftciency Pelzaeus-Merzbacher disease
Choroideremia, deafness, mental retardation Color blindness Granulomatous disease, X-linked Hemophilia A Hemophilia B Glycerol kinase deficiency, adrenal hypoplasia, hypogonadotropic hypogonadism, mental retardation Ichthyosis, chondrodysplasia punctata, Kallman syndrome, mental retardation Lesch-Nyhan syndrome
Omithine transcarbamylase
Dystrophin
PMm
PMm
LMd, LMr, LMdu LMd LMd
LMd, LMdr, LMdu LMd
PMm
Hypoxanthine guanine phosphoribosyltransferase
30800
LMd
PMm, PMn PMm, PMn
LMd, LMdu LMd LMd, LMi LMd, LMi LMd
Steroid sulfatase
Cytochrome b (-245), fl-chain Factor VIII Factor IX
1
2,3 1
2
3
1
1
2 2 1 1 2
PM, point mutations; PMi, PM in initiator codon; PMm, PM, missense type; PMn, PM, nonsense type; PMp, PM in promoter, PMrp, PM affecting RNA processing (other than splicing); PMs, PM affecting RNA splicing; PMt, PM in terminator codon; LM, length mutation; LMd, deletion; LMi, insertion; LMr, rearrangement; LMdu, duplication. Onset/detection: 1, at birth; 2, childhood; 3, adults; 4, can be asymptomatic.
31208
31060 31125
31020
30970
30810 30295
30390 30640 30670 30680 30703, 30020
30310
20 human populations, as revealed through the use of restriction enzymes, are common (e.g., Botstein et al., 1980; Cooper and Schmidtke, 1984; Jeffreys et al., 1986; N a k a m u r a et al., 1987; Mohrenweiser et al., 1989, 1990). Most of these R F L P variants do not seem to have functional significance or association with any discernible clinical phenotypes. A few of course represent mutations known to be associated with diseases (e.g., sickle-cell mutation; Kan and Dozy, 1978). Second, most Mendelian diseases are heterogeneous, phenotypically and at the molecular genetic level: genetic heterogeneity in terms of more than one locus for a single clinical phenotype as well as multiple disease phenotypes determined by a single locus have been recognized for many years. Molecular biology has taught us that heterogeneity in terms of the underlying molecular changes is also common. The latter is due to the fact that a wide variety of molecular changes is theoretically possible - and does occur, as revealed by D N A analysis although the number of clinical and biochemical phenotypes that result is often limited, as was first extensively documented in the case of globin gene mutations. Third, the kind of changes that one sees in a mutant would be expected to be dependent on the D N A sequence organization and function of the normal gene in the organism, the kind of changes it can tolerate and whether the loss of the gene or alteration of its function is compatible with viability and thus recoverable for study. When a recessive mutation first arises, it is "sheltered" by the wild-type allele in the heterozygote; therefore, for these mutations, a wide array of molecular changes is possible. These changes can be specific point mutations (base-pair changes) in the gene or more drastic events such as intragenic deletions, multilocus deletions (provided such multilocus loss is not incompatible with viability), rearrangements, etc. In contrast, for most dominant mutations, the kinds of recoverable molecular changes are likely to be less since any major disturbance in normal function due to total gene deletions or rearrangements will be incompatible with viability, i.e., there are functional constraints.
Actual obseroations As of 1989, information on gene localization
(to specific c h r o m o s o m e s / c h r o m o s o m a l regions) is available for 464 clinical disorders (Harper et al., 1989) and the molecular changes underlying 76 Mendelian conditions have been ascertained, although to different levels of precision and completeness (McKusick, 1988; Harper et al., 1989 and other published results). Following McKusick (1988), one can classify these changes into two principal groups, namely point mutations (PMs) to include base pair changes anywhere in the gene and length mutations (LMs) to include D N A deletions (small and large), insertions, rearrangements and duplications; some of the latter are fundamentally the same as microscopically detectable chromosomal aberrations, e.g., deletions, rearrangements, insertions and duplications. Table IV, based on McKusick (1988) and Harper et al. (1989) with some additions, presents the currently available information on molecular changes in Mendelian conditions, together with an indication of the age at onset (see also Mohrenweiser and Jones, 1990). It should be mentioned here that: (i) although enzyme deficiencies have been demonstrated in well over 200 recessives (autosomal and X-linked), only those for which data from molecular studies exist are included in the table; and (ii) the table does not provide information on the proportion of PM or LM type of events at any given locus but only some indication of the " m u t a t i o n a l potential" of the genes studied. Table IV permits two general conclusions. First, both PMs (of different kinds) as well as LMs have been found; however, with some genes, a considerable variety of changes has been recorded whereas with some others, PMs alone or LMs alone have been found. There are several reasons for this: clinical relevance and the amount of effort expended, rate of occurrence of mutants and availability for analysis, the specificity of the type of mutational event (i.e., whether it can occur anywhere in the gene and still give a detectable phenotype or whether it is restricted to specific codons), sequence organization, size and function of the gene. Second, most of the autosomal recessive and X-linked conditions have onset at birth or in childhood and this seems unrelated to the nature of the underlying molecular event; with autosomal dominants, however, the situation appears to be
21 TABLE V CLASSIFICATION OF MENDELIAN DISEASES ACCORDING TO THE NATURE OF THE UNDERLYING MOLECULAR DEFECT INTO THOSE WHICH ARE DUE TO POINT MUTATIONS (PM), POINT OR LENGTH MUTATIONS (PM OR LM) OR LENGTH MUTATIONS (LM) ONLY Disease classification Autosomal dominants X-linked Autosomal recessives
PM only 18 1 4 Total 23 (30.2%)
22 3 sub-total 25 (43.1%) Autosomal recessives 8 Grand total 33 (43.4%)
Autosomal dominants X-linked
PM or LM a
LM only
Total
6 4 4 14 (18.4%)
18 11 10 39 (51.3%)
42 16 18 76 (100%)
2b 2c 4 (6.9%) 0 4 (5.2%)
18 11 29 (50.0%) 10 39 (51.3%)
42 16 58 18 76 (100%)
For the top half of the table, the criterion for inclusion in this group is that at least one LM has been identified; for the bottom half, the criterion is that the frequency of LM should be higher than 5%. See text for details. b Alpha-thalassemia and familial hypercholesterolemia. c Lesch-Nyhan syndrome and ornithine transcarbamylase deficiency. a
different: those d u e to P M s m a n i f e s t themselves at birth, in c h i l d h o o d or in a d u l t s while those d u e to L M s m a n i f e s t themselves p r e d o m i n a n t l y at b i r t h or in childhood. It is easy to u n d e r s t a n d the s i t u a t i o n with respect to a u t o s o m a l a n d X - l i n k e d recessives: m o s t ( a l t h o u g h n o t all) are d u e to e n z y m e deficiencies which d i s r u p t n o r m a l m e t a b o lism a n d so are d e t e c t e d early. H o w e v e r , the m e c h a n i s m or m e c h a n i s m s t h a t p e r m i t s o m e a u t o s o m a l d o m i n a n t P M s to express themselves at a n y early stage in life while for s o m e o t h e r s onset is d e l a y e d until a d u l t h o o d , s o m e t i m e s late a d u l t h o o d , are n o t fully delineated. O n e w o u l d a p r i o r i expect that onset will b e d e p e n d e n t on the b o d i l y f u n c t i o n affected o r c o m p r o m i s e d as a result of the m u t a t i o n : for i n s t a n c e if it is a vital b o d y f u n c t i o n (e.g., the i m m u n e function) that is affected, then this is likely to c o m e to the attention of the clinician early on in the life of the patient. I n contrast, if the " n a t u r a l h i s t o r y " of the disease is such that an overt clinical m a n i f e s t a t i o n takes m a n y years (e.g., familial h y p e r c h o l e s t e r o lemia, familial a m y l o i d p o l y n e u r o p a t h y ) , clinical d e t e c t i o n m a y be d e l a y e d until later. T h e essence of the r e a s o n i n g then is that, with a u t o s o m a l d o m i n a n t PMs, onset age is m o s t p r o b a b l y r e l a t e d to the n a t u r e of the i m p a i r e d f u n c t i o n a n d is n o t necessarily d e p e n d e n t on the k i n d of u n d e r l y i n g
m o l e c u l a r defect ( P M or LM). S t a t e d differently, with respect to onset age, there m a y b e no f u n d a m e n t a l m o l e c u l a r - a l t e r a t i o n - r e l a t e d differences between a u t o s o m a l d o m i n a n t s o n the one h a n d a n d a u t o s o m a l recessive a n d X - l i n k e d ones on the other. T h e rare a u t o s o m a l d o m i n a n t c o n d i t i o n s for which o n l y L M s have b e e n r e c o v e r e d so far ( T a b l e 4) merit s o m e c o m m e n t . M o s t of these are m a n i fest at b i r t h or c h i l d h o o d . M a n y of these c o n d i tions are m i c r o d e l e t i o n ( c o n t i g u o u s gene deletion) s y n d r o m e s (Schinzel, 1988); in these, a p a r t f r o m the p u t a t i v e gene in question, a d d i t i o n a l D N A sequences that are vital for s o m e essential b o d i l y functions ( a n d not necessarily for survival) m a y also be deleted. T h e m o l e c u l a r b i o l o g y of these c o n d i t i o n s has n o t yet b e e n w o r k e d o u t in detail.
Mutational potential of different genes A n overview of the relative i m p o r t a n c e of P M s a n d L M s in M e n d e l i a n diseases s t u d i e d thus far c a n be o b t a i n e d b y r e g r o u p i n g the i n f o r m a t i o n c o n t a i n e d in T a b l e IV u n d e r the h e a d i n g s of " P M s o n l y " , " P M s or L M s " a n d " L M s " only. T h e resuits are given in the top half of T a b l e V. It c a n b e seen that a m o n g the 76 genes s t u d i e d (i) o n l y P M s have thus far b e e n f o u n d in 23 (30%), P M s or L M s in 14 (18%) a n d o n l y L M s in 39 (51%) a n d
22 (ii) there seems to be a preponderance of LMs among X-linked diseases. Of interest in the present context, however, are the relative proportions of PMs versus LMs in the different Mendelian diseases. The situation is straightforward with respect to genes at which only PMs or only LMs have actually been observed; however, those in which either PMs or LMs have been found (n = 14) need further scrutiny for ascertaining the nature of the predominant type of change. To do this, an arbitrary criterion was adopted: genes at which the recorded LMs are 5% or less were included in the " P M only" group and those at which this proportion was exceeded, in the " P M or L M " group. When the " P M or L M " group was reanalyzed with the above criterion, only four conditions "qualified" for inclusion in the above group. These are: c~-thalassemia (frequency of LMs over 80%; Antonarakis, 1985; Orkin, 1987), familial hypercholesterolemia (frequency of LMs in the range of 6-63%; see McKusick, 1988 for details and references); ornithine transcarbamylase deficiency (frequency of LMs about 7%; Rozen et al., 1985) and Lesch-Nyhan syndrome (frequency of LMs about 18%; Yang et al., 1984). For the remaining 10 entries, LMs are infrequent (around 5% or less of the events, when recorded), and therefore were included in the " P M only" group. The results of the above regrouping are given in the bottom half of Table V. Of the total of 76, 33 (43.4%) are now classified under PMs, four (5.2%) under " P M s or LMs" and 39 (51.3%) under LMs. If only the autosomal dominant and X-linked diseases are included, these percentages are nearly the same (43.1%, 6.9% and 50%, respectively). One tentative conclusion from this analysis is that in roughly one half of the genes at which spontaneous mutational events (leading to Mendelian disease) have been studied, P M is the underlying event and in the remainder, the events are DNA deletions or other gross changes.
Origin of spontaneous mutations General considerations
The question of why mutations arise "spontaneously" still cannot be answered and the term
"spontaneous" remains a thinly veiled admission of our ignorance of the phenomenon. Already early in the history of radiation genetics, Muller and Mott-Smith (1930) showed that natural background radiation can account for only a negligible fraction of spontaneous mutations in Drosophila and the same is likely to be true of humans as well. The question of whether and to what extent the very low levels of chemical mutagens present in our environment (in the food we eat, the water we drink and the air we breathe) may together contribute to spontaneous mutations has not yet been answered but it seems unlikely that a significant fraction of "spontaneous" mutations is due to these. During the last decade, there has been much discussion, although in the context of aging and cancer, on the possibility that endogenous mutagens produced by normal aerobic metabolism can contribute to naturally occurring D N A damage at least to some extent (e.g., Adelman et al., 1988; Cutler, 1986; Ames, 1988a,b, 1989; Ames and Saul, 1988; Richter et al., 1988; Simic et al., 1989). Four important endogenous processes leading to significant D N A damage are likely to be oxidation, methylation, deamination and depurination. The existence of specific D N A - r e p a i r glycosylases for oxidative, methylated and deaminated adducts and a repair system for apurinic sites produced by spontaneous depurination provide support for this premise (Ames, 1989). Perhaps, as Sargentini and Smith (1985) state " w i t h continued study, the term "spontaneous mutagenesis" will be replaced by more specific terms such as 5-methylcytosine deamination mutagenesis, fatty acid oxidation mutagenesis, phenylalanine mutagenesis and imprecise-recombination mutagenesis." On the question of how spontaneous mutations arise, there is a considerable wealth of data from diverse biological systems and these show that spontanous mutations can occur as a result of errors during D N A replication, repair and recombination. Additionally, there is now substantial evidence f r o m bacteria, yeast, maize and Drosophila which demonstrates that a significant proportion of spontaneous mutations originates from insertion of mobile genetic elements into genes (see Lambert et al., 1988). In humans, at present, the evidence for this is limited.
23 Mutational mechanisms
Table 6 (which is an expanded version of Table 4 discussed earlier) provides details on the kinds of molecular changes observed in various Mendelian diseases; they also enable one to gain some insight into the mechanisms involved in spontaneously occurring PM and LM events. These are discussed in the following paragraphs. A r e P M events distributed throughout the gene or are there specific exons / codons in which they arise?
To answer this question, genes at which almost every PM change has been ascertained and studied would represent the ideal situation; the only genes that come anywhere near this "ideal" situation are the globin genes. The less-than-ideal situation, which would permit at least some insights into major trends, would be instances of conditions due to several independent point mutations at the same gene. There are many such conditions as will be discussed below. Examples of conditions in which the PM events appear restricted to specific c o d o n s / e x o n s (Table 6) are (i) familial amyloid polyneuropathy, type I (a single PM, in exon 2 of the transthyretin gene in Portugese, Japanese, Swedish and Greek patients); (ii) familial hypercholesterolemia (the three characterized PMs in the low density lipoprotein receptor gene are in exons 14 and 17); (iii) Gaucher disease (PMs in exons 5, 9 and 10 of the glucocerebrosidase gene); (iv) phenylketonuria (PMs in exons 7, 9 and 12); (v) hemophilia A (PMs in exons 7, 18, 22-24 and 26 of the factor VIII gene); (vi) hemophilia B (PMs in exons 2, 3, 6, 8 and in IVS-3 and IVS-6 of the factor IX gene); and (vii) Lesch-Nyhan syndrome (in exons 3, 4 and 8). In fi-thalassemia, mutations in the /~-globin gene are found over the entire gene, but there seems to be a clustering of the events in the 5' part of the gene, exon 1 in particular. In severe combined immunodeficiency, the PM events appear to be distributed throughout the entire coding sequence. In osteogenesis imperfecta (OI), the PMs in the COL1A1 and COL1A2 genes are distributed over the entire gene, but the phenotypic effects vary depending on which of the procollagen chains contains the mutation, its location,
etc.; most of the PM events identified in the COL1A1 gene result in the lethal OI (type II) phenotype. Finally, PM events in the globin genes affecting practically each one of the amino acids in the a, /~, ~, and 8 chains have been discovered, but only a small proportion of these is associated with clinical effects. It would thus seem that (i) for most genes considered, PM events do not appear to be distributed at random throughout the gene and (ii) when these events appear to be distributed throughout the entire gene, either there is some clustering or only a limited number of the changes are associated with clinical effects. To what extent can one explain the spontaneous occurrence o f P M events on the basis o f the structure o f the gene?
This question can now be answered to some extent for mutations of the transition type. It has long been known (Vogel, 1972; Vogel and Kopun, 1977; Li et al., 1985) that among point mutations, transitions (A to G or G to A and C to T and T to C) occur more frequently than transversions in the genes of higher organisms. However, it was only relatively recently that the potential significance of the so-called C p G dinucleotide sequences to spontaneous mutagenesis (among others) began to attract attention. Vertebrate D N A is highly methylated at the cytosine residue and about 90% of 5-methylcytosine occurs within C p G sequences; the latter occurs, however, at only 0.25-0.2 of the frequency expected from the base composition (see Gardiner-Garden and Frommer, 1987 for a detailed discussion and citation of relevant references). At the level of the gene, C to T transitions and the corresponding G to A transitions in the complementary D N A strand occur at a high frequency within methylated gene regions (Seleker and Stevens, 1985; Savatier et al., 1985; Cooper et al., 1987; Bird et al., 1987; Long, 1987; Koeberl et al., 1989, 1990). This is thought to be due to the propensity of 5-methylcytosine to undergo spontaneous deamination to form thymine (Coulondre et al., 1978). Cooper and Youssoufian (1988) made a systematic analysis of published results to examine the role of C p G sequences in spontaneous muta-
LMd: Microdeletion in 7p21.2-.3 LMd: Microdeletion in 20pll.2-p12.1 P M s / L M d : In-frame deletion of exon 6 which codes for the amino acid residues 54-70 which includes the site at which N-proteinase normally cleaves the protein (pro-alpha-2 of type I collagen) or resulting from an RNA splicing defect LMr: A D N A rearrangement upstream from the initiator codon for translation in the gene coding for protein 4.1 PMm." His to Tyr at 58 or 87 in the alpha globin
9. Van der Woude syndrome, mental retardation
10. Craniosynostosis, mental retardation
11. Alagille syndrome, mental retardation 12. Ehlers-Danlos syndrome, type VIIA1
14. Methemoglobinemia, hemoglobinopathic, of Hb M (a-type)
13. Elliptocytosis, Rh-linked
PMm." At the cleavage site of apoA1 LMr: Inversion between 4th exon of ApoA1 and first intron of ApoC3 LMd." Microdeletion in 1q32-41; 2q34-36
8. Hypoalphalipoproteinemia
PMn: In null Bellingham allele: A to T (Lys to Stop at 217)
P M m : G to C (Trp to Cys at 402) P M n : C to T (Arg to Stop at 306) PM m : In P I * Z allele: G to A in exon 5 (Gly to Lys at 342); T to C in exon 3 (Val to Ala at 213); In PI*P: A to T in exon 3, Asp to Val at 256
PMm." T to G (Ile to Ser at 84); Thr to Ala at 60; Ser to Tyr at 77 of transthyretin P M m : T to A (Leu to Gin at 68) in cystatin C PM m : C to T (arg to Cys at 47) in antithrombin
3. Amyloidosis, neuropathic, type If
4. Amyloidosis, Icelandic, cerebrovascular type 5. Thrombophilia, familial, due to antithrombin-llI deficiency * 6. Thrombophilia, familial, due to defect in protein C* 7. al-Antitrypsin deficiency *
PMm: G to A in exon 2 (Val to Met at 30) of transthyretin gene
LMd: 3.2-kb deletion spanning the ADA promoter and the first exon
A utosomal dominants PM m : In exons 4, 7 10 and 11 and thus appear to be scattered over the coding sequence; the changes include C to T at position 396, G to A at 397, G to A at 727, C to T at 1081 and others at positions 334 and 1006 (the position numbers refer to the cDNA sequence of Wiginton et al., 1986); those at 396, 397, 727 and 1081 are at C p G dinucleotides, known to be hot spots for mutation (Cooper and Youssoufian, 1988); thus a high proportion of mutations is associated with a specific PM change; evidence for genetic compounds
Comments on mutational changes
2. Amyloidosis, neuropathic, type I *
1. Severe combined immunodeficiency (SCID) *
Disorder
See McKusick (1988)
Conboy et al. (1986)
Antonarakis (1988) Karathanasis et al. (1987) Bocian and Walker (1987); Hughes et al. (1988) Gong et al. (1976); see Harper et al. (1989) Schnittger et al. (1989) Wirtz et al. (1987); Byers (1989); Prockop et al. (1989)
Kidd et al., (1983); Nukiwa et al. (1986); Satoh et al. (1988); Fraizer et al. (1989); Farber et al. (1989)
Tawara et al. (1983); Dwulet and Benson (1984); Yoshioka et al. (1986); Saraiva et al. (1986); Maeda et al. (1986) Dwulet and Benson (1986); Wallace et al. (1986) Abrahamson et al. (1987) Duchange et al. (1986, 1987); Koide et al. (1984) Romeo et al. (1987)
Bonthron et al. (1985); Valerio et al. (1986); Akeson et al. (1987); Berkvens (1988); Berkvens et al. (1987); Markert et al. (1989)
References
DETAILS OF THE N A T U R E OF MUTATIONS IN M E N D E L I A N DISORDERS (GENERAL REFERENCES: McKUSICK, 1988; HARPER et al., 1989)
TABLE VI
17. Hereditary persistence of fetal hemoglobin, deletion type (HPFH)
16. /3-Thalassemia
15. a-Thalassemia
HPFH-3: < 70 kb, similar to the ones above; the 5' end is slightly 5' to that of HPFH-2, but the 3' end is more proximal and located about 30 kb 3' to the/3-globin gene
HPFH-2: > 70 kb, similar to the one above; the 5' and 3' end points are displaced by about 5 - 6 kb to the 5' side of the corresponding end points of HPFH-1
Deletions of /3-globin gene leading to /3-thalassemia are rare; a 619-bp deletion which removed the distal (3') third of the IVS-2 and the entire exon 3 of the gene plus 209 bp of adjacent 3' flanking D N A accounts for 1 / 3 of the /3-thalassemic genes in Asiatic Indians; a 1.3-kb deletion involving the 5' flanking DNA, exons 1 and 2 and the 5' end of IVS-2, found in Blacks; a 10-kb deletion involving the entire ,8-globin gene but sparing the 8-globin gene has been described in a Dutch family; all of these are B ° thalassemic patients HPFH-I: > 70 kb, involving deletion of both the 8- and /3-globin genes with the 5' end of the deletion located 3.75 kb to the 5' side of the ~ gene while the 3' end is located at a site greater than 60 kb to the 3' side of the /3-globin gene
4. Evidence for genetic c o m p o u n d s
3. More mutations in exon 1 than in 2 and 3.
2. B°: (a) nonsense orframeshifts (in codons 6, 8, 8 / 9 , 15, 16, 17, 39, 41/42, 71/72); (b) RNA processing defects: IVS-1, position 1, G to A, G to T; position 116, T to G; position 108, 25 bp deletion; position 117, 17 bp deletion; IVS-2, position 654, C to T; position 705, T to G
extend rightward and some left; c o m m o n deletion is 2.7 kb, rightward 1. B + : (a) transcription defects (C to G at - 87 from cap site; C to T at - 8 8 ; A to G at - 2 8 ; A to G at - 2 9 ) ; (b) RNA processing defects (codon 27, G to T; codon 26, G to A; IVS-1, position 5, G to T, G to C; position 6, T to C; position 110, G to A; IVS-2, position 745, C to G; polyadenylation signal (1000 bp from exon 3), T to C
LMd: Partial or total deletion of al a n d / o r a2 genes; some of these
PMi: A U G to ACG, alpha-2-globin gene; frameshift in codon 14 ( T G G to GG, alpha-l); PMrp: A A T A A A to A A T A A G (alpha-2); a 5-bp deletion at 5'-IVS-1 splice junction (alpha-2); PMt: T A A to C A A (Hb Constant Spring); T A A to A A A (Hb Icaria); T A A to T C A (Hb Koya Dora); T A A to G A A (Hb Seal Rock) (all the PMts are in alpha-2); unstable variant: codon 125, C T G to C G G (alpha-2); the PMs are generally rare among alpha-thalassemias
See Weatherall (1986)
See Antonarakis et al. (1985) and Weatherall (1986) and references cited therein
hemoglobin,
21. Hereditary persistence of fetal non-deletion type G 22. Hypercholesterolemia, familial *
25. Osteogenesis imperfecta (OI) *
24. Giedion-Langer syndrome
23. lmmunoglobulin kappa light chain deficiency *
hemoglobin,
fetal
20. Hereditary persistence of non-deletion type A
18. Methemoglobinemia, hemoglobinopathic, of Hb M (fl-type) 19. Sickle cell anemia
Disorder
TABLE VI (continued)
Russell et al. (1986); Lehrman et al. (1987); Langlois et at. (1988) Zannis and Breslow (1985)
PMm: G to A in exon 17; PMn: in exons 14, 17; m a n y mutations remain to be characterized LM: 2-kb insertion in IVS-1, 4-bp insertion in exon 17; 12- and 27-bp deletions in exon 4; other deletions (at least 16 known) are of various sizes ranging from 0.8 kb (in exon 5) to 11 kb (encompassing exons 4 - 8 ; evidence for genetic compounds LMdu: 14-kb duplication involving exons 2-8. PMm: T to G and C to T leading to the loss of the invariant tryptophan at position 148 on one chromosome and of the invariant cysteine at position 94 on the other; no effect on health Microdeletion in 8q23.3-q24.13
Pro-al mutations 1. Splice exon 6 ( - 24 aa) 2. Gly to Cys at 175 3. Insertion 50-70 aa 4. Gly to Val at 256 5. Gly to Arg at 391 6. Deletion exons 24-26 ( - 8 4 a a ) 7. Gly to Arg at 664 8. Gly to Cys at 748 9. Gly to Cys at 904 10. Gly to Cys at 988 11. Gly to Cys at CT3 (C-terminal telopeptide) 12. Frameshift deletion, 5 bp
OI
Consequence EDS VII OI Lethal OI Lethal OI Lethal Ol Letal OI Lethal Ol Lethal Ol Lethal OI Lethal OI OI
Most mutations involve the pro-al (COL1A1) or pro-or2 (COL1A2) gene, lead to alterations of the structure of the pro-0d (I) chain of type I procollagen and cause either osteogenesis imperfecta (OI) or EhlersDanlos syndrome (EDS)
See Weatherall (1986)
See Harper et al. (1989) for references Prockop et al. (1989); Byers (1989)
Stavnezer-Nordgren et al. (1985)
See Weatherall (1986)
Ingram (1956, 1957, 1986)
See McKusick (1988)
,4 utosomal dominants PMm: His to Tyr at 63 or 92 and Val to Glu at 67 in the fl-globin gene PMm: At the sixth codon from the 5' end of the fl-globin gene ( G A G to G T G ; Glu to Val) Rare; PMp: G to A at - 1 1 7 from the cap site of the A-T-globin gene (i.e., in close proximity to the C C A A T box); C to T at - 196 from the cap site of the A-T-globin gene Rare; PMp: C to G at - 202 from the cap site of the G-y-globin gene
References
C o m m e n t s on mutational changes
Microdeletion in 10p13 Microdeletion in 4q25-27 PM m : G to C (Glu to Asp at 104)
35. DiGeorge sequence 36. Rieger syndrome, mental retardation
37. Hemolytic anemia due to triosephosphate isomerase deficiency * 38. DiGeorge sequence, mental retardation
39. Trichorhinophalangeal syndrome, type 1, mental retardation 40. WAGR syndrome (Wilms tumor, Aniridia, Gonadoblastoma, mental retardation)
Microdeletion in 11p13
Microdeletion in 8q23.3-q24.13
Microdeletion in 22qll
Microdeletion in 8p21.1-p11.22
34. Spherocytosis, I, mental retardation
33. Retinoblastoma
Microdeletion in 7p13 Microdeletion in 15qll-q13; paternally inherited P M m : His to Asp at 10 (beta chain) Phe to Leu at 25 (beta chain) PHE to Ser at 24 Arg to His at 65 Microdeletion in 13q14
Lethal O1 Ol OI at 89) in the gene for purine
Lethal OI
OI Lethal OI Lethal Ol Lethal OI
Consequence EDS VII EDS/OI
30. Greig cephalopolysyndactyly 31. Prader-Willi syndrome 32. Diabetes mellitus, rare forms *
26. Immunodeficiency, T cell, with neurological disorder * 27. Porphyria cutanea tarda * 28. Piebald trait, mental retardation 29. Adenomatous polyposis, mental retardation
Pro-a2 mutations 1. Splice exon 6 ( - 18 aa) 2. Splice/deletion exon 11) ( - 18 aa) 3. Deletion - 20 aa 4. Splice exon 28 ( - 1 8 aa) 5. Gly to Asp at 547 6. Splice/deletion exon 33 ( - 18 aa) 7. Deletion exons 33-40 ( - 1 8 0 aa) 8. Gly to Asp at 907 9. Gly to Arg at 1012 10. Frameshift deletion of 4 bp PM m : G to A in exon 3 (Glu to Lys nucleoside phosphorylase PM m : G to A (Gly to Glu at 281) Microdeletion in 4q11-q21 Microdeletion in 5q21
de la Chapelle et al. (1981); Greenberg et al. (1988); Mascarello et al. (1989); Whitlock et al. (1989) Goldblatt and Smart (1986); Yamamoto et al. (1989) Francke et al. (1979); Porteous et al. (1989); Rose et al. (1989); Glaser et al. (1989)
Francke (1976); Cavenee et al. (1985); Lee et al. (1987) Chilcote et al. (1987); Kitatami et al. (1988) Greenberg et al. (1988) Ligutic et al. (1981); Motegi et al. (1988) Daar et al. (1986)
Verneuil et al. (1988) See Harper et al. (1989) Herrera et al. (1986); Hockey et al. (1989) Rosenkranz et al. (1989) See Harper et al. (1989) Chan et al. (1987); Shoelson et al. (1983a,b); Haneda et al. (1983); Shibasaki et al. (1985)
Williams et al. (1987)
C-II
deficiency
53. Zellweger syndrome 54. Phenylketonuria *
P M m : G to A (Val to Met at 332); C to A (Asp to Lys at 54)
52. Ornithinemia with gyrate atrophy of choroid and retina
Evidence for genetic compounds Microdeletion in 7ql 1 PMm." C to T (Arg to Try at 408) in exon 12; G to A in exon 5 (Arg to Gin at 158); T to C (Leu to Pro at 311) in exon 9, G to A in exon 7 (Ghi
PMi." G to A (Met to lie) LMd." partial deletion of the ornithine amino-transferase gene
Microdeletion in 17p13.3
51. Miller-Dieker lissencephaly syndrome
CII
L M d : a 6-kb deletion; L M d u : a 2-kb direct tandem duplication extending from within exon 6 to the 3' end of an A l u within intron 6
(Apo
termination codon Originally interpreted as a resulL of deletion of the functional gene (P450c21B; steroid cytochrome P450 21-hydroxylase) (at least 25% of cases), the current view is that these apparent deletions represent gene conversions, unequal cross-overs or polymorphism, but not physical loss of the gene; some gene deletions and duplications do occur LMd." A deletion of one base of the ACT codon for Thr at position 68 causing a frameshift and a premature termination at amino acid 68 instead of at the normal amino acid 79 P M m : ApoE2, Arg to Cys at 158 P M m : G to C in exon 5 (Ala to Pro at 149) of the fructose-l-phosphate aldolase gene P M m : T to C in exon 10 (Leu to Pro at 444); C to G in exon 9 (Pro to Arg at 415); A to G in exon 9 (Asp to Ser at 370); G to A in exon 5 (Arg to Gin at 120); C to T (Pro to Leu at 470); A to G (His to Arg at 495); evidence for genetic compounds Microdeletion in 15qll-q13 (as in Prader-Willi), but maternally inherited
A utosomal recessives L M d : A 4-bp deletion at the second codon (cDNA) upstream from the
50. Lipoprotein lipase deficiency
49. Angleman syndrome
48. Gaucher disease, types 1 to Ill *
46. Hyperlipoproteinemia, type IIl 47. Fructose intolerance, hereditary
45. Apolipoprotein Toronto)
44. Adrenal hyperplasia
43. Pyruvate dehydrogenase El deficiency
42. Adenine phosphoribosyl transferase deficiency
1988); Theo-
Naritomi et al. (1988) DiLella et al. (1986, 1987); Avigad et al. (1987); Lichter-Konecki et al.
Knoll et al. (1989); Pembrey et al. (1989) Langlois et al. (1989); Devlin et al. (1990) van Tuinen et al. (1988); Schwartz et al. (1988) Ramesh et al. (1988); Mitchell et al. (1988); Hotta et al. (1989)
Tsuji et al. (1987, phihis et al. (1989);
Smit (1987, 1989) Dazzo et al. (1989)
Cox et al. (1988)
White et al. (1987, 1988); Miller (1988); Matteson et al. (1987); Higashi et al. (1988); Rumsby et al. (1988)
Endo et al. (1989)
Kamatani et al. (1989) Hikada et al. (1987)
Shelton-lnloes et al. (1987)
A utosomal dominants L M d : 2 unrelated patients were shown to have large deletions within the
41. von Willebrand disease gene (12pter-12pl2), corresponding to at least 7.4 kb at the 3' end of cDNA; estimated size of deletion; at least 110 kb P M m : Met to Thr at 136 L M d : IN a patient with complete deficiency, in one allele, a trinucleotide deletion corresponding to phenylalanine in the deduced amino acid sequence, and in the other, a single nucleotide insertion immediately adjacent to the splice site at the 5' end of IVS-4
References
Comments on mutational changes
Disorder
TABLE VI (continued)
65. Granulomatous disease, X-linked
64. Choroideremia, mental retardation
61. Adrenal hypoplasia (Addison disease), mental retardation 62. Ocular albinism (Nettleship-Falls type OA1) and ichthyosis 63. Fabry disease
60. Propionicacidemia, type 11
59. Albinism (tyrosinase-deficient, oculocutaneous)
58. Tyrosinemia, II (tyrosinase transammase deficiency: TAT)
57. Tay-Sachs disease
56. Sandhoff disease (GM2-gangliosidosis
55. Pituitary dwarfism
LMd: in Xp21,1 and abnormal transcript
Merry et al. (1989); Cremers et al. (1989) Royer-Pokora et al. (1986); Orkin (1987)
Kornreich et al. (1988)
Schnur et al. (1989)
Microdeletion in Xp22.3 LMd." Deletions range in size from 0.4 kb to 5.5 kb; four of these had a breakpoint in IVS-2 and three of these families had breakpoints in an Atu-rich region LMdu: A partial duplication of about 8 kb encompassing exons 2 - 6 Microdeletion in Xq21
Yates et al. (1987)
Tahara et al. (1989)
Spritz et al. (1989)
Natt et al. (1987)
Tanaka et al. (1990) Myerowitz and Costigan (1988); Myerowitz and Hogikyan (1986, 1987); McDowell et al. (1989); Triggs-Raine et al. (1989)
O'Dowd et al. (1986)
Goosens et al. (1986); Braga et al. (1986)
(1988); Lyonett et al. (1989); Wang et al. (1989); John et al. (1989); Levy (1989); Okano et al. (1990); Scriver et al. (1988)
Microdeletion in Xp21
X-linked
extending 2 kb upstream past the putative promoter region of H E X - A gene encoding the alpha subunit (in French Canadians) Evidence for genetic c o m p o u n d s LMd: In one genetic compound, both T A T alleles deleted; the deletion from the mother at least 27 kb long and included the complete T A T gene; the other, inherited from the father had a de novo interstitial deletion in chromosome 16 (pter-q22 q22.3-qter) PM m : Thr to Lys (classical tyrosinase-negative OCA) which abolishes one of six putative N-linked glycosylation sites; Pro to Leu ('yellow' OCA) may interfere with normal folding of the tyrosinase polypeptide LMd." A 2-kb deletion in the gene for beta subunit of propinyl CoA carboxylase; 10 out of 30 m u t a n t alleles analyzed have this change
LMi: A 4-bp insertion in exon 11 (common in Ashkenazi Jewish patients) LMd: A 7.6-kb deletion which includes part of intron 1, all of exon 1 and
flanking the chorionic s o m a t o m a m m o t r o p i n gene; homozygosity for a 7.6-kb deletion within the growth hormone gene cluster LMd." Partial gene deletion located to the 5' end of the hexosaminidase B gene P M m : G to A (Arg to His at 178); C to T (Arg to Cys at 178) PMs: A splice junction defect in IVS-12 P M m : In exon 7 (rare)
LMd." A total of about 40 kb of D N A absent due to 2 separate deletions
Evidence for genetic c o m p o u n d s
PMi: A to G (Met to Val) PMs: G to A in intron 12 L M d. deletion of exon 3
to Lys at 280); C to T in exon 3 (Arg to Ter at 111)
70. Lesch-Nyhan syndrome
68. Glycerol kinase deficiency, adrenal hypoplasia, hypogonadotropic hypogonadism, mental retardation 69. Ichthyosis, chondrodysplasia punctata, Kallman syndrome, mental retardation
67. Hemophilia B (factor IX) *
66. Hemophilia A (factor VIII gene) *
Disorder
TABLE Vl (continued)
Severe Severe Severe Severe
PMm:C T G T G T
to A (Phe to Leu at 73) to A (Val to Asp at 129) to T (Ala to Set at 160) to G (Phe to Val at 198) to A (Cys to Tyr at 205) to C (Leu to Ser at 130)
LMd." Most patients examined have complete or partial deletions of the gene for steroid sulfatase located at Xp22.3
PMn." C to T, exon 8 (Arg to Stop at 252) C to T, exon 8 (Arg to Stop at 238) PMs: GT to T r , IVS-6, nt 1 GT to GG, IVS-3, nt2 LMd: Over 10 deletions ranging in size from about 1.5 kb up to about 115 kb LMi: Full length LINE-1 near the 5' end of Factor IX gene; A 6-kb insertion within the factor IX gene A Microdeletion in Xp21-pll.2
X-linked Change Effect P M m : A to G, exon 7 (Glu to Gly at 272) Moderately severe G to A, exon 26 (Arg to Gln at 2326) Moderately severe PMn: C to T, exon 18 (Arg to Stop at 1960) Severe C to T, exon 26 (Arg to Stop at 2326) Severe C to T, exon 22 (Arg to Stop at 2135) Severe C to T, exon 24 (Arg to Stop at 2228) Severe C to T, exon 23 (Arg to Stop at 2166) Severe All these mutations affect CpG nucleotides LMd: At least 12 deletions are known, ranging in size from about 2.5 kb to 210 kb; all except the 6-kb deletion of exon 22 (which is moderately severe) are severe LMi: Two patients with insertions of L IN E-1 sequences (3.8 kb and 2.3 kb) in exon 14 PMm.'G to A, exon 6 (Arg to His at 145) Mild A to G, exon 3 (Asp to Gly at 47) Mild G to A, exon 2 (Arg to Gin) Mild
Comments on mutational changes
Youssoufian
Shapiro et al. (1987); Bonifas et al. (1987); Ballabio et al. (1988); Bick et al. (1989) Stout and Caskey (1985, 1988); Wilson et al. (1983a,b); Wilson and Kelly (1984); Davidson et al. (1988a,b,c, 1989a,b); Cariello et al. (1988, 1989); Gibbs et al. (1989); Yang et al. (1984)
See Harper et al. (1989) for references
Antonarakis (1988); Chen et al. (1988, 1989); Ludwig et al. (1989) Scott et al. (1987); Yoshitake et al. (1985)
Kazazian et al. (1988)
Antonarakis (1988); et al. (1988a, b)
References
* Asterisks denote entries included in the analysis of Cooper and Youssoufian (1988). The references are given as a separate section in the Reference list.
L M d, LMdu: unequal exchanges between the red pigment gene and the green pigment genes
75. Pelizaeus-Merzbacher disease
76. Color blindness
LMd: Partial deletions of the OTC gene PM m : C to T (Pro to Ser at 225)
73. Norrie disease 74. Ornithine transcarbamylase deficiency *
LMd: A large number of intragenic deletions of various sizes, but these do not occur uniformly over the gene; large deletions are typically found in the first third of the gene and a large number of smaller deletions cluster in the middle of the gene; deletions appear to occur with approximately equal frequency in the D M D and the milder BMD and deletion size per se does not correlate with the severity of the disease; D M D largely results from frameshift deletions while BMD usually results from deletions that maintain the translational reading frame LMdu: Duplication of one or few exons LMr: Involves translocation of the D M D gene and the ribosomal RNA genes on 21p A microdeletion in X p l l . 3 PM m : G t o A , C t o T
71. Microphthalmia, iridoschisis, goiter, labium synechia, craniotabes 72. Muscular dystrophy, Duchenne and Becker (DMD, BMD)
C to G (His to Asp at 203) G to C (Gly to Arg at 71) C to T (Ser to Leu at 110) T to C (Leu to Pro at 41) G to A (Gly to Glu at 70) A to T (Asp to Val at 80) G to T (Ala to Ser at 161) T to G (Phe to Val at 199) T to A (Val to Asp at 130) A to G (Asp to Gly at 201) T to G (Iso to Met at 132) C to A (Ser to Arg at 103) C to U (Ser to Leu at 109) PMn: C to T (Arg to Stop at 169) LMd: A variety of very small (a few bps) to medium (a few kbs) to whole-gene deletions LMi: Insertion of one nucleotide at base 56, 57 or 58; potential insertion (GM 2227) in the region between exons 4-6. LMdu: Involves exons 2 and 3 (GM 1662) Microdeletion in Xp22
Gencic et al. (1989); Abuelo et al. (1989) Nathans et al. (1986a,b)
Donnai et al. (1988) Nussbaum et al. (1986); Rozen et al. (1985)
Monaco and Kunkel (1987, 1988); Monaco et al. (1988); Worton and Thompson (1988) and references cited in these reviews; Lindloff et al. (1989); Koenig et al., (1989); Gillard et al. (1989); den Dunnen et al. (1989); Bakker (1989); Worton et al. (1984); Hu et al. (1988, 1989) See Hu et al. (1988, 1989)
Thies et al. (1989)
32 genesis. The considerations which prompted this analysis are: (i) the now well established general observation that there is a very high frequency of RFLPs in human D N A when restiction enzymes are used whose recognition sequences contain a C p G dinucleotide; (ii) their findings and those of others that recurrent mutations occurred exclusively in CpG dinucleotides in the factor VIII gene (Youssofian et al., 1986: Antonarakis et al., 1987) and that these mutations were CG to T G or CG to CA transitions; and (iii) the strong inference from all these findings, that the presence of D N A methylation could be one cause of point mutations within human genes. Seventeen autosomal dominant and three Xlinked conditions (in which precise changes have been identified by D N A sequencing or by oligonucleotide hybridization) were included (these conditions are marked with an asterisk in Table 6 of this paper; the latter includes all but three conditions listed in Tables 1 and 2 of Cooper and Youssoufian, 1988). Excluded from their compilation were mutations causing thalassemia, because many of these remain in the population for a long time and therefore may be found in many individuals (and additionally, because of the very large number of reports) and those causing neutral polymorphisms. With these exclusions, a total of 45 independent mutations at different genes were found. For the purpose of their inquiry, they also excluded from analysis all the 11 factor VIII mutations since these mutations were not detected in an unbiased fashion (i.e., use of restriction enzymes such as TaqI which contain CpG in their recognition sequence and thus detect single basepair changes more frequently than other enzymes). The main finding was that 12 out of the remaining 34 (35%) occurred in C p G dinucleotides and of these 11 were either C to T or G to A mutations indicating the important role of naturally occurring deamination of 5-methylcytosine in the genesis of spontaneous transition mutations. Cooper and Youssoufian (1988) estimate that the rate of occurrence is about 42-fold higher than that predicted from random mutation. From a recent analysis of mutations in the factor IX gene (hemophilia B), Koeberl et al. (1990) have estimated that transitions at the CpG sites are elevated 24-fold over transitions at other sites and that the
former account for 44% of all the transitions and for 37% of all the mutations. These findings are entirely in line with the conclusion that CpG sites may be " h o t spots" for spontaneous mutations.
Mechanisms underlying naturally occurring DNA deletions and duplications The mechanisms that have so far been proposed to explain the origin of deletions and duplications in Mendelian diseases - replication slippage and recombination are related to the nucleotide sequence organization of the genome. Relevant in this context is the existence of genes in the human genome that are evolutionarily related (i.e., those that show significant sequence homologies) and of various families of repetitive D N A sequences (e.g., Alu, LINE-1 and VNTRs) within and between gene sequences.
Replication slippage In 1966, on the basis of their work with bacteriophage T4, Streisinger and colleagues proposed that frameshift mutations are most likely to occur in regions of repeats as a consequence of base mispairing (between two repeats) and slippage during D N A replication. Subsequent research in E. coli has provided ample support for this hypothesis (Farabaugh et al., 1978; Glickman et al., 1985; Schaaper et al., 1986). In this organism, a large proportion of spontaneous mutations in the lacI gene is a consequence of gain or loss of one copy of the 4-bp sequence T G G C which is tandemly repeated three times at nucleotides 621632 which also represents a hotspot for mutations of this type. Slippage during replication is one of the mechanisms that has been proposed to explain insertions and deletions in tandem repeat sequences mentioned earlier (e.g., Jeffreys et al., 1988; Wolff et al., 1989; Kovacs et al., 1990 and references cited therein). Examples of Mendelian diseases in which small deletions have been found and which have been interpreted on the basis of the replication slippage model include some fl-globin structural variants and some deletions in the retinoblastoma gene. The fl-globin structural variants were originally identified by amino acid sequence analyis (Bunn et al., 1977) and were found to lack one to eight amino acids. Comparison of the nucleotide
33 sequences in normal DNA within and adjacent to the codons for amino acids deleted in the variants revealed the presence of small 2-8-bp direct repeat sequences (Marotta et al., 1977). Efstratiadis et al. (1980) have argued that these small deletions are unlikely to be a consequence of unequal crossing-over between repeat sequences for two reasons: (i) most of the repeat sequences are very short and (ii) an unequal crossing-over event would produce one daughter molecule with three intact copies of the repeat sequence and another molecule with one intact copy; only one example of the former and very few of the latter were observed in their analysis. In studies involving deletions in the retinoblastoma gene (Canning and Dryja, 1989), sequence analysis of regions surrounding breakpoints of four deletions revealed that three deletions had termini within pairs of short, direct repeats ranging in size from four to seven base pairs. The authors have interpreted this finding as indicating that the replication slippage mechanism may predominate in the generation of deletions at this locus. Recombinational mechanisms By genetic methods, Sturtevant (1925) and Sturtevant and Morgan (1923) first inferred the occurrence of unequal recombination at the Bar locus in Drosophila. The repeat element in this case turned out to be the Bar locus itself, as was subsequently shown cytologically by Muller et al. (1936) in salivary gland chromosome preparations. These studies thus provided much of the conceptual framework for envisaging the origin of deletions and duplications in other organisms as well as in the human genome. Gene duplications have played an important role in the evolution of multigene families (e.g., haptoglobins, hemoglobins, L D L receptor, ubiquitin, etc.; see reviews by Maeda and Smithies, 1986; Fodde, 1990). Common to all multigene families are (i) the initial, largely unpredictable events that enabled single-copy genes to become ancestral to families of genes and (ii) the more predictable events (unequal recombination and gene conversion) that increased or decreased the number of copies within a family and transfer of information between its individual members. These
events, which have occurred in the course of evolution of the multigene families, still occur spontaneously and sometimes result in pathological phenotypes. For convenience, in what follows, the examples are considered under four headings: (i) homologous, unequal recombination between evolutionarily related genes; (ii) homologous, unequal recombination between DNA repeat sequences, the latter present within a n d / o r between genes; (iii) non-homologous recombination; and (iv) possible mechanisms underlying deletions/duplications in the D M D / B M D gene. Homologous unequal recombination between evolutionarily related genes. Most of the a-thalassemias result from gross D N A deletions within the a-globin gene complex and have been interpreted to arise as a result of homologous, unequal recombination between the two closely spaced a-globin genes (Higgs and Weatherall, 1983). This interpretation also holds for some complex thalassemias (unequal crossing-over between 8- and fl-globin genes or between A-T- and fl-globin genes resulting in 8-/3 (hemoglobin-Lepore), /3-8 (anti-Lepore) and 3,-/3 (hemoglobin-Kenya) fusion genes (reviewed in Weatherall, 1986)). These findings and the interpretation can be easily understood in the context of what is known about the globin genes: (i) the al- and a2-globin genes are located next to each other (in a stretch of about 30 kb), have almost identical nucleotide sequences and the sequence homologies extend beyond the coding regions (Lauer et al., 1980); (ii) the G-3,-, A-T- (fetal genes), 8- and/3-(adult) globin genes are located in a 50-kb region; (iii) the 8- and /3-globin genes are highly homologous in their coding region (Lawn et al., 1978; Efstratiadis et al., 1980); (iv) in the two fetal globin genes, the coding, intervening and flanking sequences are virtually identical (Slightom et al., 1980); and (v) there is an abundance of A lu repeats in both the a- and/3-globin gene clusters (Fritsch et al., 1980; Nicholls et al., 1987). In one type of color blindness, anomalous trichromacy, the individuals have either a 5' red-3' green or a 5' green-3' red photopigment gene; in another form of color blindness, dichromacy, the red or green photopigment is missing as a conse-
34 quence of either deletion of one of the genes or red-green gene fusion (Nathans et al., 1986a,b). The occurrence of these conditions is most easily explained by homologous but unequal recombination during meiosis. The red and green pigment genes are 96% identical, the green pigment genes vary in number (1, 2 or 3) and together with the single red pigment gene, reside in a head-to-tail tandem array among color-normal individuals (Nathans et al., 1986a,b). Homologous, unequal recombination between repeat sequences. In the a-globin cluster, one deletion has been found to be due to homologous unequal recombination between Alu repeats (Nicholls et al., 1987). Of four deletions and one duplication occurring within the low density lipoprotein receptor gene (causal in familial hypercholesterolemia) that were analyzed, three were attributed to homologous unequal recombination between Alu repeats oriented in the same direction and one from intrastrand recombination between Alu oriented in opposite directions (Lehrman et al., 1985, 1987a,b; Russell et al., 1986; Langlois et al., 1988). The remaining deletion has one breakpoint within an Alu repeat; thus nine of the 10 breakpoints are within Alu repeats. In Fabry disease, the breakpoints of four intragenic deletions were in an Alu-rich region (Kornreich et al., 1988). Other examples of Alu involvement include Tay-Sachs disease in French Canadians (Myerovitz and Hogikyan, 1987) and one reported case of A D A deficiency (Berkvens et al., 1987). A sex-chromosome rearrangement in an XX male was found to be due to homologous unequal exchange between Alu repeats present in the terminal parts of Xp and Yp (Rouyer et al., 1987). Yen et al. (1987) have suggested that the frequent occurrence of deletions of the steroid sulfatase gene (McKusick No. 30810) might be related to unequal exchanges between Alu repeats in this region. Other examples of unequal recombination between repeat sequences (although not necessarily resulting in disease phenotypes) are those at hypervariable loci. It has been suggested that the core sequences shared by many hypervariable minisatellites might serve as a recombination signal (e.g., Jeffreys et al., 1985; Chandley and
Mitchell, 1988). This view is supported by the presence of a core-related sequence at or near a meiotic recombination hot spot in the mouse major histocompatibility complex (Steinmetz et al., 1987). Non-homologous recombination. Examples of deletions arising as a result of non-homologous recombination are provided by some a-thalassemias (recombination between an al-globin gene and an Alu repeat resulting in a partially deleted a-globin gene (Orkin and Michelson, 1980; Nicholls et al., 1985)), some fl-thalassemias (Anand et al., 1988; Henthorn et al., 1990), and some complex (3'-6-fl) thalasssemia and H P F H cases (Jagadeeswaran et al., 1982; Ottolenghi and Giglioni, 1982; Vanin et al., 1983; Henthorn et al., 1986). In all these deletions (except in those studied by Henthorn et al., 1990), the 5' breakpoints mapped either in or close to Alu sequences. The interpretation is that these deletions presumably arose during D N A replication (when sequences widely separated in the linear D N A molecule might be physically close to each other as a result of anchorage to the nuclear matrix and chromatin loop formation) as a consequence of non-homologous intrachromosomal breakage and reunion events (Anand et al., 1988; Vanin et al., 1983). The models proposed by Anand et al. and Vanin et al. differ in some details. In the work of Henthorn et al. (1990) with four independent fl-globin gene deletions, none of the eight breakpoints occurred within Alu sequences. Their work and analysis of other published data permitted them to conclude that "deletion break points are more likely to occur within transcriptional units of the fl-globin gene region than would be expected by chance.., hence it is the preponderance of break points within genes, rather than in repetitive elements, that is of note in this region of the genome." However, their examination of the data for c~-globin gene deletions interpreted as having arisen as a result of non-homologous recombination suggests that four of the six breakpoints are within Alu repeats. In a case of lipoprotein lipase deficiency, Devlin et al. (1990) found a tandem duplication of approximately 2 kb in the LPE gene (from within exon 6 to the 3' end of an Alu element located within intron 6) and suggested that this probably
35 arose as a result of a non-homologous exchange event.
Duch'enne and Becker muscular dystrophies ( D M D / B M D ) . Intragenic partial deletions (well over 300 are now known) appear to be the most c o m m o n defect (accounting for about 50-70% of mutations at the D M D / B M D locus) leading to B M D or D M D (Forrest et al., 1987; den Dunnen et al., 1989; Koenig et al., 1987, 1989; Gillard et al., 1989). Much less common are intragenic duplications which appear to duplicate one or a few exons by tandem duplication of a portion of the gene, presumably by unequal crossing-over between repeat elements (Hu et al., 1988; Koenig et al., 1989). More recently, Hu et al. (1989) have published data suggesting that duplications can arise as an intrachromosomal event through unequal sister-chromatid exchange. The deletions appear to occur with approximately equal frequencies in D M D and in the milder BMD patients and are of different sizes; sequences deleted in D M D patients may also be deleted in B M D patients and there does not seem to be any correlation between the size of deletions and the severity of the condition (Hart et al., 1987; Davies et al., 1988; Darras et al., 1988; Lindlof et al., 1988). There is a striking non-rand o m distribution of deletions and the spectrum is such that large deletions are typically found within the first third of the gene and a large number of smaller deletions are clustered in the middle of the gene; the clustered deletions begin in one of the three introns in this region, suggesting that these introns may contain sequences that predispose to deletions (Koenig et al., 1987). As Koenig et al. speculated, " t h e multiple breaks could be due a sequence-sepcific rearrangement hot spot, or an extraordinarily large genomic distance spanned by this particular exon." Monaco et al. (1988) advanced the hypothesis that deletions resulting in the clinically less severe B M D bring together exons that maintain the translational reading frame of the m R N A and that such deletions should allow the production of an abnormal, but partially functional protein product; in D M D patients, however, the deletions bring together exons that disrupt the reading frame and would lead to the production of a severely trun-
cated product. Detailed analysis of deletion breakpoints published recently by Koenig et al. (1989), Gillard et al. (1989), den Dunnen et al. (1989) and Hodgson et al. (1989) lend strong support to the above "reading frame hypothesis" although there are exceptions. The mechanisms involved in the genesis of deletions in D M D / B M D are not fully understood, but they do not seem to be related to the deletions of integral copies of the repeat structural units identified in the D M D gene (Koenig et al., 1988) since a large majority of breakpoints do not involve the exons at all (den Dunnen et al., 1989). Gillard et al. (1989) suggest that the non-random clustering of breakpoints might be due to either sequence-specific (homologous) or structurespecific (non-homologous) recombination. As they stated, "non-homologous mechanisms are attractive for the relative ease with which they can account for the diversity of deletions .... By comparison, one might predict that homologous recombination mechanisms would result in a smaller number of (deletion) endpoints.., given the high frequency of deletions and the considerable heterogeneity of deletion endpoints, it seems probable that both types of mechanisms might contribute to the generation of deletions."
Molecular
requirements for
recombination.
Bearing on the general theme of recombination is the obvious requirement of sequence homology and its minimal extent, at the molecular level. Studies in bacteria have shown that as few as 20 bp of homologous sequence resulted in recombination (Watt et al., 1985). In work with a series of mutants that contained variable-sized deletions of the neomycin phosphotransferase gene in an eukaryotic-prokaryotic shuttle vector system, Ayares et al. (1986) showed that for mammalian cells, 25 bp of homologous sequence was sufficient to yield recombination products. Since the length of the homologous sequence required for recombination is much less than that of most of the repeat sequences present in the human genome, one inference is that both homologous and non-homologous recombination must be relatively c o m m o n and there must exist safeguards to prevent these from becoming excessive.
36 In experiments similar to those of Ayares et al. (1986) but using a synthetic hypervariable minisatellite sequence ((SAT)6.5), Wahtis et al. (1990) found that it stimulated homologous recombination up to 13.5-fold in the human EJ bladder carcinoma cell line. Other mutational mechanisms Gene conversions The term gene conversion was introduced by fungal geneticists to describe the non-reciprocal transfer of genetic markers from one chromosome to another (Radding, 1978; see also Whitehouse, 1982). Molecular geneticists working with D N A of higher eukaryotes use this term to describe the local transfer of D N A sequences from one gene to a related gene elsewhere in the genome in an event that resembles a double cross-over (Maeda and Smithies, 1986). Gene conversions and expansion/contraction of gene number by homologous, but unequal, crossing over have been proposed as mechanisms for concerted evolution (Hood et al., 1975) and a number of ideas have been published as to how these events could occur at the molecular level (see Maeda and Smithies, 1986 for references). Gene conversion events leading to disease phenotypes are now well-documented in the case of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (Donohoue et al., 1986; Harada et al., 1987; Higashi et al., 1988; Miller, 1988; Urabe et al., 1990). 21-Hydroxylase is mediated by a specific type of cytochrome P450 termed P450c21. In man, there are two 21-hydroxylase genes CYP21A and CYP21B, the former being a pseudogene and the latter the functional gene. Both are located in tandem immediately downstream (3') from one of the C4 genes encoding for the fourth component of serum complement on the short arm of chromosome 6 between HLA-B and H L A - D R (Carroll et al., 1985; White et al., 1985). The pseudogene contains an 8-bp deletion in the third exon, a 1-bp insertion in the seventh exon and a C to T transition in the eighth exon. These mutations generate frameshifts and a nonsense codon, respectively, which would prevent synthesis of functional protein. Therefore, if any of the mutations found in the CYP21A gene is intro-
duced into the CYP21B gene by gene conversion, it will result in deficiency for the enzyme. Such cases have in fact been reported and confirmed by molecular analysis (Higashi et al., 1988; Urabe et al., 1990). It is worth noting here that in human somatic cells in vitro, there are now data documenting a significant role of gene conversion events in the origin of spontaneous mutations at the H L A - A and thymidine kinase (TK) loci (reviewed in Sankaranarayanan, 1991b). Mutations due to insertion of mobile genetic elements There is good evidence in bacteria, yeast, maize and Drosophila and some limited evidence in the mouse indicating that insertion of mobile D N A sequences play an important role in the genesis of spontaneous mutations (Lambert et al., 1988). In man, the evidence for the occurrence of spontaneous mutations due to mobility of such elements (e.g., Alu, L I N E - l ) is quite limited at present (although that for their involvement in spontaneously occurring deletions is extensive, as reviewed in the preceding section). The situation with respect to the former has not changed much (with one exception) since the publication of the earlier review (Sankaranarayanan, 1988). Kazazian et al. (1987) found insertions of LINE-1 elements into exon 14 of the factor VIII gene in two of 240 unrelated patients with hemophilia A; in both cases the parents did not have the insertion and therefore the event must have occurred de novo. Other relevant information The fact that the overall organization of the human genome is complex and consists of different sequence classes (unique, moderately repetitive and highly repetitive sequences) has been known for a long time (Britten and Kohne, 1968; Jelinek and Schmid, 1982; Evans, 1984; Jeppsen and Bower, 1987). Considering just two classes of moderately repetitive sequences, the Alu and the LINE-1 elements, it is clear that they number around 500,000 (Alu) or 200,000-500,000 copies ( L I N E - I ; Singer, 1982; Skowronski and Singer, 1986). Likewise, the V N T R sequences (belonging to the highly repetitive sequence class) are quite abundant and appear to be dispersed throughout
37 the genome (Jeffreys et al., 1985a,b, 1986; N a k a m u r a et al., 1987). Alu and V N T R sequences are GC-rich and LINE-1 AT-rich. At the chromosomal level, using a combination of high resolution in situ hybridization with quantitative solid state imaging, Korenberg and Rykowski (1988) have shown that the Alu repeat family dominates in R (reverse bands) and the LINE-1 in G and Q (Giemsa and Quinacrine) bands. In in situ hybridization studies, Royle et al. (1988) found that six human minisatellite loci are not dispersed at random but show preferential (though not exclusive) localization to terminal Gbands of human autosomes. The functional significance of these and other repetitive elements has not been fully unravelled. F r o m circumstantial evidence, Chandley (1989; see also Chandley and Mitchell, 1988) has hypothesized that in meiosis, G-rich sequences (Alu and V N T R ) may be powerful primary sites for synaptic initiation within terminal and interstitial R-bands while the AT-rich sequences may play a more passive role in pairing being brought into alignment later, when homology is established. If essentially correct, these repeat sequences have an important functional role in meiotic pairing. (Royle et al. (1988), however, suggest that the proterminal minisatellites may arise as a by-product of generally enhanced recombination near chromosomal termini rather than being directly involved in recombination a n d / o r synapsis in these regions.) It is tempting to hypothesize that the deletions and duplications without clinical effects (e.g., those arising as a result of recombination between V N T R sequences) and those with clinical effects (e.g., the deletions and duplications in L D L gene) are the inevitable by-products of occasional mispairing. A similar line of reasoning may be applicable to "chromosomal variants" such as those involving centromeric heterochromatic regions in h u m a n chromosomes 1, 9 and 16 (the c-band polymorphisms) observed by cytogeneticists. Most of them may be innocuous; however, a small proportion may be associated with pathological effects of one kind or another (e.g., Shabtai and Halbrecht, 1979; Genest, 1979; Sudek and Sroka, 1979) but unlikely to be classified as Mendelian disease.
Conclusions The material discussed thus far with respect to mutational mechanisms that underlie Mendelian disease permits the following conclusions. (1) A variety of molecular changes (different types of point mutations, small and large intragenic deletions, multigene deletions, insertions, duplications, etc.) underlie Mendelian diseases; the lesions have been found in the coding or non-coding regions as well as in flanking sequences. (2) Point mutational events are not distributed at random throughout the gene; there is good evidence to suggest that transition mutations (C to T and the corresponding G to A transitions in the complementary D N A strand) occur at a high frequency within methylated gene regions. However, not all transition mutations are associated with C p G dinucleotides. (3) The occurrence of D N A deletions and duplications of genes is non-random and, in cases analyzed, the evidence is consistent with the view that there are at least four principal mechanisms: base mispairing (between two repeats) and slippage during D N A replication; homologous unequal recombination between genes that are related, homologous unequal recombination between Alu sequences (located within or between genes) and non-homologous intrachromosomal recombination which may involve Alu sequences; these findings suggest that Alu sequences may be deletion/duplication hotspots. However, not all deletions studied at the molecular level involve Alu sequences. (4) The repetitive sequences present in the human D N A may have a functional role in chromosome pairing and the deletions and duplications of genetic material that arise and lead to Mendelian disease may be construed as by-products of this natural process.
Acknowledgements I am grateful to my colleagues, Prof. L. Bernini and Dr. E. Bakker both at the D e p a r t m e n t of H u m a n Genetics, University of Leiden for their valuable comments on an earlier draft of this paper. I had the benefit of discussing a number of
38 ideas with Prof. W.J. Schull ( H o u s t o n ) over the past 2 - 3 years. He a n d Prof. H.J. Evans (Edinburgh) made a number of perceptive comments on the manuscript. I thank Prof. F.H. Sobels for a n u m b e r o f p r o f i t a b l e d i s c u s s i o n s , f o r his e n c o u r a g e m e n t to p r e p a r e this a n d the other p a p e r s f o r t h e s p e c i a l i s s u e a n d f o r his c r i t i c a l c o m m e n t s . I am
also i n d e b t e d
t o Dr.
N.
Yasuda
(Chiba,
J a p a n ) a n d to P r o f P . H . M . L o h m a n (Leiden) for their useful c o m m e n t s . T h e writing of this p a p e r was supported by EURATOM 6-0226-NL
C o n t r a c t N o . BI-
with the University of Leiden.
References N o t e that references to the text a n d references to T a b l e 6 a r e g i v e n s e p a r a t e l y .
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References to Table VI A utosomal dominants
Severe combined immunodeficiency Akeson, A.L., D.A. Wiginton, J.C. States, C.M. Perme et al. (1987) Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing. Proc. Natl. Acad. Sci. (U.S.A.) 84, 5947-5951. Berkvens, T.M. (1988) Normal and abnormal expression of the human adenosine deaminase gene. Ph.D. Thesis, Leiden University. Berkvens, T.M., E.J.A. Gerritsen, M. Oldenburg et al. (1987) Severe combined immunodeficiency due to a homozygous 3.2 kb deletion spanning the promoter and first exon of the adenosine deaminase gene. Nucleic Acids Res. 15, 93659378. Bonthron, D.T., A.F. Markham, D. Ginsburg, S.H. Orkin (1985) Identification of point mutation in the adenosine deaminase gene responsible for immunodeficiency. J. Clin. Invest. 76, 894-897. Cooper, D.N., H. Youssofian (1988) See References to the text. Markert, M.L., C. Norby-Slycord, F.E. Ward (1989) A high proportion of ADA point mutations associated with a specific alanine to valine substitution. Am J. Hum. Genet. 45, 354-361 (1989). Valerio, D., B.M.M. Dekker, M.G.C. Duyvesteyn et al. (1987) One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity. EMBO J. 5, 113-119. Wiginton, D.A., D.J. Kaplan, J.C. States et al. (1986) Complete sequence and structure of the gene for human adenosine deaminase. Biochemistry 25, 8234-8244. Amyloidosis, neuropathic, type I Dwulet, F.E., M.D. Benson (1984) Primary structure of an amyloid prealbumin and its plasma precursor in heredofamilial polyneuropathy of Swedish origin. Proc. Natl. Acad. Sci. (U.S.A.) 81,694-698 Maeda, S., S. Mira, S. Araki, K. Shimada (1986) Structure and expression of the mutant prealbumin gene associated with familial amyloidotic polyneuropathy. Mol. Biol. Med. 3, 329-338. Tawara, S., M. Nakazato, K. Kanagawa, H. Matsuo, S. Araki (1983) Identification of amyloid prealbumin variant in familial amyloidotic polyneuropathy (Japanese type). Biochem. Biophys. Res. Commun. 116, 880-888 (1983) Saraiva, M.J.M, P.P. Costa, D.S. Goodman (1986) Genetic expression of a transthyretin mutation in typical and late onset Portugese families with familial amyloidotic polyneuropathy. Neurology 36, 1413-1417.
Yoshioka, K., H. Sasaki, N. Yoshioka et al. (1986) Structure of the mutant prealbumin gene responsible for familial amyloidotic polyneuropathy. Mol. Biol. Med. 3, 319-328. Amyloidosis, neuropathic, type II Dwulet, F.E., M.D. Benson (1986) Characterization of a transthyretin (prealbumin) variant associated with familial amyloidotic polyneuropathy type II (Indiana/Swiss). J. Clin. Invest. 78, 880-886. Wallace, M.R., F.E. Dwulet, P.M. Conneally, M.D. Benson (1986) Biochemical and molecular genetic characterization of a new variant prealbumin associated with hereditary amyloidosis. J. Clin. Invest. 78, 6-12 (1986). Amyloidosis, Icelandic Abrahamson, M., A. Grubb, I. Olafsson, A. Lundwall (1987) Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C. FEBS Lett. 216, 229-233. Thrombophilia, familial Duchange, N., J.F. Chasse, G.N. Cohen, M.M. Zakin (1986) Antithrombin III Tours gene: identification of a point mutation leading to an arginine to cysteine replacement in a silent deficiency. Nucleic Acids Res. 14, 2408. Duchange, N., J.F. Chasse, G.N. Cohen, M.M. Zakin (1987) Molecular characterization of the antithrombin Tours deficiency. Thromb. Res. 45, 115-121. Koide, T., S. Odani, K. Takahashi, T. Ono, N. Sakuragawa (1984) Antithrombin III Toyama: replacement of arginine47 by cysteine in hereditary abnormal antithrombin III than lacks heparin binding ability. Proc. Natl. Acad. Sci. (U.S.A.) 81,289-293. Thrombophilia, familial, protein C Romeo, G., H.J. Hassan, S. Staempfli et al. (1987) Hereditary thrombophilia: identification of nonsense and missense mutations in the protein C gene. Proc. Natl. Acad. Sci. (U.S.A.) 84, 2829-2832. Alpha-l-antitrypsin deficiency Farber, J.P., S. Weidinger, H.W. Goedde, K. Olek (1989) The deficient a-l-antitrypsin phenotype is associated with an A to T transversion in exon III of the gene. Am. J. Hum. Genet. 45, 161-163. Fraizer, G.C., T.R. Harrold, M.M. Hofker, D.W. Cox (1989) In-frame single codon deletion in the Mmalton deficiency allele of a-l-antitrypsin. Am. J. Hum. Genet. 44, 894-902. Kidd, V.J., R.B. Wallace, K. Itakura, S.L.C. Woo (1983) AIpha-l-antitrypsin deficiency detection by direct analysis of the mutation in the gene. Nature 304, 230-234. Nukiwa, T., K. Satoh, M.L. Brantly, F. Ogushi et al. (1986) Identification of a second mutation in the protein-coding sequence ofthe Z type a-l-antitrypsin gene. J. Biol. Chem. 261, 15989-15994. Satoh, K., T. Nukiwa, M. Brantly, R.I. Garver et al. (1988) Emphysema associated with complete absence of a-l-antitrypsin of a stop codon in an a-l-antitrypsin coding exon. Am.J. Hum. Genet. 42, 77-83.
44 Hypocdipoproteinemia Antonarakis, S.E., Personal communication to McKusick (1988). Karathanasis, S.K., E. Ferris, I.A. Haddad (1987) D N A inversion within the apolipoproteins A I / C I I I / A I V encoding gene cluster of certain patients with p r e m a t u r e atherosclerosis. Proc. Natl. Acad. Sci. (U.S.A.) 84, 71987202. Van der Woude syndrome, M R Bocian, M., A.P. Walker (1987) Lip pits Am. J. Med. Genet. 26, 437-443. Hughes, M., F. Greenberg, E. McCabe, Does van der Woude syndrome map 36? Proc. Greenwood Genet. Center
and deletion lq32-41. D.H. Ledbetter (1988) to chromosome 2q347, 176-177.
Craniosynostosis, M R Gong, B.T., T.H. Norwood, H. Hoehn et al. (1986) Chromosome 7 short arm deletion and craniosynostosis. A 7psyndrome. Hum. Genet. 29, 117-123. Alagille syndrome, MR Schnittger, S., C. Hoefers, F. Beermann, P. Heidemann, I. H a n s m a n n (1989) Alagille-Watson syndrome is assigned to 20pll.2-p12.1 and provisionally to the region pll.23-p12.1. H G M 10, 1074. Ehlers-Danlos syndrome, type VIIA2 Byers, P.H. (1989) Inherited disorders of collagen gene structure and expression. Am. J. Med. Genet. 34, 72-80. Prockop, D.J., C.D. Constantinou, K.E. Dombrowski et al. (1989) Type 1 procollagen: the gene-protein system that harbors most of the mutations causing osteogenesis imperfecta and probably more c o m m o n heritable disorders of connective tissue. Am. J. Med. Genet. 34, 60-67. Wirtz, M.K., R.W. Glanville, B. Steinmann et al. (1987) Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-a2(1) chain. J. Biol. Chem. 262, 16376-16385. Elliptocytosis, Rh-linked Conboy, J., N. Mohandas, G. Tchernia, S.B. Shobet, Y.W. K a n (1986) Molecular basis of hereditary elliptocytosis due to protein 4.1 deficiency. New Engl. J. Med. 315, 680-685. Methemoglobinemia, of HbM, a type McKusick, V.A. (1988) See References to the text. Alpha-thalassemia, beta-thalassemia, H P F H Antonarakis, S.E., H.H. Kazazian, S.H. Orkin (1985) See References to the text. Weatherall, D.J. (1986) See References to the text. Sickle cell anemia Ingram, V.M. (1956) A specific chemical difference between the globins of normal and sickle cell anemia hemoglobin. Nature 178, 792. Ingram, V.M. (1957) Gene mutations in h u m a n hemoglobin:
the chemical difference between normal and sickle cell hemoglobin. Nature 180, 326. lngram, V.M. (1986) Sickle cell disease: molecular and cellular pathogenesis, Chapter 11. In: Hemoglobin: Molecular, Genetic and Clinical Aspects (H.F. Bunn, B.G. Forget, Eds.), W.B. Saunders, Philadelphia, PA, pp. 453-501. Hypercholesterolemia, familial Langlois, S., J.J. Kastelein, M.R. Hayden (1988) See References to the text. Lehrman, M.A., J.L. Goldstein, D.W. Russell, M.S. Brown (1987) See References to the text. Russell, D.W., M.A. Lehrman, T.C. Sudhof, T. Yamamoto, C.G. Davis, H.H. Hobbs, M.S. Brown, J.L. Goldstein (1986) See References to the text. Zannis, V.I., J.L. Breslow (1985) Genetic mutations affectinglipoprotein metabolism. Chapter 3. In: Advances in H u m a n Genetics (H. Harris, K. Hirschhorn, Eds.), Vol. 14, Plenum Press, New York, pp. 125-215 Immunoglobulin kappa light chain deficiency Stavnezer-Nordgren, J., O. Kekish, B.J.M. Zegers (1985) Molecular defects in a h u m a n immunoglobulin kappa chain deficiency. Science 230, 458-461. Giedion Langer syndrome Harper, P.S., J. Frezal, M.A. Ferguson-smith, A. Schinzel (1989) See References to the text. Osteogenesis imperfecta Byer, P.H. (1989) See Ehlers-Danlos syndrome, type VIIA2. Prockop, D.J., C.D. Constantinou, K.E. Dombrowski et al. (1989) See Ehlers-Danlos syndrome, type VIIA2. Immunodeficiency, T cell, with neurological disorder Williams, S.R., V. Gekeler, R.S. Mclvor, D.W. Martin Jr. (1987) A h u m a n purine nucleoside phosphorylase deficiency caused by a single base change. J. Biol. Chem. 263, 2332-2338. Porphyria cutanea Verneuil, H. de, J. the 281 (gly porphyria and 101-102.
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