Cli. Biochem, Vol. 24, pp. 353-361, 1991 Printed in Canada. All rights reserved.
0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Cystic Fibrosis Gene Analysis: Recent Diagnostic Applications FRANK K. FUJIMURA Nichols Institute Reference Laboratories, San Juan Capistrano, CA 92675, USA Cystic fibrosis (CF) is the most common severe genetic disease of Caucasians. Recent work by several laboratories has resulted in the identification of the CF gene and its major mutation. These findings have greatly facilitated carrier detection and prenatal diagnosis for members of families with a history of CF. This review briefly considers the historical background of CF testing, the basic features of the CF gene, and the methodologies for CF gene analysis. Examples of the application of recent technologies for analysis of CF families are discussed. In addition to family analysis, CF carrier detection in the general population is becoming more feasible. The possibility of carrier screening for CF has been the topic of much discussion recently with arguments presented both for and against. Some basic issues pertaining to population testing are presented.
KEY WORDS: genetics; mutation; linkage analysis; restriction fragment length polymorphism; polymerase chain reaction; carrier detection. Introduction
ffecting about one in 2500 births in the United States, cystic fibrosis (CF) is the most common A severe genetic disorder of Caucasians. The disease results from alteration of exocrine cell function due to a defect in regulation of chloride ion transport (1). Although the molecular basis of this defect has yet to be elucidated, the disorder leads to the secretion of excess sodium and chloride ions in the sweat of affected individuals. An elevated level of sweat sodium and chloride determined by the pilocarpine iontophoresis sweat test (2), in spite of some technical difficulties, remains the accepted diagnostic indicator for CF. The pathologic features of CF are caused by the presence of thick mucus resulting in obstruction of several organs, most often the lungs and the pancreas, b u t sometimes the intestines and gonads (3). The abnormal mucus is caused by the defect in chloride ion transport, which is required for passive transport of water into mucosal secretions.
Correspondence: Frank K. Fujimura, Nichols Institute Reference Laboratories, 32961 Calle Perfecto, San Juan Capistrano, CA 92675, USA. Manuscript received December 23, 1990; revised January 29, 1991; Accepted March 5, 1991. CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
The thickened secretions affect normal function of the affected organs leading to lung infections, deficiency in secretion of pancreatic enzymes, intestinal obstruction (meconium ileus), and sterility or reduced fertility. The severity of CF can be quite variable even within one family. Although most individuals with CF are diagnosed within the first decade of life, some individuals are not diagnosed until much later. With improved detection and appropriate therapies, the average life span of CF individuals has been increasing, with median survival age of approximately 25 years at the present time. It is estimated that there are about 20,000 to 30,000 individuals in the United States who have CF. CF is an autosomal recessive genetic disease, meaning that both parents of an affected individual must be carriers. CF carriers are asymptomatic, and, prior to DNA-based testing, identification of carriers was not possible until two carriers produced a CF offspring. As an autosomal recessive disease, each offspring of two CF carriers has a 1 in 4 risk of being affected, a 1 in 2 risk of being a carrier, and a 1 in 4 chance of being a noncarrier. There are about 8,000,000 CF carriers in the United States. The frequency of CF carriers varies with ethnic group, being about 1 in 25 in Caucasian populations. The frequency of CF carriers in American blacks is about 1 in 65 (4). The frequency in Orientals and African blacks is much lower. This discussion will concentrate on the application of DNA probe technologies for diagnosis of CF and for detection of CF carriers. Of particular interest is the recent identification and characterization of the CF gene and the major CF mutation and the impact of these discoveries on CF gene analysis. For further discussions of the applications of DNA probes for restriction fragment length polymorphism (RFLP) analysis of CF and other genetic diseases, please refer to several recent reviews on the topic (5-10). Background Some of the significant events in the clinical diagnosis of CF are listed in Table 1. Although it is 353
FUJIMURA
TABLE 1
Milestones in Cystic Fibrosis Diagnosis Year
Event
1938 1947
Clinical identification of CF Identification of CF as a genetic disease Sweat test for diagnosis of CF IRT test for neonatal screening of CF
1959 1979 1983 1985 1987 1989
MIE analysis for prenatal
diagnosis of CF Mapping of CF gene to chromosome 7 Linkage disequilibrium Identification of CF gene, major CF mutation
Reference (11) (43) (2) (13)
CFTR cDNA (6.1 kb) 5' 0 --
GENOMIC DNA
m:
1
CFTR PROTEIN (1480 aa) N TM
--
-~F508(15,16)
NBF
2
(17-19) (22,23) (24-26)
R CF
27
3
EXONS
TM 4
NBF relatively common, CF was not recognized as a distinct disease until quite recently (11). The abnormal sweat electrolyte composition in affected individuals, first recognized in 1953 by di Sant'Agnese et al. (12), led to the development of the sweat test for diagnosis of CF (2). Immunoreactive trypsinogen (IRT) analysis of dried bloodspots can be used as a neonatal screen for CF (13) and has been adopted for newborn screening by the state of Colorado. Several other states have considered newborn screening by IRT, but opposition to this effort continues to be expressed (14). The initial step toward providing prenatal diagnosis of CF in high-risk pregnancies was provided by microvillar intestinal enzyme (MIE) analysis (15,16). A major breakthrough in CF gene analysis occurred in 1985 with the mapping of the gene to chromosome 7(17-19). Subsequently, several probes linked very closely to the CF gene have been identified (20,21). These probes detect RFLPs that are useful for analysis of families with a positive history of CF. Although very powerful, linkage analysis requires testing of families and largely depends on the availability of specimen from an affected individual in the family. Linkage analysis of CF families showed that certain combinations of alleles (haplotypes) are more frequently associated with CF than others (22,23). This observation of linkage disequilibrium helped to increase the accuracy of analysis for families with a positive history, particularly in those cases where specimens were not available from an affected individual. The combination of linkage analysis coupled with linkage disequilibrium information has allowed very accurate analysis for most families. The intensive efforts to identify the CF gene culminated in 1989 with the reports (24-26) from the laboratories of Collins, Riordan and Tsui describing the cloning of the CF gene and identification of the major CF mutation. These discoveries have
354
J3.
C
i --
3'
Figure 1--CF gene structure. The locations of the met gene (18) and the probe J3.11 (19) with respect to the CF gene are shown on the genomic DNA. The CF cDNA and the CFTR protein are represented schematically (24,25). The location of the AFh08 mutation is indicated. The putative domains in the CFTR include two transmembrane (TM) domains, two nucleotide-binding folds (NBF) and a postulated regulatory (R) domain.
set the stage for direct detection of CF mutations, providing rapid and accurate carrier detection and prenatal diagnosis for CF families. CF G e n e structure
The CF gene maps to the long arm of chromosome 7, spanning 250,000 nucleotides (250 kb) and consisting of 27 exons which are processed into an mRNA of 6.1 kb (25). The cDNA contains an open reading frame encoding a protein of 1480 amino acids. This protein has been called the cystic fibrosis transmembrane regulatory protein (CFTR). The CFTR has five possible domains consisting of an aminoterminal trans-membrane domain followed by a potential ATP-binding region designated NBF for nucleotide-binding fold. These two domains are followed by a positively charged region, designated the R domain, followed by second trans-membrane and N B F domains. A cartoon illustrating the relationship of the genomic DNA, the cDNA and CFTR is shown in Figure 1. The trans-membrane and
CLINICAL BIOCHEMISTRY,VOLUME 24, AUGUST 1991
CF G E N E ANALYSIS
NBF domain structure of the CFTR is similar to several proteins involved in transport of diverse molecular species. The R domain apparently is unique to the CFTR and may serve a regulatory role in this protein. One major mutation has been identified in the CFTR gene (25). This mutation is a three nucleotide deletion within exon 10 resulting in the deletion of a phenylalanine residue at position 508, hence the designation AF508 (A for deletion, F is the single letter code for phe, codon 508). This mutation is located within the first NBF of the CFTR and comprises approximately 75% of the CF mutations in Northern European and North American populations (26-28). The fact that AF508 is so frequent initially raised the hope that the number of CF mutations might be limited, allowing for relatively simple detection of all or most CF mutations. Unfortunately, the number of other CF mutations appears to be very large and the frequency of each of these other mutations is very low (29-31). Other than AF508, no single CF mutation described to date comprises more than 1 to 2% of CF chromosomes. Clinical
applications
Prior to direct mutation detection, the main applications of DNA probes for CF gene analysis were for carrier detection of siblings of affected individuals and for prenatal diagnosis of couples with a previously affected child. Accuracy in these cases generally required analysis of several family members, including the affected individual. With the availability of AF508 mutation detection, the scope of applications has expanded. First, mutation analysis provides a powerful adjunct to RFLP analysis for carrier detection and prenatal diagnosis of CF families, particularly in those cases where a specimen is not available from the affected individual. Second, mutation analysis opens up the possibility for more accurate carrier testing of individuals with a secondary or tertiary relative affected with CF. Third, direct mutation analysis can be used as an adjunct to or in lieu of the IRT or the sweat tests for CF diagnosis in special cases (e.g., when sweat cannot be obtained from a newborn suspected of having CF, when the sweat test gives borderline results, or in cases of abnormal fetal ultrasound results suggesting the possibility of CF). It should be noted that borderline sweat results may be associated with some of the less common CF mutations and that abnormal fetal ultrasound very rarely is due to CF. Therefore, application of mutation analysis for CF diagnosis in such cases requires keeping these issues in mind. Another possible area of testing brought about with the availability of direct mutation detection is population screening for CF carriers. The issues surrounding CF screening have been discussed recently by several authors (32,33). Although there
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
is disagreement about when or if widespread population screening should be initiated, there is general agreement that increased public education regarding the medical and social issues of genetic testing is required. Furthermore, there is agreement that pilot programs are needed to evaluate CF screening. The American Society of Human Genetics (34) and a National Institutes of Health workshop (35) have issued statements concerning population screening for CF. Both statements, while acknowledging the power of CF gene analysis for testing of individuals with a family history of CF, have recommended against implementation of widespread population screening for CF carriers at the present time.
Methodology RFLPANALYSIS If the gene of interest has not been identified or if the specific mutation within a gene causing the genetic defect has not been or cannot be detected, one can still analyze families for a defect by tracking the disease gene indirectly. This is accomplished by using RFLPs detected by probes that are closely linked to the gene of interest (36). The first application of RFLP analysis for prenatal diagnosis was for sickle cell anemia by Kan and Dozy (37). The basic approach for RFLP analysis is illustrated in Figure 2A. Genomic DNA is isolated and cut with appropriate restriction enzymes detecting polymorphic sites. These sites are due to genotypic differences that occur naturally in genomic DNA with no apparent phenotypic effects. For each probe/enzyme combination, one can score for the presence or the absence of a particular restriction site. Various conventions are used to score RFLP data including - / + for the absence/presence of the site, 1/2 to represent the larger/smaller restriction fragments, or the sizes of the restriction fragments obtained. For the probe/enzyme combination shown in Figure 2, two possible fragments are generated. Using the 1/2 convention for designating RFLPs, for autesomal sites, an individual can have three possible genotypes at this site: 1 1, 1 2, or 2 2. A simple RFLP family analysis is shown in Figure 2B. The affected individual in this case is 2 2 while the parents are both 1 2. The fetus is 1 1. Assuming that the affected individual has CF and that the probe used is linked to CF, one would predict that allele 2 for each parent is tracking with the CF disease gene in this family. Because the fetus inherited allele 1 from each parent, he/she would be predicted to be unaffected and not a carrier. Note that this is an indirect analysis for the CF gene which depends upon the association of a linked marker with a disease phenotype. There is an inherent inaccuracy for indirect analysis due to
355
FUJIMURA
B)
A) E
E
I
I
E
E
E
I
I
I
1 2
m
Probe
22
11
12
11
22
Figure 2--RFLP analysis. (A) The numbers 1 and 2 represent polymorphic alleles. E represents sites on genomic DNA that are cut by restriction enzyme E. E* represents a polymorphic site for enzyme E. The probe is complementary to the regions indicated by the hatched box. The lower panel represents Southern blot results expected from three different genotypes corresponding to individuals homozygous for allele 1 (1 1) or for allele 2 (2 2) and for heterozygotes (1 2). (B) Hypothetical family analyzed with the probe/enzyme combination illustrated in panel (A) The pedigree indicates carrier parents with an affected son. The numbers under each individual in the pedigree correspond to the genotypes determined from the hypothetical results shown in the lower panel. See text for description. the possibility of recombination between the marker analyzed and the disease allele. The recombination frequency, and therefore, the accuracy of analysis will depend upon the distance between the marker allele and the disease allele. The accuracy of analysis can be increased by using several markers closely linked to and flanking the disease gene of interest. Linkage analysis, because it requires analysis of families, has certain limitations and caveats. First, it requires cooperation of family members and participation of the affected individual. Second, the analysis requires that the diagnosis on the affected individual be correct. Finally, meaningful analysis requires that paternity is known. Some of the DNA probes used in this laboratory for RFLP analysis of CF families are shown in Figure 3. These include the met probes (metD and metH) (18), XV2c (20) and KM-19 (21), which are all proximal to the CF gene, and J3.11 (D7S8, JG3C) (19) distal to the CF gene. HAPLOTYPE ANALYSIS AND LINKAGE DISEQUILIBRIUM
The allelic pattern of linked loci is defined as a haplotype. For CF, certain haplotypes have been found to have greater probability of being associated with the disease gene than other haplotypes. Beaudet et al. (23) have defined haplotypes, A through D, based on alleles of the probes XV2c using the enzyme, Taq I, and KM-19 using Pst I. 356
These haplotype definitions are shown in Table 2. Also shown in the table are the frequencies of these haplotypes in association with normal and m u t a n t CF chromosomes (27). Note that the B haplotype is associated with 85% of CF chromosomes, but with only 14% of normal chromosomes. The incorporation of this linkage disequilibrium data with linkage analysis can increase the accuracy of carrier detection and prenatal diagnosis in many CF families. However, because this is also an indirect analysis, results based on haplotype analysis are subject to similar limitations as those based on linkage analysis. DIRECT MUTATION DETECTION
With the identification of the AF508 mutation, direct detection of approximately 75% of CF mutations is possible. Current methods for CF mutation detection depend upon use of the polymerase chain reaction (PCR) (38,39) to amplify genomic DNA sequences encompassing the region of interest. After amplification, the normal and m u t a n t alleles are detected by several possible methods. Most general is the use of labelled (isotopically or otherwise) allele-specific oligonucleotides (ASOs) distinguishing the normal and m u t a n t alleles (25,38). In the case of AF508, the PCR product of the m u t a n t allele is 3 bp smaller than the PCR product of the normal allele. This size difference in PCR products can be detected by electrophoretic separation (40). CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
CF G E N E A N A L Y S I S
TABLE 2 CF Haplotype Definitions and Frequencies
Allele
Haplotype
Frequencya
XV-2c/TaqI KM19/PstI
A B C D Unk
1 1 2 2
1 2 1 2
CF Chromosomes (439)
Normal Chromosomes (433)
0.048 0.845 0.030 0.046 0.032
0.314 0.143 0.376 0.139 0.028
aHaplotypes A, B, C, and D are defined on the basis of XV-2c/TaqI and KM19/PstI alleles as described by Beaudet et al. (23). The data shown are from Lemna et al. (27) and the total number of CF and normal chromosomes analyzed are shown in parentheses. "Unk" indicates that haplotype phasing could not be established.
kb
GENES
PROBES
CF Gene analysis: examples EXAMPLE 1
0
5'-met
met 500
1i:_
met D met H
1000 I I
XV-2c KM-19
CF
EXAMPLE 2
1500
2000
JG-3C
(J3.11) 2500
Figure 3--Map of CF linkage probes with respect to the CF gene. This illustrates the relative positions of the probes used in these studies. The 2500 kb region around the CF gene on chromosome 7 is represented schematically. The positions of the met oncogene and the CF gene are shown.
CLINICAL BIOCHEMISTRY, V O L U M E
Figure 4 shows a family analysis t h a t is fully informative by RFLP analysis. The parents each have one B haplotype and the affected son inherited the B haplotype from each parent. The daughter inherited the A haplotype from the mother and the C haplotype from the father, so she would be predicted to be unaffected and not a carrier of CF. Retrospective analysis of this family for the AF508 mutation gave consistent results. Both parents are carriers of AF508. The son is homozygous positive and the daughter is negative for the AF508 mutation.
24, A U G U S T 1991
Figure 5 illustrates a more complex analysis. The affected individual is heterozygous for the AF508 mutation, indicating t h a t he/she is a compound heterozygote for CF. Approximately 37% of individuals with CF will be expected to be compound heterozygotes of AF508 and another CF gene mutation. The other mutation in this family was not identified. Haplotype analysis for this family indicated t h a t the affected boy inherited the B haplotype and the AF508 mutation from his father and a D haplotype from his mother. The fetus inherited the paternal B haplotype with the AF508 mutation, indicating t h a t it will at least be a CF carrier. Because the mother has a DD genotype, additional RFLP information is required to assess the fetal risk for CF. The mother is informative with metH/Taql and with JG-3C/Pstl, and RFLP results with these probes indicate t h a t the fetus has inherited the m a t e r n a l CF haplotype opposite from t h a t of the affected brother. This gives the fetus a low risk of being affected. Note t h a t without
357
F U ~
I-1
2 1 N 1
2 CF 1
1 2 CF 1
BC I
met D / Banl XV-2c / Taq I KM-19 / Pst I CF locus JG-3C / Pst I
2 m BA
2 1
1 1
N
N
1
1 CA
1 --
CF pos
11I
1 2 CF 1
m --
D m --
1 N neg 2
1
2 2 CF neg 2
II
2 2 2 N neg 1
DD
BA
1 2 CF 1
I1-1
1-,
BB I
Figure 4--Example 1 pedigree. The pedigree shows a family with a son affected with CF and a daughter who requested testing to determine her carrier status for CF. The bars under each individual in the pedigree show alleles for the probe/enzyme combinations shown to the lei~ of the pedigree. CF and normal (N) chromosomes and individual haplotypes (A-D) are indicated.
metH XV2c KM-19 CF l o c u s F508
JG-3C
11111112 1
2
2 CF
2 CF
pos
neg
1
informative flanking markers for the mother, the risk to the fetus of CF would have been much higher due to the possibility of recombination. EXAMPLE 3
The final example illustrated in Figure 6 illustrates a case whose resolution depended upon AFh08 analysis. The pregnant consultand and her partner initially sought testing for AFh08 because she had a brother who died of CF. Analysis of the couple showed that he, but not she, was a carrier of the AFh08 mutation. Haplotype analysis indicated a BB genotype for the consultand, giving her a carrier risk of 63% which is not significantly different from her a priori carrier risk of 67%. Analysis of the consultand's parents resolved the situation. Although both parents had BB genotypes, they also both were carriers of the AFh08 mutation. Because she was negative for the AFh08 mutation, the consultand is not a CF carrier. It should be noted that if one or both parents are negative for the AFh08 mutation, the consultand's carrier risk would increase significantly. This case illustrates two important points about DNA-based genetic analysis: first, the importance of mutation detection for accurate diagnosis, and second, the potential difficulties that can arise if a specimen is not available from one or more key individuals.
Limitations and pitfalls As with any laboratory test, CF testing by DNA probes has limitations and pitfalls. Some of these,
358
1 1
2
BD
1
2
2 CF
2 N neg 1
pos
1
BD
Figure 5--Example 2 pedigree. The pedigree shows a family with an affected son that sought prenatal diagnosis for a subsequent pregnancy. Alleles shown under each individual correspond to the probes shown in the legend to the left of the pedigree. CF and normal (N) chromosomes, individual haplotypes (A-D), and the presence (pos) or absence (neg) of the AFh08 mutation are shown.
including the possibility of specimen mix up ortechnical difficulties, are universal, while others are unique to DNA analysis. Some of the points unique to DNA analysis include: (i) the need for cooperation of family members to perform linkage analysis; (ii) the possibility of misdiagnosis in family analysis due to incorrect paternity or incorrect diagnosis of the affected individual; (iii) the possibility of error in linkage analysis due to recombination between the probe locus and the disease locus; (iv) the possibility of contamination leading to erroneous results because of the exquisite sensitivity of PCR; (v) the possibility of genotyping errors due to rare polymorphisms or other sequence variations in specimen DNA (41). Although it may seem obvious, it is worthwhile to emphasize the critical need for the laboratory to be aware of these potential problems when performing molecular genetic analyses. It is also important that these limitations and pitfalls be understood by patients prior to testing.
Population screening The desirability of population screening for CF carriers is a volatile issue with a number of points
CLINICAL BIOCHEMISTRY,VOLUME 24, AUGUST 1991
CF GENE ANALYSIS
1 2
~
CF-pos
-
2 -
m
2
2
N
CF-pos
- neg -
B
1
1
B
2
~
N -
-
neg
2 -
-
2
B
B
CF m u t a t i o n s . A p p r o x i m a t e l y 75% of CF m u t a tions in C a u c a s i a n s c a n be d e t e c t e d b y t e s t i n g for AF508. A n a l y s i s of AF508 plus t h e m o s t f r e q u e n t of t h e o t h e r m u t a t i o n s described to d a t e m a y r a i s e t h e detection level to a b o u t 80 or 90%. T a b l e 3 shows t h e d e p e n d e n c e of s e v e r a l factors on t h e level of m u t a t i o n detection. T h e s e factors are: (i) t h e CF c a r r i e r r i s k for a n i n d i v i d u a l w h o t e s t s nega t i v e ( - / - ) ; (ii) t h e r i s k to a couple for h a v i n g a CF child w h e n b o t h p a r t n e r s t e s t n e g a t i v e ( - / - ) ( - / - ) ; (iii) t h e r i s k to a couple of a CF child w h e n one p a r t n e r b u t not t h e o t h e r t e s t s positive ( + / - ) ( - / - ) ; (iv) t h e e x p e c t e d f r e q u e n c y of ( + / - ) ( - / - ) couples w i t h t h e s e different levels of m u t a t i o n detection. A t 75% m u t a t i o n detection, t h e r i s k of a C a u c a s i a n i n d i v i d u a l w i t h o u t a f a m i l y h i s t o r y of CF who t e s t s n e g a t i v e ( - / - ) is r e d u c e d to 1 in 97, down f r o m t h e a p r i o r i r i s k of 1 in 25. H o w e v e r , a t 75% m u t a t i o n detection, e v e n t h o u g h 56% of all atr i s k couples ( + / - ) ( + / - ) would be detected, a b o u t 1 in e v e r y 17 couples will h a v e one p a r t n e r who is positive a n d one p a r t n e r who is n e g a t i v e . T h e r e fore, t h e r i s k to t h e s e couples of h a v i n g a CF child is i n c r e a s e d f r o m the a p r i o r i r i s k of 1 in 2500 to 1 in 388. T h i s i n c r e a s e d r i s k m a y r a i s e t h e a n x i e t y level for a b o u t 5.8% of all couples tested, w h e n only a b o u t 1% of t h e s e ( + / - ) ( - / - ) couples a r e r e a l l y at r i s k for h a v i n g a CF child. A t e v e n 90% m u t a t i o n detection, t h e r i s k of a CF child for t h e 1 in 14 couples w h e r e one p a r t n e r b u t not the o t h e r t e s t s positive is 1 in 1000. O n l y a t a b o u t 96% detection does the r i s k of h a v i n g a CF child for t h e s e couples e q u a l the a p r i o r i risk. S o m e of t h e a r g u m e n t s a g a i n s t p o p u l a t i o n s c r e e n i n g would be e l i m i n a t e d should it be possible to detect o v e r 95% of CF m u t a t i o n s w i t h a rela t i v e l y i n e x p e n s i v e test. U n f o r t u n a t e l y , t h i s expect a t i o n m a y not be realized in t h e n e a r f u t u r e , if e v e r (42). In l i g h t of t h e c u r r e n t s i t u a t i o n , s o m e areas of i m m e d i a t e concern include: (i) t h e n e e d for g r e a t e r e d u c a t i o n of t h e p o p u l a t i o n a b o u t CF; (ii)
B
I
1
-1 - XV2c -
KM-19
-
CF
A F508
2-
-2
N-
-N -neg
neg 1-
2-
1
CF--
N
pos
-
2 -
-2 BB
- neg -
2
DA
JG-3C
Figure 6--Example 3 pedigree. The pedigree shows a family with a son affected with CF who is now deceased. The daughter and her spouse sought carrier testing for CF. Alleles shown under the pedigree correspond to the probes shown in the legend to the left of the pedigree. CF and normal (N) chromosomes, individual haplotypes (A-D), and the presence (pos) or absence (neg) of the AF508 mutation are shown. t h a t r e q u i r e clarification. One m a j o r a r g u m e n t a g a i n s t p o p u l a t i o n s c r e e n i n g is t h a t p r e s e n t m e t h ods c a n n o t detect all (or e v e n as m u c h as 95%) of
TABLE 3 CF Risks for Individuals and Couples as a Function of CF Mutation Detection Rates Risk of Having CF Child for Mutation Detection Rate (%) a priori
75 80 85 90 95 96
Carrier Risk for ( - / - ) Individual 1 in 1 in 1 in 1 in 1 in 1 in 1 in
25 97 121 161 241 481 601
(-/-) (-/-) Couple 1 in 1 in 1 in 1 in 1 in 1 in 1 in
2500 37636 58564 103684 232324 925444 1444804
(+/-) (-/-) Couple NA 1 in 388 i in 484 1 in 644 1 in 964 1 in 1924 1 in 2404
Frequency of (+/-) (-/-) Couples 1 1 1 1 1 1
NA in 17.2 in 16.1 in 15.2 in 14.4 in 13.7 in 13.5
Risks are calculated according to Ten Kate (43) for individuals and couples with no family histories of CF. ( - / - ) indicates an individual with no detectable CF mutation and ( + / - ) indicates an individual having one copy of a detectable CF mutation. NA = not applicable.
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
359
FUJIMURA
the counselling and clinical resources required to perform population screening; (iii) carrier stigmatization; (iv) standardization of test protocols and reagents; (v) quality assurance measures for clinical laboratories. There is a general consensus t h a t these and other issues will need to be addressed by pilot screening programs (34,35). In spite of the reservations expressed by m a n y geneticists about population screening for CF, the demand for v o l unt ar y screening will probably increase in the future. Media coverage of genetic testing is increasing, resulting in gr eat er public awareness. Unfortunately, the awareness does not ensure a level of understanding needed to appreciate the implications of screening. Mechanisms m u s t be established to provide this t hr ough educational programs. Finally, the controversy over population screening should not detract from the importance of DNA probe analysis for CF families. Table 1 illustrates the rapid sequence of events in this field, with an explosion in technical advances occurring during the decade of the 1980's. One can be optimistic t h a t the next decade will be as productive, allowing improved diagnostic capabilities for CF and, ultimately, improved therapeutic capabilities.
Acknowledgements I thank my associates at the Nichols Institute, especially Douglas Harrington, Jane Lee-Chen, Kimberly MacMartin, Corey Mark and Vickie Venne, for their hard work, encouragement and support. Special thanks are due to Arthur L. Beaudet, Baylor College of Medicine, for his comments on this manuscript and for many helpful discussions concerning genetic testing.
References 1. Welsh MJ, Liedtke CM. Chloride and potassium channels in cystic fibrosis airway epithelia. Nature 1986; 322: 467-70. 2. Gibson LE, Cooke RE. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 1959; 23: 545-9. 3. Boat TF, Welsh MJ, Beaudet AL. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D., eds. The metabolic basis of inherited disease. Pp. 2649-80. New York: McGraw-Hill, 1989. 4. Cutting GR, Antonarakis SE, Buetow KH, Kasch LM, Rosenstein BJ, Kazazian HH Jr. Analysis of DNA polymorphism haplotypes linked to the cystic fibrosis locus in North American black and Caucasian families supports the existence of multiple mutations of the cystic fibrosis gene. A m J H u m Genet 1989; 44: 307-18. 5. Johnson JP. Genetic counseling using linked DNA probes: cystic fibrosis as a prototype. J Pediatr 1988; 113: 957-64. 6. Kane K. Cystic fibrosis: recent advances in genetics and molecular biology. Ann Clin Lab Sci 1988; 18: 289-96. 7. LeGrys VA. Technological advances in the diagnosis 360
of cystic fibrosis. Lab Med 1988; 19: 748-52. 8. Antonarakis SE. Diagnosis of genetic disorders at the DNA level. N Engl J Med 1989; 320: 1153-63. 9. Chirgwin JM. Molecular biology for the nonmolecular biologist. Diabetes Care 1990; 13: 188--97. 10. Ostrer H, Hejtmancik JF. Prenatal diagnosis and carrier detection of genetic diseases by analysis of deoxyribonucleic acid. J Pediatr 1988; 112: 679-87. 11. Anderson DH. Cystic fibrosis of the pancreas and its relation to celiac disease. A m J Dis Child 1938; 56: 344-99. 12. di Sant'Agnese PA, Darling RC, Perea GA, Shea BA. Abnormal electrolyte composition of sweat in cystic fibrosis of the pancreas: clinical significance and relationship to the disease. Pediatrics 1953; 12: 549-63. 13. Crossley JR, Elliott RB, Smith PA. Dried blood screening for cystic fibrosis in the newborn. Lancet 1979; 1: 472-4. 14. Ad Hoc Committee Task Force on Neonatal Screening, Cystic Fibrosis Foundation. Neonatal screening for cystic fibrosis: position paper. Pediatrics 1983; 72: 741-5. 15. Carbarns NJB, Gosden C, Brock DJH. Microvillar peptidase activity in amniotic fluid: possible use in prenatal diagnosis of cystic fibrosis. Lancet 1983; 1: 329-31. 16. Brock DJH. Amniotic fluid alkaline phosphatase isoenzymes in early prenatal diagnosis of cystic fibrosis. Lancet 1983: 2: 941-3. 17. Knowlton RG, Cohen-Haguenauer O, Cong NV, et al. A polymorphic DNA marker linked to cystic fibrosis is located on chromosome 7. Nature 1985; 318: 380-2. 18. White R, Woodward S, Leppert M, et al. A closely linked genetic marker for cystic fibrosis. Nature 1985; 318: 382-4. 19. Wainwright BJ, Scambler PJ, Schmidtke J, et al. Localization of cystic fibrosis locus to human chromosome 7 cen-q22. Nature 1985; 318: 384-5. 20. Estivill X, Farrall M, Scambler, PJ, et al. A candidate for the cystic fibrosis locus isolated by selection for methylation-free islands. Nature 1987; 326: 840-5. 21. Estivill X, Scambler PJ, Wainwright BJ, et al. Patterns of polymorphism and linkage disequilibrium for cystic fibrosis. Genomics 1987; 1: 257-63. 22. Estivill X, Farrall M, Williamson R, et al. Linkage disequilibrium between cystic fibrosis and linked DNA polymorphisms in Italian families: a collaborative study. A m J H u m Genet 1988; 43: 23-8. 23. Beaudet AL, Feldman GL, Fernbach SD, Buffone GJ, O'Brien WE. Linkage disequilibrium, cystic fibrosis and genetic counselling. A m J H u m Genet 1989; 44: 319-26. 24. Rommens JM, Iannuzzi MC, Kerem B-S, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989; 245: 1059-65. 25. Riordan JR, Rommens JM, Kerem B-S, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245: 1066-73. 26. Kerem B-S, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1073-80. 27. Lemna WK, Feldman GL, Kerem B-S, et al. Mutation analysis for heterozygote detection and the prenatal diagnosis of cystic fibrosis. N Engl J Med CLINICAL BIOCHEMISTRY,VOLUME 24, AUGUST 1991
CF GENE ANALYSIS 1990; 322: 291-6. 28. The Cystic Fibrosis Genetic Analysis Consortium. Worldwide survey of the AF508 mutation-- report from the Cystic Fibrosis Genetic Analysis Consortium. A m J Hum Genet 1990; 47: 354-9. 29. Dean M, White MB, Amos J, et al. Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell 1990; 61: 863-70. 30. Cutting GR, Kasch LM, Rosenstein BJ, et al. A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein. Nature 1990; 346: 366-9. 31. Kerem B-S, Zielenski J, Markiewicz D, et al. Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene. Proc Natl Acad Sci USA 1990; 87: 8447-51. 32. Gilbert F. Is population screening for cystic fibrosis appropriate now? A m J Hum Genet 1990; 46: 394-5. 33. Beaudet AL. Invited editorial; carrier screening for cystic fibrosis. A m J Hum Genet 1990; 47: 603-5. 34. Caskey CT, Beaudet AL, Cavalli-Sforza LL. The American Society of Human Genetics statement on cystic fibrosis screening. A m J H u m Genet 1990; 46: 393. 35. Workshop on population screening for the cystic fibrosis gene. Statement from the National Institutes of Health Workshop on population screening
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36.
37. 38.
39.
40.
41. 42. 43.
for the cystic fibrosis gene. N Engl J Med 1990; 323: 70-1. Botstein D, White RL, Skolnick M, Davis RW. Construction of a linkage map in man using restriction fragment length polymorphisms. A m J Hum Genet 1980; 32: 314--31. Kan YW, Dozy AM. Antenatal diagnosis of sickle cell anemia by DNA analysis of amniotic fluid cells. Lancet 1978; 2: 910-2. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified ~-globin and HLA-DQa DNA with allele-specific oligonucleotide probes. Nature 1986; 324: 163-6. Mullis KB, Faloona FA, Scharf SJ, Saiki RK, Horn GT, Erlich HA. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51: 263-73. Rommens J, Kerem B-S, Greer W, Chang P, Tsui L-C, Ray P. Rapid nonradiactive detection of the major cystic fibrosis mutation. A m J Hum Genet 1990; 46: 395-6. Fujimura FK, Northrup H, Beaudet AL, O'Brien WE. Genotyping errors with the polymerase chain reaction. N Engl J Med 1990; 322: 61. Roberts L. CF screening delayed for a while, perhaps forever. Science 1990; 247: 1296-7. Ten Kate LP. Carrier screening for cystic fibrosis and other autosomal recessive diseases. A m J Hum Genet 1990; 47: 359-61.
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0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Cystic Fibrosis Gene Analysis: Recent Diagnostic Applications FRANK K. FUJIMURA Nichols Institute Reference Laboratories, San Juan Capistrano, CA 92675, USA Cystic fibrosis (CF) is the most common severe genetic disease of Caucasians. Recent work by several laboratories has resulted in the identification of the CF gene and its major mutation. These findings have greatly facilitated carrier detection and prenatal diagnosis for members of families with a history of CF. This review briefly considers the historical background of CF testing, the basic features of the CF gene, and the methodologies for CF gene analysis. Examples of the application of recent technologies for analysis of CF families are discussed. In addition to family analysis, CF carrier detection in the general population is becoming more feasible. The possibility of carrier screening for CF has been the topic of much discussion recently with arguments presented both for and against. Some basic issues pertaining to population testing are presented.
KEY WORDS: genetics; mutation; linkage analysis; restriction fragment length polymorphism; polymerase chain reaction; carrier detection. Introduction
ffecting about one in 2500 births in the United States, cystic fibrosis (CF) is the most common A severe genetic disorder of Caucasians. The disease results from alteration of exocrine cell function due to a defect in regulation of chloride ion transport (1). Although the molecular basis of this defect has yet to be elucidated, the disorder leads to the secretion of excess sodium and chloride ions in the sweat of affected individuals. An elevated level of sweat sodium and chloride determined by the pilocarpine iontophoresis sweat test (2), in spite of some technical difficulties, remains the accepted diagnostic indicator for CF. The pathologic features of CF are caused by the presence of thick mucus resulting in obstruction of several organs, most often the lungs and the pancreas, b u t sometimes the intestines and gonads (3). The abnormal mucus is caused by the defect in chloride ion transport, which is required for passive transport of water into mucosal secretions.
Correspondence: Frank K. Fujimura, Nichols Institute Reference Laboratories, 32961 Calle Perfecto, San Juan Capistrano, CA 92675, USA. Manuscript received December 23, 1990; revised January 29, 1991; Accepted March 5, 1991. CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
The thickened secretions affect normal function of the affected organs leading to lung infections, deficiency in secretion of pancreatic enzymes, intestinal obstruction (meconium ileus), and sterility or reduced fertility. The severity of CF can be quite variable even within one family. Although most individuals with CF are diagnosed within the first decade of life, some individuals are not diagnosed until much later. With improved detection and appropriate therapies, the average life span of CF individuals has been increasing, with median survival age of approximately 25 years at the present time. It is estimated that there are about 20,000 to 30,000 individuals in the United States who have CF. CF is an autosomal recessive genetic disease, meaning that both parents of an affected individual must be carriers. CF carriers are asymptomatic, and, prior to DNA-based testing, identification of carriers was not possible until two carriers produced a CF offspring. As an autosomal recessive disease, each offspring of two CF carriers has a 1 in 4 risk of being affected, a 1 in 2 risk of being a carrier, and a 1 in 4 chance of being a noncarrier. There are about 8,000,000 CF carriers in the United States. The frequency of CF carriers varies with ethnic group, being about 1 in 25 in Caucasian populations. The frequency of CF carriers in American blacks is about 1 in 65 (4). The frequency in Orientals and African blacks is much lower. This discussion will concentrate on the application of DNA probe technologies for diagnosis of CF and for detection of CF carriers. Of particular interest is the recent identification and characterization of the CF gene and the major CF mutation and the impact of these discoveries on CF gene analysis. For further discussions of the applications of DNA probes for restriction fragment length polymorphism (RFLP) analysis of CF and other genetic diseases, please refer to several recent reviews on the topic (5-10). Background Some of the significant events in the clinical diagnosis of CF are listed in Table 1. Although it is 353
FUJIMURA
TABLE 1
Milestones in Cystic Fibrosis Diagnosis Year
Event
1938 1947
Clinical identification of CF Identification of CF as a genetic disease Sweat test for diagnosis of CF IRT test for neonatal screening of CF
1959 1979 1983 1985 1987 1989
MIE analysis for prenatal
diagnosis of CF Mapping of CF gene to chromosome 7 Linkage disequilibrium Identification of CF gene, major CF mutation
Reference (11) (43) (2) (13)
CFTR cDNA (6.1 kb) 5' 0 --
GENOMIC DNA
m:
1
CFTR PROTEIN (1480 aa) N TM
--
-~F508(15,16)
NBF
2
(17-19) (22,23) (24-26)
R CF
27
3
EXONS
TM 4
NBF relatively common, CF was not recognized as a distinct disease until quite recently (11). The abnormal sweat electrolyte composition in affected individuals, first recognized in 1953 by di Sant'Agnese et al. (12), led to the development of the sweat test for diagnosis of CF (2). Immunoreactive trypsinogen (IRT) analysis of dried bloodspots can be used as a neonatal screen for CF (13) and has been adopted for newborn screening by the state of Colorado. Several other states have considered newborn screening by IRT, but opposition to this effort continues to be expressed (14). The initial step toward providing prenatal diagnosis of CF in high-risk pregnancies was provided by microvillar intestinal enzyme (MIE) analysis (15,16). A major breakthrough in CF gene analysis occurred in 1985 with the mapping of the gene to chromosome 7(17-19). Subsequently, several probes linked very closely to the CF gene have been identified (20,21). These probes detect RFLPs that are useful for analysis of families with a positive history of CF. Although very powerful, linkage analysis requires testing of families and largely depends on the availability of specimen from an affected individual in the family. Linkage analysis of CF families showed that certain combinations of alleles (haplotypes) are more frequently associated with CF than others (22,23). This observation of linkage disequilibrium helped to increase the accuracy of analysis for families with a positive history, particularly in those cases where specimens were not available from an affected individual. The combination of linkage analysis coupled with linkage disequilibrium information has allowed very accurate analysis for most families. The intensive efforts to identify the CF gene culminated in 1989 with the reports (24-26) from the laboratories of Collins, Riordan and Tsui describing the cloning of the CF gene and identification of the major CF mutation. These discoveries have
354
J3.
C
i --
3'
Figure 1--CF gene structure. The locations of the met gene (18) and the probe J3.11 (19) with respect to the CF gene are shown on the genomic DNA. The CF cDNA and the CFTR protein are represented schematically (24,25). The location of the AFh08 mutation is indicated. The putative domains in the CFTR include two transmembrane (TM) domains, two nucleotide-binding folds (NBF) and a postulated regulatory (R) domain.
set the stage for direct detection of CF mutations, providing rapid and accurate carrier detection and prenatal diagnosis for CF families. CF G e n e structure
The CF gene maps to the long arm of chromosome 7, spanning 250,000 nucleotides (250 kb) and consisting of 27 exons which are processed into an mRNA of 6.1 kb (25). The cDNA contains an open reading frame encoding a protein of 1480 amino acids. This protein has been called the cystic fibrosis transmembrane regulatory protein (CFTR). The CFTR has five possible domains consisting of an aminoterminal trans-membrane domain followed by a potential ATP-binding region designated NBF for nucleotide-binding fold. These two domains are followed by a positively charged region, designated the R domain, followed by second trans-membrane and N B F domains. A cartoon illustrating the relationship of the genomic DNA, the cDNA and CFTR is shown in Figure 1. The trans-membrane and
CLINICAL BIOCHEMISTRY,VOLUME 24, AUGUST 1991
CF G E N E ANALYSIS
NBF domain structure of the CFTR is similar to several proteins involved in transport of diverse molecular species. The R domain apparently is unique to the CFTR and may serve a regulatory role in this protein. One major mutation has been identified in the CFTR gene (25). This mutation is a three nucleotide deletion within exon 10 resulting in the deletion of a phenylalanine residue at position 508, hence the designation AF508 (A for deletion, F is the single letter code for phe, codon 508). This mutation is located within the first NBF of the CFTR and comprises approximately 75% of the CF mutations in Northern European and North American populations (26-28). The fact that AF508 is so frequent initially raised the hope that the number of CF mutations might be limited, allowing for relatively simple detection of all or most CF mutations. Unfortunately, the number of other CF mutations appears to be very large and the frequency of each of these other mutations is very low (29-31). Other than AF508, no single CF mutation described to date comprises more than 1 to 2% of CF chromosomes. Clinical
applications
Prior to direct mutation detection, the main applications of DNA probes for CF gene analysis were for carrier detection of siblings of affected individuals and for prenatal diagnosis of couples with a previously affected child. Accuracy in these cases generally required analysis of several family members, including the affected individual. With the availability of AF508 mutation detection, the scope of applications has expanded. First, mutation analysis provides a powerful adjunct to RFLP analysis for carrier detection and prenatal diagnosis of CF families, particularly in those cases where a specimen is not available from the affected individual. Second, mutation analysis opens up the possibility for more accurate carrier testing of individuals with a secondary or tertiary relative affected with CF. Third, direct mutation analysis can be used as an adjunct to or in lieu of the IRT or the sweat tests for CF diagnosis in special cases (e.g., when sweat cannot be obtained from a newborn suspected of having CF, when the sweat test gives borderline results, or in cases of abnormal fetal ultrasound results suggesting the possibility of CF). It should be noted that borderline sweat results may be associated with some of the less common CF mutations and that abnormal fetal ultrasound very rarely is due to CF. Therefore, application of mutation analysis for CF diagnosis in such cases requires keeping these issues in mind. Another possible area of testing brought about with the availability of direct mutation detection is population screening for CF carriers. The issues surrounding CF screening have been discussed recently by several authors (32,33). Although there
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
is disagreement about when or if widespread population screening should be initiated, there is general agreement that increased public education regarding the medical and social issues of genetic testing is required. Furthermore, there is agreement that pilot programs are needed to evaluate CF screening. The American Society of Human Genetics (34) and a National Institutes of Health workshop (35) have issued statements concerning population screening for CF. Both statements, while acknowledging the power of CF gene analysis for testing of individuals with a family history of CF, have recommended against implementation of widespread population screening for CF carriers at the present time.
Methodology RFLPANALYSIS If the gene of interest has not been identified or if the specific mutation within a gene causing the genetic defect has not been or cannot be detected, one can still analyze families for a defect by tracking the disease gene indirectly. This is accomplished by using RFLPs detected by probes that are closely linked to the gene of interest (36). The first application of RFLP analysis for prenatal diagnosis was for sickle cell anemia by Kan and Dozy (37). The basic approach for RFLP analysis is illustrated in Figure 2A. Genomic DNA is isolated and cut with appropriate restriction enzymes detecting polymorphic sites. These sites are due to genotypic differences that occur naturally in genomic DNA with no apparent phenotypic effects. For each probe/enzyme combination, one can score for the presence or the absence of a particular restriction site. Various conventions are used to score RFLP data including - / + for the absence/presence of the site, 1/2 to represent the larger/smaller restriction fragments, or the sizes of the restriction fragments obtained. For the probe/enzyme combination shown in Figure 2, two possible fragments are generated. Using the 1/2 convention for designating RFLPs, for autesomal sites, an individual can have three possible genotypes at this site: 1 1, 1 2, or 2 2. A simple RFLP family analysis is shown in Figure 2B. The affected individual in this case is 2 2 while the parents are both 1 2. The fetus is 1 1. Assuming that the affected individual has CF and that the probe used is linked to CF, one would predict that allele 2 for each parent is tracking with the CF disease gene in this family. Because the fetus inherited allele 1 from each parent, he/she would be predicted to be unaffected and not a carrier. Note that this is an indirect analysis for the CF gene which depends upon the association of a linked marker with a disease phenotype. There is an inherent inaccuracy for indirect analysis due to
355
FUJIMURA
B)
A) E
E
I
I
E
E
E
I
I
I
1 2
m
Probe
22
11
12
11
22
Figure 2--RFLP analysis. (A) The numbers 1 and 2 represent polymorphic alleles. E represents sites on genomic DNA that are cut by restriction enzyme E. E* represents a polymorphic site for enzyme E. The probe is complementary to the regions indicated by the hatched box. The lower panel represents Southern blot results expected from three different genotypes corresponding to individuals homozygous for allele 1 (1 1) or for allele 2 (2 2) and for heterozygotes (1 2). (B) Hypothetical family analyzed with the probe/enzyme combination illustrated in panel (A) The pedigree indicates carrier parents with an affected son. The numbers under each individual in the pedigree correspond to the genotypes determined from the hypothetical results shown in the lower panel. See text for description. the possibility of recombination between the marker analyzed and the disease allele. The recombination frequency, and therefore, the accuracy of analysis will depend upon the distance between the marker allele and the disease allele. The accuracy of analysis can be increased by using several markers closely linked to and flanking the disease gene of interest. Linkage analysis, because it requires analysis of families, has certain limitations and caveats. First, it requires cooperation of family members and participation of the affected individual. Second, the analysis requires that the diagnosis on the affected individual be correct. Finally, meaningful analysis requires that paternity is known. Some of the DNA probes used in this laboratory for RFLP analysis of CF families are shown in Figure 3. These include the met probes (metD and metH) (18), XV2c (20) and KM-19 (21), which are all proximal to the CF gene, and J3.11 (D7S8, JG3C) (19) distal to the CF gene. HAPLOTYPE ANALYSIS AND LINKAGE DISEQUILIBRIUM
The allelic pattern of linked loci is defined as a haplotype. For CF, certain haplotypes have been found to have greater probability of being associated with the disease gene than other haplotypes. Beaudet et al. (23) have defined haplotypes, A through D, based on alleles of the probes XV2c using the enzyme, Taq I, and KM-19 using Pst I. 356
These haplotype definitions are shown in Table 2. Also shown in the table are the frequencies of these haplotypes in association with normal and m u t a n t CF chromosomes (27). Note that the B haplotype is associated with 85% of CF chromosomes, but with only 14% of normal chromosomes. The incorporation of this linkage disequilibrium data with linkage analysis can increase the accuracy of carrier detection and prenatal diagnosis in many CF families. However, because this is also an indirect analysis, results based on haplotype analysis are subject to similar limitations as those based on linkage analysis. DIRECT MUTATION DETECTION
With the identification of the AF508 mutation, direct detection of approximately 75% of CF mutations is possible. Current methods for CF mutation detection depend upon use of the polymerase chain reaction (PCR) (38,39) to amplify genomic DNA sequences encompassing the region of interest. After amplification, the normal and m u t a n t alleles are detected by several possible methods. Most general is the use of labelled (isotopically or otherwise) allele-specific oligonucleotides (ASOs) distinguishing the normal and m u t a n t alleles (25,38). In the case of AF508, the PCR product of the m u t a n t allele is 3 bp smaller than the PCR product of the normal allele. This size difference in PCR products can be detected by electrophoretic separation (40). CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
CF G E N E A N A L Y S I S
TABLE 2 CF Haplotype Definitions and Frequencies
Allele
Haplotype
Frequencya
XV-2c/TaqI KM19/PstI
A B C D Unk
1 1 2 2
1 2 1 2
CF Chromosomes (439)
Normal Chromosomes (433)
0.048 0.845 0.030 0.046 0.032
0.314 0.143 0.376 0.139 0.028
aHaplotypes A, B, C, and D are defined on the basis of XV-2c/TaqI and KM19/PstI alleles as described by Beaudet et al. (23). The data shown are from Lemna et al. (27) and the total number of CF and normal chromosomes analyzed are shown in parentheses. "Unk" indicates that haplotype phasing could not be established.
kb
GENES
PROBES
CF Gene analysis: examples EXAMPLE 1
0
5'-met
met 500
1i:_
met D met H
1000 I I
XV-2c KM-19
CF
EXAMPLE 2
1500
2000
JG-3C
(J3.11) 2500
Figure 3--Map of CF linkage probes with respect to the CF gene. This illustrates the relative positions of the probes used in these studies. The 2500 kb region around the CF gene on chromosome 7 is represented schematically. The positions of the met oncogene and the CF gene are shown.
CLINICAL BIOCHEMISTRY, V O L U M E
Figure 4 shows a family analysis t h a t is fully informative by RFLP analysis. The parents each have one B haplotype and the affected son inherited the B haplotype from each parent. The daughter inherited the A haplotype from the mother and the C haplotype from the father, so she would be predicted to be unaffected and not a carrier of CF. Retrospective analysis of this family for the AF508 mutation gave consistent results. Both parents are carriers of AF508. The son is homozygous positive and the daughter is negative for the AF508 mutation.
24, A U G U S T 1991
Figure 5 illustrates a more complex analysis. The affected individual is heterozygous for the AF508 mutation, indicating t h a t he/she is a compound heterozygote for CF. Approximately 37% of individuals with CF will be expected to be compound heterozygotes of AF508 and another CF gene mutation. The other mutation in this family was not identified. Haplotype analysis for this family indicated t h a t the affected boy inherited the B haplotype and the AF508 mutation from his father and a D haplotype from his mother. The fetus inherited the paternal B haplotype with the AF508 mutation, indicating t h a t it will at least be a CF carrier. Because the mother has a DD genotype, additional RFLP information is required to assess the fetal risk for CF. The mother is informative with metH/Taql and with JG-3C/Pstl, and RFLP results with these probes indicate t h a t the fetus has inherited the m a t e r n a l CF haplotype opposite from t h a t of the affected brother. This gives the fetus a low risk of being affected. Note t h a t without
357
F U ~
I-1
2 1 N 1
2 CF 1
1 2 CF 1
BC I
met D / Banl XV-2c / Taq I KM-19 / Pst I CF locus JG-3C / Pst I
2 m BA
2 1
1 1
N
N
1
1 CA
1 --
CF pos
11I
1 2 CF 1
m --
D m --
1 N neg 2
1
2 2 CF neg 2
II
2 2 2 N neg 1
DD
BA
1 2 CF 1
I1-1
1-,
BB I
Figure 4--Example 1 pedigree. The pedigree shows a family with a son affected with CF and a daughter who requested testing to determine her carrier status for CF. The bars under each individual in the pedigree show alleles for the probe/enzyme combinations shown to the lei~ of the pedigree. CF and normal (N) chromosomes and individual haplotypes (A-D) are indicated.
metH XV2c KM-19 CF l o c u s F508
JG-3C
11111112 1
2
2 CF
2 CF
pos
neg
1
informative flanking markers for the mother, the risk to the fetus of CF would have been much higher due to the possibility of recombination. EXAMPLE 3
The final example illustrated in Figure 6 illustrates a case whose resolution depended upon AFh08 analysis. The pregnant consultand and her partner initially sought testing for AFh08 because she had a brother who died of CF. Analysis of the couple showed that he, but not she, was a carrier of the AFh08 mutation. Haplotype analysis indicated a BB genotype for the consultand, giving her a carrier risk of 63% which is not significantly different from her a priori carrier risk of 67%. Analysis of the consultand's parents resolved the situation. Although both parents had BB genotypes, they also both were carriers of the AFh08 mutation. Because she was negative for the AFh08 mutation, the consultand is not a CF carrier. It should be noted that if one or both parents are negative for the AFh08 mutation, the consultand's carrier risk would increase significantly. This case illustrates two important points about DNA-based genetic analysis: first, the importance of mutation detection for accurate diagnosis, and second, the potential difficulties that can arise if a specimen is not available from one or more key individuals.
Limitations and pitfalls As with any laboratory test, CF testing by DNA probes has limitations and pitfalls. Some of these,
358
1 1
2
BD
1
2
2 CF
2 N neg 1
pos
1
BD
Figure 5--Example 2 pedigree. The pedigree shows a family with an affected son that sought prenatal diagnosis for a subsequent pregnancy. Alleles shown under each individual correspond to the probes shown in the legend to the left of the pedigree. CF and normal (N) chromosomes, individual haplotypes (A-D), and the presence (pos) or absence (neg) of the AFh08 mutation are shown.
including the possibility of specimen mix up ortechnical difficulties, are universal, while others are unique to DNA analysis. Some of the points unique to DNA analysis include: (i) the need for cooperation of family members to perform linkage analysis; (ii) the possibility of misdiagnosis in family analysis due to incorrect paternity or incorrect diagnosis of the affected individual; (iii) the possibility of error in linkage analysis due to recombination between the probe locus and the disease locus; (iv) the possibility of contamination leading to erroneous results because of the exquisite sensitivity of PCR; (v) the possibility of genotyping errors due to rare polymorphisms or other sequence variations in specimen DNA (41). Although it may seem obvious, it is worthwhile to emphasize the critical need for the laboratory to be aware of these potential problems when performing molecular genetic analyses. It is also important that these limitations and pitfalls be understood by patients prior to testing.
Population screening The desirability of population screening for CF carriers is a volatile issue with a number of points
CLINICAL BIOCHEMISTRY,VOLUME 24, AUGUST 1991
CF GENE ANALYSIS
1 2
~
CF-pos
-
2 -
m
2
2
N
CF-pos
- neg -
B
1
1
B
2
~
N -
-
neg
2 -
-
2
B
B
CF m u t a t i o n s . A p p r o x i m a t e l y 75% of CF m u t a tions in C a u c a s i a n s c a n be d e t e c t e d b y t e s t i n g for AF508. A n a l y s i s of AF508 plus t h e m o s t f r e q u e n t of t h e o t h e r m u t a t i o n s described to d a t e m a y r a i s e t h e detection level to a b o u t 80 or 90%. T a b l e 3 shows t h e d e p e n d e n c e of s e v e r a l factors on t h e level of m u t a t i o n detection. T h e s e factors are: (i) t h e CF c a r r i e r r i s k for a n i n d i v i d u a l w h o t e s t s nega t i v e ( - / - ) ; (ii) t h e r i s k to a couple for h a v i n g a CF child w h e n b o t h p a r t n e r s t e s t n e g a t i v e ( - / - ) ( - / - ) ; (iii) t h e r i s k to a couple of a CF child w h e n one p a r t n e r b u t not t h e o t h e r t e s t s positive ( + / - ) ( - / - ) ; (iv) t h e e x p e c t e d f r e q u e n c y of ( + / - ) ( - / - ) couples w i t h t h e s e different levels of m u t a t i o n detection. A t 75% m u t a t i o n detection, t h e r i s k of a C a u c a s i a n i n d i v i d u a l w i t h o u t a f a m i l y h i s t o r y of CF who t e s t s n e g a t i v e ( - / - ) is r e d u c e d to 1 in 97, down f r o m t h e a p r i o r i r i s k of 1 in 25. H o w e v e r , a t 75% m u t a t i o n detection, e v e n t h o u g h 56% of all atr i s k couples ( + / - ) ( + / - ) would be detected, a b o u t 1 in e v e r y 17 couples will h a v e one p a r t n e r who is positive a n d one p a r t n e r who is n e g a t i v e . T h e r e fore, t h e r i s k to t h e s e couples of h a v i n g a CF child is i n c r e a s e d f r o m the a p r i o r i r i s k of 1 in 2500 to 1 in 388. T h i s i n c r e a s e d r i s k m a y r a i s e t h e a n x i e t y level for a b o u t 5.8% of all couples tested, w h e n only a b o u t 1% of t h e s e ( + / - ) ( - / - ) couples a r e r e a l l y at r i s k for h a v i n g a CF child. A t e v e n 90% m u t a t i o n detection, t h e r i s k of a CF child for t h e 1 in 14 couples w h e r e one p a r t n e r b u t not the o t h e r t e s t s positive is 1 in 1000. O n l y a t a b o u t 96% detection does the r i s k of h a v i n g a CF child for t h e s e couples e q u a l the a p r i o r i risk. S o m e of t h e a r g u m e n t s a g a i n s t p o p u l a t i o n s c r e e n i n g would be e l i m i n a t e d should it be possible to detect o v e r 95% of CF m u t a t i o n s w i t h a rela t i v e l y i n e x p e n s i v e test. U n f o r t u n a t e l y , t h i s expect a t i o n m a y not be realized in t h e n e a r f u t u r e , if e v e r (42). In l i g h t of t h e c u r r e n t s i t u a t i o n , s o m e areas of i m m e d i a t e concern include: (i) t h e n e e d for g r e a t e r e d u c a t i o n of t h e p o p u l a t i o n a b o u t CF; (ii)
B
I
1
-1 - XV2c -
KM-19
-
CF
A F508
2-
-2
N-
-N -neg
neg 1-
2-
1
CF--
N
pos
-
2 -
-2 BB
- neg -
2
DA
JG-3C
Figure 6--Example 3 pedigree. The pedigree shows a family with a son affected with CF who is now deceased. The daughter and her spouse sought carrier testing for CF. Alleles shown under the pedigree correspond to the probes shown in the legend to the left of the pedigree. CF and normal (N) chromosomes, individual haplotypes (A-D), and the presence (pos) or absence (neg) of the AF508 mutation are shown. t h a t r e q u i r e clarification. One m a j o r a r g u m e n t a g a i n s t p o p u l a t i o n s c r e e n i n g is t h a t p r e s e n t m e t h ods c a n n o t detect all (or e v e n as m u c h as 95%) of
TABLE 3 CF Risks for Individuals and Couples as a Function of CF Mutation Detection Rates Risk of Having CF Child for Mutation Detection Rate (%) a priori
75 80 85 90 95 96
Carrier Risk for ( - / - ) Individual 1 in 1 in 1 in 1 in 1 in 1 in 1 in
25 97 121 161 241 481 601
(-/-) (-/-) Couple 1 in 1 in 1 in 1 in 1 in 1 in 1 in
2500 37636 58564 103684 232324 925444 1444804
(+/-) (-/-) Couple NA 1 in 388 i in 484 1 in 644 1 in 964 1 in 1924 1 in 2404
Frequency of (+/-) (-/-) Couples 1 1 1 1 1 1
NA in 17.2 in 16.1 in 15.2 in 14.4 in 13.7 in 13.5
Risks are calculated according to Ten Kate (43) for individuals and couples with no family histories of CF. ( - / - ) indicates an individual with no detectable CF mutation and ( + / - ) indicates an individual having one copy of a detectable CF mutation. NA = not applicable.
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the counselling and clinical resources required to perform population screening; (iii) carrier stigmatization; (iv) standardization of test protocols and reagents; (v) quality assurance measures for clinical laboratories. There is a general consensus t h a t these and other issues will need to be addressed by pilot screening programs (34,35). In spite of the reservations expressed by m a n y geneticists about population screening for CF, the demand for v o l unt ar y screening will probably increase in the future. Media coverage of genetic testing is increasing, resulting in gr eat er public awareness. Unfortunately, the awareness does not ensure a level of understanding needed to appreciate the implications of screening. Mechanisms m u s t be established to provide this t hr ough educational programs. Finally, the controversy over population screening should not detract from the importance of DNA probe analysis for CF families. Table 1 illustrates the rapid sequence of events in this field, with an explosion in technical advances occurring during the decade of the 1980's. One can be optimistic t h a t the next decade will be as productive, allowing improved diagnostic capabilities for CF and, ultimately, improved therapeutic capabilities.
Acknowledgements I thank my associates at the Nichols Institute, especially Douglas Harrington, Jane Lee-Chen, Kimberly MacMartin, Corey Mark and Vickie Venne, for their hard work, encouragement and support. Special thanks are due to Arthur L. Beaudet, Baylor College of Medicine, for his comments on this manuscript and for many helpful discussions concerning genetic testing.
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