Prenatal diagnosis of hereditary disorders.

Symposium on Medical Genetics

Prenatal Diagnosis of Hereditary Disorders Judith H. Miles, M.D., Ph.D.,* and Michael M. Kaback, M.D.t

Dramatic biomedical achievements over the past two decades now provide accurate and relatively safe methods for the intrauterine assessment of a variety of spectlic fetal parameters in early pregnancy. Such advances are revolutionizing the way families, physicians, medical geneticists, and the general public can now approach certain recognized reproductive problems. By both direct and indirect biochemical, cytologic, and structural approaches, it is now possible to detect a variety of serious hereditary disorders in early fetal life. With relatively straightforward tissue culture techniques, amniotic fluid cells obtained by midtrimester amniocentesis can be cultivated and applied to the detection of virtually all fetal cytogenetic aberrations, and to the diagnosis of more than 70 inborn metabolic errors. Major structural defects of the central nervous system (anencephaly, meningomyelocele, etc.) can be addressed through measurement of amniotic fluid alpha-fetoprotein levels, or indirectly screened by quantitation of this fetal specific protein in maternal serum during early pregnancy. Direct visualization techniques, such as radiography, ultrasonography, and fetoscopy also are rapidly evolving as important and invaluable tools for the assessment of selected pregnancies. Certain major structural defects already have been identtlied by radiographic or ultrasonographic techniques. Other serious disorders such as Duchenne muscular dystrophy, beta thalassemia, and sickle cell anemia, can be approached through fetal blood sampling, obtained either by placental aspiration or by direct sampling of a vessel on the inner placental surface under direct fetoscopic visualization. The steady increase in public acceptance of intrauterine diagnosis reflects changes in the social and legal attitudes of our society. As many couples elect to limit family size,216 they come to place a premium value on each conception. Women who continue to seek educational and career "'Fellow, Division of Medical Genetics, UCLA School of Medicine, Harbor General Hospital, Torrance, California tProfessor, Departments of Pediatrics and Medicine, UCLA School of Medicine; Associate Chief, Division of Medical Genetics, Harbor General Hospital, Torrance, California

Pediatric Clinics of North America-Vol. 25, No.3, August 1978

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JUDITH

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MILES AND MICHAEL M. KABACK

opportunities are more likely to delay childbearing until the later part of their reproductive years, and it is this group of women who are at a relatively increased risk for having children with chromosomal and neural tube abnormalities. The recent advances in prenatal diagnosis and the United States Supreme Court decision permitting therapeutic abortion have facilitated the use of genetic services for the prevention of serious congenital defects. Fetal medicine is in its infancy. Although specific treatment for genetic diseases in fetal life remains primarily a promise of the future, our current ability to prevent genetic disease represents an effective method of halting the drain that birth defects place on families and society. Prevention in this context implies that a couple found to have an affected pregnancy be given the option of terminating that pregnancy by elective abortion. Fortunately, the vast majority of monitored pregnancies are unaffected, and parents are relieved of the anxiety caused by the risk of a serious disorder. Indeed, many pregnancies that would have been terminated on the basis of the risks alone now continue. In this light, prenatal diagnosis has become widely regarded as an important preventive instrument in genetic counseling, and one which, in many instances, may be life-saving.

AMNIOCENTESIS Midtrimester amniocentesis is now the primary diagnostic tool for prenatal diagnosis, allowing the geneticist to take a diagnostic "peek" at the genetic constitution of the fetus early in pregnancy. Initiallyintroduced in the 1930's as an obstetric tool for the management of erythroblastosis fetalis in the third trimester,t26 amniocentesis became an accepted procedure with its safety and risks well defined. 59,172 In the 1950's amniotic fluid cells were used to diagnose fetal sex prenatally using the Barr-body technique. 64 • 191 A decade later, studies revealed that second trimester amniotic fluid cells of fetal origin were viable and could be grown in tissue culture in a quantity sufficient for karyotype analysis. 199 These advances culminated in Jacobson and Barter's diagnosis of a balanced DID translocation in utero in 1967,93 and Nadler's demonstration of the efficacy of determining enzyme deficiencies from the amniotic fluid cells in 1968. '54

Technique Amniocentesis is best performed at about 16 weeks' gestation, when the uterine fundus is midway between the symphysis pubis and the umbilicus. At that time, amniotic fluid volume is approximately 200 mP13 and ensures an adequate "target size." Though success in obtaining amniotic fluid has been found to be directly proportional to gestational age,'95 obstetricians experienced in Inidtrimester amniocentesis are able to obtain fluid from greater than 95 per cent of women on a single tap at this stage. 210 Equally important, when amniocentesis is performed at 16 weeks, adequate time remains for completion of the required studies, since pregnancy termination, if indicated, must be performed by 24 weeks' gestation. In addition to the time required for growing the cells (2

j

PRENATAL DIAGNOSIS OF HEREDITARY DISORDERS

595

to 4 weeks) and performing cytogenetic and biochemical analyses, a number of taps must be repeated. In four of the largest reported series, amniocentesis was repeated in 10, 11, 13, and 14 per cent of the cases. 30, 148,159,195 Poor cell growth followed by failure to obtain fluid and the need to substantiate inconclusive results were the most frequent indications for repeat taps. Gestational age is initially ascertained from a detailed recent menstrual history, including the normalcy of the last menstrual period, regularity of menses, and whether oral contraceptives (known to delay ovulation) were used just prior to conception. Routine ultrasonic scanning prior to amniocentesis is then used to confirm gestational age as well as to assess the possibility of multiple pregnancies, localize the placenta, evaluate fetal gestational age (by fetal head measurement), and assess the possibility of other important obstetric conditions (such as myoma) that could complicate the procedure. Whether ultrasound reduces the percentage of unsuccessful taps 150, 1SB. 195 or the percentage of bloody taps30. 66. 156 is still controversial. Certainly, earlier questions of possible biohazards of diagnostic ultrasound with respect to either chromosomal damage or interference with the growth of cultured cells have not been substantiated.1:l3,150 Amniocentesis is performed as an outpatient procedure. Immediately prior, maternal blood samples are obtained for serum alpha-fetoprotein, relevant enzymatic analyses, and maternal karyotype. If the tap is bloody, a second maternal blood sample is obtained one hour after the tap for a Kleihower-Betke determination offetal cells in the maternal circulation. All Rh negative women are evaluated with sensitization titers and Kleihower-Betke analyses before and after amniocentesis. Whether to administer Rh immune globulin (Rhogam) following amniocentesis to women at risk for sensitization is still unanswered because there have been no clinical trials to test either its efficacy or possible harmful effects on the fetus. 36 The possibility of sensitization based on the husband's Rh status and the Kleihower-Betke analysis must be weighed against the possible harmful effects of Rhogam on the fetus. We are presently involved in a collaborative effort designed to answer this question. On arrival at the laboratory, the supernatant from each tube is studied for alpha-fetoprotein, and the sedimented material is prepared for cell culture. The cells obtained are primarily desquamated epithelial cells from the fetal skin and amnion, but also fetal cells from the gastrointestinal, respiratory, and genitourinary tracts. 89 At the 16 week stage, approximately 20 per cent of these cells are viable. 211 Cytogenetic 30 . 144,152 and blood group analyses 63 have clearly determined that most cells obtained by amniocentesis are of fetal origin; however, it now seems certain that amniotic fluid samples do occasionally become contaminated with maternal cells. The origin of these cells is obscure; they may represent either placental macrophages or maternal cells picked up by the amniocentesis needle. Maternal cell contamination obviously can present a serious problem. In the collaborative study of the Institute of Child Health and Development at the National Institutes of Health (NICHD), two of the three diagnostic errors in sex assignment were inaccurate female predictions, suggesting that maternal cells inadvertently had been used for the predic-

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tion of the fetal genetic condition. Likewise, in Milunsky's series of 1600 aIlllliocenteses, seven erroneous female sex assignments were made. l44 Thus it appears that as many as 0.8 per cent of amniotic fluid cell cultures could be derived from maternal rather than fetal cells. In order to circumvent this potential problem, separate cultures are set up from each syringe of amniotic fluid and then subcultures are cloned from single cells so that multiple cultures are established, grown, and analyzed independently. In addition, fetal and maternal karyotypes are compared for differences in chromosome polymorphisms which are expected to be informative in nearly all cases. 12 These procedures both rule out errors based on maternal contamination and ensure against being misled by changes that can occur in the genetic material of some cells in vitro. The detailed cell culture technique has been described previously and will not be reiterated here. 96 Nevertheless, the importance of successful amniotic fluid cell cultivation cannot be overstated since the vast majority of prenatal diagnostic studies are performed on cultured cells. Both cytogenetic and quantitative biochemical studies depend on a population of actively dividing cells, and to perform such studies on a population of predominantly non viable cells, has been shown to clearly be hazardous. 97 At this time, only the neural tube detection test for alpha-fetoprotein and rapidly adhering cell types are performed on uncultured amniotic fluid. 28 Risks It is imperative that the risks of amniocentesis be clearly delineated. The literature is replete with isolated reports of problems following amniocentesis, most of which refer to third trimester procedures. Until recently no controlled prospective data on mid trimester aIlllliocentesis were available. In 1971, the NICHD initiated a prospective collaborative study involving nine American institutions to obtain the data needed to assess the risks of midtrimester amniocentesis. 159 This study involved 1040 subjects who underwent amniocentesis between 1971 and 1973, and 992 controls matched for age, race, income, and previous pregnancy history. When the results were analyzed, no significant difference was found between the number of subsequent fetal deaths in the aIlllliocentesis group (3.5 per cent) and the control group (3.2 per cent). The aIlllliocentesis group contained a significantly higher number of older women, who would be expected to have a higher spon taneous abortion frequency. 214 No evidence of fetal injury was found, though it was carefully looked for. Approximately 1 per cent of women experienced inconsequential vaginal bleeding or vaginal aIlllliotic fluid leakage; there were no significant differences in any other complications of pregnancy, labor, or delivery. Apgar scores at one and five minutes, prematurity, birth weight, perinatal . complications, and infant deaths during the first year were similarly unaffected by the procedure. At one year of age detailed physical and neurological examinations detected no difference in the number of abnormal findings in the children born to the two groups of women. Actually, the scores were higher on the Denver Developmental screening test in the aIlllliocentesis group. Most, but not all, of this difference could be accounted for by the higher number of infants with Down's syndrome in the control group. These results from the NICHD collaborative study provided the first prospective controlled data that midtrimester amniocentesis is a safe procedure.

1


597

Similiar encouraging results were found by the Canadian collaborative amniocentesis study which collected data on 1020 pregnancies from 13 centers across Canada between January 1973 and February 1976. Their frequency of 1 per cent spontaneous abortions and 1 per cent stillbirths did not differ significantly from control data obtained from various sources, including census records, vital statistics, and special studies using obstetrical records. 195 From these controlled studies we conclude that the combined maternal and fetal risks from mid trimester amniocentesis are no greater than 1 in 200 when the procedure is performed in experienced centers. Accuracy The NICHD collaborative study clearly established amniocentesis to be an accurate medical diagnostic procedure with an overall accuracy rate of 99.4 per cent. Of the six erroneous diagnoses, sex was identified incorrectly in three instances: a child was diagnosed as having galactosemia, but proved normal at birth; and two infants with trisomy 21 were diagnosed as normal fetuses of the opposite sex. There were seven errors or misdiagnoses in the Canadian collaborative study, and 10 errors in Milunsky's series (1973), for accuracy rates of 99.3 and 89.4 per cent respectively. The source of the error could not be accurately identified in all cases, although maternal cell contamination and overgrowth, or sample interchange,could account for most of the discrepancies. Hopefully, recent advances in the delineation of chromosome polymorphisms, allowing distinction between fetal and maternal karyotypes, will eliminate most of these problems. Certain cytogenetic findings, however, do present diagnostic dilemmas; one of the most distressing is the discovery of mosaicism in cultured amniotic fluid. 108, 147 In some instances, presumed mosaicism has not been confirmed in the fetus, 122.160 but in other cases apparent mosaicism in culture has been followed by the abortion of an abnormal fetus. zo , 117 The detection of tetraploidy or polyploidy in amniotic fluid cultures presents a second type of dilemma, since tetraploid cells occur naturally in the human amnion. 114 Thus, tetraploid cells observed in culture are generally felt to represent successful cultivation of cells derived from the amnion. Nevertheless, a tetraploid/diploid infant with multiple congenital anomalies has been born,116 and a triploid fetus was diagnosed prenatally and confirmed at termination ofpregnancy.21 Fortunately, these are rare occurrences. Additional problems infrequently encountered in cytogenetic analysis of cultured amniotic fluid cells involve the interpretation of small variations in chromosome size or the size of satellites, and the interpretation of the significance of balanced translocations. Often karyotype analysis of the parents will provide a clue to the significance of these findings.

INDICATIONS FOR PRENATAL DIAGNOSIS

Chromosomal Abnormalities Chromosomal abnormalities represent a major cause of aberrations in development. Indeed, it is estimated that 50 per cent of spontaneously

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aborted fetuses have a chromosomal abnormality, though considerable variation exists in the prevalence of unbalanced karyotypes reported from various studies. Investigations of abortuses expelled through the end of the second trimester revealed 20 to 30 per cent abnormal karyotypes,42, 48 whereas those restricted to earlier gestational age show 50 to 60 per cent abnormalities. 24, 102, 123. 207 Thus, it appears that the frequency of abortuses with chromosomal abnormalities decreases from over 60 per cent in the earlier stages of pregnancies to below 5 per cent by the end of the sixth month. Correspondingly, about 5 per cent of stillborn infants 130 and 0.6per cent to 0.7 per cent of liveborn infants 53,92 have abnormal karyotypes. Obviously, the great majority of abnormal fetuses are aborted spontaneously; in fact, it is estimated that approximately 65 per cent of Down's fetuses and perhaps 95 per cent of Turner's (XO) conceptuses are spontaneously abortedY This point is worthy of emphasis since the sensitive and delicate issues associated with elective abortion of a chromosomally abnormal fetus are cast into a somewhat different light when one is cognizant of the natural selection that occurs in most cases of chromosomal abnormality. ADVANCED MATERNAL AGE. The increased relative risk of producing children with chromosomal trisomies in women over 35 is the most common indication for amniocentesis, accounting for 47 per cent of the women tested in the NICHD collaborative study, 159 and more than 50 per cent of the women tested in the Canadian collaborative study.195 Actually, the positive correlation between the incidence of Down's syndrome and older mothers was recognized long before the chromosomal basis of the syndrome was discovered. Empiric live birth data indicate that for women between 35 and 39 years of age, the chance of having a child with Down's syndrome is 1 in 290 (0.3 per cent); it is 1 in 100 (1 per cent) for women 40 to 45 years of age, and 1 in 50 (2 per cent) over age 45. 39 The other chromosomal trisomies, including trisomy 18 and trisomy 13, which also result from meiotic nondisjunction, are similarly increased in the offspring of older mothers. 159 Recent data culled from experience with amniocentesis at 16 weeks' gestation indicates that the risk of producing a chromosomally abnormal offspring for women 35 to 39 years of age approximates 2.2 per cent. The risk rises to 3.4 per cent at 40 years, and to about 10 per cent at the age of 45. 144 The higher percentages found at midtrimester amniocentesis are felt to represent both more complete ascertainment and to reflect a certain degree of mid trimester loss of trisomic fetuses, which normally occurs spontaneously. Since the risks gradually increase with age, there can be no absolute definition of advanced maternal age. However, in light of the clear delineation of the low procedural risks of amniocentesis supplied by the collaborative studies, we believe that all women over age 35 should be couns~led concerning the risk of producing a child with a chromosomal abnormality and the availability of amniocentesis. PREVIOUS CHILD WITH A CHROMOSOMAL ABNORMALITY. The second most frequent indication for prenatal diagnosis is the previous birth of a child with trisomy 21. For these families the recurrence risk is from 1 to 2 per cent, based on data from both retrospective and prospective studies. 67, 141, 144 For trisomy 18 or 13, or even for a sex chromosome abnormality, the risks of recurrence are largely unknown, though evidence is


599

accumulating to indicate that the predisposition to nondisjunction is nonspecific. In some families high numbers of trisomic births suggest either the existence of single gene mutations, which predispose to nondisjunction in gamete formation, or possible gonadal mosaicism in one of the parents. In any event, families with a previous chromosomally abnormal child generally approach future pregnancies with such apprehension that their anxiety is often sufficient reason to consider prenatal diagnosis. BALANCED TRANSLOCATION CARRIER PARENTS. When one parent is a balanced translocation carrier, there is a considerable risk of producing an abnormal child. Empiric data obtained from couples undergoing amniocentesis for this indication reveals that from 15 per cent to 20 per cent will produce an abnormal fetus. 67, 144 Most families in this situation are ascertained after the birth of a child with congenital anomalies due to an unbalanced translocation. Though more than 50 per cent of translocations arise de novo in the affected child, in the remainder one parent is found to carry the translocation in a balanced state. When the father carries the translocation, the risk is less (2 to 4 per cent), presumably because of the selection against abnormal sperm prior to or at the time of conception. 91, 171 HABITUAL ABORTERS. Prenatal and preferably preconception genetic consultation is indicated for couples with a history of three or more spontaneous abortions. From a review of 10 earlier studies, Carr3 2 concluded that chromosomal anomalies were about 12 times higher in this group than in the general population. Once these women have had a thorough 0 bstetric evaluation to exclude medical causes ofhabitual abortion, such as thyroid disease, diabetes, incompetent cervix or uterine malformation, a significant group of those remaining will be found to carry a balanced translocation in one of the parents. Kim,111 in a study of 50 couples with fetal wastage, discovered three (6 per cent) with a balanced translocation. Stenchever200 found that 31 per cent of 16 couples with three or more abortions carried a balanced translocation. In this way, it is possible to detect risk situations before the birth of a chromosomally abnormal liveborn infant. Intrauterine monitoring of future pregnancies can ensure that this does not occur, and these families can be counseled that they have an excellent chance of having a normal child. MULTIPLE CONGENITAL ANOMALIES IN PREVIOUS OFFSPRING. It is generally agreed that consideration of amniocentesis is appropriate for families with a history of multiple congenital anomalies in a previous offspring when cytogenetic evaluation had not been performed. One investigation of consecutive late abortions (20 to 28 weeks), stillbirths, and neonatal deaths revealed that 7.2 per cent were chromosomally abnormal; more than one half were trisomic. ' :3 In addition, 5.6 per cent of unselected dying infants studied by Machin '30 had chromosomal aberrations; when only those dying with malformations were considered, 13 per cent were chromosomally abnormal. Of those infants who died of other causes where malformations were not recognized, 2.5 per cent had abnormal karyotypes, five times higher than the incidence of chromosomal abnormalities in series of consecutive liveborn infants. n, 129 Certainly the empiric risk of recurrence in any of these families, if there is no compounding history, will be relatively low. In many cases, however, fear of recurrence is extremely high and will often preclude future pregnancies unless prenatal diagnosis is available.

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OTHER INDICA TIONS. Perhaps the most important aspect ofprenatal diagnosis and counseling is that each family and situation require individual consideration and counseling. Families with Down's syndrome in a second and third degree relative are statistically at a very low risk for producing a chromosomally aneuploid child. Likewise, diagnostic x-ray exposure and certain drugs such as LSD are no longer believed to cause chromosomal abnormalities. Nevertheless, a combination of multiple low risk factors may sometimes cloud the risks sufficiently to make amniocentesis a realistic alternative action. In addition, in certain cases, excessive parental anxiety sometimes justifies taking the risk of amniocentesis. Certainly there are instances where reassurance of chromosomal normalcy has saved a fetus from abortion.

X-Linked Recessive Disorders For many severe X-linked recessive disorders, such as hemophilia A and B, Brutons and Swiss type agammaglobulinemia, X-linked mental retardation, X-linked hydrocephalus, and until recently, Duchenne muscular dystrophy, there are no precise biochemical means to distinguish affected from nonaffected fetuses in utero. Because the gene for each of these disorders is carried on the X chromosome, the conditions are generally expressed only in males. Rare exceptions are women who have only one X chromosome (e.g., Turner's syndrome) and women who inherit anX chromosome with the same mutant gene from each parent. In addition, female carriers of a recessive X-linked mutant gene can sometimes express the condition due to random chance inactivation or lyonization of predominantly the normal X chromosome in early fetal life. For families in which the wife is a known carrier of one of these conditions, it is only possible at this time to offer iden tification of the sex of the fetus prenatally, realizing that if a male fetus is identified there is a 50 per cent chance it will be affected. Obviously this is an extremely imperfect system since the family can only be assured of having unaffected children by electing to abort all male fetuses. Nevertheless, it does allow some families a means of having unaffected children. In the NICHD collaborative study, 22 pregnancies were monitored because ofthe risk of an X-linked disorder, and 11 male fetuses were identified with a 50 per cent chance of being affected. Eight of the eleven mothers chose to have an elective abortion. One had a male unaffected by hemophilia, another had a male unaffected by severe combined immunodeficiency disease, and a third had a male at risk for Duchenne muscular dystrophy (his status remains unknown). That relatively fewer families will abort a male fetus with a 50 per cent chance of being affected is well recognized. Interestingly, the decision to terminate such a pregnancy is only poorly correlated with the severity of the disease. 197 Though sex identification can generally be accomplished by determining the presence of sex chromatin or Barr bodies in uncultivated amniotic fiuid,4. 64 and though the accuracy ofthis technique approaches 100 per cent in some studies,1 this procedure alone is not recommended. Even when Y body determinations are made concurrently, 110 there is an inherent degree of error even in the most experienced laboratories. 2 II Since karyotype analysis allows the most accurate method offetal sexing,


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it is advised that it be used to confirm the sex chromatin and Ybody studies in all cases in which termination of the pregnancy is contemplated. Recently, attempts have been made to determine fetal sex by looking for fetal cells in the maternal blood, since it is known by 14 weeks' gestation there is a fetal-maternal transfer oflymphocytes. Grosset et al. 76 were able to predict fetal sex correctly in 86 per cent of 86 women between 14 and 18 weeks' gestation. Certainly this method is not advised in pregnancies at risk for X-linked disorders at this time, but it does emphasize the importance of looking to maternal blood for the evaluation of fetal problems in the future.

Inborn Errors of Metabolism Diseases caused by the absence or deficiency of a normal functioning enzyme are commonly known as inborn errors of metabolism and are inherited as either autosomal or X-linked recessive conditions. Though most of these disorders are relatively rare to uncommon, considered as a group they cause a significant amount of neonatal and childhood morbidity and mortality. It is estimated that 0.8 per cent of newborn infants have an inherited disorder of metabolism, one third of which are defined as serious. For couples identified to be at risk either because they have previously had an affected child or because they were identified through a heterozygote screening program, there is a 25 per cent chance that each subsequent pregnancy will result in an affected child. The delineation of the biochemical basis of many of these disorders, the capability to identify heterozygous carriers of these recessive genes, and the subsequent ability to perform prenatal diagnosis represent major contributions of biochemical genetics to modern genetic counseling. At this time nearly all definitive prenatal diagnostic techniques are performed on dividing cells cultured from amniotic fluid. Though both uncultivated amniotic fluid cells67.158 and amniotic fluid 60. 137 have been used to make some diagnoses, the great variation in amniotic fluid cell concentrations, origin, and viability, plus a host of other variables makes the use of uncultured cells unreliable. 97. 175. 185 Recently developed microbiochemical techniques may, however, in the future make it possible to circumvent the time-consuming process of cell culture which is now necessary for accurate in utero enzyme analysis. 65. 87. 177 Before it is possible to diagnose a metabolic disease in utero, a number of basic prerequisites must be satisfied. 98 Obviously, the implicated enzymatic or metabolic reaction must be expressed in amniotic fluid cells cultured from mid trimester control pregnancies. Certain enzymes, including phenylalanine hydroxylase, which is deficient in patients with phenylketonuria, and glucose-6-phosphatase, which is deficient in type I glycogen storage disease, are not expressed in cultured amniotic fluid cells. Therefore, neither condition can presently be identified by midtrimester amniocentesis. Another critical consideration is that the investigator must be able to distinguish clearly between enzyme levels found in the affected and the non affected fetus during the midtrimester. For a number of reasons it is often not possible to predict the levels of enzyme activity in amniotic fluid cells. One factor is that certain metabolic or enzyme steps may be de-

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velopmental; that is, they may increase quantitatively as a function of age from fetal to neonatal to adult life. For example, arylsulfatase A and arginosuccinase, the deficient enzymes in metachromatic leukodystrophy and arginosuccinic aciduria, are lower in specific activity in normal mid trimester amniotic fluid cells than in skin fibroblasts cultivated from either the neonate or the adult. 95. 193 Obviously, normal values must be established in a large series of control pregnancies at various stages of gestation. A second consideration is that the non affected heterozygote will often have an enzyme level intermediate between that of the normal and the homozygous affected fetus. Thus, without prior knowledge of

Table 1. Prenatal Diagnosis of Inborn Errors of Lipid Metabolism* DISORDER

Cholesterol ester storage disorder Fabry's disease Farber's disease Gaucher's disease GM, Gangliosidoses Type I-generalized gangliosidosis Type II-juvenile GM, gangliosidosis GM, Gangliosidoses Type I-TaySachs disease Type II-Sandhoff's disease Type III-juvenile GM, gangliosidosis Krabbe's disease (globoid cell leukodystrophy) Lactosyl ceramidosis Metachromatic leukodystrophy Mucolipidosis type II (I cell disease) Mucolipidosis III (pseudopolydystrophy) Mucolipidosis IV Niemann-Pick disease Refsum's disease Wolman's disease

ENZYME ACTIVITY

PBENATAL

BEFEBENCE

DEFICIENCY

DIAGNOSIS

NO.

Acid lipase

Possible

14

a-galactosidase A Ceramidase J3-glucosidase

Achieved Potentially possible Achieved

26, 65 203 188

{3-galactosidase (A, B, and C)

Achieved

98,99, 128

J3-galactosidase (B and Conly)

Achieved

23

Hexosaminidase A

Achieved

163, 187

Hexosarninidase A and B Partial deficiency

Achieved

47

Possible

161, 164

Galactocere broside ,8-galactosidase

Achieved

51,204

Lactosyl ceraInidase Aryl sulfatase A

Possible Achieved

46 95,212

Multiple lysosomal hydrolases Multiple lysosomal hydrolases

Achieved

169, 215

Possible

208

Multiple lysosomal hydrolases Sphingomyelinase

Achieved

115

Achieved

52

Phytanic acid a-hydrolase Acid lipase

Possible

81

Possible

121

*The inheritance of each of these disorders is autosomal recessive except Fabry's disease which is X-linked recessive.

603


Table 2. Mucopolysaccharidoses that can Be Diagnosed Prenatally':' DISORDER

Type I H-Hurler syndrome Type I S-Scheie syndrome Type I H/SH urlerlScheie Type II-Hunter syndrome" Type IIIa-Sanfilippo A Type IITh-Sanfilippo B Type IV-Morquio syndrome Type VI - Marateaux-Lamy Type VII-~ glucuronidase deficiency

ENZYME ACTIVITY DEFICIENCY

PRENATAL DIAGNOSIS

REFERENCE NO.

a-L-iduronidase

Achieved

40, 58, 80

a-L-iduronidase

Possible

9

a-L-iduronidase

Possible

9

Iduronic acid sulfatase Heparin sulfamidase

Achieved

8, 58

Achieved

78

Possible

162

Possible

49, 136

Achieved

113

Possible

196

a-N-acetylglucosaminidase N-acetylgalactosamine6-sulfatase Arylsulfatase B ,a-glucuronidase

''The inheritance is autosomal recessive for each disorder except for Type II-Hunter syndrome, which is X-linked recessive.

these levels, the carrier fetus might be confused with the homozygous affected one. With appropriate control studies prior to prenatal evaluation these problems can be avoided. It is not the purpose of this discussion to review each metabolic disorder which is presently amenable to prenatal diagnosis, as this information can be found in several excellent recent reviews. 145. 156 It is important to note the ever increasing number of disorders for which the criteria for detection have been established. Defects in the metabolism of lipids, mucopolysaccharides, carbohydrates, amino acids, and nucleic acids, as well as a number of other cellular reactions, can now be approached by prenatal study. Table 1 outlines the currently recognized disorders oflipid metabolism, most of which cause severe disability or death in early years. The mucopolysaccharidoses are another group of lysosomal storage disorders in which the specific enzyme deficiency has been described (Table 2). Each of these disorders can be diagnosed in utero by assaying for the missing enzyme in cultured amniotic fluid fibroblasts. In addition, normal individuals and heterozygotes can be distinguished from affected ones by comparing the rates of radioactive sulfate (35 80 4 ) accumulation in cell culture. 57 A significant number of disorders of carbohydrate and amino acid metabolism are also presently amenable to prenatal study (Tables 3 and 4). In Table 5 miscellaneous disorders of metabolism are listed, each of which is potentially identifiable in utero by assaying for the specific abnormalities in cultured cells.

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Table 3. Prenatal Diagnosis of Inborn Errors of Carbohydrate Metabolism':' DISORDER

Fucosidosis Galactokinase deficiency Galactosemia

Glucose-6-phosphate dehydrogenase deficiency· Glycogen storage diseases Type 11Pompe's disease Type 111Debrancher deficiency Type 1VBrancher deficiency Glycogen storage disease with phosphorylase kinase deficiency* Mannosidosis Phosphohexose isomerase deficiency Pyruvate decarboxylase deficiency Pyruvate dehydrogenase deficiency

ENZYME ACTIVITY

PRENATAL

REFERENCE

DEFICIENCY

DIAGNOSIS

NO.

a-fucosidase Galactokinase

Possible Potentially possible

170 15

Galactose-lphosphate uridyl transferase Glucose-6-Pdehydrogenase

Achieved

154

Possible

155

a-l,4-glucosidase (acid maltase)

Achieved

54

Amylo-l, 6glucosidase

Possible

94

Amylo-l, 4 to 1, 6trans glucosidase

Possible

88

Phosphorylase kinase

Possible

140

a-mannosidase Phosphohexose isomerase

Possible Possible

7, 22 120

Pyruvate decarboxylase

Potentially possible

17

Pyruvate dehydrogenase


18

"The inheritance is autosomal recessive except for glucose-6-phosphate dehydrogenase deficiency and glycogen storage disease with phosphorylase kinase deficiency, in which it is X-linked recessive.

Many inherited disorders are not expressed in skin fibroblasts and cannot be diagnosed by analysis of cultivated amniotic fluid fibroblasts. Most notable in this group are the various hemoglobinopathies including sickle cell anemia and beta thalassemia. The first step toward prenatal detection of these diseases was the demonstration that adult hemoglobin is synthesized by the fetus as early as the first trimester. 85 Subsequently, the time course of beta chain synthesis during gestation has been elucidated,as, 109 and methods have been developed for obtaining small samples of fetal blood through fetoscopy (discussed subsequently). The culmination of these efforts has been the recent diagnosis of both beta thalassemia and sickle cell anemia in utero. 3 , :J7, 84. 104-106

L

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Table 4. Prenatal Diagnosis of Amino Acid and Related Metabolic Disorders':' DISORDER

Arginosuccinic aciduria Asparty19lucosaminuria Citrullinemia Cystathioninuria Cystinosis Histidinemia Homocystinuria Hypervalinernia Maple syrup urine disease Severe infantile form Intermittent form Methylmalonic acidemia I B12 unresponsive Methylmalonic acidemia II B 12 responsive Ornithinemia

Propionic acidemia (ketonic hyperglycinemia)

ENZYME ACTIVITY DEFICIENCY

PRENATAL DIAGNOSIS

REFERENCE NO.

Arginosuccinase

Achieved

74

Aspartylglucosaminidase Argininosuccinate synthetase Cystathionase t Intracellular cystine content Histidase Cystathionine synthetase Valine transaminase

Possible

6

Achieved

180

Potentially possible Achieved

62 189. 198

Possible Achieved

139


43

Branched-chain ketoacid decarboxylase Branched -chain ketoacid decarboxylase Methylmalonic-CoA mutase

Achieved

217


44

Achieved

134

Partial defect in vitamin B 12 coenzyme

Achieved

5

Ornithine-aketoacid transaminase Propionyl CoA carboxylase

Possible

194

Achieved

73

55

*The inheritance of each of these disorders is autosomal recessive.

Included in the group of miscellaneous disorders is Duchenne muscular dystrophy, a relatively common and thoroughly debilitating X-linked recessive disorder for which no effective therapy currently exists. Families at risk of producing an affected son have generally prevented the birth of male offspring either by curtailing all future pregnancies or by the elective abortion of male fetuses. 9o Though Duchenne muscular dystrophy has recently been diagnosed in utero by measurement of fetal serum creatine phosphokinase levels,132 the ability to obtain micro samples of fetal blood must still be regarded as an experimental technique limited to a very few centers. Neural Tube Defects Neural tube defects comprise the spectrum of birth defects including

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Table 5. Other Metabolic Disorders in Which Prenatal Diagnosis is Applicable DISORDER

Acatalasemia Adenosine deaminase deficiency Alpha-thalassemia Andogenital syndrome (congenital adrenal hyperplasia) Chediak-Higashi disease Congenital erythropoietic porphyria Congenital nephrotic syndrome Familial hypercholesterolemia

Hypophosphatasia Lesch-Nyhan syndrome*

Lysosomal acid phosphatase deficiency Menkes' disease' Myotonic dystrophy'

Orotic aciduria

Sulfite oxidase deficiency Xeroderma pigmentosa

OBSERVED

PRENATAL

REFERENCE

ABNORMALITY

DIAGNOSIS

NO.

t Catalase tAdenosine deaminase

Potentially possible Achieved

119 82

Deletion of a-globin structural genes 21-hydroxylase deficiency (one of many types)

Achieved

103

Possible

149

Not known


45

t Uroporphyrinogen III cosynthetase

Achieved

181, 186

t a-Fetoprotein amniotic fluid

Achieved

146

Possible

72

Achieved Achieved

19, 176, 182 25, 77

Achieved

157

tCopper accumulation Linkage analysis possible in some families t Ototidylic pyrophosphorylase t Ototidylic decarboxylase t Sulfite oxidase

Achieved

86

Possible

190


118

Possible

192

Defective DNA repair

Achieved

174

Abnormal feedback suppression of 3hydroxy-3-methyl glutaryl coenzyme A reductase t Alkaline phosphatase t Hypoxanthineguanine phosphoribosyl transferase t Lysosomal acid phosphatase

*Inheritance is autosomal recessive except for Lesch-Nyhan syndrome and Menkes' disease in which it is X-linked recessive, and myotonic dystrophy in which it is autosomal dominant.

anencephaly and meningomyelocele which result from the incomplete closure of the neural tube (see the article on Neural Tube Defects in this symposium).


607

METHODS OF FETAL VISUALIZATION The development of techniques that allow visualization of the fetus and permit sampling of fetal blood in midtrimester are extremely important and relevant to the future course of prenatal medicine. They not only allow us to improve the accuracy and extend the scope of in utero diagnosis, but of perhaps greater significance, they are the stepping stones to prenatal treatment and ultimately cure of fetal disease. Each technique presently available for fetal visualization, including ultrasound, radiography, fetography, amniography, and fetoscopy, has both specific advantages and definite limitations. In most instances the solution to each question referred for prenatal diagnosis will benefit from the judicious use of a combination of the available technical modalities. Ultrasound Ultrasound, because of the total lack of ionizing irradiation and the relative ease of examination, has become the primary noninvasive method for visualizing the fetus. The two techniques presently available are B mode gray scale, which permits delineation of both external and internal structures, and real time ultrasound, which allows assessment of fetal movement. Thus, in addition to its supporting role in preparation for amniocentesis and fetoscopy, ultrasound has developed as a diagnostic tool useful in the delineation of many fetal abnormalities. For example, since the alpha-fetoprotein assay is not specific for neural tube defects, an elevated alpha-fetoprotein can only suggest a fetal abnormality, whereas ultrasonic examination in many cases can actually demonstrate the lesion. This is particularly true for anencephaly in which by 18 to 19 weeks' gestation, failure to define the fetal skull after a careful and thorough examination is almost pathognomonic for anencephaly. Though spinal abnormalities are more difficult to visualize, Campbell31 was able to demonstrate 7 of 10 cases of spina bifida detected by an elevated alphafetoprotein. Hydrocephalus, a frequent companion to neural tube abnormalities, can also be recognized by comparing the biparietal diameter of the skull to the maximum thoracic diameter. Since these measurements normally remain equal ± 0.5 cm, a skull diameter more than 1 cm greater than the thorax strongly suggests hydrocephalus. 167 To families faced with the report of an elevated alpha-fetoprotein, the additional often specific information provided by ultrasound is of immeasurable assistance. Polyhydramnios and intrauterine growth retardation are two relatively frequent prenatal complications for which ultrasound is the initial diagnostic procedure of choice. For the patient with increased uterine size, ultrasound can distinguish multiple pregnancy from a pelvic mass or excessive amniotic fluid. In addition, many etiologies of polyhydramnios including intestinal atresia,50 omphalocele, and neural tube defects are approachable with ultrasound. Ifintrauterine growth retardation is suspected, consecutive ultrasonic examinations with comparisons of the biparietal diameter may allow one to narrow down the diagnostic possibilities, since it is well appreciated that many congenital anomalies, including chromosomal abnormalities, are associated with intrauterine

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growth retardation in the midtrimester.69 Recently, dysplastic kidneys and oligohydramnios have been identified during the second trimester in pregnancies at risk for three genetic syndromes featuring renal abnormalities (Meckel syndrome, Robert's syndrome, and bilateral renal agenesis ).101 Certainly, further experience with ultrasound will allow precise diagnosis of many other structural anomalies.

Fetoscopy Fetoscopy alone permits direct access to the fetus. 100 Samples offetal blood obtained during fetoscopy have for the first time extended prenatal diagnosis to three of the world's most prevalent and most severe inherited diseases, that is, beta thalassemia,3, 37. 84, 104, 106 sickle cell anemia,3, 84, 106 and Duchenne muscular dystrophy.132 With direct visualization of the fetus, the Ellis van Creveld and TAR (thrombocytopenia with absent radii) syndromes have been diagnosed in the midtrimester. As currently performed,83, 84, 125, 168 fetoscopy involves the abdominal percutaneous introduction of a small fiberoptic telescope (Needlescope) into the amniotic cavity under local anesthesia. For fetal blood sampling a 27 gauge needle is inserted through a side arm and advanced under direct visualization into a vessel coursing over the fetal surface of the placenta. Despite the theoretical simplicity, certain major limitations exist. First, because the endoscope is inflexible, it is in some instances impossible to approach an anterior placenta, which is estimated to occur in 17 to 66 per cent of pregnancies. 84,105 An alternative method of blind placental aspiration has been utilized 105 , 107 as a means of circumventing this difficulty. Although the number of patients studied is still small, blind placental aspiration has been associated with higher fetal mortality rates than has fetoscopy. In three reported series there have been a total of 7 fetal losses in 47 patients undergoing blind placental aspiration (14.9 per cent). 3,84, 105 At present, however, placental aspiration is the only procedure available for the totally anterior placenta. Perhaps development of a flexible endoscope will permit access to the amniotic surface of anterior placentas. An additional limitation of fetoscopy is that samples obtained are rarely 100 per cent fetal blood. Though hemoglobin analyses require only a small amount of fetal hemoglobin, and though methods have been developed for separation and concentration of fetal cells and hemoglobin, other potential prenatal diagnostic techniques such as determining the fetal blood concentration of various hormones and metabolites are not feasible with impure samples. Another important consideration is the difficulty encountered in simply visualizing the fetus. Benzie 1" reports the fetoscope was used in 25 cases before they had any real idea of which fetal part was being seen. The intrinsic problems of fetal movement, pulsations of the cord, the veil of amnion, and any clouding of the amniotic fluid with blood or meconium are compounded by the narrow visual field of the fetoscope. To illustrate, the hand of the 18 week fetus completely fills the visual field of the N eedlescope. Accurate visualization of fetal parts requires not only massive experience and expertise, but also fortuitous positioning of the fetus. The critical concern is the safety of the fetus. Certainly fetoscopy carries all the risks of amniocentesis plus danger from fetal blood loss


609

during sampling, fetal scarring, and any possible effects of an intense light source. Currently, fetal complications appear to be minimized by using an instrument 2 to 3 mm in diameter and a 27 gauge needle for blood sampling, and by locating the placenta prior to fetoscopy. In the largest series of28 patients who underwent 34 fetoscopies in the second trimester there was no evident fetal or maternal morbidity associated with the procedure. 84 This, however, is clearly insufficient data to establish the risks of fetoscopy either for viewing the fetus or for withdrawal of fetal blood samples. Therefore, at present, fetoscopy should be considered clinically for a limited number of serious fetal conditions that cannot be detected by other means.

Fetal Radiography, Amniography, and Fetography For many malformation syndromes, precise biochemical or chromosomal markers are not yet available. Some of these do, however, have specific skeletal abnormalities that may serve as diagnostic markers in the midtrimester. Indeed, by 17 to 19 weeks, the fetal skeleton is sufficiently well ossified to permit visualization of most tubular bones, including the long and short bones of the limbs, the clavicles, and the ribs. 165 This would indicate that for conditions in which a long bone is absent, such as TAR syndrome, Fanconi syndrome, or ulnar-fibular dysplasia, radiographic diagnosis migh t be feasible. Absence or dysplasia of the small bones such as the clavicle in cleidocranial dysplasia, or the thumb, radius, or clavicle in Holt-Dram syndrome would be expected to be more difficult. Though it is impossible to predict a priori the degree of success in picking out abnormalities in each of the severe dwarfing syndromes, one might anticipate success since these disorders preferentially affect the long bones and the ribs. Pregnancies at risk for a number of syndromes with theoretically informative skeletal abnormalities have been monitored by classical direct radiography, including the TAR syndrome, achondrogenesis, thanatophoric dwarfism, short-rib polydactyly dwarfing syndrome, homozygous achondroplasia, and cleidocranial dysplasia. 165, 166 In each case the fetus appeared normal and a normal infant was delivered at term. Likewise, normal long bones have been visualized at 20 weeks in two fetuses at risk for lethal osteogenesis imperfecta; one infant born at term is normal and the outcome of the second pregnancy is not yet known. HZ One fetus affected with Saldino-Noonan dwarfism was studied at 19 weeks and no long bones were visualized. Unfortunately, owing to technical limitations, the authors found it impossible to clearly distinguish fetal abnormality, and the diagnosis was not unequivocally made until the 29th week. Direct radiography, however, failed to diagnose achondroplasia,fiR Ellis van Creveld dwarfism,75 and osteopetrosis 71 when they were present. Obviously, the inability to visualize specific fetal bones at 20 weeks may be difficult to interpret and to distinguish from delayed calcification or the technical limitations of the procedure. Further delineation of the normal development of ossification ofthe long bones and the spine 10, 11 and increasing experience that is being accumulated with each specific disorder are expected to expand the application of prenatal diagnostic radiography.

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Two methods of contrast radiography that incorporate the introduction of dyes into the amniotic fluid may be employed to visualize better the fetal outline and to delineate an inability of the fetus to swallow. In amniography, a water soluble dye is used to opacify the amniotic fluid; in fetography, an oil soluble dye is injected which tends to adhere to the vernex caseosa, thereby clarifying the detail of the fetal outline. Both techniques have been used successfully to demonstrate a number of fetal abnormalities in late pregnancy.35. 173. 205. 218 Recently, Golbus et al. 70 used amniography at 20 weeks to correctly diagnose the severe dwarfism in a fetus with achondrogenesis, and Frigoletto and Griscom 61 correctly demonstrated the absence of a myelomeningocele at 20 weeks. With all radiographic procedures, the major hazards are the known, and perhaps even more importantly, the unknown effects of ionizing irradiation on the developing fetus and its progeny. Though the estimated radiation exposure of 0.1 to 1 rad to these fetuses has not been found to be teratogenic in animals or man, 153. 183. 184,206 an in utero exposure of 1 rad has been shown perhaps to double the incidence of childhood leukemia and other malignancies. 131, 202 In one retrospective series, children who received approximately 1 rad of intrauterine exposure had an absolute risk ofl in 1700 of developing cancer in the first nine years oflife. 201 Obviously, this risk must be compared with the 1 in 4 risk from a serious autosomal recessive disorder such as the TAR syndrome. In addition to the risks of amniocentesis and radiation, the fetus undergoing contrast radiography is subject to possible untoward effects of the contrast media. Though extensive clinical experience has shown the procedure to be relatively safe in the third trimester, some neonates who underwent amniofetography using two dyes showed a transient impairment of fetal thyroid function;179 others studied with one dye did not. l5l Clearly the delineation of the risks of these procedures requires further study since extrapolation to the midtrimester is inadequate. As with biochemical and cytogenetic prenatal diagnosis, there should be a reasonable conviction that the suspected disorder will demonstrate early intrauterine malformations, that the risks of the abnormality completely outweigh the risks of the procedure, and finally that other less invasive techniques have been used preferentially.

PRENATAL DIAGNOSIS IN GENETIC COUNSELING Prenatal diagnosis adds a new dimension to genetic counseling by providing many families an alternative, and in many instances a positive course of action. Families at risk for an increasing number of disorders that can be detected by intrauterine diagnosis need no longer fear the risk of recurrence of a specific genetic disease in their children. Because the risks rarely exceed 25 per cent, as in autosomal recessive conditions, they have an excellent chance through prenatal diagnosis of having unaffected offspring. This implies, of course, that such families would elect to monitor their pregnancies by amniocentesis and electively to terminate those in which an affected fetus is identified.


611

Genetic counseling has been described as a proc ess of comm unication during which the physician or other trained counselor helps the family to understand better the genetic and medical aspects of an inherited disease. 56 Before the advent of modern prenatal diagnosis, families who clearly understood their risk for a serious genetic disorder often chose not to have other children. 34. 124 Their options were contraception, sterilization, and in some instances artificial insemination. If an unplanned pregnancy did occur, the fetus was often aborted on the basis of the risk. In addition, anger and seemingly unjustified guilt felt by parents in such situations not infrequently led to family disruption and to lifelong stigmatization of other healthy members of the family. We have delineated a number of circumstances in which consideration of prenatal diagnosis should be offered. Nevertheless, no absolute indications for these procedures exist. Because of the enormous emotional, ethic al, and religious issues involved, each family must make their own decision based on the clear knowledge provided by the counselor. Counseling must include not only a discussion of the risks and burdens of having a genetically defective child, but also address the hazards of the procedures and the diagnostic accuracy of the tests, and explore any reservations the family might have. The husband is urged to be present and to participate in these decisions. When the family has reached a decision, the physician has a further obligation to facilitate the selected course of action. The goals of prenatal counseling are to provide families with complete and appropriate information, to aid them in making a decision that is in the best interests of their family, and to help them to make the best possible adjustment to that decision.

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46. Dawson, G., and Stein, A. 0.: Lactosyl ceramidosis: Catabolic enzyme defect of glycosphingolipid metabolism. Science, 170:556, 1970. 47. Desnick, R. J., Ramon, M. K, Bendel, R P., et al.: Prenatal diagnosis of glycosphingolipidoses: Sandhoff's and Fabry's diseases. J. Pediat., 83:149,1973. 48. Dhadial, R K, Machin, A. M., and Tait, S. M.: Chromosomal anomalies in spontaneously aborted human fetuses. Lancet, 2:20, 1970. 49. DiFerranti, N., Ginsberg, L. C., Donnelly, P. V., et al.: Deficiencies of glucosamine-6sulfate or galactosamine-6-sulfate sulfatases are responsible for different mucopolysaccharidoses. Science, 199:79, 1977. 50. Duenhoelter, J. M., Ramos, R S., Rosenfeld, C. R., et al.: Prenatal diagnosis ofgastrointestinal tract obstruction. Obstet. Gynecol., 47:618, 1976. 51. Ellis, W. G., Schneider, E. L., McCulluch, J. R, et al.: Fetal globoid cell leukodystrophy (Krabbe disease). Arch. Neurol., 29:253, 1973. 52. Epstein, C. J., Brady, R. 0., Schneider, E. L., et al.: In utero diagnosis of Niemann-Pick disease. Am. J. Hum. Genet., 23:533, 1971. 53. Evans, H. J.: Chromosome anomalies among live births. J. Med. Genet., 14:309, 1977. 54. Fensom, A. H., Benson, P. F., Blunt, S., et al.: Amniotic cell 4-methyumbelliferyl-aglucosidase activity for prenatal diagnosis of Pompe's disease. J. Med. Genet., 13: 148, 1976. 55. Fleisher, L. D., Longhi, R C., Tallan, H. H., et al.: Homocystinuria: Investigations of cystathionine synthase in cultured fetal cells and the prenatal determination of genetic status. J. Pediat., 85:677, 1974. 56. Fraser, F. C.: Genetic counseling. Am. J. Hum. Genet., 26:636,1974. 57. Fratantoni, J. C., Hall, C. W., and Neufeld, E. F.: The defect in Hurler's and Hunter's syndromes: Faulty degradation of mucopolysaccharide. Proc. Nat. Acad. Sci. U.S.A., 60:699, 1968. 58. Fratantoni, J. C., Neufeld, E. F., Uhlendorf, B. W., et al.: Intrauterine diagnosis of the Hurler and Hunter syndromes. New Engl. J. Med., 280:686, 1969. 59. Freda, V. J.: Recent obstetrical advances in the Rh problem. Bull. N.Y. Acad. Med., 42:474, 1966. 60. Friedland, J., Perle, G., Saifer, A., et al.: Screening for Tay-Sachs disease in utero using amniotic fluid. Proc. Soc. Exp. BioI. Med., 136:1297, 1971. 61. Frigoletto, F. D., and Griscom, N. T.: Amniography for the detection offetal myelomeningocele. Obstet. Gynecol., 44:286, 1974. 62. Frimpter, G. W., Greenberg, A. J., Hilgartner, M., et al.: Cystathioninuria: Management. Am. J. Dis. Child., 113:115, 1967. 63. Fuchs, F., Freiesleben, E., Knudsen, E., et al.: Determination of fetal blood groups. Lancet, 1 :996, 1956. 64. Fuchs, F., and Riis, P.: Antenatal sex determination. Nature (Lond.), 177:330,1956. 65. Galjaard, H., Niermeijer, M. F., Hahnemann, N., et al.: An example of rapid prenatal diagnosis of Fabry's disease using microtechniques. Clin. Genet., 5:368, 1974. 66. Gerbie, A. B., and Shkolnik, A. A.: Ultrasound prior to amniocentesis for genetic counseling. Obstet. Gynecol., 46:716, 1975. 67. Golbus, M. S.: The antenatal detection of genetic disorders: Current status and future prospects. Obstet. Gynecol., 48:497, 1976. 68. Golbus, M. S., and Hall, B. D.: Failure to diagnose achondroplasia in utero. Lancet, 1 :629, 1974. 69. Golbus, M. S., Hall, B. D., and Creasy, R K: Prenatal diagnosis of congenital anomalies in an intrauterine growth retarded fetus. Hum. Genet., 32:349, 1976. 70. Golbus, M. S., Hall, B. D., Filly, R A., et al.: Prenatal diagnosis of achondrogenesis. J. Pediat., 91 :464, 1977. 71. Golbus, M. S., Koerper, M. A., and Hall, B. D.: Failure to diagnose osteopetrosis in utero. Lancet, 2:1246,1976. 72. Goldstein, J. L., Harrod, M. J. E., and Brown, M. S.: Homozygous familial hypercholesterolemia: Specificity of the biochemical defect in cultured cells and feasibility of prenatal detection. Am. J. Hum. Genet., 26:199, 1974. 73. Gompertz, D., Goodey, P. A., Thorn, H., et al.: Antenatal diagnosis of propionic acidemia. Lancet, 1 : 1009, 1973. 74. Goodman, S. I., Mace, J. W., Turner, B., et al.: Antenatal diagnosis of arginosuccinic aciduria. Clin. Genet., 4:236, 1973. 75. Griscom, N. T.: Possible radiographic approaches to fetal diagnosis and therapy. Clin. Perinatal., 11 :435, 1974. 76. Grosset, L., Barrelet, V., and Odartchenko, N.: Antenatal sex determination from maternal blood during early pregnancy. Am. J. Obstet. Gynecol., 120:60, 1974. 77. Halley, D., and Heukels-Dully, M. J.: Rapid prenatal diagnosis of the Lesch-Nyhan syndrome. J. Med. Genet., 14:100,1977. 78. Harper, P. S., Laurence, K M., Parkes,A., et al.: Sanfilippo A disease in the fetus. J. Med. Genet., 11 :123,1974.

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