451
Archives ofDisease in Childhood 1991; 66: 451-454
REGULAR REVIEW
Prenatal diagnosis of
enzyme
defects
an
update
Bryan Winchester, Elisabeth Young
The trends in prenatal diagnosis of enzyme defects predicted last year,' namely an increase in diagnosis in the first trimester by chorionic villus sampling and early amniocentesis and greater exploitation of DNA analysis and the diagnosis of genetic errors in preimplantation embryos, have been fulfilled. These developments were also reflected in the papers presented at the 5th International Congress on Early Fetal Diagnosis, held in Prague in July 1990.
sis to be made by direct assay on chorionic villi before 8 weeks and subsequent termination of a pregnancy with antiprogestins and prostaglandins rather than surgical methods. The safety of this procedure is being assessed, especially with regard to fetal abnormalities. It will be necessary to establish the levels of enzyme activities at this stage of development. In a few women, sampling by the transcervical and transabdominal routes is difficult or impossible for anatomical reasons. However, it is possible to obtain chorionic villi by transvaginal needle aspiration under the guidance of an intravaginal ultrasound probe.9 Although it is a more lengthy procedure, accurate sampling is achieved and it could be very useful in these difficult cases. Sampling may be carried out as early as 6-8 weeks. It seems inevitable that, with wider experience of sampling, technical improvements in assay procedures and the perception that chorionic villus sampling is no more dangerous than amniocentesis, more enzymic defects will be diagnosed in the first trimester by chorionic villus analysis.
Chorionic vilus sampling The move towards demonstrating a deficiency of enzynic activity directly in a biopsy specimen of chorionic villi has continued as more centres gain experience of chorionic villus sampling. In our own laboratory over 50% of all prenatal diagnoses of enzyme defects are now made by direct assay of chorionic villi. The proportion is even higher (75-80%) in other centres, although measurement of the concentration of metabolites in amniotic fluid is sometimes carried out to support a diagnosis (WJ Kleijer, personal communication). More assay procedures previously used for cultured amniocytes have been shown to be valid for Early amniocentesis chorionic vili. Interestingly, several enzymes Many enzyme defects and metabolic disorders considered to be tissue specific and thought not have been diagnosed by the detection of specific to be expressed in chorionic vili can, in fact, be metabolites in amniotic fluid obtained at 16 detected by the use of more sensitive radioisoto- weeks of gestation. Recently it has been shown pic assays. For example, the five activities of the that it is possible to detect some of these metaurea cycle have been assayed in chorionic vili bolites in amniotic fluid obtained at 11-12 and prenatal diagnosis of deficiencies of three of weeks' gestation, thereby permitting diagnosis the enzymes has been made.2 The experience of and termination in the first trimester. Propionic several centres with prenatal diagnosis of acidaemia,'0 tyrosinaemia type I,1" and arginienzyme defects by chorionic villus sampling has nosuccinic aciduria'2 have all been diagnosed at been reported recently.3 4 11-12 weeks' gestation by measurement of the Two large surveys in North America sug- specific metabolite in amniotic fluid by stable gest that the risk of fetal loss is not significantly isotope dilution gas chromatography-mass greater for transcervical chorionic villus sam- spectrometry with selected ion monitoring. The pling than for amniocentesis when carried out organicacidurias and aminoacidurias that can be in experienced centres."7 Another survey came diagnosed by quantitative measurement of to the conclusion that transcervical and transab- metabolites in amniotic fluid obtained in the dominal chorionic villus sampling are equally first and second trimester have been reviewed. 13 safe procedures in experienced hands.8 These By obtaining a sample of amniotic fluid at the ent of Dep Clnical Biochemistry, conclusions should allay the fear that chorionic same time as transabdominal sampling for chorIstitute of Chd Health villus sampling is a more risky procedure than ionic villi it will be possible to confirm a direct and Hospitals for amniocentesis. enzynic diagnosis by measurement of metaboSick Chdren, 30 Guilford Street, Although the usually accepted limit for chor- lites in amniotic fluid. London WC1N 1EH ionic villus sampling is 9-12 weeks' gestation, it Culture of cells from amniotic fluid taken at Bryan Winchester is possible to obtain adequate samples as early as 7-11 weeks' gestation is proving to be more difElisabeth Young 6-7 weeks by transabdominal needling under ficult than from fluid sampled in the second Correspondence to: ultrasound guidance. This would allow diagno- trimester. Methods are being developed and Dr Winchster.
452
Winchester, Young
modified to increase the success rate of these mutation because it is uncommon or new. Altercultures and provided enzyme activities are natively, RFLP analysis of DNA from the expressed in these amniocytes, it should be pos- fetus, affected child, and parents may be inforsible to make an earlier diagnosis of those mative. It has been predicted that the latter enzymic defects which cannot be made directly approach can be applied to 90% of white famion chorionic vifli but require culture of cells. It lies with phenylketonuria. Another disorder in which DNA analysis has has also been possible to make a diagnosis of I cell disease in a fetus by the demonstration of been used for prenatal diagnosis is Tay-Sachs an increase in lysosomal enzymic activities in disease, which results from a deficiency of hexamniotic fluid taken at 10-12 weeks' ges- osaminidase A and has an occurrence of 1/3600 tation.'4 Work is currently in progress to see in the unscreened population of Ashkenazi if the concentrations and patterns of glycosami- Jews. Three mutations in the gene encoding the noglycans in early amniotic fluid can be used to a subunit of hexosaminidase account for over 90% of the mutant alleles in this group.2' 22 diagnose the mucopolysaccharidoses. Application of this information was possible when the parents of an affected child were both found to carry the same one of these mutations, DNA analysis It is possible to investigate the molecular basis a four base pair insertion. This mutation can be of over 250 different genetic diseases using readily detected if the sequence containing it is recombinant DNA techniques. The application amplified by the polymerase chain reaction and of these techniques to prenatal diagnosis is then digested with a restriction enzyme because advancing rapidly. This is illustrated very one of the resultant fragments is four bases lonclearly by the progress made on the prenatal ger from the mutant allele than from the normal diagnosis of cystic fibrosis in the period of just allele. A subsequent pregnancy was tested for over a year since the defective gene was first dis- this mutation by analysis of DNA from chorcovered. Among enzyme defects DNA technol- ionic villi. The fetus was found to be heterozyogy has found greatest application so far for gous and this diagnosis was confirmed by phenylketonuria and closely related disorders. enzyme assay both prenatally and postnatally.23 DNA analysis may be useful, even when a This is because of the relatively high incidence of the disease in white people and the previous disorder can be diagnosed by direct enzymic lack of a reliable method for prenatal diagnosis. analysis, if only a very small sample is available At the same time as providing a reliable prenatal for analysis. Identification of a particular mutadiagnosis for the first time for some cases, tion in an affected fetus may have prognostic however, DNA methods have confirmed the value for other affected members of the family. About 20 lysosomal enzymes have now been great heterogeneity in this disorder.'5 At least 18 mutations have been reported in the phenyla- cloned and molecular genetical analysis of cases lanine hydroxylase gene (the enzyme affected in of the different lysosomal storage diseases has revealed tremendous heterogeneity. Therefore phenylketonuria). 6 Several restriction fragment length polymor- it seems that each kindred or subpopulation will phisms (RFLPs) have been found in the region require specific DNA tests for a single or small of the phenylalanine hydroxylase gene. Cur- number of mutations. It will be interesting to rently haplotypes are defined by the pattern of see what ingenious strategies are devised to cope alleles at eight sites produced with seven restric- with this problem. tion endonucleases. 17 There are appreciable ethnic differences in the prevalence of these haplotypes. Although certain mutations have Fetal celis in maternal circulation been found to occur predominantly in particular Another source of fetal material for analysis of haplotypes, as yet no mutation has been shown enzymic defects may be fetal cells in the materto be exclusively linked to a specific haplotype. nal circulation. It has been claimed that lymThese polymorphisms may be informative phocytes from a male fetus can be detected in within a family with phenylketonuria, however, the maternal circulation using flow cytometry and prenatal diagnosis has been carried out on and antibodies against paternal antigens not present in the mother24 and that fetal metathis basis.'8 19 It is now possible to detect known mutations phases can be detected in maternal peripheral using allele specific oligonucleotides. Alterna- blood.25 However, other groups have not been tively the polymerase chain reaction can be used able to confirm these observations.26 27 to amplify the sequence in the genomic DNA Recently the polymerase chain reaction has containing the mutations, which can be been used to demonstrate Y chromosome specidetected by sequencing, restriction enzyme fic sequences in the blood of women carrying cleavage, chemical mismatch analysis, or selec- male fetuses.28 This technique, which requires tive hybridisation with oligonucleotides.20 Thus stringent control of conditions to avoid contamiin a family with phenylketonuria if the mutation nation, is now being evaluated in other laborin the affected child has been established, it atories. If the detection or even isolation of fetal cells should be possible to monitor a subsequent pregnancy by analysis of DNA from chorionic villi from the maternal circulation can be established for the specific mutation. If the mutation has reliably, it will provide a non-invasive method not been established, at least two options are for prenatal diagnosis of genetic defects with available. Known mutations could be screened essentiaully no risk to the fetus. The sex of the for in fetal DNA, taking into account ethnic fetus could be investigated using Y chromosome prevalences but there is a risk of not detecting a specific probes and the polymerase chain reac-
Prenatal diagnosu of enyme defects-an update
453
tion or perhaps in situ hybridisation for X enzymes of clinical relevance, for example, hexlinked disorders. Theoretically, it would be pos- osaminidase have also been found to be low at sible to detect specific mutations in an enzyme this stage of development. DNA analysis may be possible for specific gene using the polymerase chain reaction or to measure enzynic activity directly in a very human mutations after amplification of the small number of isolated fetal cells. DNA in the blastomere (approximately 6 pg of Of related interest is the observed increased DNA); P thalassaemia can be diagnosed by activity of some lysosomal enzymes in the serum amplification of DNA from a single blastomere of pregnant women compared with the activity from a mouse preimplantation embryo.34 in the same women when not pregnant-for An exciting new extension of this procedure example, iduronate sulphate sulphatase (the to unfertilised human oocytes and to the first enzyme defective in Hunter's disease). If a fetus polar bodies isolated from them offers the possiis affected with Hunter's disease, however, no bility of detection of defects before fertilisation, increase in this enzyme is found in the maternal with the benefit of not testing the embryo or serum, whereas with a normal or heterozygous fetus at any stage.35 Preliminary results have fetus the rise is observed. The origin of this shown that it is possible to amplify the DNA increased activity needs further investigation as sequence containing the sickle cell mutation site does the whole subject of fetal material in the from human oocytes and polar bodies. Further maternal circulation. work is necessary to show whether both sequences are amplified in a heterozygote.
Testing of preimplantation embryos Another way of avoiding the development of a fetus carrying a genetic defect is to diagnose the defect in preimplantation embryos and only transfer unaffected embryos to the uterus.29 Recently important ethical and practical steps have been taken towards making this a feasible proposition for some genetic defects. The acceptance by the British parliament of a clause in the Human Fertilization and Embryology Bill permitting carefully regulated research on embryos up to 14 days from conception has removed the legal constraint. It is now clear that it is technically possible to remove one or two cells for analysis from a human embryo obtained by in vitro fertilisation or uterine lavage without affecting the development of the fetus.30 31 Therefore if the microanalytical techniques developed for other small fetal samples can be applied to this material it will be possible to detect genetic defects at the levels of the chromosome, DNA, or gene product-that is, enzyme or specific protein in preimplantation embryos. To date this strategy has been used successfully for X linked disorders by sexing embryos using Y specific DNA amplification and only transferring female embryos.32 Two couples at risk for adrenoleukodystrophy and X linked mental retardation have given birth to female dizygotic twins after sexing of in vitro fertilised preimplantation embryos and transfer of female embryos, although one baby was stillborn. At least three other pregnancies have been monitored after this form of prenatal diagnosis. The X linked enzymopathy, Lesch-Nyhan syndrome, resulting from a deficiency of hypoxanthine/guanine phosphoribosyl transferase, is a candidate for this approach to prenatal diagnosis. The activity of this enzyme can be measured in cultures derived from a single blastomere from a mouse preimplantation embryo by a very sensitive assay using a radiolabelled substrate.33 Unfortunately the enzyme does not appear to be expressed at a sufficiently high level in corresponding cultures of human blastomeres to distinguish carried over maternal activity from fetal activity,' thereby excluding prenatal diagnosis by enzyme assay on preimplantation embryos. The levels of several other
Fetal treatment of enzymic defects For a small number of enzyme defects treatment in utero is an alternative to prenatal diagnosis and the option of termination.36 This may take the form of vitamin supplementation for disorders responsive to additional cofactor, for example, cobalamin E disease, dietary control as in phenylketonuria, or drug administration to suppress a harmful secondary metabolic activity resulting from the primary enzymic defect, for example, the suppression by glucocorticoid of adrenal production of adrenogenic steroids in adrenal hyperplasia (due to a deficiency of steroid 21-hydroxylase). In theory the earlier in pregnancy the diagnosis is made and treatment is started, the more effective it should be. The reliable diagnosis of adrenal hyperplasia in the first trimester by DNA analysis permits initiation of treatment at about 9-10 weeks before masculinisation of the genitalia begins. A recent survey of 15 female infants with congenital adrenal hyperplasia who had been treated prenatally from the first trimester, however, shows that only five had normal genitalia.37 Many factors affect the efficacy of the administered hormone in the fetus and close monitoring of the pregnancy is necessary to avoid complications. These observations emphasise the need for more detailed knowledge of the biochemical events in fetal development to formulate rational chemical strategies for fetal treatment. Addendum to list' of enzyme defects that have been diagnosed prenatally Considerable progress has been made in clarifying the defects in peroxisomal disorders and in developing methods for their prenatal diagnosis in the first trimester.38 3 Prenatal diagnosis has been carried out for non-ketotic hyperglycinaemia (McKusick No 23830) in chorionic villi by enzymatic analysis of the glycine cleavage system4 and for Canavan's disease (McKusick No 27190) (R Matalon, personal communication). A pregnancy at risk for the new inborn error of metabolism due to a deficiency of aromatic L-amino acid decarboxylase (EC 4.1.1.28)4' has been monitored by analysis
Winchester, Young
454
of fetal liver.42 Two disorders where the methodology for prenatal diagnosis would appear to be available are the defect of mitochondrial fatty acid , oxidation due to a deficiency of long chain 3-hydroxyacyl-CoA dehydrogenase43 and atypical phenylketonuria due to a deficiency of glutamyl transpeptidase cyclohydrolase I (McKusick No 23391)." 1 Winchester B. Prenatal diagnosis of enzyme defects. Arch Dis Child 1990;65:59-67. 2 Shin YS, Endres W. First trimester diagnosis of inherited metabolic diseases by biochemical methods. Proceedings of the 5th Interna al Congress on Early Fetal Diagnosts. Prague, July 1990:251. 3 Fowler B, Giles L, Cooper A, SardharwaUla IB. Chorionic viUus sampling: diagnostic uses and limitations of enzyme assays. J Inherited Metab Dis 1989;12 (suppl 1):105-17. 4 Besley GTN, Broadhead DM. Prenatal diagnosis of inherited metabolic disease by chorionic villus analysis: the Edinburgh experience. J Inherited Metab Dis 1989;12 (suppl 2): 263-6. 5 Rhoads GG, Jackson LG, Schlesselman SE, et al. The safety and efficacy of chorionic villus sampling for early prenatal diagnosis of cytogenetic abnormalities. N EnglJ Med 1989; 320:609-17. 6 Canadian Collaborative CVS-Amniocentesis Clinical Trial Group. Multicentre randomized clinical trial of chorion viUus sampling and amniocentesis. First report. Lancet
1989;i:1-6.
7 Goldberg JD, Porter AE, Golbus MS. Current assessment of fetal losses as a direct consequence of chorionic vilus sampling. Am J Med Genet 1990;35:174-7. 8 Jackson L. Transcervical and transabdominal chorionic vilus sampling are comparably safe procedures for first trimester prenatal diagnosis: preliminary analysis. Am Jf Hum Genet 1990;47 (suppl):278. 9 Sidransky E, Black SH, Soenksen DM, Jones SL, Dorfmann AD, Schulman JD. Transvaginal chorionic viUus sampling. Prenat Diagn 1990;10:583-6. 10 Chadefaux B, Rabier D, Bonnefont JP, et al. Prenatal diagnosis of propionic acidaemia: amniocentesis at the 11th week of pregnancy. Prenat Diagn 1989;9:448. 11 Jakobs C, Stellard F, Kvittingen EA, Henderson M, Lilford R. First trimester prenatal diagnosis of tyrosinaemia type I by amniotic fluid succinylacetone determination. Prenat Diagn 1990;10:133-4. 12 Chadefaux B, Ceballos I, Rabier M, et al. Prenatal diagnosis of argininosuccinic aciduria by assay of argininosuccinate in amniotic fluid at the 12th week of gestation. Am 7 Med Genet 1990;35:594. 13 Jakobs C, Ten Brink HJ, Stellard F. Prenatal diagnosis of inherited metabolic disorders by quantitation of characteristic metabolites in amniotic fluid: facts and future. Prenat Diagn 1990;10:265-71. 14 Besley GTN, Broadhead DM, Nevin NC, Nevin J, Dorman JC. Prenatal diagnosis of mucolipidosis II by early amniocentesis. Lancet 1990;335:1164-5. 15 Cotton RGH. Heterogeneity of phenylketonuria at the clinical, protein and DNA levels. J Inherited Metab Dis
1990;13:739-50. phenylketonuria. Biochemisty 1989;28:1-6. 17 Chakraborty R, Lidsky AS, Daiger SP, et al. Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria. Hum Genet 1987;76:40-6. 18 Lidsky AS, Guittler F, Woo SLC. Prenatal diagnosis of cassic phenylketonuria by DNA analysis. Lancet 1985i:549-51. 19 Speer A, Bellman F, Michel A, et al. Prenatal diagnosis of classical phenylketonuria by linked restriction fragment
16 Woo SLC. Molecular basis and population genetics of
length polymorphism analysis. Prenat Diagn 1986;6: 447-50.
20 Reiss J, Cooper DN. Application of the polymerase chain
reaction to the diagnosis of human genetic disease. Hum Genet 1990;85:1-8. 21 Paw BH, Phuong TT, Kaback M, Lim J, Neufeld EF. Frequency of three hex A mutant alleles among Jewish and non-Jewish carriers identified in a Tay-Sachs screening program. Am J Hum Genet 1990;47:698-705. 22 Triggs-Raine BL, Feigenbaum ASJ, Natowicz M, et al. Screening for carriers of Tay-Sachs disease among Ashkenazi Jews. N Engi J Med 1990;323:6-12. 23 Triggs-Raine BL, Archibald A, Gravel RA, Clarke JTR. Prenatal exclusion of Tay-Sachs disease by DNA analysis. Lancet 1990;335:1164. 24 Herzenberg LA, Bianchi DW, Schroder J, et al. Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. Proc Nad Acad Sci USA 1979;76:1453-5. 25 Selypes A, Lorencz R. A non-invasive method for determination of the sex and karyotype of the fetus from the maternal blood. Hun Genet 1988;79:357-9. 26 Tharapel A, Jaswaney V, Dockder M, et al. Can fetal cells in maternal blood be selected through cytogenetic means? Am J Hum Geset 1989;45:A271. 27 YouseffM, Shulman LP, Tharapel AT, Simpson JL, Elias S. Failure to document fetal cells in maternal circulation using the Selypes-Lorencz 'air culture' cytogenetic technique. Hum Genet 1990;85:133-4. 28 Lo Y-MD, Wainscoat JS, Gillmer MDG, Patel P, Sampietro M, Fleming KA. Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancet 1989; ii:1363-5. 29 Penketh R, McLaren A. Prospects for prenatal diagnosis during preimplantation human development. In: Rodeck C, ed. Clinical obstetrics and gynaecology. London: Bailliere Tindall, 1987:747-4. 30 Hardy K, Martin KL, Leese HJ, et al. Human preimplantation development in vitro is not adversely affected by biopsy at 8-cell stage. Human Reproduction 1990;5:708-14. 31 Buster JE, Carson SA. Genetic diagnosis of the preimplantation embryo. Am J Med Genet 1989;34:211-6. 32 Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344:768-70. 33 Monk M, Handyside A, Hardy K, Whittingham D. Preimplantation diagnosis of deficiency of hypoxanthine phosphoribosyl transferase in a mouse model for LeschNyhan syndrome. Lancet 1987;ii:423-5. 34 Holding C, Monk M. Diagnosis of ,-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos. Lancet 1989;i:532-5. 35 Monk M, Holding C. Amplification of ,B-haemoglobin sequence in individual human oocytes and polar bodies. Lancet 1990;335:985-8. 36 Schulman JD. Treatment of the embryo and the fetus in the first trimester. Am J Med Genet 1990;35:197-200. 37 Pang S, Pollack MS, Marshall RN, Immken L. Prenatal treatment of congenital adrenal hyperplasia due to 21hydroxylase deficiency. N Engl J Med 1990;322:111-5. 38 Schutgens RBH, Schrakamp G, Wanders RJA, Heymans HSA, Tager JM, Van den Bosch H. Prenatal and perinatal diagnosis of peroxisomal disorders. J Inherited Metab Dis 1989;12 (suppl 1):118-34. 39 Wanders RJA, Van Roermund CWT, Schutgens RBH, et al. The inborn errors of peroxisomal ,B-oxidation: a review. J Inherited Metab Dis 1990;13:4-36. 40 Hayasaka K, Tada K, Fueki N, Aikawa J. Prenatal diagnosis of nonketotic hyperglycinemia: enzymatic analysis of the glycine cleavage system in chorionic villi. J Pediatr 1990;
116:444-5.
41 Hyland K, Clayton PT. Aromatic aminoacid decarboxylase deficiency in twins. J Inherited Metab Dis 1990;13:301-4. 42 Hyland K, Clayton PT. Aromatic aminoacid decarboxylase
deficiency. Pteridines 1990;2:40.
43 Wanders RJA, Ijlst L, Van Gennip AH, et al. Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: identification of a new inborn error of mitochondrial fatty acid ,Boxidation. J Inherited Metab Dis 1990;13:311-4. 44 Schoedon G, Redweik U, Curtius H-C. Purification of GTP cyclohydrolase I from human liver and production of specific monoclonal antibodies. Eur J Biochem 1989;178: 627-34.
Archives ofDisease in Childhood 1991; 66: 451-454
REGULAR REVIEW
Prenatal diagnosis of
enzyme
defects
an
update
Bryan Winchester, Elisabeth Young
The trends in prenatal diagnosis of enzyme defects predicted last year,' namely an increase in diagnosis in the first trimester by chorionic villus sampling and early amniocentesis and greater exploitation of DNA analysis and the diagnosis of genetic errors in preimplantation embryos, have been fulfilled. These developments were also reflected in the papers presented at the 5th International Congress on Early Fetal Diagnosis, held in Prague in July 1990.
sis to be made by direct assay on chorionic villi before 8 weeks and subsequent termination of a pregnancy with antiprogestins and prostaglandins rather than surgical methods. The safety of this procedure is being assessed, especially with regard to fetal abnormalities. It will be necessary to establish the levels of enzyme activities at this stage of development. In a few women, sampling by the transcervical and transabdominal routes is difficult or impossible for anatomical reasons. However, it is possible to obtain chorionic villi by transvaginal needle aspiration under the guidance of an intravaginal ultrasound probe.9 Although it is a more lengthy procedure, accurate sampling is achieved and it could be very useful in these difficult cases. Sampling may be carried out as early as 6-8 weeks. It seems inevitable that, with wider experience of sampling, technical improvements in assay procedures and the perception that chorionic villus sampling is no more dangerous than amniocentesis, more enzymic defects will be diagnosed in the first trimester by chorionic villus analysis.
Chorionic vilus sampling The move towards demonstrating a deficiency of enzynic activity directly in a biopsy specimen of chorionic villi has continued as more centres gain experience of chorionic villus sampling. In our own laboratory over 50% of all prenatal diagnoses of enzyme defects are now made by direct assay of chorionic villi. The proportion is even higher (75-80%) in other centres, although measurement of the concentration of metabolites in amniotic fluid is sometimes carried out to support a diagnosis (WJ Kleijer, personal communication). More assay procedures previously used for cultured amniocytes have been shown to be valid for Early amniocentesis chorionic vili. Interestingly, several enzymes Many enzyme defects and metabolic disorders considered to be tissue specific and thought not have been diagnosed by the detection of specific to be expressed in chorionic vili can, in fact, be metabolites in amniotic fluid obtained at 16 detected by the use of more sensitive radioisoto- weeks of gestation. Recently it has been shown pic assays. For example, the five activities of the that it is possible to detect some of these metaurea cycle have been assayed in chorionic vili bolites in amniotic fluid obtained at 11-12 and prenatal diagnosis of deficiencies of three of weeks' gestation, thereby permitting diagnosis the enzymes has been made.2 The experience of and termination in the first trimester. Propionic several centres with prenatal diagnosis of acidaemia,'0 tyrosinaemia type I,1" and arginienzyme defects by chorionic villus sampling has nosuccinic aciduria'2 have all been diagnosed at been reported recently.3 4 11-12 weeks' gestation by measurement of the Two large surveys in North America sug- specific metabolite in amniotic fluid by stable gest that the risk of fetal loss is not significantly isotope dilution gas chromatography-mass greater for transcervical chorionic villus sam- spectrometry with selected ion monitoring. The pling than for amniocentesis when carried out organicacidurias and aminoacidurias that can be in experienced centres."7 Another survey came diagnosed by quantitative measurement of to the conclusion that transcervical and transab- metabolites in amniotic fluid obtained in the dominal chorionic villus sampling are equally first and second trimester have been reviewed. 13 safe procedures in experienced hands.8 These By obtaining a sample of amniotic fluid at the ent of Dep Clnical Biochemistry, conclusions should allay the fear that chorionic same time as transabdominal sampling for chorIstitute of Chd Health villus sampling is a more risky procedure than ionic villi it will be possible to confirm a direct and Hospitals for amniocentesis. enzynic diagnosis by measurement of metaboSick Chdren, 30 Guilford Street, Although the usually accepted limit for chor- lites in amniotic fluid. London WC1N 1EH ionic villus sampling is 9-12 weeks' gestation, it Culture of cells from amniotic fluid taken at Bryan Winchester is possible to obtain adequate samples as early as 7-11 weeks' gestation is proving to be more difElisabeth Young 6-7 weeks by transabdominal needling under ficult than from fluid sampled in the second Correspondence to: ultrasound guidance. This would allow diagno- trimester. Methods are being developed and Dr Winchster.
452
Winchester, Young
modified to increase the success rate of these mutation because it is uncommon or new. Altercultures and provided enzyme activities are natively, RFLP analysis of DNA from the expressed in these amniocytes, it should be pos- fetus, affected child, and parents may be inforsible to make an earlier diagnosis of those mative. It has been predicted that the latter enzymic defects which cannot be made directly approach can be applied to 90% of white famion chorionic vifli but require culture of cells. It lies with phenylketonuria. Another disorder in which DNA analysis has has also been possible to make a diagnosis of I cell disease in a fetus by the demonstration of been used for prenatal diagnosis is Tay-Sachs an increase in lysosomal enzymic activities in disease, which results from a deficiency of hexamniotic fluid taken at 10-12 weeks' ges- osaminidase A and has an occurrence of 1/3600 tation.'4 Work is currently in progress to see in the unscreened population of Ashkenazi if the concentrations and patterns of glycosami- Jews. Three mutations in the gene encoding the noglycans in early amniotic fluid can be used to a subunit of hexosaminidase account for over 90% of the mutant alleles in this group.2' 22 diagnose the mucopolysaccharidoses. Application of this information was possible when the parents of an affected child were both found to carry the same one of these mutations, DNA analysis It is possible to investigate the molecular basis a four base pair insertion. This mutation can be of over 250 different genetic diseases using readily detected if the sequence containing it is recombinant DNA techniques. The application amplified by the polymerase chain reaction and of these techniques to prenatal diagnosis is then digested with a restriction enzyme because advancing rapidly. This is illustrated very one of the resultant fragments is four bases lonclearly by the progress made on the prenatal ger from the mutant allele than from the normal diagnosis of cystic fibrosis in the period of just allele. A subsequent pregnancy was tested for over a year since the defective gene was first dis- this mutation by analysis of DNA from chorcovered. Among enzyme defects DNA technol- ionic villi. The fetus was found to be heterozyogy has found greatest application so far for gous and this diagnosis was confirmed by phenylketonuria and closely related disorders. enzyme assay both prenatally and postnatally.23 DNA analysis may be useful, even when a This is because of the relatively high incidence of the disease in white people and the previous disorder can be diagnosed by direct enzymic lack of a reliable method for prenatal diagnosis. analysis, if only a very small sample is available At the same time as providing a reliable prenatal for analysis. Identification of a particular mutadiagnosis for the first time for some cases, tion in an affected fetus may have prognostic however, DNA methods have confirmed the value for other affected members of the family. About 20 lysosomal enzymes have now been great heterogeneity in this disorder.'5 At least 18 mutations have been reported in the phenyla- cloned and molecular genetical analysis of cases lanine hydroxylase gene (the enzyme affected in of the different lysosomal storage diseases has revealed tremendous heterogeneity. Therefore phenylketonuria). 6 Several restriction fragment length polymor- it seems that each kindred or subpopulation will phisms (RFLPs) have been found in the region require specific DNA tests for a single or small of the phenylalanine hydroxylase gene. Cur- number of mutations. It will be interesting to rently haplotypes are defined by the pattern of see what ingenious strategies are devised to cope alleles at eight sites produced with seven restric- with this problem. tion endonucleases. 17 There are appreciable ethnic differences in the prevalence of these haplotypes. Although certain mutations have Fetal celis in maternal circulation been found to occur predominantly in particular Another source of fetal material for analysis of haplotypes, as yet no mutation has been shown enzymic defects may be fetal cells in the materto be exclusively linked to a specific haplotype. nal circulation. It has been claimed that lymThese polymorphisms may be informative phocytes from a male fetus can be detected in within a family with phenylketonuria, however, the maternal circulation using flow cytometry and prenatal diagnosis has been carried out on and antibodies against paternal antigens not present in the mother24 and that fetal metathis basis.'8 19 It is now possible to detect known mutations phases can be detected in maternal peripheral using allele specific oligonucleotides. Alterna- blood.25 However, other groups have not been tively the polymerase chain reaction can be used able to confirm these observations.26 27 to amplify the sequence in the genomic DNA Recently the polymerase chain reaction has containing the mutations, which can be been used to demonstrate Y chromosome specidetected by sequencing, restriction enzyme fic sequences in the blood of women carrying cleavage, chemical mismatch analysis, or selec- male fetuses.28 This technique, which requires tive hybridisation with oligonucleotides.20 Thus stringent control of conditions to avoid contamiin a family with phenylketonuria if the mutation nation, is now being evaluated in other laborin the affected child has been established, it atories. If the detection or even isolation of fetal cells should be possible to monitor a subsequent pregnancy by analysis of DNA from chorionic villi from the maternal circulation can be established for the specific mutation. If the mutation has reliably, it will provide a non-invasive method not been established, at least two options are for prenatal diagnosis of genetic defects with available. Known mutations could be screened essentiaully no risk to the fetus. The sex of the for in fetal DNA, taking into account ethnic fetus could be investigated using Y chromosome prevalences but there is a risk of not detecting a specific probes and the polymerase chain reac-
Prenatal diagnosu of enyme defects-an update
453
tion or perhaps in situ hybridisation for X enzymes of clinical relevance, for example, hexlinked disorders. Theoretically, it would be pos- osaminidase have also been found to be low at sible to detect specific mutations in an enzyme this stage of development. DNA analysis may be possible for specific gene using the polymerase chain reaction or to measure enzynic activity directly in a very human mutations after amplification of the small number of isolated fetal cells. DNA in the blastomere (approximately 6 pg of Of related interest is the observed increased DNA); P thalassaemia can be diagnosed by activity of some lysosomal enzymes in the serum amplification of DNA from a single blastomere of pregnant women compared with the activity from a mouse preimplantation embryo.34 in the same women when not pregnant-for An exciting new extension of this procedure example, iduronate sulphate sulphatase (the to unfertilised human oocytes and to the first enzyme defective in Hunter's disease). If a fetus polar bodies isolated from them offers the possiis affected with Hunter's disease, however, no bility of detection of defects before fertilisation, increase in this enzyme is found in the maternal with the benefit of not testing the embryo or serum, whereas with a normal or heterozygous fetus at any stage.35 Preliminary results have fetus the rise is observed. The origin of this shown that it is possible to amplify the DNA increased activity needs further investigation as sequence containing the sickle cell mutation site does the whole subject of fetal material in the from human oocytes and polar bodies. Further maternal circulation. work is necessary to show whether both sequences are amplified in a heterozygote.
Testing of preimplantation embryos Another way of avoiding the development of a fetus carrying a genetic defect is to diagnose the defect in preimplantation embryos and only transfer unaffected embryos to the uterus.29 Recently important ethical and practical steps have been taken towards making this a feasible proposition for some genetic defects. The acceptance by the British parliament of a clause in the Human Fertilization and Embryology Bill permitting carefully regulated research on embryos up to 14 days from conception has removed the legal constraint. It is now clear that it is technically possible to remove one or two cells for analysis from a human embryo obtained by in vitro fertilisation or uterine lavage without affecting the development of the fetus.30 31 Therefore if the microanalytical techniques developed for other small fetal samples can be applied to this material it will be possible to detect genetic defects at the levels of the chromosome, DNA, or gene product-that is, enzyme or specific protein in preimplantation embryos. To date this strategy has been used successfully for X linked disorders by sexing embryos using Y specific DNA amplification and only transferring female embryos.32 Two couples at risk for adrenoleukodystrophy and X linked mental retardation have given birth to female dizygotic twins after sexing of in vitro fertilised preimplantation embryos and transfer of female embryos, although one baby was stillborn. At least three other pregnancies have been monitored after this form of prenatal diagnosis. The X linked enzymopathy, Lesch-Nyhan syndrome, resulting from a deficiency of hypoxanthine/guanine phosphoribosyl transferase, is a candidate for this approach to prenatal diagnosis. The activity of this enzyme can be measured in cultures derived from a single blastomere from a mouse preimplantation embryo by a very sensitive assay using a radiolabelled substrate.33 Unfortunately the enzyme does not appear to be expressed at a sufficiently high level in corresponding cultures of human blastomeres to distinguish carried over maternal activity from fetal activity,' thereby excluding prenatal diagnosis by enzyme assay on preimplantation embryos. The levels of several other
Fetal treatment of enzymic defects For a small number of enzyme defects treatment in utero is an alternative to prenatal diagnosis and the option of termination.36 This may take the form of vitamin supplementation for disorders responsive to additional cofactor, for example, cobalamin E disease, dietary control as in phenylketonuria, or drug administration to suppress a harmful secondary metabolic activity resulting from the primary enzymic defect, for example, the suppression by glucocorticoid of adrenal production of adrenogenic steroids in adrenal hyperplasia (due to a deficiency of steroid 21-hydroxylase). In theory the earlier in pregnancy the diagnosis is made and treatment is started, the more effective it should be. The reliable diagnosis of adrenal hyperplasia in the first trimester by DNA analysis permits initiation of treatment at about 9-10 weeks before masculinisation of the genitalia begins. A recent survey of 15 female infants with congenital adrenal hyperplasia who had been treated prenatally from the first trimester, however, shows that only five had normal genitalia.37 Many factors affect the efficacy of the administered hormone in the fetus and close monitoring of the pregnancy is necessary to avoid complications. These observations emphasise the need for more detailed knowledge of the biochemical events in fetal development to formulate rational chemical strategies for fetal treatment. Addendum to list' of enzyme defects that have been diagnosed prenatally Considerable progress has been made in clarifying the defects in peroxisomal disorders and in developing methods for their prenatal diagnosis in the first trimester.38 3 Prenatal diagnosis has been carried out for non-ketotic hyperglycinaemia (McKusick No 23830) in chorionic villi by enzymatic analysis of the glycine cleavage system4 and for Canavan's disease (McKusick No 27190) (R Matalon, personal communication). A pregnancy at risk for the new inborn error of metabolism due to a deficiency of aromatic L-amino acid decarboxylase (EC 4.1.1.28)4' has been monitored by analysis
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of fetal liver.42 Two disorders where the methodology for prenatal diagnosis would appear to be available are the defect of mitochondrial fatty acid , oxidation due to a deficiency of long chain 3-hydroxyacyl-CoA dehydrogenase43 and atypical phenylketonuria due to a deficiency of glutamyl transpeptidase cyclohydrolase I (McKusick No 23391)." 1 Winchester B. Prenatal diagnosis of enzyme defects. Arch Dis Child 1990;65:59-67. 2 Shin YS, Endres W. First trimester diagnosis of inherited metabolic diseases by biochemical methods. Proceedings of the 5th Interna al Congress on Early Fetal Diagnosts. Prague, July 1990:251. 3 Fowler B, Giles L, Cooper A, SardharwaUla IB. Chorionic viUus sampling: diagnostic uses and limitations of enzyme assays. J Inherited Metab Dis 1989;12 (suppl 1):105-17. 4 Besley GTN, Broadhead DM. Prenatal diagnosis of inherited metabolic disease by chorionic villus analysis: the Edinburgh experience. J Inherited Metab Dis 1989;12 (suppl 2): 263-6. 5 Rhoads GG, Jackson LG, Schlesselman SE, et al. The safety and efficacy of chorionic villus sampling for early prenatal diagnosis of cytogenetic abnormalities. N EnglJ Med 1989; 320:609-17. 6 Canadian Collaborative CVS-Amniocentesis Clinical Trial Group. Multicentre randomized clinical trial of chorion viUus sampling and amniocentesis. First report. Lancet
1989;i:1-6.
7 Goldberg JD, Porter AE, Golbus MS. Current assessment of fetal losses as a direct consequence of chorionic vilus sampling. Am J Med Genet 1990;35:174-7. 8 Jackson L. Transcervical and transabdominal chorionic vilus sampling are comparably safe procedures for first trimester prenatal diagnosis: preliminary analysis. Am Jf Hum Genet 1990;47 (suppl):278. 9 Sidransky E, Black SH, Soenksen DM, Jones SL, Dorfmann AD, Schulman JD. Transvaginal chorionic viUus sampling. Prenat Diagn 1990;10:583-6. 10 Chadefaux B, Rabier D, Bonnefont JP, et al. Prenatal diagnosis of propionic acidaemia: amniocentesis at the 11th week of pregnancy. Prenat Diagn 1989;9:448. 11 Jakobs C, Stellard F, Kvittingen EA, Henderson M, Lilford R. First trimester prenatal diagnosis of tyrosinaemia type I by amniotic fluid succinylacetone determination. Prenat Diagn 1990;10:133-4. 12 Chadefaux B, Ceballos I, Rabier M, et al. Prenatal diagnosis of argininosuccinic aciduria by assay of argininosuccinate in amniotic fluid at the 12th week of gestation. Am 7 Med Genet 1990;35:594. 13 Jakobs C, Ten Brink HJ, Stellard F. Prenatal diagnosis of inherited metabolic disorders by quantitation of characteristic metabolites in amniotic fluid: facts and future. Prenat Diagn 1990;10:265-71. 14 Besley GTN, Broadhead DM, Nevin NC, Nevin J, Dorman JC. Prenatal diagnosis of mucolipidosis II by early amniocentesis. Lancet 1990;335:1164-5. 15 Cotton RGH. Heterogeneity of phenylketonuria at the clinical, protein and DNA levels. J Inherited Metab Dis
1990;13:739-50. phenylketonuria. Biochemisty 1989;28:1-6. 17 Chakraborty R, Lidsky AS, Daiger SP, et al. Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria. Hum Genet 1987;76:40-6. 18 Lidsky AS, Guittler F, Woo SLC. Prenatal diagnosis of cassic phenylketonuria by DNA analysis. Lancet 1985i:549-51. 19 Speer A, Bellman F, Michel A, et al. Prenatal diagnosis of classical phenylketonuria by linked restriction fragment
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reaction to the diagnosis of human genetic disease. Hum Genet 1990;85:1-8. 21 Paw BH, Phuong TT, Kaback M, Lim J, Neufeld EF. Frequency of three hex A mutant alleles among Jewish and non-Jewish carriers identified in a Tay-Sachs screening program. Am J Hum Genet 1990;47:698-705. 22 Triggs-Raine BL, Feigenbaum ASJ, Natowicz M, et al. Screening for carriers of Tay-Sachs disease among Ashkenazi Jews. N Engi J Med 1990;323:6-12. 23 Triggs-Raine BL, Archibald A, Gravel RA, Clarke JTR. Prenatal exclusion of Tay-Sachs disease by DNA analysis. Lancet 1990;335:1164. 24 Herzenberg LA, Bianchi DW, Schroder J, et al. Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. Proc Nad Acad Sci USA 1979;76:1453-5. 25 Selypes A, Lorencz R. A non-invasive method for determination of the sex and karyotype of the fetus from the maternal blood. Hun Genet 1988;79:357-9. 26 Tharapel A, Jaswaney V, Dockder M, et al. Can fetal cells in maternal blood be selected through cytogenetic means? Am J Hum Geset 1989;45:A271. 27 YouseffM, Shulman LP, Tharapel AT, Simpson JL, Elias S. Failure to document fetal cells in maternal circulation using the Selypes-Lorencz 'air culture' cytogenetic technique. Hum Genet 1990;85:133-4. 28 Lo Y-MD, Wainscoat JS, Gillmer MDG, Patel P, Sampietro M, Fleming KA. Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancet 1989; ii:1363-5. 29 Penketh R, McLaren A. Prospects for prenatal diagnosis during preimplantation human development. In: Rodeck C, ed. Clinical obstetrics and gynaecology. London: Bailliere Tindall, 1987:747-4. 30 Hardy K, Martin KL, Leese HJ, et al. Human preimplantation development in vitro is not adversely affected by biopsy at 8-cell stage. Human Reproduction 1990;5:708-14. 31 Buster JE, Carson SA. Genetic diagnosis of the preimplantation embryo. Am J Med Genet 1989;34:211-6. 32 Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344:768-70. 33 Monk M, Handyside A, Hardy K, Whittingham D. Preimplantation diagnosis of deficiency of hypoxanthine phosphoribosyl transferase in a mouse model for LeschNyhan syndrome. Lancet 1987;ii:423-5. 34 Holding C, Monk M. Diagnosis of ,-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos. Lancet 1989;i:532-5. 35 Monk M, Holding C. Amplification of ,B-haemoglobin sequence in individual human oocytes and polar bodies. Lancet 1990;335:985-8. 36 Schulman JD. Treatment of the embryo and the fetus in the first trimester. Am J Med Genet 1990;35:197-200. 37 Pang S, Pollack MS, Marshall RN, Immken L. Prenatal treatment of congenital adrenal hyperplasia due to 21hydroxylase deficiency. N Engl J Med 1990;322:111-5. 38 Schutgens RBH, Schrakamp G, Wanders RJA, Heymans HSA, Tager JM, Van den Bosch H. Prenatal and perinatal diagnosis of peroxisomal disorders. J Inherited Metab Dis 1989;12 (suppl 1):118-34. 39 Wanders RJA, Van Roermund CWT, Schutgens RBH, et al. The inborn errors of peroxisomal ,B-oxidation: a review. J Inherited Metab Dis 1990;13:4-36. 40 Hayasaka K, Tada K, Fueki N, Aikawa J. Prenatal diagnosis of nonketotic hyperglycinemia: enzymatic analysis of the glycine cleavage system in chorionic villi. J Pediatr 1990;
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43 Wanders RJA, Ijlst L, Van Gennip AH, et al. Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: identification of a new inborn error of mitochondrial fatty acid ,Boxidation. J Inherited Metab Dis 1990;13:311-4. 44 Schoedon G, Redweik U, Curtius H-C. Purification of GTP cyclohydrolase I from human liver and production of specific monoclonal antibodies. Eur J Biochem 1989;178: 627-34.
