Archives ofDisease in Childhood 1990; 65: 59-67 59 Arch Dis Child: first published as 10.1136/adc.65.1_Spec_No.59 on 1 January 1990. Downloaded from CURRENT TOPIC Prenatal diagnosis of enzyme defects B Winchester About 250 enzyme deficiencies have been the case of X linked disorders, a woman who demonstrated in the 750 or so recognised has an affected brother or whose sister has given human recessive disorders.' Approximately 100 birth to an affected child may also request of these enzymopathies can be diagnosed pre- testing. The reliability of the test is crucial and natally. Undoubtedly this number will increase these tests should only be carried out in as more methods are developed for the detection experienced laboratories. The precise diagnosis of enzymic defects in fetal samples (particularly of the enzymic defect in the index case is a in chorionic villus samples obtained in the first prerequisite for undertaking such investigations. trimester). There are two main limitations to Because it is selective, prenatal diagnosis of the application of the enzyme assay procedures enzymic defects is a highly effective preventative used in postnatal diagnosis to fetal samples. measure. Experience from our laboratory (un- Firstly the amount of material available for published observations) and others,2-4 shows investigation is very small. Therefore the that 18-25% of the fetuses tested are affected enzymic assay procedure has to be very sensitive, with the suspected defect, very close to the which may necessitate modification of postnatal expected 25%. If a pregnancy is terminated as a methods. Secondly the enzyme under investi- result of testing, it is important to confirm the gation may not be expressed in fetal samples diagnosis on the aborted fetus to assess the that are readily accessible, for example, chorionic efficiency of the diagnostic procedure and to villi or cultured amniotic cells, because it is reassure the parents. If possible a cell culture tissue specific. Although sampling of some should be established and specimens preserved specialised tissues is possible, the enzyme may from the fetus for future reference. Similarly still not be expressed at a sufficiently early stage prediction of an unaffected fetus can be con- of gestation to allow diagnosis and termination. firmed postnatally by biochemical tests but this These problems can, in principle, be overcome is usually only carried out now at the parents' by the use of recombinant DNA technology, request or if there is any concern over the health which detects or tracks mutations in genomic of the baby. http://adc.bmj.com/ DNA. As this does not require expression of the enzyme protein, it can be applied to any nucleated cell. Currently, however, DNA probes Molecular basis and heterogeneity of enzymic are being used for the prenatal diagnosis of a defects very small number of enzymic defects (see A complex sequence of events takes place before appendix) and modifications of conventional an enzyme can function correctly in vivo. The The assay procedures are used predominantly. gene encoding the enzyme has to be transcribed on September 24, 2021 by guest. Protected copyright. expansion of DNA analysis depends upon in a controlled manner and the RNA transcript progress in cloning genes encoding enzymes and edited before translation into a precursor in elucidation of the molecular genetical basis of enzymic protein. Subsequently most newly enzymopathies. This article will review the synthesised proteins undergo some form of range of enzymic defects that can be detected post-translational modification or maturation prenatally and discuss how the method of before or during transport to a specific intracel- detection depends upon the molecular basis of lular or extracellular site of action. The mature the defect, the tissue and subcellular site of enzymic protein may require a cofactor or action of the enzyme, and the nature of the fetal association with one or more proteins, encoded biopsy available. by other genes, to be fully active. The complexity of the biosynthesis and intracellular localisation of an enzyme affords many opportunities for Indications for prenatal diagnosis of an genetic errors. Accordingly an enzyme deficiency enzyme defect leading to a characteristic pathological pheno- Prenatal diagnosis of an enzymic defect is type may be due to several different underlying Institute of Child Health, generally only carried out for pregnancies known molecular defects. In many disorders there is London to be at high risk for a severe disorder for which residual enzymic activity, the amount of which Correspondence to: there is no effective treatment. Most requests may be related to the age of onset or severity of Dr B Winchester, Department of Ciinical for prenatal monitoring of a pregnancy will be the disease. It is generally possible to differen- Biochemistry, Institute from parents who have previously had a child tiate prenatally among the variant forms of a of Child Health, 30 Guilford Street, with an enzyme deficiency and for whom disease due to different mutations if the mole- London WC1N IEH. termination of the pregnancy is acceptable. In cular basis of the defect has been clearly 60 Winchester established in the index case. However this will Fresh intact chorionic villi can also be used Arch Dis Child: first published as 10.1136/adc.65.1_Spec_No.59 on 1 January 1990. Downloaded from often involve a more elaborate detection proce- to study enzymic activities by the uptake and dure than a simple assay of enzymic activity in a utilisation of radioactively labelled metabolic crude tissue homogenate. precursors and for the extraction of DNA for diagnosis using recombinant DNA techniques (see below). For pregnancies at high risk Fetal sampling chorionic villus sampling has become the The demonstration ofan enzymic defect requires method of choice because most physicians and removal offetal material for biochemical analysis. parents consider the advantage of early diag- This is carried out under the guidance of nosis to compensate for the increased risk. ultrasonography or by fetoscopy. Transabdominal removal of chorionic villi from the placenta has been successfully carried out in the second and third trimester for diagnosis of CHORIONIC VILLUS SAMPLING genetic abnormalities by direct karyotyping Chorionic villus sampling without ultrasound without cell culture.15 Such samples could also was first used in China in 1975 for diagnostic be used for enzymic assays. fetal sexing,5 but the first measurement of enzymic activities in chorionic villi was in 1982.6 Currently chorionic villus sampling is AMNIOCENTESIS carried out with the assistance of ultrasono- Enzymic defects can be investigated both in graphy by transcervical aspiration7 or biopsy amniotic fluid by detection of specific metabo- with forceps8 between eight and 12 weeks' lites and in cultured amniotic fluid cells by gestation but optimally between nine and 10 enzyme assay or incorporation studies. Amnio- weeks. Transabdominal aspiration is an alterna- centesis under the guidance of ultrasonography tive that decreases the risk of infection and is a well established and reliable procedure with permits sampling between 12 and 14 weeks a risk of miscarriage of about 0 5% in centres obviating waiting for amniocentesis at 16 weeks with experienced staff."6 A sample of 10-20 ml or later. The risk to pregnancy of chorionic of amniotic fluid is removed by transabdominal villus sampling is 2-4%, about 2% higher than puncture at 15-16 weeks' gestation, the optimal that for spontaneous abortion and about twice time with regard to cell number and viability. that for amniocentesis.9" As more centres gain The amniotic fluid and cells are separated and a experience of chorionic villus sampling this risk culture initiated of the cells. Amniotic cells may decrease. Over the last three years cannot be used directly because many are non- chorionic villus sampling has become more viable and in various states of decay. It can take widespread and popular because of the advan- up to six weeks to obtain sufficient viable cells tage of early diagnosis. This has stimulated the for some enzyme determinations but three to development of suitable enzyme assay proce- four weeks is more usual. Experience of cell dures. A major problem is the contamination culture, especially of amniotic cells, is necessary of the chorionic villi biopsy specimen with to assess the state of readiness of the cultures for maternal material. The sample should be biochemical analysis. 17 Appropriate controls examined under a low power dissecting micro- must always be available and analysed at the http://adc.bmj.com/ scope and maternal material, mainly decidua, same time. A wide range of enzymic defects has removed. A typical sample of 10-40 mg wet been diagnosed by analysis of amniotic material. weight will provide enough material for the Although most of these disorders can now be assay of the enzyme under investigation and a diagnosed on chorionic villus samples, amnio- reference enzyme and for the measurement of centesis will still be widely used until more protein. All the enzymic defects that have been centres gain experience of chorionic villus detected in cultured amniotic fluid cells can be sampling, for late presentations and because of on September 24, 2021 by guest. Protected copyright. detected in chorionic villi cells. Most enzymes the lower risk associated. In some situations a can be assayed directly in a homogenate of the diagnosis made in the first trimester by chorionic biopsy specimen enabling a diagnosis to be villus sampling, particularly of an unaffected made within one to three days of sampling. For fetus, is confirmed by amniocentesis. DNA can a few enzymes, whose activity is very low in be extracted from cultured amniotic fluid cells normal chorionic villi, it is necessary to obtain a for analysis. culture of cells from the sample,2 3 12 thereby delaying the result for two to three weeks.