Prenatal diagnosis of citrullinaemia: Review of a 10-year experience including recent use of DNA analysis

Prenatal diagnosis of citrullinaemia has been accomplished by three different methods to date: (1) enzyme assay of cultured fetal cells; (2) quantification of citrullirie in amniotic fluid supernatant; and (3) incorporation of [¹⁴C]citrulline into protein by cultured fetal cells. Our laboratory has used these methods to perform prenatal diagnosis for 28 fetuses over a 10-year period. More recently, DNA polymorphisms were used for prenatal diagnosis by linkage analysis. Of the 28 fetuses studied, 23 were predicted to be unaffected, four were predicted to be affected, and results were conflicting in one case where [¹⁴C]citrulline incorporation erroneously indicated an affected fetus but linkage analysis correctly predicted an unaffected fetus. Because of low levels of enzyme activity in heterozygotes and in certain amniotic fluid cell types, biochemical diagnosis of citrullinaemia is complicated by the risk of false affected results, although [¹⁴C]citrulline incorporation is relatively reliable. When informative, linkage analysis is the preferable method for cases with a 25 per cent risk. The risk of false affected results makes prenatal diagnosis for cases with less than 25 per cent risk of questionable value.     

Prenatal diagnosis of medium-chain acyl-coenzyme A dehydrogenase deficiency

A fatal case of medium-chain acyl-coenzyme A dehydrogenase deficiency is described in a patient who presented with hypoglycaemia and a gross non-ketotic dicarboxylic aciduria. Cultured skin fibroblasts released ¹⁴CO₂ from [1–¹⁴C] octanoic acid at half the normal rate. Prenatal diagnosis was undertaken in a subsequent pregnancy in which cultured amniotic fluid cells revealed a marked reduction in octanoate oxidation indicative of an affected fetus. The pregnancy was terminated and the diagnosis was confirmed by enzyme analysis of skin fibroblasts taken from the fetus. The high residual octanoate oxidation by affected fibroblasts together with the absence of any characteristic abnormality of amniotic fluid organic acids are a potential limitation to the reliability of this type of prenatal diagnosis.     

Prenatal diagnosis of the hurler syndrome: Report on 40 pregnancies at risk

In 40 pregnancies at risk for the Hurler syndrome 13 affected fetuses were detected by the demonstration of an α-L -iduronidase deficiency and an increased level of ³⁵S-sulphate incorporation. The diagnoses were confirmed by the analysis of fetal tissues and/or cultured fetal skin fibroblasts. Microassays for α-L -iduronidase, using phenyl α-L -iduronide and more recently 4–methyl-umbelliferyl α-L -iduronide, enabled a reliable diagnosis to be made within 15 to 18 days after amniocentesis. ³⁵S-sulphate incorporation has been a valuable adjunct in cases with a low (heterozygote) enzyme activity.     

First report of prenatal diagnosis of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency in a pregnancy at risk

Prenatal diagnosis of long-chain 3-hydroxyacyl-CoA dehydrogenase (3-HAD) deficiency was performed in a family at risk. The diagnosis of an affected fetus was carried out by enzyme assay in cultured chorionic villus cells.     

Prenatal diagnosis of isovaleric acidaemia by enzyme and metabolite assay in the first and second trimesters

Isovaleric acidaemia (IVA) is caused by a deficiency of isovaleryl CoA dehydrogenase. The diagnosis can be established biochemically by the demonstration of increased levels of isovalerylglycine (IVG) and 3-hydroxyisovaleric acid in urine and by the deficiency of incorporation of radiolabel from [¹⁴C]isovaleric acid in macromolecules in cultured fibroblasts. This paper reports a consecutive series of 24 prenatal diagnoses in pregnancies at high risk, using both methods-metabolite and indirect enzyme assay. Affected fetuses were diagnosed in four pregnancies: three in the second trimester and one recent case in the first trimester. The latter represents the first reported case of a first-trimester diagnosis of IVA by direct analysis of chorionic villi. We also report the first demonstration of strongly accumulated IVG in the amniotic fluid in the 12th week of an affected pregnancy.     

Prenatal diagnosis of congenital enzymopenic methaemoglobinaemia with mental retardation due to generalized cytochrome b5 reductase deficiency: First report of two cases

Prenatal diagnosis of congenital enzymopenic methaemoglobinaemia (CEM) with mental retardation was performed in two fetuses at risk for generalized NADH-cytochrome b₅ reductase deficiency. In the first case the enzyme activity of cultured amniotic cells was in the heterozygous to normal range. The mother delivered a normal baby with normal enzyme activity in cord blood cells. In the second case, the amniotic cells were almost completely enzyme deficient. The pregnancy was terminated, and the diagnosis of homozygous NADH-cytochrome b₅ reductase deficiency was confirmed in cord blood cells, in several different tissues and in cultured fibroblasts from the aborted fetus.     

Prenatal tests for Sanfilippo disease type B in four pregnancies

We report the prenatal diagnosis of two fetuses with Sanfilippo disease type B. In both pregnancies there were excessive amounts of heparan sulphate in amniotic fluid and the activity of N-acetyl-α-D-glucosaminidase was undetectable in cultured amniotic fluid cells. The predictions were confirmed by enzyme assay of cultured skin fibroblasts from the aborted fetus or the affected infant. The disorder was excluded for two other pregnancies at risk and the predictions are considered to be correct because of the normal progress of the healthy children.     

First trimester monitoring of a pregnancy at risk for glucose phosphate isomerase deficiency

First trimester prenatal diagnosis was offered to the mother of a child affected by severe haemolytic anaemia due to glucose phosphate isomerase (GPI) deficiency. The mutant enzyme was characterized by an increased thermal lability. Both parents had 50 per cent normal red cell GPI activity. We have shown that the homozygous and heterozygous genotypes can be clearly distinguished from each other and controls by combinations of the measurement of enzyme activity and enzyme thermal lability. Examination of trophoblast cells obtained at 9 weeks of gestation led to the diagnosis of a GPI heterozygous fetus. The result was confirmed by analysis on uncultured and cultured amniotic fluid cells sampled at 16 weeks and by red blood cell studies of the healthy newborn. Prenatal diagnosis of GPI deficiency is indicated in families with previous cases resulting in severe haemolysis and mainly with the conservative view of arranging appropriate therapeutic measures for affected fetuses.     

Prenatal diagnosis of Lesch-Nyhan syndrome: Experience with three fetuses at risk

Our experience with the prenatal detection of the Lesch-Nyhan syndrome (LNS; hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency) in three fetuses at risk is reported. Enzyme activities were measured in cultured amniocytes in two pregnancies, and in tissues and cultures obtained from chorionic villus sampling (CVS) in a third pregnancy. In all tissues the specific activities of HGPRT and adenine phosphoribosyltransferase (APRT) were determined and APRT/HGPRT ratios were calculated. In addition to the enzyme assays, the rate of purine synthesis de novo was assessed in the two amniocyte cultures, and the rate of [¹⁴C]hypoxanthine incorporation into nucleotides and sensitivity to azaguanine were measured in one of the amniocyte cultures. We report the diagnosis of normal fetuses by study of amniocytes in two pregnancies and of LNS using CVS in one pregnancy. In all three cases the diagnosis was confirmed.     

Prenatal diagnosis of glutaryl-CoA dehydrogenase deficiency: Experience using first-trimester chorionic villus sampling

We have performed prenatal diagnosis for glutaryl-CoA dehydrogenase (GDH) deficiency in 16 pregnancies at risk by measuring the enzyme activity in chorionic villus samples. In most cases, GDH activity was measured both in uncultured chorionic villus samples and in cultured chorionic cells. In 4 of the 16 cases, an affected fetus was predicted, while the remaining cases were found to be normal. In three of the four affected cases, GDH activity was measured in both uncultured and cultured chorionic cells and the correct diagnosis established by both measurements. In the fourth case, only cultured cells were investigated because the chorionic villus sample was too small for the direct assay. All four pregnancies predicted to be affected were interrupted and the diagnoses confirmed on the aborted material in three of the cases. In the fourth case, no material was available for investigation. Of the 12 pregnancies predicted to be unaffected, ten cases resulted in the birth of healthy unaffected babies while two pregnancies are still in progress.     

Prenatal diagnosis of citrullinemia: Elevated levels of citrulline in the amniotic fluid in the three affected pregnancies

In three pregnancies at risk for citrullinemia affected fetuses were predicted both by strongly increased levels of citrulline in the amniotic fluid and by the reduced incorporation of ¹⁴C-citrulline into TCA-precipitable material in cultured amniotic fluid cells. The prenatal diagnoses of affected fetuses were confirmed after termination of the pregnancies by direct and indirect assays of argininosuccinate synthetase in the fetal livers and fibroblasts respectively. Measurement of the citrulline concentration in amniotic fluid appears to be a valuable adjunct in the prenatal diagnosis of citrullinemia.     

First trimester diagnosis of methylmalonic aciduria

We have studied methylmalonyl CoA mutase activity in control chorionic villi to establish the potential use of assays performed directly on this tissue for prenatal diagnosis of methylmalonic aciduria. We report the detection of a fetus affected with the apo-mutase deficient form of this condition at 9 weeks' gestation. Methylmalonyl CoA mutase was markedly deficient in chorionic villi, approximately 2–5 per cent of the mean control value. However, incorporation of label from [¹⁴C]-propionate into protein was 10 and 40 per cent of the mean control value, respectively, in two portions of the same biopsy, highlighting potential problems in the use of this indirect assay. Normal results were obtained in chorionic villus samples from four other pregnancies ‘at risk’ for methylmalonic aciduria which were subsequently shown to be unaffected with this condition. The diagnosis in the affected pregnancy was confirmed by demonstration of a marked deficiency of methylmalonyl CoA mutase activity in villi obtained at termination and in cultured fetal fibroblasts. Reduced incorporation of [¹⁴C]-propionate label into protein was also found in these tissues.     

First-trimester prenatal diagnosis of aspartylglucosaminuria

Fetal aspartylglucosaminuria (AGU) was studied during the first trimester of pregnancy in six at-risk pregnancies using chorionic villus samples. The activity of aspartylglucosaminidase (AGA) was high in five cases, indicating an unaffected fetus. This was confirmed through delivery of healthy newborns with a normal pattern of urinary oligosaccharides. Low enzyme activity in an uncultured biopsy specimen and in cultured amniotic fluid cells in one case demonstrated that the fetus was affected. The pregnancy was terminated and the prenatal diagnosis was confirmed by showing reduced AGA activity in cultured fibroblasts of the fetus.     

First-trimester diagnosis of an unusual case of α-mannosidosis

Chorionic villi obtained in the first trimester from a pregnancy at risk for α-mannosidosis were shown to have reduced α-mannosidase (EC 3.2.1.24) activity. The pregnancy was terminated and subsequent enzyme studies of the fetal tissues were consistent with the diagnosis of α-mannosidosis. Like the enzyme in the child's fibroblast, α-mannosidase of the chorionic villi from a pregnancy at risk for α-mannosidosis was activated by high substrate concentration and by Zn^{2 +}, and displayed a K_m value two-fold higher than normal. Our results confirm that chorionic villi can be used for early prenatal diagnosis of α-mannosidosis.     

Prenatal diagnosis of a heterozygote for mucopolysaccharidosis type VII (β-glucuronidase deficiency)

We had the opportunity of investigating a case (BK) of a severe form of mucopolysacchari- dosis with nearly total deficiency of β-glucuronidase in serum, leucocytes and fibroblasts. We here report results obtained by prenatal diagnosis of a clinically normal child (BK's sister), and point out the difficulty in interpreting a heterozygous level of β-glucuronidase activity in cultured amniotic cells. Four successive passages of amniotic cells were tested for β-glucuronidase and α-mannosidase activity in at-risk and control cells. In different passages, enzyme activity was between 8 and 49 per cent of controls but 2 to 18 times higher than fibroblasts from the affected brother (BK). The highest activity was observed in the first passage and the lowest in the third. The electrophoretic separation of GAGS from at-risk amniotic fluid showed a normal pattern. We discuss the correlation between enzyme levels in different passages of cultured cells and that found in leucocytes and fibroblasts from the propositus and parents. From a practical point of view, we conclude that the first passage gives the most reliable results for prenatal diagnosis.     

The cerebro-hepato-renal (Zellweger) syndrome: Prenatal detection based on impaired biosynthesis of plasmalogens

Prenatal diagnosis of the cerebro-hepato-renal (Zellweger) syndrome has been performed in 10 pregnancies at risk by measuring both the activity of acyl CoA: dihydroxyacetonephosphate acyltransferase (DHAP-AT) and the de novo plasmalogen biosynthesis, either in cultured amniotic fluid cells or in fibroblasts cultured from a chorionic villus biopsy. In 7 of the pregnancies both tests indicated no abnormality. All 7 continued to term and normal infants were delivered. However, in amniotic fluid cells from 2 fetuses affected by Zellweger syndrome unequivocal differences from control values were found. The activity of DHAP-AT was clearly deficient and the de novo plasmalogen biosynthesis was impaired. In one pregnancy at risk prenatal diagnosis was performed during the first trimester by measuring both the DHAP-AT activity and the de novo plasmalogen biosynthesis in fibroblasts cultured from a chorionic villi biopsy. From the deficient DHAP-AT activity and the impaired de novo plasmalogen biosynthesis it was concluded that the fetus was affected. This was confirmed biochemically after induced abortion. It can be concluded that measurement of the DHAP-AT activity and the de novo plasmalogen biosynthesis provides convenient methods for the early prenatal detection of Zellweger syndrome.     

Prenatal diagnosis of sanfilippo disease type C using a simple fluorometric enzyme assay

A new fluorogenic substrate, 4-methylumbelliferyl β-D-glucosaminide, was used for the assay of acetyl CoA:glucosaminide N-acetyltransferase in chorionic villi, cultured villus cells, and amniocytes. Optimal conditions for the assay and the ranges of enzyme activity were established for the various types of fetal cells. This simple fluorometric assay provides a reliable method for early prenatal diagnosis of Sanfilippo disease type C which is more convenient than current methods using radiolabelled substrates. The method was applied to amniotic fluid cells and fetal fibroblasts from an at-risk pregnancy in which an affected fetus was diagnosed by two-dimensional electrophoresis of glycosaminoglycans in the amniotic fluid.     

Mucolipidosis IV: Prenatal diagnosis by electron microscopy

Mucolipidosis IV (ML 1V) is a lysosomal storage disease presenting in infancy with cloudy cornea and psychomotor retardation. Our experience with 12 pregnancies at risk for ML IV, monitored by transmission electron microscopy (TEM) studies of cultured amniotic fluid cells, is presented. The prenatal diagnoses were confirmed in the 3 affected and the 8 un- affected pregnancies. In the one pregnancy where no definite diagnosis was reached the pregnancy was terminated. TEM examination of fetal tissues from this pregnancy showed no abnormal lysosomal storage bodies and a review of the cultured amniotic fluid cell sections revealed that the diagnosis of a normal fetus could have been made.     

First-trimester prenatal diagnosis of cystic fibrosis using fibroblasts from a deceased index child to establish haplotypes

First-trimester prenatal diagnosis of cystic fibrosis (CF) using linked DNA markers is usually only possible if there is an index affected child to establish the haplotype of the parental chromosomes. We describe a prenatal diagnosis where fibroblasts, cultured from the skin of a deceased affected child and then held in frozen storage for 3 years, were used as the starting point for tracking the CF gene. The fetus was diagnosed as a homozygous normal and the diagnosis confirmed by immunoreactive trypsin testing after birth. It was also possible to establish heterozygosity in the aunt of the affected child.     

Prenatal diagnosis of mucopolysaccharidosis by two-dimensional electrophoresis of amniotic fluid glycosaminoglycans

Amniotic fluid from 72 pregnancies at risk for mucopolysaccharidosis have been analysed for glycosaminoglycans (GAG) by means of two-dimensional electrophoresis. Definite results were obtained in all cases and required only 3 days to report. Of the 66 cases for which GAG analysis was accompanied by enzyme assays on cultured amniotic fluid cells, there was agreement of results in 65. In the one case of disagreement the result of GAG analysis proved to be correct. Fourteen pregnancies were predicted to be affected and the predictions were confirmed by studies on fetal tissues and/or cultured cells, or in one case the birth of an affected infant. Of the 58 pregnancies predicted to be unaffected, 48 have so far gone to term and produced healthy infants. There have been no false negative predictions. The analytical method is simple, rapid, and, in this study, has been found completely reliable for prenatal diagnosis.