DNA-based prenatal diagnosis for severe and variant forms of multiple acyl-CoA dehydrogenation deficiency

OBJECTIVES: Multiple acyl-CoA dehydrogenation deficiency (MADD) is a clinically heterogeneous disorder of mitochondrial fatty acid, amino acid, and choline oxidation due to mutations in the genes encoding electron transfer flavoprotein (ETF) or ETF ubiquinone oxidoreductase (ETFQO). So far, prenatal diagnosis of MADD has relied mostly on second-trimester biochemical analyses of amniotic fluid or cultured amniocytes. We report here on an alternative DNA-based approach for prenatal diagnosis in pregnancies at risk of MADD. METHODS: We used our knowledge of the mutational status in three unrelated families with a history of MADD to perform direct sequencing for the familial mutations using genomic DNA isolated from chorionic villus samples (CVS) at gestational week 10 to 11. RESULTS: Within two days, we were able to carry out accurate DNA-based prenatal testing in one pregnancy at risk of severe MADD, and in two pregnancies at risk of variant forms of MADD. CONCLUSION: This is the first report of DNA-based prenatal diagnosis of MADD. Our molecular approach is suitable for fast and reliable first-trimester prenatal diagnosis in pregnancies at risk of severe and variant forms of MADD.     

First prenatal molecular diagnosis in a family with holocarboxylase synthetase deficiency

OBJECTIVES: We report on the first prenatal molecular diagnosis of holocarboxylase synthetase (HLCS) deficiency in the fourth pregnancy of an at-risk family. This disorder is a rare autosomal recessive inborn error of metabolism, leading to a multiple carboxylase defect (MCD). HLCSD diagnosis was performed postmortem in the proband on DNA from autoptic biological material. Molecular analysis of the proband's entire HLCS gene by direct sequencing identified the R508W amino acid change, at the homozygous status. METHODS: Fetal DNA was isolated from chorionic villus sampling at 11 weeks of gestation. Direct sequencing of exon 6 of the fetal HLCS gene was performed. RESULTS: The R508W mutation was identified in the fetal DNA at the homozygous level. The genetic lesion was confirmed on abortive tissue. CONCLUSION: Molecular diagnosis has several advantages over enzymatic activity assay of carboxylases in chorionic villi or amniocytes. It can be performed earlier, is faster, and the response time is shorter.     

Mucolipidosis III type C: first-trimester biochemical and molecular prenatal diagnosis

OBJECTIVES: Mucolipidosis IIIC (MLIIIC) is a rare autosomal recessive lysosomal storage disease resulting from defective mannose 6-phosphate-dependent lysosomal enzyme trafficking; mutations of the gamma subunit of N-acetylglucosamine-1 phosphotransferase (GINAcPT) were recently found to cause its pathogenesis. We report here for the first time prenatal diagnosis (PND) for MLIIIC by means of chorionic villous sampling (CVS). METHODS AND RESULTS: A fetus in a large Bedouin-Moslem family was found to be homozygous for the founder haplotype and the mutational SSCP pattern of MLIIIC. The diagnosis was confirmed by markedly reduced lysosomal enzyme activities in cultured chorionic villi. The molecular identification of the disease-causing mutation in this large Bedouin-Moslem kindred permitted, for the first time, identification of carriers and couples at risk. CONCLUSIONS: The feasibility of early PND for a progressive disabling disease is important for its prevention. Nevertheless, the feasibility of PND raises a serious dilemma since affected individuals might have a variable phenotype and the disease is progressive and non-lethal. In addition, religious and social constraints are important factors to be taken into consideration in the genetic counseling of couples at risk.     

DNA based prenatal testing for the skin blistering disorder epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a skin fragility disorder in which mild physical trauma leads to blistering. The phenotype of the disorder is variable, from relatively mild affecting only the hands and/or feet, to very severe with widespread blistering. For the severest forms of EBS there is a demand for prenatal diagnosis which until now has involved a fetal skin biopsy in the second trimester. The identification of mutations in the genes encoding keratins K5 and K14 as the cause of EBS opens up the possibility of much earlier diagnosis of the disease. We report here four cases in which prenatal testing was performed. In three of the cases the genetic lesions were unknown at the start of the pregnancy, requiring the identification of the causative mutation prior to testing fetal DNA. In two of the four cases novel mutations were identified in K14 and in the two remaining families, a previously identified type of mutation was found. Fetal DNA, obtained by chorionic villus sampling or amniocentesis, was analysed for the identified mutations. Three of the DNA samples were found to be normal; a mutant K14 allele was identified in the fourth case and the pregnancy was terminated. These results demonstrate the feasibility of DNA-based prenatal testing for EBS in families where causative mutations can be found.     

Fetal muscle biopsy as a diagnostic tool in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a relentless progressive disorder, leading to severe disability during childhood and death in adolescence or early adulthood. In most families, prenatal diagnosis is readily achieved by molecular detection of DNA deletions using chorionic villi or amniocytes, or by linkage analysis. In some cases, however, molecular methods fail to provide a definitive diagnosis and in such cases in utero fetal muscle biopsy may serve as a diagnostic option. We describe three families in whom fetal muscle biopsy was performed, focusing on the prenatal diagnostic dilemmas, the indications and timing for in utero fetal muscle biopsy, and the difficulties encountered.     

Genetic approach to prenatal diagnosis in urea cycle defects

OBJECTIVE: To demonstrate the feasibility of prenatal diagnosis by molecular genetics in all urea cycle defects in order to improve and standardize the current approaches. METHODS: Deceased index patients who had suffered from a urea cycle disorder were investigated for mutations of the biochemically most likely affected gene. If no material of index patients was available, parental DNA was studied for obligate carrier status. Fetal cells of 15 pregnancies, either chorionic villi or amniotic fluid cells, were used for direct sequence analysis of the respective mutations. Thirteen families were investigated, of which two were affected by N-acetylglutamate synthase deficiency, four by carbamoylphosphate synthetase 1 deficiency, one by ornithine transcarbamylase deficiency, three by argininosuccinate synthetase deficiency, two by argininosuccinate lyase deficiency, and one by arginase deficiency. RESULTS: Molecular genetics allowed the determination of the fetal status in all cases. Besides 14 known mutations, we detected the novel mutation c.544delC of the N-acetylglutamate synthase gene, the novel missense mutation c.721G>A (E241K) of the argininosuccinate lyase gene, and the novel double mutated allele comprising the known mutation c.703G>A (G235R) and the novel insertion c.712ins[GGACC](2) (254X) of the arginase 1 gene. CONCLUSION: Direct genetic analysis of chorionic villi or amniotic fluid cells is feasible, fast, and specific, and can be regarded as the method of choice for prenatal diagnosis in urea cycle disorders.     

Prenatal diagnosis of carnitine palmitoyltransferase 2 deficiency in chorionic villi: a novel approach

Carnitine palmitoyltransferase 2 (CPT2) deficiency, the most common autosomal recessive inherited disease of the mitochondrial long-chain fatty acid (LCFA) beta-oxidation, may result in three distinct clinical phenotypes, namely, a mild adult muscular form, a severe infantile hepatocardiomuscular disease, and a neonatal form, which includes dysmorphic features in addition to hepatocardiomuscular symptoms. Both the latter forms are life-threatening diseases, and prenatal diagnosis (PND) can be offered to couples at a one-fourth risk of having an affected child. PND of CPT2 deficiency hitherto relied mostly on mutation detection from fresh chorionic villi (10 weeks' gestation), since CPT2 activity could be assayed on cultured amniocytes only (16-17 weeks' gestation).We devised a CPT2 activity assay from 10 mg of chorionic villi sampling (CVS). Combining this enzymatic assay to haplotype study using polymorphic markers linked to the CPT2 gene, we were able to carry out within 2 days, CPT2 deficiency PND, in two unrelated families, using a CVS performed at the 11th week of gestation.     

Prenatal paternity testing with deoxyribonucleic acid techniques

OBJECTIVES: Our purpose was to determine the feasibility and optimal techniques for prenatal paternity testing. STUDY DESIGN: Since January 1989 we have offered prenatal paternity testing by deoxyribonucleic acid testing. We analyzed the ability to complete the testing and the time required to complete the testing and developed polymerase chain reaction - based tests to speed test results. RESULTS: Before April 1990 only five of nine cases could be completed. Since that time 28 consecutive cases were successfully completed before delivery. Introduction of polymerase chain reaction - based testing has allowed us to perform testing on uncultured chorionic villi and to derive results within 3 weeks. CONCLUSION: Analysis of uncultured chorionic villi allows prenatal paternity testing to be completed within the first trimester of pregnancy. Prenatal paternity testing can also be performed on cultured amniocytes and chorionic villi. (Am J Obstet Gynecol 1996;174:1849-54.)     

Prenatal diagnosis of beta-thalassemia in Southern China

OBJECTIVE: To control the birth of thalassemic children in Southern China. STUDY DESIGN: DNA-based diagnosis was offered on fetal tissues in pregnancies when beta-globin gene mutations were identifiable in both parents using polymerase chain reaction (PCR)-reverse dot blot (RDB) assay. An automated high-performance liquid chromatography (HPLC) system was used to analyze fetal hemoglobin in pregnancies when mutation was unidentified in at least one parent. Fetal samplings were collected by chorionic villi sampling (CVS) in the first trimester, and by amniocentesis or cordocentesis in the second trimester. Maternal contamination of fetal DNA was ruled out by short tandem repeats (STR) analysis. RESULTS: Five hundered and forty-five fetuses of 540 at-risk pregnancies were performed prenatal diagnosis. Out of 540 fetuses tested by DNA analysis, 150 were found to be normal, 257 were carriers, whereas 133 were affected. Out of five fetuses diagnosed by HPLC, one fetus was affected and four were unaffected. Totally, 133 pregnancies with affected fetuses, except for one twin pregnancy, were voluntarily terminated, leading to a marked reduction of severe beta-thalassemia in this region. CONCLUSIONS: Our prenatal diagnosis strategy proved to be highly effective. DNA- and HPLC-based testing could enable prenatal diagnosis of beta-thalassemia in all at-risk pregnancies.     

First-trimester prenatal diagnosis of Roberts syndrome.

We present a case of prenatal detection of premature centromere separation on chorionic villi sampled at 8 weeks' gestation from a woman at risk of recurrence of Roberts syndrome. The same cytogenetic characteristic was confirmed on amniocytes at 14 weeks when ultrasound examination showed morphological anomalies of the fetus. To our knowledge, this is the first report of early prenatal diagnosis of Roberts syndrome.     

Prenatal analysis of insulin receptor autophosphorylation in a family with leprechaunism

We describe a method for the isolation and functional characterization of insulin receptors from chorionic villi and cultured amniotic fluid cells. The functionality of these receptors is assayed by measuring the insulin-induced stimulation of autophosphorylation of the receptor beta-chain. The method is expected to allow the prenatal diagnosis of those forms of leprechaunism and related diseases which are the result of a decreased stimulation by insulin of receptor autophosphorylation. A pregnancy at risk for leprechaunism was examined and an unaffected child was correctly predicted by study of the functionality of the insulin receptor on cultured amniocytes and by echoscopic examination.     

Prenatal diagnosis of a lethal form of Netherton syndrome by SPINK5 mutation analysis

Netherton syndrome (NS) is a severe autosomal recessive ichthyosis with no specific treatment or prenatal diagnosis available at present. The recent identification of SPINK5, which encodes a serine protease inhibitor, as the defective gene enables DNA based prenatal diagnosis to be carried out. Here we report the first direct molecular prenatal diagnosis of a lethal form due to a recurrent SPINK5 mutation in three consanguineous Turkish families. XmnI restriction enzyme digestion and DNA sequencing demonstrated that each deceased affected child was homozygous for mutation 153delT inherited from each parent. Analysis of fetal DNA from amniotic fluid cells in Family 1 and from a chorionic villus sampling in Family 3 showed that the fetus was heterozygous for 153delT in both cases. The pregnancies were carried to term and the newborns were unaffected. In Family 2, fetal DNA analysis from chorionic villus biopsy showed in a first pregnancy that the fetus was homozygous for 153delT. The pregnancy was terminated at 13 weeks and DNA analysis of fetal keratinocytes confirmed the prenatal prediction. In a second pregnancy in Family 2, fetal DNA analysis showed heterozygosity for 153delT, and the pregnancy was continued. Direct SPINK5 mutation analysis in families at risk for NS represents the first early, rapid and reliable method for prenatal diagnosis of this life threatening form of ichthyosis.     

First-trimester diagnosis of late-infantile neuronal ceroid lipofuscinosis (LINCL) by tripeptidyl peptidase I assay and CLN2 mutation analysis

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a progressive neurodegenerative disorder caused by the deficiency of lysosomal tripeptidyl peptidase I (TPP-I) encoded by the CLN2 gene. We report the first case of early prenatal diagnosis of LINCL by combined enzyme and mutation analysis. TPP-I activity in chorionic villi (CV) was less than 2% of the mean normal control level and g.1946A > G and g.3670C > T mutations were demonstrated, as in the two previously affected children. After termination of pregnancy, TPP-I deficiency was confirmed in cultured CV cells and in the fetal skin fibroblasts. The expression of unequivocal TPP-I deficiency in CV demonstrates that enzyme assay is a reliable option for prenatal diagnosis of LINCL.     

Prenatal diagnosis of a novel COL1A1 mutation in osteogenesis imperfecta type I carried through full term pregnancy

Prenatal diagnosis was performed in a family where the father has osteogenesis imperfecta (OI) type I, with a novel mutation in the COL1A1 gene: a C to T change at position c3076 (c.3076C-->T) leading to a change of arginine at codon 848 to a stop codon (R848X). Prenatal diagnosis by chorionic villous sampling (CVS) was performed during the fourth pregnancy, and revealed that the fetus is a carrier of the same COL1A1 mutation. The possibility of phenotypic variability was discussed with the parents. They elected to carry the pregnancy to term, and a male child with mild OI was born. This is the first reported case where OI was diagnosed prenatally, and the parents opted to carry the pregnancy to term. It illustrates the potential use of DNA-based analysis for early prenatal diagnosis of OI, and the complexities of genetic counselling.     

Prenatal exclusion of Leigh syndrome due to T8993C mutation in the mitochondrial DNA

Leigh syndrome (LS) is a mitochondrial encephalopathy that is caused by a mutation either in the mitochondrial DNA (mtDNA) or in the nuclear encoded genes of the mitochondrial proteins. Prenatal diagnosis of defects in the mtDNA is usually problematic because of mtDNA heteroplasmy and tissue specificity. However, the mutations T8993 G/C in the ATP synthase subunit 6 gene of the mtDNA show a more even tissue distribution and do not appear to change significantly over time. There are only few reports of prenatal diagnosis of the T8993G mutation in Leigh disease. Here we describe the first prenatal genetic testing of T8993C in a fetus of a mother whose previous child had died of Leigh syndrome due to the T8993C mutation. Mutant load in the chorionic villus sample (CVS) as well as in amniocytes was undetectable, thus predicting a very high likelihood of an unaffected outcome, indicative of a healthy baby. The diagnosis was confirmed after birth. Gathering data on the prenatal diagnosis of mtDNA mutations is of great importance so that prenatal diagnosis of both T8993G and T8993C mutations can be offered routinely.     

A case of discordant related abnormal karyotypes from chorionic villi and amniocytes.

A case of three discordant cell lines in prenatal diagnosis is described, of which two were abnormal related structural abnormalities of chromosome 11. One of the abnormal cell lines was seen in all metaphases examined from direct preparations of chorionic villi, the cultured preparations showing an apparently normal male karyotype; the other abnormal cell line was seen in conjunction with a normal cell line in cultured amniocytes. Prenatal diagnosis offered solely on chorionic villus sampling would have yielded a mistakenly normal result on the basis of established criteria for distinguishing true mosaicism from confined placental mosaicism.     

Clinical consequences of an increasing trend of preferential use of cultured villi for molecular diagnosis by CVS

OBJECTIVE: To compare the use of uncultured versus cultured villus cells for DNA-based prenatal diagnosis. METHODS: A retrospective review of molecular testing of chorionic villus sampling (CVS) cases from 1988-2007. Method of analysis, gestational age (GA) at CVS and at diagnosis, time from procedure to results, results of maternal contamination studies, and the laboratory employed were abstracted from patient charts. Trends in laboratory practices over time were analyzed. RESULTS: Time from CVS to diagnosis was longer when cultured cells were used. Average GA at diagnosis was 14-6/7 weeks with cultured cells vs 13-0/7 weeks with uncultured villi (p < 0.001). Recently, laboratories are more frequently requiring cultured cells, resulting in significantly greater delays in time to diagnosis and GA at results. CONCLUSIONS: 'Direct' DNA extraction saves 2 weeks from CVS to results. More women are afforded the option of an earlier, safer pregnancy termination if uncultured villi are used for molecular diagnosis. Implementation of standardized DNA extraction protocols and sample-size requirements can optimize the use of uncultured villi for molecular prenatal diagnosis. Increased awareness of the importance of rapid results and the advantages of 'direct' DNA extraction from uncultured villi can lead to improvements that are of clinical significance for patients undergoing early prenatal diagnosis.     

Prenatal diagnosis of Morquio disease type A using a simple fluorometric enzyme assay

A new fluorogenic substrate, 4 methylumbelliferyl beta-D-6-sulphogalactoside, was used for the assay of galactose-6-sulphate sulphatase activity in chorionic villi, cultured villus cells, and amniocytes. The fluorometric assay is much more convenient than the conventional assay using radiolabelled, sulphated oligosaccharides. Both types of substrate were used in the prenatal diagnosis of three pregnancies at risk for Morquio type A disease using amniocytes. These enzyme tests, as well as electrophoresis of glycosaminoglycans in the amniotic fluid, indicated affected fetuses in two pregnancies and a non-affected fetus in one.     

Prenatal diagnosis of metabolic diseases on chorionic villi obtained before the ninth week of pregnancy.

Nine pregnancies at risk for various metabolic disorders were monitored by prenatal diagnosis on chorionic villi obtained between the sixth and ninth weeks of pregnancy. A diagnosis of an affected fetus was made in five cases (Sandhoff, Tay-Sachs (2), Pompe's, GM1), while metachromatic leukodystrophy, GM1 (2), and Pompe's were excluded in four cases. It is concluded that chorionic villi are a reliable tissue for prenatal diagnosis of metabolic disorders also when obtained before the ninth week.