Prenatal diagnosis of 21-hydroxylase deficiency by rflp analysis of the 21-hydroxylase,complement C4, and HLA class II genes

In 19 pregnancies at risk for 21-hydroxylase deficiency (21OHD) in 18 families with at lea one affected child, prenatal diagnosis was performed by RFLP analysis using the enzymi Taq I and EcoRI and the DNA probes specific for the 21 OH genes, the closely linke complement C4 genes and the highly polymorphic HLA class II genes DRB, DQB, and DPI For fetal DNA analysis either chorionic villi or cultivated amniotic cells were used. In all 1 cases, a clear prenatal diagnosis was possible either with the 21OH probe alone or in mo cases, by combining the results of the different closely linked loci.     

Early prenatal diagnosis of the fragile site AT Xq27.3 associated with Martin-Bell syndrome

Early prenatal diagnosis of the fragile X was attempted in 44 pregnancies, including one twin pregnancy at risk of Martin-Bell (MB) syndrome. The sex ratio was 24M:21F. The fragile site was reproducibly demonstrated in cultured chorionic villus (CV) cells in eight male and five female fetuses. Six of the male and three of the female fetuses were terminated. Simultaneous RFLP analysis provided confirmative data with flanking DNA markers in 3 of 13 analysed cases. Recombination and/or non-informativeness at available distal and/or proximal loci were found in nine cases. In one male fetus, discordance between the haplotype and cyto-genetics (fragile-X-negative) suggested the presence of a normal male transmitter, a double meiotic cross-over within the region, or a false-negative cytogenetic diagnosis. However, discordance between prenatal and post-termination/postnatal cytogenetic findings was not observed in this series. The use of excess thymidine for induction of the fragile X in cultured CV cells provided in the majority of cases a safe and rapid method for cytogenetic diagnosis, with options for early induced termination in fragile-X-positive pregnancies, for simultaneous RFLP analysis, and for subsequent second-trimester analysis of fetal blood in complicated cases.     

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. Copyright © 2002 John Wiley & Sons, Ltd.     

Early prenatal diagnosis of cartilage-hair hypoplasia (CHH) with polymorphic DNA markers

Cartilage-hair hypoplasia (CHH) is an autosomal recessive disorder resulting in short stature and hypoplasia of hair. Associated features include impaired T-cell-mediated immunity, deficient erythropoiesis, gastrointestinal dysfunction, and an increased risk of malignancies. As the condition may, in some cases, be severe or even fatal during childhood, families with a previous history of CHH may wish to have prenatal diagnosis. We have previously assigned the gene for CHH to the proximal 9p by linkage analysis using several polymorphic DNA markers. Here we report the prenatal testing for CHH in three Finnish and one Australian family using three DNA markers closely linked to the CHH gene. In three cases a fetus unaffected with CHH was predicted at the probability level of more than 94 per cent. In one case, an affected fetus was predicted. The results were in concordance with ultrasonography performed for all fetuses. The three children born to date were unaffected as predicted. The DNA marker-based analysis thus provides a useful method for early prenatal testing for CHH.     

Prenatal diagnosis of friedreich ataxia: Improved accuracy by using new genetic flanking markers

Friedreich ataxia is a neurodegerative disorder with autosomal recessive inheritance. Since the gene causing mutation has not yet been identified, prenatal, predictive, and carrier diagnoses are based on indirect haplotype analysis with closely linked markers. Until recently, only distal markers were available and their physical distance to the Friedreich ataxia (FRDA) gene remained elusive. The identification of close flanking markers that mark out the boundaries of the FRDA locus and reduce the critical genomic region which contains the gene allows for the first time misdiagnosis due to undetectable recombination to be avoided and diagnosis accuracy to be greatly improved. In this sense, we have verified a prenatal diagnosis in which the fetus was diagnosed as an unaffected carrier last year with a confidence of 95 per cent. By using the new flanking markers, the diagnosis improved and confidence reached almost 100 per cent.     

Prenatal diagnosis of Huntington's disease (HD): Experiences with six cases and PCR

In the course of a 2-year predictive testing programme for Huntington's disease (HD), six couples from a total of 52 applicants requested prenatal testing. In each case, the pregnancy was in the first or second trimester when the couples were referred for DNA diagnosis. In five cases, exclusion testing was offered; in one case, a person at risk with an increased risk of being a gene carrier requested prenatal diagnosis. In all cases, informative markers for prenatal testing could be determined. Whenever possible, the newer technique of polymerase chain reaction (PCR) for D4S125 was applied to perform rapid prenatal diagnosis. Two couples withdrew before chorionic villus sampling was undertaken; prenatal diagnosis was completed in the remaining four cases. After exclusion testing, two pregnancies were determined to have an increased risk and two fetuses to have a low risk of being HD gene carriers.     

First trimester prenatal diagnosis of haemophilia A: Two years' experience

We evaluated the feasibility, reliability, and acceptability of prenatal diagnosis of haemophilia A by DNA analysis of chorionic villi. Twenty-two women at risk to transmit the abnormal gene were referred for prenatal diagnosis, two of them twice. Two of the 22 women appeared to be non-carriers by DNA analysis. In one of these women, the results were known only after chorionic villus sampling had been carried out. Thirteen of the twenty carriers were heterozygous for an intragenic (Bell or Xbal) marker; six women were only heterozygous for the extragenic DXS52 (Stl4) locus. One of the women was homozygous for all the presently known DNA markers within or closely linked with the factor VIII locus. Twelve of the 22 fetuses at risk were male, ten were female. Seven of the 12 male fetuses were shown to be affected and were subsequently aborted. Four male fetuses appeared to be not affected. In one case, the diagnosis was made by use of an extragenic marker. The woman rejected fetal blood sampling to confirm the diagnosis. After birth, a normal factor VIII level was found in three of the four cases. The fourth pregnancy is still continuing. In one of the 12 male fetuses, no diagnosis at the gene level was possible. DNA analysis is expected to provide maximum certainty as to the phenotype of the fetus for approximately 60 per cent of the women; for another 37 per cent a rate of misdiagnosis of 4–5 per cent applies. In only 3 per cent of the cases will no diagnosis at the gene level be possible as yet. The new possibility of a prenatal diagnosis in the first trimester of pregnancy enabled some of these women to have a family of their own and was appreciated in particular by the women who underwent fetoscopy in an earlier pregnancy.     

First-trimester prenatal diagnosis of spinal muscular atrophy using microsatellite markers

Twenty-five pregnancies at risk for spinal muscular atrophy I (SMA I) have been monitored by first-trimester prenatal diagnosis. Microsatellite markers were used in all cases to amplify polymorphic regions at the D5S125, D5S435, D5S39, D5S127, and D5S112 loci. All families, including 12 SMA I pedigrees with a deceased index child, were fully informative for DNA analysis. Three fetuses were predicted to be affected and 22 fetuses were predicted to be unaffected. Twenty-two newborns were unaffected by clinical examination at birth. These results support the accuracy of SMA I prenatal diagnosis based on linkage analysis.     

Prenatal molecular diagnosis of gaucher disease

Prenatal diagnosis of Gaucher disease, the most prevalent glycolipid storage disease, is based on a reliable enzyme assay of cells from amniocentesis or chorionic villous samples. However, this method cannot differentiate among the various forms of the disease. This report details four cases of prenatal diagnosis of Gaucher disease, three of which predate the use of molecular diagnosis. DNA mutation analysis to determine the genotype was predictive of the phenotypic status of the fetus and conformed to the genotype of an affected proband where available.     

First-trimester prenatal diagnosis in haemophilia A and B families—10 years experience from a centre in India

During the last 10 years (1995–2005) we have offered diagnosis in 438 families out of 502 families referred, by chorionic villus sampling procedure between 10 and 12 weeks of pregnancy in severe haemophilia A and B families. Sixty four families could not be offered a diagnosis in the first trimester either due to non-informativeness with the available techniques or due to the absence of affected members in the family and were subsequently offered diagnosis in the second trimester by direct analysis of factor VIII/IX clotting activity and antigen in the fetal blood samples. For first-trimester diagnosis in the chorionic villus samples, we have used both direct and indirect methods, that is, intron 22 and 1 inversions in the factor VIII gene, a multiplex PCR for the detection of gross deletions in the factor IX gene and RFLP analysis using a battery of markers within and outside the factor VIII/IX gene. The informativeness of all these techniques was found to be 92.21% in case of haemophilia A and 83.91% in case of haemophilia B. We followed up 122 children out of 326 (diagnosed unaffected prenatally) after birth and the diagnosis was adequately reconfirmed by both factor assays and by DNA analysis. Only one case of misdiagnosis was detected so far (0.22%), where the diagnosis was based only on the extragenic marker of the factor VIII gene. Copyright © 2006 John Wiley & Sons, Ltd.     

First trimester diagnosis of β-thalassaemia in a twin pregnancy

This paper reports the results of first trimester prenatal diagnosis in a twin pregnancy at risk for homozygous β°−thalassaemia (β°−39 mutant). Trophoblast samples from both twins were obtained at 10 weeks gestation with a forceps guided by ultrasound. Trophoblast DNA analysis, carried out with the oligonucleotide technique, revealed that one fetus was homozygous and the other heterozygous for the β−39 mutant. This diagnosis was confirmed at 17 weeks gestation by amniocyte DNA analysis. DNA polymorphism analysis within the α-globin gene provided useful genetic markers for twin differentiation.     

Chorionic villus sampling and acceptance rate of prenatal diagnosis

In this paper, we compared the acceptance rate of fetal diagnosis for β-thalassemia in three group of couples of Sardinian descent; the first counselled before DNA analysis was available, the second presenting after DNA analysis was introduced but too late for chorionic villus sampling and thus monitored by amniocyte DNA analysis and the third presenting within the first trimester after DNA analysis was introduced and thus in time for trophoblast DNA analysis. A higher proportion of couples from the latter group opted for fetal testing as compared to the 1st and 2nd group. These results indicate that in this population, introduction of 1st trimester diagnosis made prenatal testing acceptable to practically all counselled couples at risk.     

Prenatal diagnostic testing for familial dysautonomia using linked genetic markers

Familial dysautonomia (FD), a recessively inherited disease, has been mapped to chromosome 9q31. Highly polymorphic dinucleotide repeat markers flanking the genetic locus and at the same genetic location have been identified. We describe the prenatal diagnosis of FD using linkage and linkage disequilibrium analyses with these markers. Twelve families were analysed for informativeness and of these, seven went on to have prenatal testing (a total of eight fetuses tested). All of these fetuses were predicted to be heterozygous unaffected (FD carriers). Seven fetuses have come to term and are normal. In the absence of a recombinant proband, a panel of three proximal and three distal markers is sufficient to provide informative flanking markers and an 87–96 per cent likelihood of a highly predictive test. In an additional family at 1:4 risk for FD, no DNA was available from the propositus. This family was analysed using linkage disequilibrium to the #18 allele of the tightly linked marker D9S58 in conjunction with linkage analysis using data from two unaffected children. Prenatal diagnosis in this family indicated an affected fetus.     

Prenatal diagnosis of Fukuyama congenital muscular dystrophy

Fukuyama congenital muscular dystrophy (FCMD) is characterized by infantile hypotonia, symmetrical generalized muscle weakness, and neuronal migration disturbances that result in changes consistent with cobblestone lissencephaly with cerebral and cerebellar cortical dysplasia. FCMD is recognized as an autosomal recessive genetic defect. Genetic counselling is recommended for parents at risk of having a child with FCMD. Given the high risk and overwhelming prospect of having another child with this incurable devastating condition leads many couples to consider prenatal diagnosis. In Japanese families, haplotype analysis using microsatellite markers is available. In non-Japanese families, DNA sequence analysis is available. Both disease-causing alleles of an affected family member must be identified before prenatal testing can be performed. Copyright © 2006 John Wiley & Sons, Ltd.     

Five years' experience of prenatal diagnosis of cystic fibrosis in the former U.S.S.R.

From a total of 490 cystic fibrosis (CF) high-risk families under supervision (mostly Russian Slavs from the European part of the country), DNA data including both direct screening for some CF gene(CFTR)mutations(deIF508, G551D and 1677delTA) and allelic polymorphism studies with tightly CF linked DNA markers were collected from 261 families. All full families (129) and 86 CF families with a deceased index child were found to be either fully (42 per cent) or partially (40 per cent) informative for DNA analysis. Prenatal diagnosis (PD) was carried out in 161 CF families. Microvillar enzyme (MVE) assay was applied to all 140 PD at the second trimester either as a single test (88) or in conjunction with DNA analysis (52). The frequency of false-negative results of the MVE assay was 1.3 percent and that of false-positive results, as judged by the albumin meconium test, was 5.0 per cent. Ambiguous results of MVE analysis were found in 30 cases, 12 of which were verified by DNA analysis. Molecular diagnosis of CF at the first trimester was carried out in 21 cases and four pregnancies were terminated. Altogether, 39 pregnancies with a predicted high risk of CF fetuses were terminated. The low average frequency of delF508 in CF chromosomes of Russian Slavs (50 per cent), its remarkable inter-population variation, and the significant proportion of at-risk families without an affected child determine the necessity of combined molecular and biochemical (MVE assay) approaches for efficient prenatal diagnosis of CF in the former U.S.S.R.     

Circulating ‘trophoblast’ cells in pregnancy have maternal genetic markers

A syncytiotrophoblast-associated antigen identified by the monoclonal antibody (McAb) H315 is detectable on the surface of a low proportion of peripheral blood cells in pregnant women, raising the possibility of a new approach to prenatal diagnosis of genetic disorders. We aimed at verifying the trophoblastic origin of H315⁺ cells and their use for prenatal diagnosis of β-thalassaemia. H315 ⁺ cells were separated from the peripheral blood of pregnant women: the DNA obtained from these cells in two selected cases was shown to have genetic markers indistinguishable from those of the mother and definitely different from the fetus. Our results suggest that H315 antigen is expressed by maternal cells and that prenatal diagnosis on peripheral blood of the mother using H315 McAb is not feasible.     

Microsatellite analysis provides efficient confirmation of fetal trophoblast isolation from maternal circulation

Fetal trophoblasts can be found in maternal circulation from an early stage of pregnancy and thus provide a potential source of DNA for non-invasive prenatal diagnosis. We have developed a two-step method for trophoblast isolation between the 8th and 12th week of pregnancy. Blood was sampled from 14 women undergoing termination of pregnancy or spontaneous abortion. Immunomagnetic beads precoated with HLA class I and II, and with anti-cytokeratin-18 monoclonal antibodies, were used to remove CD8+ and other maternal cells, and to select for fetal trophoblasts, respectively. Microsatellite analysis was performed on DNA extracted from the isolated, maternal, paternal and placental cells after PCR amplification. Recovery of the trophoblasts was confirmed in 13/14 cases (93%) by the identification of an identical microsatellite pattern for fetal and placental cells. Further evidence was the presence of heterozygous alleles of both maternal and paternal origin. The correct prediction of gender in all five male fetuses was an additional confirmation of trophoblast recovery. We conclude that trophoblasts can be effectively isolated from maternal blood in the first trimester, and by using polymorphic microsatellite markers to confirm sample purity, this method has potential future application in prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

Dystrophin protein and rflp analyis for fetal diagnosis and carrier confirmation of duchenne muscular dystrophy

A pregnant woman with indeterminate Duchenne muscular dystrophy (DMD) carrier status, but with DMD diagnosed in her deceased brother (unavailable for study), presented for prenatal diagnosis, intending to continue the pregnancy only if proven unaffected with DMD with near absolute certainty. Creatine kinase (CK) assays to clarify carrier status were inconclusive. Male sex in the fetus was identified, but DNA restriction fragment length polymorphism (RFLP) analysis was not yet available to this centre to investigate the possible transmission of the DMD gene, and the pregnancy was terminated. Tissue histology and dystrophin protein analysis demonstrated the absence of DMD. In a situation with proven maternal carrier status, future fetal inheritance of the opposite maternal X chromosome would indicate the presence of DMD. However, maternal carrier status remained in doubt through a second pregnancy, even with RFLP studies, and was finally established when dystrophin analysis confirmed the presence of DMD in the second fetus. Histologic findings are presented, contrasting features in the two fetuses. The value of dystrophin analysis for establishing the diagnosis of fetal DMD, in this case proving maternal carrier status in a difficult situation, and for demonstrating DMD gene:RFLP haplotype relationships is illustrated.     

Prenatal detection of an Arg→Ter mutation at codon 111 of the pah gene using DNA amplification

A CGA→TGA mutation at codon 111 in exon 3 of the phenylalanine hydroxylase (PAH) gene was recently identified in a Chinese phenylketonuria (PKU) patient. This paper reports the prenatal diagnosis of a Chinese fetus at risk for PKU using DNA amplification with PCR and oligonucleotide hybridization. RFLP analysis revealed that the fetus had inherited a PKU gene from his mother, but his paternal PAH gene was uninformative. PCR amplification of 300 bp which included exon 3 plus the flanking intronic sequences of the PAH gene was performed. The amplified DNA was hybridized with a pair of allele-specific oligonucleotide probes. The results indicated that the fetal DNA carried a PAH 111 Arg→Ter mutant gene inherited from his father. Thus, the fetus was predicted to be affected with PKU.     

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.