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 for dystrophia myotonica using the polymerase chain reaction

The polymerase chain reaction has been used to detect an abundant class of short repeat DNA families of the form (dC-dA)n.(dG-dT)n, known as microsatellites. These units are found throughout the human genome and have been characterized for several loci including APOC2 on chromosome 19ql2-ql3.2. The locus APOC2 is linked to the gene for dystrophia myotonica and a microsatellite within this locus was used to derive polymorphisms in a family to predict the inheritance of the disease. Chorionic villus sampling (CVS) was performed at 151/2 weeks' gestation. Following DNA extraction from the CVS material and parental blood samples, microsatellite analysis was carried out by the polymerase chain reaction.     

Amniotic fluid calcium concentration in the prenatal diagnosis of cystic fibrosis

Calcium concentrations were measured in supernatant amniotic fluid in order to establish whether they may be used as a marker for cystic fibrosis. No difference in values were found, whether the sample was derived from a normal pregnancy or from a pregnancy which resulted in a baby affected with cystic fibrosis.     

Microvillar enzyme analysis in amniotic fluid and the prenatal diagnosis of cystic fibrosis

The activities of two microvillar enzymes, gamma-glutamyltranspeptidase (GGTP) and alkaline phosphatase (ALP) have been determined in amniotic fluid (AF) samples from 39 pregnancies with a 1-in-4 risk of cystic fibrosis. Seventeen of these were investigated prospectively. A reduced proportion of the fetal specific intestinal ALP isoenzyme was found in 7 of a total of 13 pregnancies with cystic fibrosis and in one pregnancy of confirmed normal outcome. Eight of the affected pregnancies were tested for AF GGTP activity and depressed levels were found in 15. None of the 3 liveborn cystic fibrosis cases in the prospective series was identified by the ALP assay although 2 had significantly reduced GGTP activity. There were several amniotic fluid samples from cases of cystic fibrosis, trisomy 18 and normal outcome which had discordant GGTP and ALP results. Four of the 6 cases of cystic fibrosis misclassified by the ALP assay had amniocentesis at 15 or 16 weeks gestation. Evidence is presented which confirms a previous suggestion that amniocentesis after 17 weeks gestation improves the predictability of the ALP isoenzyme assay for the prenatal diagnosis of cystic fibrosis.     

Disaccharidase deficiency in amniotic fluid from cases of cystic fibrosis

The activities of the disaccharidases maltase and sucrase in 4 amniotic fluid from cystic fibrosis (CF) pregnancies have been compared to those of 120 non CF-pregnancies. Very low levels were found in 3 of the CF-fluids. The fourth CF-fluid was normal in all measured microvillar enzyme activities. Elevated levels of disaccharidases in meconium from one of the patients born with CF, supports the idea that these enzymes are trapped in the intestinal cavity by sticky meconium.     

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.     

Amniotic fluid disaccharidases in the prenatal detection of cystic fibrosis

Intestinal disaccharidases in amniotic fluid were studied in 41 pregnancies with a recurrence risk for cystic fibrosis (CF). In 11 out of 13 pregnancies with CF fetuses the maltase and sucrase activities were either below the control range (8 cases) or below the 10th percentile of control values (3 cases). Trehalase and lactase were slightly less informative indicators of CF. Of the other 28 pregnancies 3 had low amniotic fluid activities of several intestinal enzymes and were terminated, 12 resulted in the birth of a healthy child and 13 are continuing. The findings in fetal CF suggest an impairment of the defaecation of intestinal contents into the amniotic fluid. Reduced or low amniotic fluid disaccharidase activities were also found in other fetal disorders with demonstrated or presumed intestinal anomalies: e.g. anal atresia (2 cases), anencephaly (3 our of the 7 cases), trisomy 13 (5 cases), trisomy 18 (3 of the 5 cases) and trisomy 21 (19 of the 22 cases). Reduced amniotic fluid disaccharidase activities, although not specific for CF, are highly informative in pregnancies at high risk for CF. Using the 10th percentile of the normal range for amniotic fluid disaccharidase activities as an action line, the sensitivity of CF detection is estimated at 80 to 90 per cent, which could in high risk pregnancies reduce the risk of having another affected child from 1 in 4 to 1 in 20.     

Prenatal diagnosis of cystic fibrosis: I. Prospective study of 51 pregnancies

A prenatal diagnosis was performed in 51 pregnancies with a 1-in-4 risk of having a child with cystic fibrosis. The criteria for determining an affected fetus were based on the results of alkaline phosphatase (ALP) residual activity after inhibition by phenylalanine and by homoarginine, of total ALP activity, and of gamma-glutamyltranspeptidase (GGTP) activity in the amniotic fluid taken between 16 and 19 weeks of pregnancy. The chromosomal analysis of amniotic fluid cells showed trisomy 13 in one case which was excluded from the analysis of biochemical assays. The biochemical assays were in the normal ranges in the amniotic fluid of 35 pregnancies: 26 have reached term and a normal infant has been born, 9 are still in progress. A deficiency of the ALP phenylalanine-inhibitable form, depressed values of total ALP and GGTP were observed in the amniotic fluid of 15 pregnancies: one pregnancy went to term and the infant had CF, in 14 cases the pregnancy was terminated, and meconium ileus was observed in ten of these cases. It was observed that the changes towards abnormal values became more significant with advancing gestational age and that 18 weeks appeared to be the optimum time for diagnostic amniocentesis.     

Effect of introducing prenatal diagnosis on the reproductive behaviour of families at risk for cystic fibrosis. A Cohort study

We studied 101 couples to determine how far their reproductive behaviour was affected by the diagnosis of cystic fibrosis (CF) on its first occurrence in the couple's progeny and by the availability of prenatal diagnosis (PD). The couples were all resident in the Veneto and Trentino regions and attending the Verona CF Centre. CF had been diagnosed in the first affected child, during the period 1 January 1980-1 July 1990, before the age of 1 year. Couples received a questionnaire regarding socio-demographic status, reproduction data, and awareness of PD. Reproductive history was divided into three phases: prior to diagnosis of CF in the first affected child; from this time until PD was made available; and after the couples had learned of PD. In phase 2 (awareness of the genetic risk but not of PD), 54 couples showed a marked decrease in reproduction, none of the few pregnancies that occurred being taken to term. When couples became aware of PD, some resumption of reproductive activity occurred and 11 per cent of the 101 couples had another child; PD was used in 65 per cent of pregnancies and the abortion rate decreased to 35 per cent. All couples who opted for PD had no children without CF.     

Polymerase chain reaction analysis of the cystic fibrosis ΔF508 mutation in human blastomeres following oocyte injection of a single sperm from a carrier

The efficiency of the polymerase chain reaction (PCR) in detecting the cystic fibrosis (CF) ΔF508 mutation (which is the most common mutation of CF) was assessed in single human blastomeres. Twenty-one human immature oocytes (germinal-vesicle-stage oocytes) that had been donated for research were matured in vitro and a single spermatozoon from a carrier of the CF ΔF508 mutation was injected into the ooplasm. Fourteen embryos were obtained after intracytoplasmic sperm injection (ICSI). PCR analysis was carried out on 70 single blastomeres isolated from these 14 embryos. The results showed that the efficiency of DNA amplification by PCR in single nucleate blastomeres was 94 per cent (59/63). There were no false-positive results since none of the blank samples or the blastomeres without a nucleus showed an amplified signal. We found that nine embryos were homozygous for the unaffected genotype and that four embryos were heterozygous since they contained both the unaffected and the ΔF508 genotype. In a four-cell embryo, we observed the homozygous unaffected genotype in one blastomere and a heterozygous ΔF508/unaffected genotype in the other three blastomeres.     

Amniotic fluid protease activity and the prenatal detection of cystic fibrosis

Changes in the protease activity in amniotic fluid has been proposed as a valid method for the prenatal detection of cystic fibrosis (CF). We have studied by quantitative and qualitative procedures, sixty four amniotic fluids: two of them from CF-affected fetuses. Interpretation of the benzoyl arginine ethyl ester (BAEE)-staining patterns after isoelectric focusing was often difficult, and repeated experiments gave variable results. In order to improve gel discrimination, we performed amniotic fluid electrofocusing in the presence of detergents: 0.1 per cent Triton X-100, 0.1 per cent DOC, or 0.1 per cent SDS. In these conditions, the pattern revealed by BAEE was modified, but no differences were observed between CF and normal amniotic fluids.     

Prenatal diagnosis of cystic fibrosis. II. Meconium ileus in affected fetuses

Meconium ileus was the presenting feature of cystic fibrosis in 46 per cent of the couples which have been referred for prenatal diagnosis. In fetuses which have been aborted on the basis of alkaline phosphatase isoenzymes assays, meconium ileus represented the only pathological feature of cystic fibrosis, and was observed in three fourths of the cases. Real-time sonographic examination of fetuses at the time of amniocentesis was able to show an echogenic mass in the abdomen corresponding to the meconium ileus, and thus may afford a complementary means of diagnosis.     

Prenatal diagnosis of cystic fibrosis: Ultrasonographic appearance of meconium ileus in the fetus

Four often fetuses carrying a risk of 1:4 for cystic fibrosis were found to have low levels of microvillar enzymes in the amniotic fluid obtained between 17 and 18 weeks' gestational age. On sonography performed prior to the amniocentesis, three fetuses showed enlarged bowel loops. At autopsy, meconium ileus was detected. Enlarged bowel loops are a sign which has not been described previously so early in pregnancies.     

Prenatal diagnosis of cystic fibrosis by methylumbelliferylguanidinobenzoate protease titration in amniotic fluid

Amniotic fluids were obtained from 19 mothers who had previously given birth to a child with cystic fibrosis. Measurement of methylumbelliferyl guanidinobenzoate (MUGB) reactive proteases suggested that all 19 would have unaffected babies. Amongst the first 10 cases to come to term there were 5 infants with cystic fibrosis. It is concluded that MUGB protease titration is not suitable for the early prenatal diagnosis of cystic fibrosis.     

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.     

Discriminant analysis for assessing the value of amniotic fluid microvillar enzymes in the prenatal diagnosis of cystic fibrosis

We have analysed the sensitivity, specificity, and reliability of biochemical diagnosis based on microvillar membrane enzyme assay and using discriminant analysis in amniotic fluid samples obtained from 54 pregnancies at high risk for cystic fibrosis and 125 normal pregnancies. Our results show that amniotic fluid trehalase, alkaline phosphatase, alkaline phosphatase isoenzymes and gamma-glutamyltransferase enzyme activities measured during 16–20 gestational weeks, in spite of their non-specificity for cystic fibrosis, have a very good predictive value for fetal cystic fibrosis or exclude the possibility of the disease. Overall enzyme activity analysis provided over 90 per cent reliability of the method.     

In vitro amplification of the α1-antitrypsin gene: Application to prenatal diagnosis

Discrimination of the M, Z, and S alleles of α₁-antritrypsin (AAT) has been carried out using in vitro gene amplification with the polymerase chain reaction (PCR). Amplification of 90 nucleotides surrounding the Z mutation site and 120 nucleotides surrounding the S mutation site dramatically improves the sensitivity and reliability of allele-specific oligonucleotide (ASO) hybridization for direct detection of these alleles. Analysis is performed using Southern blots or dot blots hybridized with 19 base oligonucleotides and differentially washed for allele specificity. Amplification of the Z and S mutation sites can be combined in one PCR to allow detection of both mutations when analysed by gel electrophoresis and Southern transfer. This technique can be performed reliably using less than 0·1 μg of genomic DNA or less than 100 amniocytes or white blood cells. This technique has been used to perform prenatal diagnosis on a chorionic villus sample (CVS) in a fetus at risk for the ZZ Pi type form of AAT deficiency.