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.     

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.     

First-trimester prenatal diagnosis of cystic fibrosis using the polymerase chain reaction: Report of eight cases

Eight pregnancies at risk for cystic fibrosis have been monitored by first-trimester prenatal diagnosis with DNA amplification analysis. The polymerase chain reaction (PCR) was used in all cases to amplify the region detected by KM 19. In two cases, the region detected by CS·7, another DNA probe tightly linked to the CF locus, was also examined. The results of the PCR determinations were confirmed using the Southern blotting procedure, by segregation analysis of restriction fragment length polymorphisms (RFLPs) relative to XV-2c, J3·11, metH, metD, and KM19 probes.     

Usefulness of linkage disequilibrium of KM-19 and XV-2c DNA probes for genetic counselling in a high-risk cf family

A French couple with an individual risk of carrying the cystic fibrosis (CF) mutation of 1/2 sought genetic counselling. From the DNA haplotypes generated by XV-2c and KM-19 RFLPs, it could be deduced that only one subject was a carrier, lowering the risk of having a CF baby from 1/16 to 1/200. The strong linkage disequilibrium between these RFLPs and the CF allele observed in France reduced the risk to 1/1600.     

Cystic fibrosis prenatal diagnosis: Confirmation of an equivocal microvillar enzyme result by direct analysis of the common gene mutation

A child was tentatively diagnosed as having cystic fibrosis, based on neonatal presentation with severe gastrointestinal complications; the diagnosis was not confirmed biochemically and no tissues were available for DNA analysis. The mother presented in her subsequent pregnancy, and microvillar enzyme analysis of cell-free amniotic fluid at both 18 and 20 weeks gestation gave equivocal results. The pregnancy was terminated voluntarily because of a trend towards abnormal enzyme assay results on the second amniocentesis. Retrospectively, fetal tissues were found to be homozygous for the most common mutation of the cystic fibrosis gene (AF₅₀₈), which confirmed the prenatal assessment and suggested that the first infant of the couple was probably also affected by the disease.     

Biochemical analysis of meconium in fetuses presumed to have cystic fibrosis

A series of proteins (albumin, transferrin, a₁- antitrypsin, alpha-fetoprotein and pancreatic oncofetal antigen) and enzymes (γ-glutamyltranspeptidase, aminopeptidase M, alkaline phosphatase, a-glucosidase and protease) was measured in fetal meconium extracts. There were 19 fetuses thought to have cystic fibrosis (CF), 13 with neural tube defects, three with chromosome abnormalities and 19 normal controls, all with gestational ages between 18 and 21 weeks. With the exception of alpha-fetoprotein, all the proteins and enzymes were significantly elevated in the CF meconium extracts. The most definitive indicator of a CF fetus was the albumin concentration, where the mean level was five times that found in the control groups. However, five of 19 fetuses assumed to have CF had albumin in the normal range. In these cases the meconium protease levels were grossly elevated. Furthermore, in the same five fetuses meconium concentration of pancreatic oncofetal antigen, a protein synthesized in the fetal pancreas, was also greatly raised. We suggest that post-mortem examination of a fetus thought to have CF should include measurement of meconium albumin, protease and pancreatic oncofetal antigen.     

Prenatal diagnosis of cystic fibrosis using a monoclonal antibody specific for intestinal alkaline phosphatase

A monoclonal antibody (AAP-1), specific for the intestinal isoenzyme of alkaline phosphatase (ALP), has been used to develop an immunoassay for amniotic fluid samples. Values in the immunoassay correlated closely with those obtained by direct determination of phenylalanine-inhibitable ALP. A panel of 124 control second-trimester amniotic fluids and 21 fluids with a 1 in 4 risk of a cystic fibrosis fetus were examined in the immunoassay. Eight of 10 affected cases had values below an arbitrary cut-off of one third median, while all the non-affected cases were above this level. Almost identical results were obtained by enzymatic determination of phenylalanine-inhibitable ALP. However, in both systems the false positive rate (control fluids with values below one third median), was unacceptably high. It is pointed out that at present the most effective system for the prenatal diagnosis of cystic fibrosis is achieved by measuring the ratio of intestinal to total ALP in amniotic fluid supernatants. This is probably best effected by enzymatic assay in the presence of phenylalanine and homoarginine inhibition.     

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.     

Hyperechoic fetal bowel: The perinatal consequences

A number of publications have reported an association between the finding of hyperechoic fetal bowel on prenatal sonogram and disorders such as aneuploidy and cystic fibrosis. To define more precisely the significance of this finding, we systematically reviewed the published material on the subject. Based on a total of 357 reported cases, we documented a high prevalence of cystic fibrosis (25·6 per cent) and chromosome abnormality (12·4 per cent) associated with increased bowel echogenicity in the fetus. High rates of intrauterine growth retardation (14·9 per cent), fetal demise (9·0 per cent), and prematurity (15·3 per cent) were also found. The data were obtained from a population at high a priori risk for aneuploidy and included fetuses at 1 in 4 risk for cystic fibrosis reported in two studies. This increased the bias towards an adverse outcome. The rate of complications when a hyperechoic abdomen is noted in a low-risk fetal population has so far not been delineated. Although the high frequency of complications found is of concern and warrants investigation, extrapolation of these risk figures to a fetal population at low a priori risk may not be appropriate.     

Mid–trimester hyperechogenic bowel in a fetus of Japanese origin carrying a new mutation of CFTR gene (L548Q)

We present a case of a fetus with hyperechogenic bowel, in which the L548Q mutation was detected in the mother of Japanese origin and the ΔF508 mutation in the father of Caucasian origin. The fetus proved to be compound heterozygous. Research into cystic fibrosis transmembrane conductance regulator (CFTR) mutations in this case was triggered by the fact that the fetus had a characteristic hyperechogenic bowel image with normal karyotype and no indications of intrauterine infections. Hyperechogenic bowel is highly indicative of a CFTR gene mutation. The incidence of cystic fibrosis (CF) in fetuses with mid-trimester hyperechogenic bowel is 5%, but once the most frequent mutations have been accounted for, rarer mutations must be investigated. Copyright © 2006 John Wiley & Sons, Ltd.     

Community attitudes to cystic fibrosis carrier testing in England: A pilot study

Inexpensive and accurate carrier testing for cystic fibrosis (CF) will be possible in the near future. There are no existing studies on the attitudes of English persons in the community to carrier testing for CF or any other recessive disorder. We have conducted a trial study of 166 persons at two schools, two doctors' surgeries, and a family planning clinic. Only a minority had clear pre-existing knowledge of cystic fibrosis and its genetic nature. However, over 80 per cent of those questioned expressed interest in knowing their carrier status. Although it is well known that uptake can only be assessed when a service is in place, and while further studies are required to confirm that testing will be of interest to couples of reproductive age with no previous knowledge of CF, the data strongly suggest that there will be interest in communitywide testing for CF carrier status when such a test becomes available in the United Kingdom.     

Cystic fibrosis: selecting the prenatal screening strategy of choice

Cystic fibrosis is a serious disorder. Research into the treatment of affected individuals is in progress, but a cure is not expected in the near future. In this review, we demonstrate that prenatal screening for cystic fibrosis meets the requirements for a worthwhile screening programme. We explain the reasons that have led us to conclude that one approach (‘couple screening’) is the method of choice. The couple-based approach calls for reporting results to the couple as a unit. Only if both parents are found to be carriers is the result designated screen-positive and an amniocentesis or chorionic villus sampling offered. This offers a substantial reduction in the proportion of women with unaffected pregnancies with positive results (the false-positive rate) compared with other methods without reducing the detection of affected pregnancies. It also avoids creating a screen-positive group for which no definitive diagnosis is available. This is a problem with other screening methods. The couple method can achieve a 72% detection rate for a 0.1% false-positive rate. The screening method is simple, non-invasive, reliable, safe and reasonably cost effective. Existing programmes have shown that screening using this method is acceptable to health care professionals and patients. Setting up a national prenatal screening programme for cystic fibrosis is timely and should be implemented using the couple screening method. Copyright © 2003 John Wiley & Sons, Ltd.     

Amniotic fluid microvillar enzyme activities in the early detection of fetal abnormalities

Measurement of the microvillar enzymes, γ-glutamyltranspeptidase (GGTP), aminopeptidase M (APM) and alkaline phosphatase (ALP), in amniotic fluid supernatant has been proposed as a method for the early prenatal diagnosis of cystic fibrosis. The activities of these enzymes in a series of other fetal abnormalities have now been examined. GGTP activities were below the 5th percentile in 28 out of 54 cases of trisomy 21 and 9 of 14 cases of trisomy 18, while APM values were below this cut-off in 26 of 54 cases of trisomy 21 and 8 of 14 cases of trisomy 18. Abnormal ALP isoenzyme ratios were found in 6 of 54 cases of trisomy 21 and 4 of 14 cases of trisomy 18. If prenatal cytogenetic studies are routinely carried out on amniotic fluid cells, the occasional confounding effect of abnormal microvillar enzymes associated with fetal trisomies rather than with cystic fibrosis should be avoided.     

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.     

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.     

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.     

Prenatal diagnosis of cystic fibrosis using linked DNA probes

This paper presents data collected in Europe on 107 prenatal diagnoses of cystic fibrosis (CF) using linked DNA markers. To date, 38 children have been born without CF, as predicted, demonstrating the present rapid move from research to clinical genetic service.     

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 abdominal cystic hygroma

We present a case of fetal abdominal cystic hygroma that presented at 19 weeks of gestation. Ultrasonographic evaluation of the fetus revealed soft tissue enlargement of the left leg and a retroperitoneal mass in the left pelvis and abdomen. This represents the first reported case of prenatal diagnosis of abdominal cystic hygroma.     

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.