Prenatal diagnosis of cystic fibrosis by assay of amniotic fluid microvillar enzymes

Summary Activities of the microvillar enzymes -glutamyl-transpeptidase (GGTP), aminopeptidase M (APM), phosphodiesterase and maltase have been examined in second-trimester amniotic fluid as possible aids to the early prenatal diagnosis of cystic fibrosis (CF). The two peptidases, GGTP and APM, gave best results. If the fifth percentile of the normal range is used as an action line, the sensitivity of a positive test (low GGTP value) is 78% and the predictability 84%. At the tenth percentile the sensitivity is 100% and the predictability 77%. These approximate figures apply only to pregnancies where there has been a previous affected child. Until the primary protein defect in CF is discovered, this may prove an acceptable form of prenatal diagnosis to the high-risk mother.     

Prenatal diagnosis of cystic fibrosis using linked DNA markers and microvillar intestinal enzyme analysis

Summary Prenatal dignosis was performed for 47 pregnancies with 1 in 4 risk of cystic fibrosis, including 7 cases analyzed with linked DNA markers, 16 cases analyzed by microvillar intestinal enzyme testing, and 24 cases where both methods of testing were attempted. DNA was obtained by chorionic villus sampling in 10 cases and by amniocentesis in 21 cases, and diagnosis was based on analysis with the tightly linked DNA markers D7S8 and met. DNA analysis using these probes was fully informative in 74.4% of 90 couples with 1 in 4 risk. In 18 cases where both DNA results and microvillar intestinal enzyme data were diagnostic, there was agreement regarding the predicted status of the fetus. No adequate diagnosis was achieved in two cases where both diagnostic tests were attempted. Ourcome is known for 24 pregnancies including 10 where DNA analysis was diagnostic, and no errors in diagnosis were detected. Prenatal diagnosis of cystic fibrosis using DNA markers is highly informative and accurate, but microvillar intestinal enzyme analysis remains a valuable part of a complete diagnostic program.     

Prenatal diagnosis of cystic fibrosis. Analytical evaluation of microvillar enzyme determinations in amniotic fluid

The activity of gamma-glutamyltranspeptidase and total alkaline phosphatase and its isoenzymes has been determined in 261 amniotic fluid samples taken from pregnant women with known normal outcome and in 30 amniotic fluid samples from pregnant women with a 1:4 risk for cystic fibrosis (CF). Preliminarily, 114 amniotic fluid samples were assayed in parallel in three different laboratories, and a good correlation was found even though different assays were used. From the results obtained in control amniotic fluids, normal range and CF-predictive cutoff values were established. No false-negative results were found in this study. Among the predicted affected pregnancies 7 were terminated, and 3 went to term: 1 resulting in a CF-affected child and the other 2 in healthy children. CF was confirmed in all the aborted fetuses. In 1 case the results were inconclusive. In this study numerical results obtained for samples with a 1:4 risk of CF analyzed in the three laboratories were always virtually identical.     

Prenatal diagnosis in 200 pregnancies with a 1-in-4 risk of cystic fibrosis

Summary Prenatal diagnosis of cystic fibrosis was performed in 200 pregnancies with a 1-in-4 risk, and was based on significant modifications in amniotic fluid taken at 17, 18, 19 weeks of pregnancy, of six enzymatic assays: gamma-glutamyl-transpeptidase, aminopeptidase M, and alkaline phosphatase (total and isoenzymes). On the basis of normal values, normal outcome was predicted in 135 pregnancies reaching term, all the babies were normal. On the basis of significantly abnormal enzymatic values, an affected fetus was predicted in 56 pregnancies, 53 were terminated, and 3 went to term; the infants were affected. There were discrepancies in enzymatic values in nine cases, in eight cases normal outcome was predicted, six babies were normal and two were affected; in one case an affected baby was predicted, the pregnancy went to term and the baby is normal. Criteria giving evidence for cystic fibrosis in fetuses have been described: macroscopic observation of a typical meconium ileus, significant increase of albumin content in the meconium, and PAS-positive mucus-like material in some pancreatic acini. Using these criteria, diagnosis of cystic fibrosis has been confirmed in all the examined fetuses. The recurrence rate of cystic fibrosis was 22.5% in 147 diagnoses in which the index case had cystic fibrosis without a history of meconium ileus at birth, but was 47.5% when the index case had meconium ileus. The results of the study suggest that prenatal diagnosis of cystic fibrosis can be performed with an accuracy of 98%.     

Prenatal diagnosis by amniocentesis in 800 pregnancies

Prenatal diagnoses by amniocentesis have been made in 800 pregnancies at the Genetics Division of the Los Angeles County-University of Southern California Medical Center. This experience has indicated that the amniocentesis procedure is safe and highly accurate. Amniocentesis was not associated with significant morbidity or mortality for either infant or mother. The observed complications are assumed to be related to the high-risk nature of these pregnancies, the patients having been selected primarily on the basis of advanced maternal age or a previous abnormal child. The needle puncture marks, which occurred in 2.4 percent of the live births, resulted in no serious developmental or cosmetic effects to the infants. No errors in cytogenetic diagnosis are known to have occurred in this series.     

Prenatal diagnosis and linkage disequilibrium with cystic fibrosis for markers surrounding D7S8

Summary Three polymorphic DNA markers surrounding the D7S8 locus were tested for their usefulness in the diagnosis of cystic fibrosis (CF) by linkage analysis. The markers correspond to the loci D7S424 and D7S426. These polymorphisms were studied by centers in the U.S., the United Kingdom, the Netherlands, and Italy, using samples from populations throughout Europe and North America. The additional information provided by these probes increased the heterogeneity of the region from 50% to 58% and was essential for a completely informative diagnosis in one family. A very high degree of linkage disequilibrium was found between these markers, which span a distance of approximately 250kb. In addition, linkage disequilibrium with CF was noted. Significant heterogeneity of linkage disequilibrium was found among the populations, both for the marker-marker pairs and between the markers and CF.     

Prospects for prenatal diagnosis of cystic fibrosis: Induction of biochemical abnormalities in fibroblasts from patients with cystic fibrosis by a urinary glycoprotein

Alkaline phosphatase activity, assayed on a per cell basis with an ultra micro-method, can be increased up to 7-fold in fibroblast cultures derived from patients with cystic fibrosis by induction with a urinary glycoprotein. Fibroblasts from normal and heterozygous individuals are not significantly induced. There is a suggestion of heterogeneity among cases as reflected in varying baseline alkaline phosphatase and α-glucosidase levels.     

Technology platforms for molecular diagnosis of cystic fibrosis

Cystic fibrosis (CF) is one of the most common recessive genetic diseases in North America. So far, 1200 mutations causing CF have been identified. Several techniques such as allele specific oligonucleotide (ASO) dot-blot, reverse dot-blot, amplification refractory mutation (ARMS), and an oligo-ligation assay, are available to detect the most common mutations. However, detecting compound heterozygotes between DeltaF508, the most common disease causing mutation, and other mutations which are rare is difficult as some mutations are common only to particular ethnic groups. Therefore, new diagnostic tests such as restriction enzyme assays and single stranded conformational polymorphism (SSCP) have been designed to recognize rare and population-specific mutations. This review will describe the most commonly used CF mutation detecting diagnostic techniques, as well as novel assays and techniques currently in development that might be employed in future.     

Biochemical implications of sequence comparisons of the cystic fibrosis transmembrane conductance regulator

System A, the Na(+)-dependent amino acid transport activity, is encoded by the ATA2 gene and up-regulated following partial hepatectomy (PH), and its competitive inhibition interferes with liver regeneration. Rabbit polyclonal antibody was raised against a portion of the ATA2 gene product followed by immunodetection of ATA2 in isolated liver plasma membrane and lysate. The level of ATA2 increased in the plasma membrane following PH, while the relatively high quantity of ATA2 found in liver lysate remained constant. We also have shown that Northern analysis of steady-state ATA2 mRNA revealed no significant change following PH. These data show that ATA2-mediated transport is not regulated by the steady-state level of ATA2 mRNA but is regulated by the amount of ATA2 and redistribution to the plasma membrane. We hypothesize that ATA2 activity is regulated by recruitment of ATA2 protein from an intracellular compartment. In addition, the pattern of expression of System A activity in oocytes, transport kinetics, and sensitivity to chemical modification indicate the presence of a second System A isoform in liver that differs substantially from ATA2.     

Effects of hyperoxia on oxygen uptake kinetics in cystic fibrosis patients as determined by pseudo-random binary sequence exercise

Patients with cystic fibrosis (CF) have been shown to exhibit impaired oxygen uptake (VO2) kinetics independent of their physical fitness. This study investigated whether oxygen supplementation improves VO2 kinetics in CF as determined by cycle ergometry at submaximal exercise intensities using a pseudo-random binary sequence exercise test i.e. a simultaneous application of different frequencies of sinusoidal work. The subjects were 9 CF patients and 13 healthy controls (HC) and they exercised while breathing humidified and heated air with a fractional concentration of oxygen in inspired air (F(I)O2) of either 0.21 or 0.40. With a F(I)O2 of 0.21 the respiratory exchange ratio (R) was higher in CF than in HC both at rest (0.91 vs 0.81) and during exercise (0.97 vs 0.89). Oxygen saturation (SO2) was slightly lower in CF. but remained above 90% during exercise (92.7% vs 95.2%). Spectrum analysis revealed that in CF, the amplitude ratio (AR) between V02 and exercise intensity was lower over a wide frequency range (P < 0.05). In addition, CF showed a larger negative phase shift (PS) at lower frequencies (P < 0.005). With a F(I)O2 of 0.40, SO2 increased to about 97% in both groups; while R remained higher in CF (0.92) compared to HC (0.81). In the control group, the O2 supplement raised AR but the VO, kinetics of the CF patients were not significantly affected. In HC the enhanced AR during oxygen supplementation would suggest a cardiopulmonary limitation of VO2 at the onset of submaximal exercise. In CF patients low AR and PS would indicate an attenuated VO2 response attributable to an impaired oxygen utilization in the muscles because the oxygen supplement normalised SO2 but failed to improve R and VO2 kinetics.     

Prenatal diagnosis of type III congenital cystic adenomatoid malformation of the lung

The authors report a case of type III Congenital Cystic Adenomatoid Malformation (CCAM) of the lung in a stillborn with ultrasound and pathological findings. CCAM is an unusual morphological entity and the solid pattern (type III) is the rarest. Antenatal demonstration may allow to salvage these infants by surgical removal in immediate postnatal period.     

DNA sequence analysis of the KM19 locus linked to cystic fibrosis

Summary The PstI polymorphism detected by probe KM19 is a highly informative marker in linkage disequilibrium with the cystic fibrosis locus and has been used extensively for prenatal diagnosis. The currently available primers used for polymerase chain reaction- (PCR-) based analysis of this locus have been shown to produce spurious amplification products. In this report, we describe the sequence of the KM19 locus and the major contaminating PCR product. We have used this information to design a more specific amplification procedure for analysis of the KM19 locus.     

Use of linkage disequilibrium data in prenatal diagnosis of cystic fibrosis

Summary The authors report a case of 11;17 translocation associated with recurrent spontaneous abortions, and request contact with colleagues who have observed similar cases.     

Use of linkage disequilibrium data in prenatal diagnosis of cystic fibrosis

Summary Parents at risk of bearing a child with cystic fibrosis, and who have no living affected child, often use prenatal diagnosis based on microvillar enzyme assay in second-trimester amniotic fluid samples. If enzyme levels are abnormal and the pregnancy is terminated, it is possible in principle to use the fetal tissues to establish the phase relationship of linked DNA markers for a subsequent first-trimester prenatal diagnosis. However, the probability of a fetus being affected after an abnormal microvillar enzyme test may be no greater than 80%. The strong linkage disequilibrium between haplotypes at the D7S23 locus and the cystic fibrosis gene may be used to increase this probability. If fetal tissues are homozygous for the 6.6-kb band defined by pKM.19 and PstI and also homozygous for the 2.1-kb band with pXV-2c and TaqI, the chance of being affected increases from 80% to between 95% and 97%. We regard this as being sufficiently certain for use in phase determination.