A case of prenatal paternity discrimination

A case is described in which prenatal paternity determination was required, on account of suspected incest. Chromosome heteromorphisms were uninformative, but discrimination was achieved by HLA typing.     

Prenatal diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency by steroid analysis in the amniotic fluid of mid-pregnancy: Comparison with HLA typing in 17 pregnancies at risk for CAH

Amniotic fluid (AF) levels of 17-hydroxyprogesterone (17OHP) and testosterone (T) were determined at 16–17 weeks in 17 pregnancies at risk for CAH and results compared to 75 normal controls. The fetus was predicted to be unaffected in 12 cases on the findings of normal AF levels of both 17OHP and T and the latter allowed a correct prediction of fetal sex in all instances. HLA typing confirmed normality in 12 cases revealing 5 carriers, 5 homozygous normal and 2 indeterminate. Steroid levels of the 2 groups were similar. Three fetuses were predicted to be CAH affected on unambiguously high levels of 17OHP and T (in female only). HLA typing was in agreement, and the diagnosis was confirmed in 2 abortuses and a female newborn by physical and hormonal studies. In the last 2 cases AF levels of OHP and T were normal but HLA (A/B/C) genotypes were identical to the CAH affected siblings. Normal physical and hormonal findings in the 2 aborted fetuses would exclude the possibility of an in utero virilizing form of CAH. The discrepancy could be explained on the basis that the fetuses had an allelic form of 21-hydroxylase deficiency or on the basis of recombination (not fully tested). It is concluded that a fully informative prenatal diagnosis of CAH should not rely entirely on HLA typing but on hormonal studies.     

Prenatal diagnosis of congenital adrenal hyperplasia (21-OH deficiency type) by HLA typing

The close genetic linkage between HLA-B and congenital adrenal hyperplasia due to 21-hydroxylase deficiency permits prenatal diagnosis of an affected fetus by HLA typing of amniotic fluid cells in pregnancies at risk. Some families at risk, especially those with an affected girl with ambiguous genitalia, will only plan another pregnancy if a prenatal diagnosis is possible. After HLA typing of the index case, parents and eventually grandparents, the family were informed of the possibility of a prenatal diagnosis. Fibroblast cell lines were initiated from skin biopsies of the index cases and parents and were used as controls in the tests. HLA typing of the fetus was done on amniotic fluid cells grown in vitro using first, a microcytotoxicity test and second quantitative microabsorption test. Ten prenatal diagnoses are reported. In two cases the HLA genotype indicated an affected fetus, examination of the aborted fetuses was in agreement with the diagnosis. In one case an affected male fetus was diagnosed, the pregnancy is in progress. In seven cases an unaffected infant was predicted (four carriers and three homozygous normal infants).     

HLA-A,B,C,DR typing and 17-OHP determination for second trimester prenatal diagnosis of 21-hydroxylase deficient CAH

In 18 families at risk for the HLA-linked, 21-hydroxylase deficient form of autosomal recessive congenital adrenal hyperplasia (CAH), prenatal diagnosis (PD) was performed using two methods: (1) HLA-A,B,C typing and in the latter 11 cases also DR typing of cultured amniotic fluid cells (AFC) using the standard microcytotoxicity assay, and (2) measurement of second trimester amniotic fluid 17-hydroxyprogesterone (17-OHP) concentration using gel chromatography and radioimmunoassay. The accuracy of the prenatal predictions was confirmed by postnatal HLA typing of umbilical cord blood lymphocytes and by clinical evaluation. In 16/18 families, both HLA typing of AFC and 17-OHP measurements proved informative for PD. The predictions of both methods were concordant in 14/16 families (88 per cent). In ten of these families, a normal fetus was predicted, and in four, an affected fetus; all pregnancies were carried to term and all predictions were confirmed postnatally. In 2/16 cases (12 per cent), however, the predictions were discordant: the prenatal HLA typing indicated an affected fetus, whereas the 17-OHP values predicted a normal fetus. Both pregnancies were continued and two healthy boys were delivered. The discordance proved to be due to a ‘missed’ HLA antigen in one case and to serologically cross-reactive HLA antigens in the second. Finally, in 2/18 cases, prenatal assessment of fetal genotype had to rely on HLA typing alone as 17-OHP measurement was not performed in one family and in the second family the 17-OHP values obtained were not informative due to inadvertent continuation of hormone therapy to the date of amniocentesis. In both cases, the HLA typing data accurately predicted a normal fetus. In conclusion, a combination of HLA typing of cultured AFC and 17-OHP measurements of amniotic fluid permits accurate prenatal diagnosis of CAH in most cases (88 per cent). In addition, the supplementary use of HLA-DR typing of AFC as presented here for the first time proved helpful in families with HLA-A.B homozygosity due to parental sharing of antigens and can be informative for identifying HLA-B/21-OH recombinant haplotypes.     

Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency by amniotic fluid steroid analysis

The concentration of 17OH-progesterone was measured in second trimester amniotic fluid samples from 12 mothers who previously had had an infant with congenital adrenal hyper-plasia due to 21-hydroxylase deficiency. In 4 affected pregnancies, the concentrations were more than 2 S.D. higher than those determined in 44 samples from normal pregnancies (mean ± S.D., 8·1 ± 2·4 nmol/1). The remaining 8 pregnancies were predicted to be unaffected based on the results of amniotic fluid concentrations within the normal range. In each instance, the infant was normal. The results indicate that measurement of amniotic fluid 17OH-progester-one concentrations during the second trimester is an accurate prenatal test for 21-hydroxylase deficiency. The results should be supplemented with determination of fetal sex by karyotype analysis on the amniotic fluid cells.     

Early prenatal diagnosis of 21-hydroxylase deficiency using amniotic fluid 17-hydroxyprogesterone determination and DNA probes

The results of early prenatal diagnoses of congenital adrenal hyperplasia are reported. The determination of 17-hydroxyprogesterone values in amniotic fluid taken transabdominally at 11 weeks of gestation enabled prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency. There is a clear-cut difference between normal and pathological values at that time of pregnancy. This method of diagnosis can be combined with genotyping of the fetus by HLA-DNA probes on chorionic villus sampling or can be used alone. Prenatal diagnosis with a 21-OH probe is possible when a preliminary study has demonstrated that the index case is homozygous for the deletion.     

Rapid prenatal diagnosis of aneuploidy from uncultured amniotic fluid cells using five-colour fluorescence in situ hybridization

In this paper we describe the use of five-colour fluorescence in situ hybridization for prenatal diagnosis of aneuploidy using uncultured amniotic fluid cells. The analysis is based on ratio mixing of dual-labelled probes and digital imaging for the detection and visualization of five different probes specific for the five target chromosomes, 13, 18, 21, X, and Y. A retrospective blind analysis of 30 coded uncultured amniotic fluid samples correctly detected fetal sex and five trisomy 21 cases. Multicolour fluorescence in situ hybridization used in this way allows rapid and simultaneous detection of the most frequent aneuploidies.     

Citrulline in amniotic fluid and the prenatal diagnosis of citrullinemia

We report relatively high citrulline concentration in amniotic fluid of a citrullinemic fetus suggesting that prenatal detection of this condition could be done on this basis in conjunction with a direct or an indirect determination of argininosuccinate synthetase activity in amniotic fluid cells.     

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Accuracy of amniotic fluid testing before 21 weeks' gestation in prenatal diagnosis of congenital cytomegalovirus infection

Cytomegalovirus (CMV) is the most common cause of intrauterine infection. Recent publications show amniocentesis to have an 81–100 per cent sensitivity in antenatal diagnosis after 21 weeks' gestation. Testing before 21 weeks' gestation is less well documented. We performed 36 amniocenteses between 14 and 20 weeks' gestation. The sensitivity was 45 per cent and the specificity 100 per cent. Implications and possible causes of this low sensitivity are discussed.     

Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders: A methodological study

The influence of culture conditions on the ultrastructure and enzyme activities of amniotic fluid cells are reported. Morphological changes were determined as a function of the number of lysosomal-like inclusion bodies per cell, and these results correlated to the activity of Thiexosaminidase, a-mannosidase, β-glucuronidase, arylsulphatase C and 5′ nucleotidase. The parameters examined were pH of the culture media, type of media, increasing cell passage and day of harvest. Our results indicate that enzyme activities are less sensitive to changes in culture conditions as compared to ultrastructural changes. We therefore recommend that in order to obtain reliable ultrastructural results for the diagnosis of storage disorders, cultures should be grown in MEM as the culture medium, the pH of the medium carefully monitored to remain below pH 7·4, examining the cultures no later than the eighth cell passage and no later than the 10th day after subculture.     

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.     

False-positive diagnosis of trisomy 21 using fluorescence in situ hybridisation (FISH) on uncultured amniotic fluid cells

An amniocentesis was performed on a 22-week pregnancy following the detection of foetal abnormalities on ultrasound. A rapid aneuploid screen using fluorescence in situ hybridisation on uncultured amniotic fluid cells revealed 3 signals for chromosome 21, consistent with trisomy 21. In situ metaphase cultures revealed a 46,XY normal male karyotype. These discordant results may be explained by a sub-standard batch of the commercially available probe or alternatively, a very specific variation within the sample interacting with the probe. Alternative strategies are proposed in order to safeguard against inappropriate clinical action as a consequence of discordant results. Copyright © 2003 John Wiley & Sons, Ltd.     

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.     

European collaborative study on prenatal diagnosis: Mosaicism,pseudomosaicism and single abnormal cells in amniotic fluid cell cultures

A report is given of the results of a European collaborative study on mosaicism, pseudomosaicism and single abnormal cells in amniotic fluid cell cultures. The mean frequency of cases with mosaicism was 0.10 per cent, with pseudomosaicism 0.64 per cent and with single abnormal cells 2.83 per cent in a series of 44 170 amniotic fluid samples. There was no significant difference between the colony (in situ) and the flask method with regard to the frequency of mosaicism. Pseudomosaicism and single abnormal cells were more frequent in cases studied with the flask method probably due to other factors than the method of cultivation of the cells. The frequency of maternal cell contamination was 0.17 per cent and the frequency of wrong sex assignment was 0.11 per cent. A more correct estimation is obtained if these frequencies are doubled. There was a considerable variation between laboratories with regard to the frequencies given above. One reason for this variation is that there are no sharp limits between mosaicism, pseudomosaicism and single abnormal cells. Thus the material contained cases diagnosed as having pseudomosaicism which turned out to be mosaics at birth and to have an abnormal phenotype. These cases were very rare but pose a definite problem in prenatal cytogenetic diagnosis.     

Prenatal diagnosis of citrullinemia: Elevated levels of citrulline in the amniotic fluid in the three affected pregnancies

In three pregnancies at risk for citrullinemia affected fetuses were predicted both by strongly increased levels of citrulline in the amniotic fluid and by the reduced incorporation of ¹⁴C-citrulline into TCA-precipitable material in cultured amniotic fluid cells. The prenatal diagnoses of affected fetuses were confirmed after termination of the pregnancies by direct and indirect assays of argininosuccinate synthetase in the fetal livers and fibroblasts respectively. Measurement of the citrulline concentration in amniotic fluid appears to be a valuable adjunct in the prenatal diagnosis of citrullinemia.     

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.