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).     

Technical and theoretical considerations in the HLA typing of amniotic fluid cells for prenatal diagnosis and paternity testing

HLA typing of amniotic fluid cells has been used for the prenatal diagnosis of the HLA linked diseases congenital adrenal hyperplasia (21-OH-deficiency (21-OH-def) type) and complement C4 deficiency and it has also been used for the prenatal de termination of paternity. There are, however, technical difficulties in this test associated with the weak expression of some B locus antigens on amniotic fluid cells, and theoretical difficulties related to associations between particular HLA antigens and the 21-OH-def allele. Since certain HLA-B locus antigens are found in significantly increased frequencies among patients with 21-OH-def, there is a relatively high incidence of HLA-B homozygosity among the patients and over 40 percent of the parents of these patients share one or more HLA-B locus antigens. Results of some prenatal HLA typing tests may thus be difficult to interpret, and supplementary tests should be used whenever possible. HLA typing of amniotic cells is, however, the only available procedure for prenatal diagnosis of C4 deficiency and it is the best available procedure for prenatal determination of paternity. A modification of our original procedure allows HLA typing to be performed with increased numbers of HLA typing sera, and sera with optimum reactivity for amniotic fluid cells have now been selected for the definition of most of the more commonly expressed HLA antigens. Although amniotic fluid cells do not express DR antigens, amniotic fluid cells can be typed for the HLA-linked marker glyoxalase I (GLO) and this may be the informative for prenatal diagnosis in some cases.     

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.     

Prenatal diagnosis,fetal pathology,and cytogenetic analysis of mosaic trisomy 14

While true mosaicism occurs in only 0–25 per cent of genetic amniocenteses, nearly 2–5 per cent of amniotic fluid cell cultures contain a second cell line. In the common practice of prenatal diagnosis, an aberrant cell line confined to a single colony is usually disregarded. We present a case of mosaic trisomy 14 which was not detected on initial chromosome analysis. At birth, multiple malformations were apparent. Newborn cytogenetic studies revealed mosaicism [46,XX/46,XX,-14,+i(14q)] with an isochromosome 14 in 37 per cent of lymphocytes. Additional cells from the initial amniotic fluid culture were analysed post-delivery and the isochromosome 14 identified in only one of 12 total colonies. This case illustrates two important lessons in prenatal diagnosis. First, amniotic fluid cell cultures may not accurately reflect the relative distribution of the normal and abnormal cell lines within a mosaic fetus. Second, while it is generally reasonable to disregard mosaicism confined to a single colony, this policy will, on rare occasion, result in diagnostic error. This should be taken into consideration, particularly when dealing with autosomal trisomies potentially compatible with livebirth.     

Cytogenetic analysis of 2928 CVS samples and 1075 amniocenteses from randomized studies

We report cytogenetic results from a randomized Danish chorionic villus sampling (CVS) and amniocentesis (AC) study including 2928 placental and 1075 amniotic fluid specimens processed in the same laboratory. The results are presented in groups comparing CVS with amniocentesis and transabdominal (TA) CVS with transcervical (TC) CVS as randomized. More abnormalities and more ambiguous diagnostic problems were found in placental tissues than in amniotic cells. There were no diagnostic errors and no incorrect sex predictions. Mosaicism was detected in 1 per cent of all cases of CVS (discordancies included). When confirmation studies were done, 90 per cent were found to be confined to the placenta. Eight cases (0.7 per cent) of mosaicism/pseudomosaicism were seen in amniotic fluid specimens, and two cases of five with confirmation studies were confirmed in the fetus. The rate of mosaicism/pseudomosaicism in CVS and AC specimens differed (P <0.05). The rate of pseudomosaicism in cultures of villi and amniotic fluid cells was 0.5 and 0.6 per cent, respectively. Single-cell aneuploidy was observed in 1.8 per cent of villi and 1.4 per cent of amniotic fluid cell specimens. Maternal cell contamination (MCC) was seen more often after TC sampling (4.5 per cent) compared with TA sampling (1.5 per cent), but posed no problems in interpretation. Compared with the processing of cultured specimens, the short-term method of preparation of villi in our laboratory doubled the technicians' workload. For practical and economic reasons we have ceased the routine use of short-term preparations.     

Prenatal tests for Sanfilippo disease type B in four pregnancies

We report the prenatal diagnosis of two fetuses with Sanfilippo disease type B. In both pregnancies there were excessive amounts of heparan sulphate in amniotic fluid and the activity of N-acetyl-α-D-glucosaminidase was undetectable in cultured amniotic fluid cells. The predictions were confirmed by enzyme assay of cultured skin fibroblasts from the aborted fetus or the affected infant. The disorder was excluded for two other pregnancies at risk and the predictions are considered to be correct because of the normal progress of the healthy children.     

Amniotic fluid digestive enzymes: Diagnostic value in fetal gastrointestinal obstructions

The diagnostic value of amniotic fluid gamma-glutamyl-transpeptidase (GGTP) and intestinal alkaline phos-phatase (iALP) was evaluated in 55 patients who underwent amniocentesis for karyotyping because fetal gastric or small bowel dilatation had been detected by ultrasound. Gastrointestinal malformation was confirmed in 46 cases and there was no gastrointestinal anomaly in nine cases. Prenatal ultrasound was suggestive of gastroduodenal dilatation in 34 cases (group I) and small bowel dilatation in 21 cases (group II). In group I, amniotic fluid GGTP above the 99th percentile was 71 per cent sensitive and 100 per cent specific for a true anatomical defect of the digestive tract (mainly duodenal atresia). In group II, high levels of GGTP and/or iALP were 69 per cent sensitive and 83 per cent specific for a fetal digestive tract anomaly. In other words, when digestive tract dilatations were diagnosed by prenatal sonography, abnormal amniotic fluid enzyme activities were strongly suggestive of such an anomaly, the possibility of which was not precluded by normal amniotic fluid iALP and GGTP activities. But amniotic fluid digestive enzyme activities do not help in defining the prognosis.     

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.     

Prenatal diagnosis of trisomy 9 mosaicism: Two new cases

We present two prenatal cases of trisomy 9 mosaicism, both of which presented intrauterine growth retardation (IUGR) and other abnormal ultrasound findings. In case A, mosaicism was found in amniotic fluid cell cultures, of which 65 per cent were trisomic cells, on average. In case B, trisomic cells were present in amniotic fluid cell cultures (12 per cent) but none were found in fetal cord blood. After autopsy, cytogenetic findings were confirmed in different tissue cultures. It is concluded that echographic indicators are a very useful tool for a correct prenatal diagnostic interpretation of trisomy 9. Suspected trisomy 9 mosaicism always requires further investigation and fetal cord blood cytogenetic analysis may not be considered as providing an accurate diagnosis of fetal trisomy 9.     

First trimester monitoring of a pregnancy at risk for glucose phosphate isomerase deficiency

First trimester prenatal diagnosis was offered to the mother of a child affected by severe haemolytic anaemia due to glucose phosphate isomerase (GPI) deficiency. The mutant enzyme was characterized by an increased thermal lability. Both parents had 50 per cent normal red cell GPI activity. We have shown that the homozygous and heterozygous genotypes can be clearly distinguished from each other and controls by combinations of the measurement of enzyme activity and enzyme thermal lability. Examination of trophoblast cells obtained at 9 weeks of gestation led to the diagnosis of a GPI heterozygous fetus. The result was confirmed by analysis on uncultured and cultured amniotic fluid cells sampled at 16 weeks and by red blood cell studies of the healthy newborn. Prenatal diagnosis of GPI deficiency is indicated in families with previous cases resulting in severe haemolysis and mainly with the conservative view of arranging appropriate therapeutic measures for affected fetuses.     

Prenatal diagnosis of PIBIDS

In a well-documented PIBIDS family, two investigations of DNA excision repair showed a severe defect in lymphocytes from the index case (residual repair activities were 10.6–12.1 per cent). The values for the mother, father, and sister were within the normal range when compared with a healthy control. In the pregnant mother, a prenatal diagnosis of PIBIDS was made by measuring UV-induced unscheduled DNA synthesis in cultivated amniotic fluid cells. Results ranged between 12.5 and 26.1 per cent depending on the UV doses applied and were consistent with an affected fetus. The parents opted for a termination of pregnancy. Following a therapeutic abortion, fetal skin fibroblasts were tested and showed a severe DNA excision-repair defect of 9.2–13.5 per cent of residual activity.     

Chromosomal prenatal diagnosis: Study of 936 cases of intrauterine abnormalities after ultrasound assessment

Nine hundred and thirty-six prenatal chromosomal analyses were performed by four cytogenetic centres after ultrasound diagnosis of fetal abnormalities, amniotic fluid disorders, fetal growth retardation, and fetal or placental abnormalities. During the same period, 6515 fetal karyotypes were analysed because of maternal age. Frequencies of chromosomal aberrations in each case were respectively 4·4, 6·7 and 15·8 per cent, compared with 3·18 per cent when the fetal karyotype was performed because of maternal age. High rates of chromosomal aberrations are observed in cases of cervical hygroma, limb abnormalities, omphaloceles, duodenal stenosis, hydrocephalus, and facial abnormalities. In the case of polymalformations, this rate was 29·2 per cent. When malformations were seen together with an amniotic fluid disorder or growth retardation, 21·5 per cent chromosomal aberrations were observed. This frequency was 10·4 per cent when growth retardation was associated with an amniotic fluid disorder. Trisomy 13, 18, 21 and monosomy X accounted for 4/5 of all abnormalities in which we observed a high rate of triploidies (4·9 per cent) and balanced (3·3 per cent) or unbalanced (9·8 per cent) non-Robertsonian structural abnormalities. Sonographic ascertainment of these aberrations and prenatal characteristics of major anomalies are discussed.     

Evaluation of inorganic pyrophosphate in amniotic fluid as a mode of prenatal diagnosis of osteogenesis imperfecta

Using a modified procedure by Solomons and Styner (1969), an evaluation of inorganic pyrophosphate (PPi) was performed on the amniotic fluid of two fetuses at risk for osteogenesis imperfecta (OI) at 14½ weeks gestation. The parents of both cases had a previous child with OI, Type II. The normal control group at 14–16 weeks gestation had PPi values ranging from 22.0–59.2 ug/100 ml, with a mean of 38.6±9.51 ug/100 ml. In each at-risk fetus, the amniotic fluid PPi value was within normal range. The first baby was born phenotypically normal at term. Intrauterine radiographic and fetal sonograms were done on the second fetus at approximately 19 weeks gestation. Both showed evidence of OI, Type II. The pregnancy was terminated at 21 weeks. Radiologic studies of the aborted fetus were consistent with OI, Type II. Our results indicate that the evaluation of PPi levels in amniotic fluid is not the method of choice for prenatal diagnosis of IO.     

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.     

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.     

Non-ketotic hyperglycinaemia: Glycine/serine ratio in amniotic fluid–an unreliable method for prenatal diagnosis

We describe a prenatal diagnosis of a fetus at risk for non-ketotic hyperglycinaemia based on the glycine/serine ratio in amniotic fluid at 16 weeks of gestation. Although the glycine level and the glycine/serine ratio in amniotic fluid were within the normal range, the fetus was affected and therefore a false-negative prediction was made. The reliability of this method is questioned.     

Prenatal diagnosis of Chediak-Higashi syndrome

We report the first prenatal diagnosis of an affected fetus with Chediak-Higashi syndrome (CHS). Diagnosis was accomplished via fetal blood sampling at 17 menstrual weeks and was confirmed after birth. Retrospective measurement of the largest acid phosphatase-positive lysosomes in cultured amniotic fluid cells and chorionic villus cells showed that in CHS these lysosomes are significantly larger than those in normal cells. This method may be used for prenatal diagnosis of CHS by amniocentesis and chorionic villus sampling (CVS).     

A comparative study of microvillar enzyme activities in the prenatal diagnosis of cystic fibrosis

The potential of four enzyme-based analytical systems has been compared in the secondtrimester prenatal diagnosis of cystic fibrosis (CF). Direct activity measurements were made of γ-glutamyltranspeptidase (GGTP), aminopeptidase M (APM) and the intestinal isoenzyme of alkaline phosphatase (ALP). In the fourth system the proportions of total ALP inhibited by phenylalanine and homoarginine, respectively, were assessed. Each system was applied to amniotic fluid samples from 94 pregnancies with al in 4 risk of CF, divided into retrospective (36) and prospective (58) series. No system gave an absolute separation of affected from unaffected cases. Measurement of APM and intestinal ALP (phenylalanine-inhibitable ALP) gave a better detection rate for CF (35 of 41 cases, 85 per cent) than did measurement of GGTP (63 per cent) or assessment of ALP proportions (76 per cent). APM had a lower false positive rate (4 per cent) than intestinal ALP (8 per cent). For both the latter systems the detection rate of CF rose to 96 per cent (25 of 26), if gestations less than 17 weeks were excluded.     

Prenatal diagnosis of trisomy 18 mosaicism

Trisorny 18 mosaicism was found in multiple primary cultures of amniotic fluid cells and subsequently confirmed by chromosome analysis of several tissues derived from the aborted fetus. The overall frequency of the minority cell line was 25 per cent in the amniotic fluid cultures and 28 per cent in the fetal tissues although much intertissue variations were noted.