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 (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.     

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.     

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 and prenatal imaging features of fetal monosomy 1p36

Deletion of the distal end of the short arm of chromosome 1 (1p36) is thought to be a common terminal chromosomal deletion. However, few cases prospectively diagnosed prenatally have been reported. In this case, prenatal ultrasound at 21 weeks of gestation noted the fetus to have mild ventriculomegaly (Vhanterior = 11 mm and Vhposterior = 12 mm) and increased nuchal edema (6 mm). Maternal serum α-fetoprotein was normal unlike in a majority of previously described cases. The prenatal ultrasound features were further clarified with fetal MRI. Chromosome analysis following amniocentesis demonstrated a 1p36 deletion, which was confirmed by fluorescence in situ hybridization (FISH). The syndrome associated with 1p36 deletion is well described in infants and is characterized by typical facial features (prominent forehead, straight eyebrows. deep-set eyes, flat nasal bridge and a pointed chin). Other associated features are neurodevelopmental delay, seizures, cardiomyopathy and neurosensory hearing impairment. This case supplements our knowledge of the prenatal features of 1p36. Identification of this deletion by direct chromosomal analysis can be technically difficult and vigilance is required to improve diagnosis. FISH analysis is an important diagnostic adjunct where the diagnosis is suspected following classical G-banding techniques. However, in this chromosomal anomaly there remain few characteristic prenatal signs that are readily diagnosed with prenatal imaging. Copyright © 2007 John Wiley & Sons, Ltd.     

Congenital nephrosis in low-risk pregnancies

Congenital nephrosis is an autosomal recessive disorder requiring neonatal renal transplant for survival. The postnatal diagnosis rests upon the electron microscopic evaluation of the epithelial foot processes and basal membrane of the glomeruli. The prenatal diagnosis can be suspected in the presence of a positive family history with an amniotic fluid (AF) alpha-fetoprotein level greater than 5 standard deviations (SD) above the population mean accompanied by a negative AF acetylcholinesterase, absent haemoglobin F, and an unremarkable fetal sonographic examination. We reviewed our series of seven cases of congenital nephrosis fulfilling the above criteria; four cases had negative family histories, and in two cases the diagnosis of congenital nephrosis was further supported by the presence of elevated AF albumin concentrations. We conclude that (1) the prenatal diagnosis of congenital nephrosis is feasible in a low-risk population, and (2) an elevated AF albumin concentration may represent an additional marker for the diagnosis of congenital nephrosis, even though false-negative results have been reported.     

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.     

Pseudomosaicism for trisomy 13: Three case reports

Pseudomosaicism is of particular concern in prenatal diagnosis when it involves mosaicism for a cytogenetic abnormality associated with a clinical syndrome which is compatible with postnatal life, such as trisomies for chromosomes 13, 18, and 21. The lack of data regarding the outcome of pregnancies involving these specific kinds of pseudomosaicism makes genetic counselling difficult. Three cases of prenatal diagnosis of pseudomosaicism for trisomy 13, each of which had a normal outcome, will be presented (Tables 1 and 2). The three main areas for consideration are: (1) the genetic counselling issues, (2) the additional prenatal diagnostic options available to evaluate the status of the fetus in an attempt to identify some of the clinical features of trisomy 13, and (3) the outcome of the pregnancies.     

Increased nuchal translucency as a prenatal manifestation of congenital adrenal hyperplasia

We present two cases of pregnant women with a previous history of congenital adrenal hyperplasia. In both cases the only abnormal feature in the initial pregnancy had been increased nuchal translucency at 10–14 weeks of gestation. The fetal karyotype was normal and a diagnosis of congenital adrenal hyperplasia was made after delivery. In their current pregnancies, both fetuses also had increased nuchal translucency and normal fetal karyotype. Diagnosis of 21-hydroxylase deficiency was made prenatally by DNA analysis. These findings in four affected fetuses suggest that congenital adrenal hyperplasia should be added to the list of genetic anomalies associated with an increase in nuchal translucency. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis of a de novo supernumerary marker derived from chromosome 16

Marker chromosomes are supernumerary chromosomes of unknown origin and are seldom found in prenatal diagnosis. Application of fluorescent in situ hybridization (FISH) allows the identification of the chromosomal origin of markers. Estimation of the risk of an abnormal phenotype outcome can be enabled by collecting data on phenotypes associated with markers of the same chromosomal origin. So far only very few cases of prenatal diagnosis of de novo supernumerary markers derived from chromosome 16 have been reported. Here the prenatal diagnosis of a de novo supernumerary marker chromosome 16 is described and the relevant literature discussed. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature

Since 1993, the position of the American College of Medical Genetics (ACMG) has been that prenatal interphase fluorescence in situ hybridization (FISH) is investigational. In 1997, the FDA cleared the AneuVysion^® assay (Vysis, Inc.) to enumerate chromosomes 13, 18, 21, X and Y for prenatal diagnosis. Data is presented from the clinical trial that led to regulatory clearance (1379 pregnancies) and from retrospective case review on 5197 new pregnancies. These studies demonstrated an extremely high concordance rate between FISH and standard cytogenetics (99.8%) for specific abnormalities that the AneuVysion assay is designed to detect. In 29 039 informative testing events (6576 new and 22 463 cases in the literature) only one false positive (false positive rate=0.003%) and seven false negative results (false negative rate=0.024%) occurred. A historical review of all known accounts of specimens tested is presented (29 039 using AneuVysion and 18 275 specimens tested with other probes). These performance characteristics support a prenatal management strategy that includes utilization of FISH for prenatal testing when a diagnosis of aneuploidy of chromosome 13, 18, 21, X or Y is highly suspected by virtue of maternal age, positive maternal serum biochemical screening or abnormal ultrasound findings. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis of Huntington's disease (HD): Experiences with six cases and PCR

In the course of a 2-year predictive testing programme for Huntington's disease (HD), six couples from a total of 52 applicants requested prenatal testing. In each case, the pregnancy was in the first or second trimester when the couples were referred for DNA diagnosis. In five cases, exclusion testing was offered; in one case, a person at risk with an increased risk of being a gene carrier requested prenatal diagnosis. In all cases, informative markers for prenatal testing could be determined. Whenever possible, the newer technique of polymerase chain reaction (PCR) for D4S125 was applied to perform rapid prenatal diagnosis. Two couples withdrew before chorionic villus sampling was undertaken; prenatal diagnosis was completed in the remaining four cases. After exclusion testing, two pregnancies were determined to have an increased risk and two fetuses to have a low risk of being HD gene carriers.     

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.     

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.     

Molecular prenatal diagnosis of Smith–Lemli–Opitz syndrome is reliable and efficient

Smith–Lemli–Opitz (RSH) syndrome (SLOS, OMIM 270400) is a relatively common, autosomal recessive disorder of cholesterol biosynthesis with a broad spectrum of phenotypic abnormalities caused by mutations of the 7-dehydrocholesterol reductase gene (DHCR7) on chromosome 11. Prenatal diagnosis can be established by detection of elevated 7-dehydrocholesterol or of SLOS-causing mutations in the DHCR7 gene. We report here our experience with molecular prenatal diagnosis of SLOS. Mutation analysis of the DHCR7 gene was performed in chorionic villus samples of 13 pregnancies of couples with a family history of SLOS and known SLOS genotypes. This approach is accurate and reliable. If facilities for biochemical analysis are not available, or in cases with ambiguous biochemical patterns, molecular prenatal diagnosis is an attractive, alternative option. Copyright © 2002 John Wiley & Sons, Ltd.     

A pipette method for rapid karyotyping in prenatal diagnosis

The 'pipette method' is introduced as a method of prenatal diagnosis which is in competition with the 'in situ' and the 'trypsinization' technique. It is sufficiently standardized for routine diagnosis and the banding techniques currently used in prenatal diagnosis (G, Q, C-banding and NOR) have been adapted for it. In 180 cases from 27 different centres, the 'pipette method' was employed for chromosomal harvesting in order to save time. An average of 6·6 days was taken to achieve a result. There was a pathological karyotype in 28 cases (16·1 per cent) and this high proportion can be related to cases where ultrasound scan has led to a diagnosis of 'suspected chromosomal abnormality'. This technique is also of use in advanced stages of pregnancy. The early recognition of the fetal karyotype can contribute to the future management of the pregnancy. The 'pipette method' can also be used in chromosomal harvesting of tumour cells and fibroblast cultures.     

Comparison of chorionic villus sampling and amniocentesis: Current status of prenatal diagnosis in Japan

Data were tabulated and compared for cases involving prenatal diagnostic procedures performed in the 8 years between January 1985 and December 1992. Of a total of 2781 pregnancies, 2546 cases (91·5 per cent) were diagnosed by amniocentesis, and 235 (8·5 per cent) by chorionic villus sampling (CVS). These findings reflect the fact that amniocentesis has taken deep root as the prenatal diagnostic procedure widely acknowledged by society, whereas even the existence of CVS has yet to be generally recognized. Analysis of our clinical and laboratory results and complication rates, however, suggests that CVS is a safe and accurate alternative to amniocentesis which should and can be positively presented to all patients who are candidates for prenatal diagnosis.     

Prenatal diagnosis of mosaicism 46, XX/46, XX, −21, +t(21q21q)

Mosaicism for a structural chromosome abnormality in amniotic cell cultures indicative of true fetal mosaicism is a rare event. In addition to the laboratory findings the clinical interpretation for counselling in such cases is based on observation of the same abnormality in liveborns as well as previous experience with prenatal diagnosis of the same or similar abnormalities. We report here the prenatal diagnos is of 46,XX/46,XX,−21,+t(21q21q) which was confirmed in fetal skin cell and amnion cell cultures.     

Prenatal diagnosis of haemoglobin disorders by cordocentesis at 12 weeks' gestation

Prenatal diagnosis of haemoglobin disorders is accepted to be a useful procedure to avoid births of infants with homozygous diseases. Advances in sampling and molecular techniques, such as polymerase chain reaction (PCR) and chorionic villus sampling (CVS), have made earlier and safer first-trimester prenatal diagnosis possible. However, these procedures need previous studies of at-risk couples, which can be very time-consuming when a number of different β-thalassaemia mutations occur in the region. We describe the possibility of making a first-trimester prenatal diagnosis by cordocentesis and fetal blood analysis at the 12th week of gestation. We found no statistically significant difference (p>0.05) between β/γ values in fetuses at the 12th and 18th weeks of gestation. In seven affected fetuses aborted at the 12th week of gestation, the diagnosis was confirmed in all cases by PCR analysis. These findings suggest that early cordocentesis could be an alternative procedure to CVS and PCR analysis.