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.     

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.     

Evaluation of X,Y, 18, and 13/21 alpha satellite DNA probes for interphase cytogenetic analysis of uncultured amniocytes by fluorescence in situ hybridization

The major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. This prospective study evaluated the use of four commercially available centromeric DNA probes (DXZ1, DYZ1, D18Z1, and D13Z1/D21Z1) for direct analysis of uncultured amniocytes. One hundred and sixteen amniotic fluid samples were analysed by FISH and standard cytogenetics. This evaluation demonstrated that FISH with, X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory. In contrast, the 13/21 alpha satellite DNA probe hybridizing both chromosomes 13 and 21 was unreliable for prenatal diagnosis in uncultured amniocytes.     

Familial X centromere variant resulting in false-positive prenatal diagnosis of monosomy X by interphase FISH

Interphase fluorescent in situ hybridization (FISH) analysis performed on uncultured amniotic fluid cells from a female fetus revealed a single signal using an X chromosome alpha-satellite probe, and the absence of any signal using a Y chromosome alpha-satellite probe. This result was initially interpreted as monosomy for the X chromosome in the fetus. Subsequent chromosome analysis from the cultured amniotic fluid cells showed two apparently normal X chromosomes. FISH using the X alpha-satellite probe on metaphase spreads revealed hybridization to both X chromosomes, although one signal was markedly reduced compared to the other. The same hybridization pattern was observed in the mother of the fetus. This is the first report of a rare familial X centromere variant resulting in a false-positive diagnosis of monosomy X by interphase FISH analysis for prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

Parental mosaic trisomy 21 detected following maternal cell contamination of an amniotic fluid specimen from a normal male pregnancy

We report a case of maternal mosaic trisomy 21 ascertained at prenatal diagnosis as a result of maternal cell contamination of an amniotic fluid sample. A 34 year old female was referred for karyotyping because of a previous trisomy 21 pregnancy. Chromosome analysis of primary in situ cultures showed a karyotype of 47,XX, + 21[6]/46,XY[32]/46,XX[2]. Molecular testing demonstrated maternal cell contamination of the amniotic fluid sample and G-banded karyotyping of maternal blood showed that 3/200 cells had trisomy 21, consistent with the mother being a Down syndrome mosaic. A normal male baby with a 46,XY chromosome complement was delivered at 30 weeks. This case emphasises the need for close collaboration between cytogenetic and molecular genetics laboratories in resolving unusual cases of mosaicism. Copyright © 2007 John Wiley & Sons, Ltd.     

Cytogenetic studies of amniotic fluid taken before the 15th week of pregnancy for earlier prenatal diagnosis: A report of 114 consecutive cases

One hundred and fourteen samples of amniotic fluid taken before 15 weeks of gestation were cultured for cytogenetic studies. The results of culturing these early amniotic fluid (EAF) samples were compared with the results of culturing 114 standard amniotic fluid (SAP) samples taken after 15 weeks of gestation matched for maternal age and received in the laboratory within the same week. Cell culture was successful in all 114 of the EAF samples and in 111 SAP samples. There was no significant difference in the days to harvesting and days to reporting in the two groups. Three samples of SAP failed to grow and two EAF samples produced tetraploid karyotypes, so that in these five cases amniocentesis had to be repeated. These problems were attributed to toxicity of a fungicide used in the culture medium. Pseudomosaicism was noted in two EAF samples and one SAP sample; and maternal cell contamination was noted in one EAF and one SAP sample. Thus, culturing and karyotyping cells harvested from EAF and SAP are similar, indicating that EAF samples from 12–14-week pregnancies could be used for prenatal diagnosis.     

Prenatal diagnosis by in situ hybridization on uncultured amniocytes: Reduced sensitivity and potential risk of misdiagnosis in blood-stained samples

Maternal cell contamination was assessed in 18 macroscopically blood-stained amniotic fluid samples from male fetuses. The samples were analysed by double-target fluorescent in situ hybridization (ISH) with Y and X chromosome-specific probes. The only sample with an aberrant karyotype (47, XY, +18) was also analysed by hybridization with a chromosome 18-specific probe. An interpretation of extensive maternal cell contamination was made in two samples, one of which was the sample with trisomy 18. ISH with the chromosome 18-specific probe on this latter sample showed that the sensitivity of the ISH method for chromosome enumeration of uncultured amniotic fluid samples may be reduced in bloodstained samples. It was calculated that by using ISH for chromosome enumeration of the two extensively contaminated samples, a case of trisomy 21 might have been overlooked in both samples, while a case of trisomy 18 might only have been overlooked in one of the samples. It is concluded that ISH should not be used for chromosome enumeration of uncultured amniotic fluid samples that are macroscopically blood-stained without further technical developments.     

Rapid prenatal diagnosis of 14 cases of triploidy using fish with multiple probes

Fluorescence in situ hybridization (FISH) of chromosome-specific probes to interphase nuclei can rapidly identify aneuploidies in uncultured amniotic fluid cells. Using DNA probe sets specific for chromosomes 13, 18, 21, X, and Y, we have identified 14 fetuses where the hybridization pattern was consistent with a triploid chromosome constitution. In each case, the identification of fetal abnormalities by ultrasound examination initiated a request for rapid determination of ploidy status via prenatal FISH analysis of uncultured amniocytes. FISH produced a three-signal pattern for the three autosomes in combination with signals indicating an XXX or XXY sex chromosome complement. This hybridization pattern was interpreted to be consistent with triploidy. Results were reported to the physician within 2 days of amniocentesis and subsequently confirmed by cytogenetics. These cases demonstrate the utility of FISH for rapid prenatal identification of triploidy, particularly when fetal abnormalities are seen with ultrasonographic examination.     

First-trimester prenatal diagnosis of aspartylglucosaminuria

Fetal aspartylglucosaminuria (AGU) was studied during the first trimester of pregnancy in six at-risk pregnancies using chorionic villus samples. The activity of aspartylglucosaminidase (AGA) was high in five cases, indicating an unaffected fetus. This was confirmed through delivery of healthy newborns with a normal pattern of urinary oligosaccharides. Low enzyme activity in an uncultured biopsy specimen and in cultured amniotic fluid cells in one case demonstrated that the fetus was affected. The pregnancy was terminated and the prenatal diagnosis was confirmed by showing reduced AGA activity in cultured fibroblasts of the fetus.     

46,XX/46,XY chromosome complement in amniotic fluid cell culture followed by the birth of a normal female child

Experience indicates that the most likely explanation for a mixture of 46,XX/46,XY cells in an amniotic fluid sample is that of maternal cell contamination and that a normal male child is to be expected at birth. We report the birth of a normal female child following prenatal diagnosis of such a mixture. Extensive postnatal studies failed to reveal an XY cell line. The possible sources of the XY cell line are discussed, as are the various techniques that were applied in an effort to discover it's origin. Cross-contamination of samples could be ruled out and there was no evidence of an unsuspected twin pregnancy. It is clear from this case that not all 46,XX/46,XY results obtained in amniotic fluid can be assumed to represent maternal cell contamination and some effort should be made to eliminate other potential sources for such a mixture.     

Alpha-fetoprotein and acetylcholinesterase are not predictive of fetal junctional epidermolysis bullosa,Herlitz variant

Junctional epidermolysis bullosa, Herlitz variant (junctional EB-Herlitz) is a lethal autosomal recessive skin disorder currently amenable to prenatal diagnosis only by direct analysis of fetal skin. However, elevated levels of alpha-fetoprotein, as well as the presence of acetylcholinesterase in amniotic fluid, have been associated with other severe fetal genodermatoses. Fetal skin samplings were performed in ten pregnancies at risk for fetal junctional EB-Herlitz, with three fetuses affected on the basis of electron microscopic detection of blisters within the lamina lucida and abnormal hemidesmosomes. In neither affected nor unaffected pregnancies were maternal serum or amniotic fluid alpha-fetoprotein levels elevated. Moreover, alphafetoprotein levels in both maternal serum and amniotic fluid were not statistically different comparing affected and unaffected fetuses. Acetylcholinesterase was not present in the amniotic fluid samples of the three affected pregnancies. Unlike other severe fetal genodermatoses, neither alpha-fetoprotein nor acetylcholinesterase was predictive of junctional EB-Herlitz.     

The cerebro-hepato-renal (Zellweger) syndrome: Prenatal detection based on impaired biosynthesis of plasmalogens

Prenatal diagnosis of the cerebro-hepato-renal (Zellweger) syndrome has been performed in 10 pregnancies at risk by measuring both the activity of acyl CoA: dihydroxyacetonephosphate acyltransferase (DHAP-AT) and the de novo plasmalogen biosynthesis, either in cultured amniotic fluid cells or in fibroblasts cultured from a chorionic villus biopsy. In 7 of the pregnancies both tests indicated no abnormality. All 7 continued to term and normal infants were delivered. However, in amniotic fluid cells from 2 fetuses affected by Zellweger syndrome unequivocal differences from control values were found. The activity of DHAP-AT was clearly deficient and the de novo plasmalogen biosynthesis was impaired. In one pregnancy at risk prenatal diagnosis was performed during the first trimester by measuring both the DHAP-AT activity and the de novo plasmalogen biosynthesis in fibroblasts cultured from a chorionic villi biopsy. From the deficient DHAP-AT activity and the impaired de novo plasmalogen biosynthesis it was concluded that the fetus was affected. This was confirmed biochemically after induced abortion. It can be concluded that measurement of the DHAP-AT activity and the de novo plasmalogen biosynthesis provides convenient methods for the early prenatal detection of Zellweger syndrome.     

A simple and effective approach for detecting maternal cell contamination in molecular prenatal diagnosis

The presence of maternal cells in fetal samples constitutes a serious potential source for prenatal misdiagnosis. Here we present our approach for detecting maternal cell contamination (MCC) at prenatal diagnosis for eight monogenic disorders (autosomal recessive: β-thalassaemia, sickle-cell anaemia, cystic fibrosis, prelingual deafness; autosomal dominant: achondroplasia, Huntington disease, myotonic dystrophy, neurofibromatosis type I; X-linked: spinobulbar muscular atrophy). Our aim was to apply a simple and low-cost approach, which would easily and accurately provide information on the fetal tissue MCC status. MCC testing was applied to cases of recessive inheritance where the primary mutation screening of the fetus revealed the presence of the maternal mutation, to cases concerning dominant inheritance and to cases of multiple gestation. The potential presence of maternal cells was determined by the amplification of the 3′-HVR/APO B, D1S80, THO1 and VNTRI of vWf polymorphic loci, which have previously demonstrated high heterozygosity in Caucasians. Among 135 prenatal diagnoses, 44 finally needed to be tested for MCC (32.6%). MCC was detected in four cases, where DNA was isolated directly from chorionic villi samples (CVS), and in one case with DNA isolated directly from amniotic fluid (AF). In almost 90% of cases a simple test of one polymorphic locus provided sufficient information about MCC. The choice of the appropriate locus is therefore essential, while the simultaneous screening of both parents provides the means for distinguishing non-informative sites about MCC. Copyright © 2002 John Wiley & Sons, Ltd.     

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 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.     

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.     

Correct prenatal diagnosis of a hurler fetus where amniotic fluid cell cultures were of maternal origin

Contamination of amniotic fluid cell cultures by maternal cells can be expected to lead to misdiagnosis of fetal genotype in 0·1 to 0·5/100 cultures, when assays are carried out directly on cultured cells. Chemical analysis of the cell-free amniotic fluid supernatant may overcome this source of error and has the added advantages of speed and independence from amniotic cell culture failure. We describe a pregnancy at risk for Hurler's disease where amniotic cells cultured at amniocentesis had a female karyotype and an α-iduronidase activity towards both phenyl and 4-methylumbelliferyl substrates at the lower end of the normal range, suggesting a heterozygous fetus. An affected fetus was predicted, however, because of a high concentration of dermatan sulphate in the amniotic fluid. The discrepancy between these findings was shown to be due to maternal cell contamination of amniotic fluid cell cultures by the birth of a male infant with Hurler's disease.     

Prenatal diagnosis of mucolipidosis type II on first-trimester amniotic fluid

We investigated the possibility of prenatal diagnosis of mucolipidosis type II (ML II) by lysosomal enzyme determination on amniotic fluid obtained at 11 weeks of gestation in three pregnancies at risk. Diagnosis of ML II was made in one case on the basis of increased levels of five lysosomal enzymes tested. The diagnosis was confirmed on cultured chorionic cells, their cultured medium, 17–week amniotic fluid, and fetal plasma obtained for confirmation prior to the termination of pregnancy.     

Detection and enumeration of colonic mucosal cells in amniotic fluid using a colon epithelial-specific monoclonal antibody

Since its introduction, prenatal diagnosis of chromosomal and metabolic disorder by mid-trimester amniocentesis has relied upon the use of a mixture of fetal cells obtained from amniotic fluid. Little knowledge has been gained in the sorting of these cells for diagnosis of tissue-specific disorders. In an attempt to determine the contribution of fetal colonic mucosal cells to the overall amniocyte population, we used the colonic epithelial-specific monoclonal antibody (MC-Ab) 7E₁₂H₁₂, IgM isotype. Specimens of the small intestine, colon, buccal mucosa, kidney, urinary bladder, and umbilical cord were obtained from electively aborted normal fetuses of 12–28 weeks' gestation. All of these specimens were examined with 7E₁₂H₁₂ by the immunoperoxidase technique. The MC-Ab reacted with the colonic epithelial cells but not with any of the other tissues. In addition, 40 amniotic fluid samples obtained from women between 16 and 18 weeks of gestation, who underwent amniocentesis because of advanced maternal age, were tested using a fluorescent activated cell sorter. Among the amniotic fluid specimens examined, 18·4 ± 10·3 percent cells reacted with 7E₁₂H₁₂. Double immunofluorescence studies revealed that all Mc-Ab-stained cells contained secretory component, confirming that they were epithelial in origin. All fetuses whose amniotic fluid was analysed had normal karyotypes and amniotic fluid alpha-fetoprctein levels that were also normal. This study demonstrates that cell-specific Mc-Ab can be used to detect colon cells in the amniotic fluid and that colon cells contribute significant numbers in the mixture of amniotic fluid cells. This technique could be helpful in the prenatal diagnosis of disorders in which the flow of amniotic fluid through the fetal intestine is impaired, such as cystic fibrosis, imperforate anus, Hirschsprung aganglionic megacolon, and intestinal atresia.     

Prenatal diagnosis of glutaryl-CoA dehydrogenase deficiency: Experience using first-trimester chorionic villus sampling

We have performed prenatal diagnosis for glutaryl-CoA dehydrogenase (GDH) deficiency in 16 pregnancies at risk by measuring the enzyme activity in chorionic villus samples. In most cases, GDH activity was measured both in uncultured chorionic villus samples and in cultured chorionic cells. In 4 of the 16 cases, an affected fetus was predicted, while the remaining cases were found to be normal. In three of the four affected cases, GDH activity was measured in both uncultured and cultured chorionic cells and the correct diagnosis established by both measurements. In the fourth case, only cultured cells were investigated because the chorionic villus sample was too small for the direct assay. All four pregnancies predicted to be affected were interrupted and the diagnoses confirmed on the aborted material in three of the cases. In the fourth case, no material was available for investigation. Of the 12 pregnancies predicted to be unaffected, ten cases resulted in the birth of healthy unaffected babies while two pregnancies are still in progress.