Prenatal detection of trisomy 13 from amniotic fluid by quantitative fluorescent polymerase chain reaction

Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. However, this requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. An alternative method, which is rapid, inexpensive and suitable for diagnosing trisomies, even from single fetal cells, is the fluorescent polymerase chain reaction (PCR) using polymorphic small tandem repeats (STRs). In this paper, we present the method of rapid prenatal detection of trisomy 13 from amniotic fluid using fluorescent PCR and two highly polymorphic STRs (D13S258 and D13S631). The results obtained by quantitative fluorescent PCR amplification of fetal DNA were concordant with amniocyte karyotyping results in all cases. Two cases of trisomy 13 were detected from 212 amniotic fluids and the results obtained from D13S631 and D13S258 amplification are presented. In the first trisomy 13 case, a triallelic pattern was detected by both markers, and in the second case, D13 markers showed a characteristic 2:1 dosage allele ratio, both of which demonstrate trisomy 13 status. All other heterozygous disomic samples showed an allele intensity ratio of 1:1.     

Prenatal diagnosis of triploidy and trisomy 21 through fetal erythroblasts isolated from maternal blood

BACKGROUND: A long-sought goal of medical genetics has been the development of prenatal diagnostic procedures that do not endanger the conceptus. The safety of noninvasive methods for prenatal diagnosis would be especially attractive because they could be extended to all pregnant women, regardless of their ages or histories. Noninvasive prenatal diagnosis for the entire population might be possible recovering fetal cells from maternal blood. For this purpose, we have studied fetal erythroblasts. MATERIALS AND METHODS: To evaluate the potential of the method for clinical use, we studied maternal blood samples from 11 women referred to us for prenatal diagnosis between 15 and 20 weeks of gestation. For simple and effective enrichment of fetal nucleated erythrocytes from peripheral maternal blood, we combined a triple density gradient and magnetic-activated cell sorting (MACS) of anti-CD71 transferrin receptor antibody labeled cells. The isolated cells were analysed by using dual-colour interphase fluorescent in situ hybridization (FISH) with X-, Y-, 18- and 21-specific DNA probes. RESULTS: Chromosomal abnormalities detected on enriched fetal cells include trisomy 21 and triploidy. CONCLUSIONS: Based on the current results it is suggested that the technique described here is a simple, fast, efficient and reliable method for non invasive prenatal diagnosis.     

Rapid genetic diagnosis at 7-9 weeks gestation: diagnosis of sex, single gene defects and DNA fingerprint from coelomic samples

Prenatal diagnosis (chorionic villus sampling (CVS) or amniocentesis) is performed at a relatively late stage of pregnancy (11-18 weeks). Such tests have significant disadvantages including increased risk of miscarriage and delay before results are known. Earlier prenatal diagnosis (< 11 weeks) has been discontinued because of the risk of fetal abnormalities. Recently fetal cells have been recovered from the coelomic cavity at 7-12 weeks gestation (coelocentesis). This study has established that highly sensitive fluorescent polymerase chain reaction (PCR) can provide rapid (4-5 h), reliable and accurate multiple genetic diagnoses (sexing and single-gene diagnosis) from coelomic cells. As prenatal diagnosis has a significant risk of contamination, we have also shown that coelomic cells can be simultaneously DNA fingerprinted to determine that contamination has not occurred. This earlier method of prenatal diagnosis would be very valuable, as it may overcome some problems of later conventional prenatal diagnosis and allow reassurance/treatment to be undertaken at a much earlier stage. Successful application of these techniques may supersede alternative methods of prenatal diagnosis. Although these techniques appear very promising, extensive clinical trials must be undertaken to determine safety of coelocentesis, diagnostic reliability and accuracy in a clinical setting.     

Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.     

Prenatal diagnosis by minimally invasive first-trimester transcervical sampling is unreliable

OBJECTIVE: We investigated whether reliable prenatal diagnosis is possible from fetal cells harvested transcervically in first-trimester pregnancies. STUDY DESIGN: Fetal cells were obtained transcervically from 87 women undergoing pregnancy termination. Fetal gender was determined in 51 pregnancies with three different polymerase chain reaction techniques and in 36 pregnancies with fluorescent in situ hybridization. In known male pregnancies the number of male fetal cells present was also determined. RESULTS: Polymerase chain reaction detected male deoxyribonucleic acid in up to 79% of cases in male pregnancies and up to 45% of cases in female pregnancies. Fetal gender was correctly predicted in up to 72% of cases with fluorescent in situ hybridization. However, fetal cells were identified in < 40% of informative male pregnancies and were present in low numbers-0.7% to 3.4% in swabs and 4.4% to 24.8% in flushes. CONCLUSION: The use of fetal cells obtained by minimally invasive first-trimester transcervical sampling is unreliable for prenatal diagnosis.     

Fetal cells in the maternal blood: a step towards non-invasive prenatal diagnosis? Review of the literature

Prenatal diagnosis of genetic abnormalities requires nucleated fetal cells which are currently obtained by invasive techniques such as amniocentesis, chorionic villus sampling and percutaneous umbilical blood sampling. Each of these entails a risk to the foetus and sometimes to the mother. Nucleated fetal cells have been reported to be present in maternal blood. Recovery of fetal cells from maternal blood would allow a noninvasive prenatal diagnosis. Their rarity (1 fetal cell for 10(6) to 10(8) maternal cells) presents a technical challenge. Due to the small number of fetal cells, sensitive analysis techniques such as PCR and FISH are necessary. Some degree of fetal cells enrichment in the maternal blood sample often precedes the analysis. Different techniques are used for the enrichment: discontinuous density gradient, magnetic activated cell sorting, fluorescence activated cell sorting, micromanipulator.... Several prenatal diagnosis have already been performed from maternal venous blood samples: diagnosis of gender, RhD blood genotype, Duchenne muscular dystrophy and hemoglobinopathy by PCR, diagnosis of gender and chromosome aneuploidy by FISH. Many teams are working on this subject. It is difficult to compare the studies because the techniques of enrichment and analysis vary. We review the different strategies chosen for prenatal diagnosis from maternal blood and discuss the results.     

Rapid detection of aneuploidies by microsatellite and the quantitative fluorescent polymerase chain reaction

Several studies have been performed to assess the diagnostic value of using small tandem repeat (STR) markers and quantitative fluorescent polymerase chain reaction (QF-PCR) assays for the rapid detection of aneuploidies involving chromosomes 21, 18, 13 (Mansfield, 1993; Pertl et al., 1994, 1996; Adinolfi et al., 1995a). The results of these investigations have documented the diagnostic advantages of this approach to perform prenatal tests using amniotic and chorionic samples, or fetal nucleated cells retrieved from peripheral maternal blood or endocervical samples. The use of two or more STR markers for each autosome facilitates the diagnosis of aneuploidies, while avoiding the need to employ internal non-polymorphic markers. Multiplex quantitative fluorescent analyses can be performed in about six hours from the collection of the samples and, although targeted to specific abnormalities, they can exclude the presence of the most frequent chromosomal disorders. QF-PCR can be exploited to analyse DNA present in single or clumps of cells and thus to perform prenatal diagnoses on maternal peripheral blood or transcervical cell samples and on preimplantation embryos.     

Serum homocysteine level and protein intake are related to risk of microalbuminuria: the Hoorn Study

OBJECTIVE: Currently, prenatal diagnosis of chromosome abnormalities requires invasive techniques such as amniocentesis and chorionic villus sampling that carry small but finite risks of fetal loss. A noninvasive approach is to isolate fetal cells from maternal blood by flow sorting followed by genetic interphase analysis with fluorescence in situ hybridization. Because the ratio of fetal to maternal cells is relatively low after flow sorting and to detect 90% to 95% of fetal aneuploidies associated with serious birth defects, a 5-color fluorescent in situ hybridization strategy is necessary for simultaneous detection of chromosomes X, Y, 13, 18, and 21 in all flow-sorted nuclei recovered from a specimen. STUDY DESIGN: Fetal nucleated red blood cells were isolated from maternal blood in 40 cases (10.4 to 27.0 weeks' gestation) by flow cytometry on the basis of positive selection of CD71+ (transferrin receptor), CD45-, and LDS751 staining. Each case was evaluated for 5-color fluorescent in situ hybridization efficiency by determining the percentage of flow-sorted nuclei containing 8 hybridization signals for chromosomes X, Y, 13, 18, and 21. RESULTS: A total of 42,312 flow-sorted nuclei from maternal blood samples were analyzed. In 5 of 16 (31%) cases with a male fetus, 0.16% of nuclei scored were identified as fetal by the presence of 1 signal each for chromosomes X and Y. Fetal trisomy 21 nuclei were accurately detected in 2 cases with a female fetus, each of which was subsequently confirmed. CONCLUSIONS: Five-color interphase fluorescent in situ hybridization analysis can be used to effectively analyze rare fetal aneuploid nuclei in enriched flow-sorted cells isolated from maternal blood.     

Rapid prenatal diagnosis of Down's syndrome in the first trimester of pregnancy by fluorescence in situ hybridization

OBJECTIVE: To assess whether fluorescence in situ hybridization (FISH) with chromosome 21, specific DNA probe is applicable as a prenatal diagnostic tool for Down's syndrome. METHOD: We used FISH with chromosome 21 specific probe on 30 uncultured chorionic villi cell samples to detect the Down's fetus, and we also performed the conventional chromosome analysis of chorion cells from parallel samples. RESULTS: In samples with disomic karyotype, an average of 1 percent (0-5 percent) of the nuclei had three hybridization signals. By contrast, in the samples of trisomy 21 fetus, an average of 86 percent (78-91 percent) of the nuclei displayed three signals. CONCLUSION: FISH can provide a rapid and accurate method for the first trimester prenatal diagnosis of Down's syndrome.     

Molecular, cytogenetic, and clinical characterisation of six XX males including one prenatal diagnosis

Cytogenetic analysis, fluorescent in situ hybridisation (FISH), and molecular amplification have been used to characterise the transfer of Yp fragments to Xp22.3 in six XX males. PCR amplification of the genes SRY, RPS4Y, ZFY, AMELY, KALY, and DAZ and of several other markers along the Y chromosome short and long arms indicated the presence of two different breakpoints in the Y fragment. However, the clinical features were very similar in five of the cases, showing a male phenotype with small testes, testicular atrophy, and azoospermia. All these patients have normal intelligence and a stature within the normal male range. In the remaining case, the diagnosis was made prenatally in a fetus with male genitalia detected by ultrasound and a 46,XX karyotype in amniocytes and fetal blood. Molecular analysis of fetal DNA showed the presence of the SRY gene. FISH techniques also showed Y chromosomal DNA on Xp22.3 in metaphases of placental cells. To our knowledge, this is the second molecular prenatal diagnosis reported of an XX male.     

Trisomy 21 placentas: histopathological and immunohistochemical findings using proliferating cell nuclear antigen

OBJECTIVE: The cause of growth retardation in trisomy 21 and other autosomal trisomies is not known, but may be the result of defective cell proliferation, slowing of the cell cycle, or placental structural abnormalities. Abnormalities of the fetal cell cycle may be reflected in placental growth and can be detected using proliferating cell nuclear antigen (PCNA). METHODS: Twelve second-trimester and six third-trimester trisomy 21 placentas were examined histopathologically and stained immunohistochemically using antibodies to PCNA. Normal age-matched placentas were used as controls. RESULTS: The second-trimester trisomy 21 placentas all exhibited many large irregular hypovascular villi. The third-trimester trisomy 21 placentas showed two patterns: (i) many large, irregular hypovascular villi, and (ii) relatively normal-appearing villi with only a few abnormal villi and focal hypervascularity. PCNA staining was significantly greater in second-trimester placentas when compared to third-trimester placentas for both trisomy 21 and controls. There was no significant difference in PCNA staining in trisomy 21 placentas when compared to the normal age-matched controls. CONCLUSIONS: PCNA staining indicates no significant differences in proliferation between normal and trisomy 21 placentas. Trisomy 21 placentas show villus abnormalities, including hypovascularity.     

Fetal cells in maternal blood: the use of primed in situ (PRINS) labelling technique for fetal cell detection and sex assessment

Prenatal diagnosis is presently performed following invasive procedures with variable risks of fetal loss; non-invasive procedures using fetal cells in maternal blood would be welcome for the early detection of fetal sex or aneuploidy. We describe a simple and rapid protocol to detect fetal cells and thus to assess fetal sex. In a first step, nucleated blood cells were separated into mononuclear and polynuclear cells using a double density gradient centrifugation. In a second step primed in situ (PRINS) labelling technique was performed to label Y-chromosomes. 15 samples were studied and correct gender assignment was made in 13/15. The number of labelled nuclei was higher in polynuclear cell phases than in mononuclear cell phases. Moreover, the polylobular aspect of labelled nuclei from polynuclear cell phases strongly suggested that they could belong to fetal polynuclear cells. The PRINS technique combines some advantages of FISH, such as visual assessment of in situ chromosome labelling and the powerful specificity and sensitivity of PCR. In association with a simple enrichment procedure it constitutes a rapid protocol for fetal cell detection, non-invasive early prenatal sex assessment, and could further be applied to detect the main viable aneuploidies.     

Potential role of transforming growth factor-beta 1 in terminating the immune response in patients with Guillain-Barré syndrome

Currently, amniocentesis, chorionic villus sampling (CVS) and fetal blood sampling are used to obtain fetal cells for genetic diagnosis. These invasive procedures pose a small but not negligible risk for the fetus. Efforts have been directed towards the enrichment of fetal cells, such as erythroblasts, from maternal blood and progress has been made in the diagnosis of some chromosomal disorders and in sex determinations. We now report the detection of point mutations in single gene disorders using this method of prenatal diagnosis by enriching fetal cells from maternal blood by magnetic cell sorting followed by isolation of pure fetal cells by microdissection. In two pregnancies at risk for sickle cell anaemia and beta-thalassaemia, we successfully identified the fetal genotypes. Thus, prenatal diagnosis of single gene disorders by recovering fetal cells from maternal circulation appears to be a feasible approach.     

Morphological classification of nuchal skin in human fetuses with trisomy 21, 18, and 13 at 12-18 weeks and in a trisomy 16 mouse

An increase in the nuchal translucency that can be detected at 10-14 weeks of gestation by ultrasound forms the basis for a screening test for chromosomal abnormality. Several mechanisms leading to this increase in skin thickness have been proposed, including changes of the extracellular matrix, cardiac defects and abnormalities of the large vessels. This study examines the composition of the extracellular matrix of the skin in gestational age-matched fetuses with trisomy 21, 18 and 13 from 12-18 weeks. Immunohistochemistry was applied with monoclonal and polyclonal antibodies against collagen type I, III, IV, V and VI and against laminin and fibronectin. Collagen type VI gene expression was further studied by in situ hybridization to detect differences in expression patterns of COL6A1, COL6A3 and COL1A1 between normal fetuses and those with trisomy 21. The ultrastructure of tissue samples was studied by transmission electron microscopy (TEM) and additionally by immunogold TEM. Further, we examined the morphology of the skin in an animal model for Down's syndrome, the murine trisomy 16, by light and TEM. The dermis of trisomy 21 fetuses was richer in collagen type VI than that of normal fetuses and other trisomies, and COL6A1, located on chromosome 21, was expressed in a wider area than COL6A3, which is located on chromosome 2. Collagen type I was less abundant in the skin of trisomy 18 fetuses, while the skin of all three trisomies contained a dense network of collagen type III and V in comparison with normal fetuses. Collagen type IV, of which two genes are located on chromosome 13, was expressed in the basement membranes of the skin in all fetuses and additionally in the dermal fibroblasts only of trisomy 13 fetuses. Likewise, laminin was present in all basement membranes of normal and trisomic fetuses as well as in dermal fibroblasts of fetuses with trisomy 18. LAMA1 and LAMA3 genes are located on chromosome 18. Dermal cysts were found in the skin of trisomy 18 and 13, but not in trisomy 21 and normal fetuses. Ultrastructural findings showed that an extracellular precipitate containing glycosaminoglycans was regularly present in the skin of trisomy 21 fetuses and murine trisomy 16 embryos. In conclusion, this study suggests that the skin edema in fetal trisomies is characterized by specific alterations of the extracellular matrix that may be attributed to gene dosage effects as a result of a genetic imbalance due to the condition of fetal trisomy.     

Preimplantation genetic diagnosis: current status and new developments

Preimplantation genetic diagnosis (PGD) is a very early form of prenatal diagnosis aimed at eliminating embryos carrying serious genetic diseases before implantation. To this end, two major technologies are in use: the polymerase chain reaction (PCR) for monogenic diseases and fluorescent in-situ hybridization (FISH) for chromosomal aberrations. In this review, a number of problems arising from the use of these technologies, as well as their possible solutions and new developments, are discussed. Concerning PCR, the phenomenon of allelic drop-out, as well as methods to reduce this problem, such as fluorescent PCR, are described. The advantages and disadvantages of sperm separation by flow cytometry as an adjunct to sex determination for the avoidance of X-linked disease are discussed. The application of FISH for aneuploidy detection is commented upon and the advances in cell recycling, in which PCR and FISH are combined, are analysed. Finally, diseases for which PGD is currently possible are summarized.     

Prenatal diagnosis of congenital cytomegalovirus infection

We report here the results of a study on the prenatal diagnosis of congenital cytomegalovirus (CMV) infection. The study was carried out by both PCR and virus isolation from amniotic fluid (AF) for 82 pregnant women at risk of transmitting CMV for the detection of (i) seroconversion to CMV immunoglobulin G (IgG) positivity during the first trimester of pregnancy, (ii) symptomatic CMV infection in the mother during the first trimester of pregnancy or intrauterine growth retardation detected by ultrasound or abnormal ultrasonographic findings suggestive of fetal infections, and (iii) seropositivity for CMV-specific IgM. For 50 women, fetal blood (FB) was also obtained and tests for antigenemia and PCR were performed. The results indicate that AF is better than FB for the prenatal diagnosis of CMV infection. PCR with AF has a sensitivity (SNS) of 100%, a specificity (SPE) of 83.3%, a positive predictive value (PPV) of 40%, and a negative predictive value (NPV) of 100%; rapid virus isolation with the same material has an SNS of 50%, an SPE of 100%, a PPV of 100%, and an NPV of 94.7%. Fewer than 10% of the women positive for IgM by enzyme immunoassay (EIA) had a congenitally infected fetus or newborn infant. When EIA IgM positivity was confirmed by Western blotting (WB) and the WB profile was considered, the percent transmission detected among women with an "at-risk" profile was higher than that observed among IgM-positive women and was the same as that among women who seroconverted during the first trimester of pregnancy (transmission rates of 29 and 25%, respectively).     

Prenatal medicine: development, current status and future perspectives

Prenatal diagnosis and therapy are based mainly on the progress of diagnostic ultrasound and laboratory methods in genetics. There is a general tendency to replace second trimester fetal diagnoses by first trimester approaches. Transvaginal sonography and chorionic villus sampling in particular have been proven helpful in this context. The aim of all prenatal diagnostic measures is fetal therapy in time to prevent untreatable abnormalities. There has been some progress in this area; in particular, the application of stem cells for the correction of single cell diseases seems to be promising. Because all prenatal interventions involve a risk to the mother and especially the fetus, there is a concentrated effort to develop non-invasive screening or diagnostic methods. Extensive work on the isolation of fetal cells from the maternal circulation has revealed that such cells are present physiologically in pregnancy, but further trials need to show whether this method is safe enough for routine diagnostic use.     

Cytogenetic and molecular genetic characterization of trisomy 20 mosaicism in fetal blood and tissues

We report a case of mosaic trisomy 20, the most common autosomal mosaicism identified in amniocytes, ascertained in a woman referred for amniocentesis because of abnormal ultrasound at 18.1 weeks' gestation which revealed short femurs and nuchal thickening. Metaphase analysis of 98 clones revealed 47,XY, +20 in 96 cells (98 per cent). Trisomy 20 was demonstrated in 6 cells (12 per cent) in a total of 50 cells from two fetal blood cultures obtained after pregnancy termination. Fluorescence in situ hybridization (FISH) analysis of interphase nuclei utilizing a chromosome 20 alpha-satellite centromeric DNA probe revealed three signals in 57/546 nuclei (10 per cent) in fetal blood. Metaphase analysis of 167 cells from seven different fetal tissue sources revealed trisomy 20 in 32 cells (19.2 per cent). The percentage of trisomy 20 cells varied with tissue type, with the highest percentage (13/25 cells, 52 per cent) identified in the small intestine and lymph nodes and the lowest percentage (1/34 cells, 2.9 per cent) identified in a specimen of chorionic villi. Molecular genetic analyses utilizing polymerase chain reaction (PCR)-formated dinucleotide repeat polymorphisms demonstrated that the non-disjunctional event most likely occurred post-zygotically and that the origin of the extra chromosome 20 was maternal. This study is the first to demonstrate trisomy 20 cells in fetal blood, suggesting that mosaic trisomy 20 can be embryonic in origin. In cases of prenatally detected mosaic trisomy 20, examination of fetal blood should be considered, as well as study of placental membranes, skin, and urine sediment to confirm the karyotype and determine its significance.     

Attitudes of women of childbearing age towards prenatal diagnosis in southeastern France

The objective of this study was to explore women's attitudes towards prenatal diagnosis of trisomy 21 and to examine some of the factors possibly responsible for these attitudes before implementing in real practice serological screening of pregnant women at risk for trisomy 21. We carried out a telephone survey on a representative sample of women who had recently had a normal livebirth delivery in the Marseille district in 1990. The participation rate was 80 per cent and the average age of the mothers was 28.9 years. Among the 514 women interviewed, 78 per cent stated that they would ask for an amniocentesis for a 1 per cent risk of trisomy 21 at their next pregnancy. When adjusting for confounding factors, the decision to have or not to have an amniocentesis was found to depend not only on the women's attitude towards induced abortion, but also on their understanding of the risk involved and on the social context (knowing a handicapped child, discussion with the father). It also depended on the women's age and on what they knew about amniocentesis from the medical point of view. The risk of miscarriage can influence a woman's choice but this objection was not found to affect the women's decisions significantly in our survey. The data showed the existence of a high potential demand for fetal karyotyping.     

Primed in situ labeling for rapid prenatal diagnosis

OBJECTIVE: Our purpose was to assess the feasibility of primed in situ labeling for analysis of prenatal diagnostic specimens. STUDY DESIGN: Prenatal diagnostic specimens were chosen at random for analysis without knowledge of clinical indication. Primed in situ labeling with primers for chromosomes 18, 21, X, and Y was performed separate from conventional cytogenetic analyses. All clinical management considerations were based solely on conventional cytogenetic analyses. RESULTS: Forty-one samples were analyzed by primed in situ labeling: 35 direct preparations of chorionic villi and 6 uncultured amniotic fluid samples. In all cases analysis confirmed the particular chromosome number determined by conventional cytogenetic analysis. CONCLUSIONS: Although conventional metaphase studies remain the standard for prenatal cytogenetic analyses, the preliminary feasibility study finds primed in situ labeling to be a rapid and reliable adjunctive diagnostic technique applicable for prenatal diagnosis in certain clinical situations. Further study is needed to assess the efficacy of primed in situ labeling in comparison to fluorescent in situ hybridization and conventional cytogenetic analyses for prenatal diagnoses.