Dataset Information

Molecular detection of trisomy 21 by bicolor competitive fluorescent PCR.

ABSTRACT: OBJECTIVE:To develop a reliable and specific method for rapid prenatal diagnosis of Trisomy 21 (Down syndrome). METHODS:We established a dual color competitive fluorescent Polymerase Chain Reaction (PCR) to measure the gene dosage of Down syndrome critical region (DSCR), a single copy sequence in chromosome 21. Another unique single copy sequence located on chromosome 2 (USC2) but not glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chose as reference gene. RESULTS:The DSCR3/USC2 ratio of peripheral blood in trisomy 21 syndrome patients to normal subjects was 1.41?1.74 to 0.93?1.15, respectively (p < 0.01). Dual color competitive fluorescent PCR technique effectively differentiates the normal subjects from the Down syndrome patients. Next, according to the dual color competitive fluorescence quantitative PCR, among the 46 pregnant women, 3 cases were Down syndrome and 43 cases were normal, and these were confirmed by cytogenetic karyotype analysis. CONCLUSION:This indicated that the new technique may be a reliable and specific method for the rapid prenatal diagnosis of Trisomy 21.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6807619 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Molecular detection of trisomy 21 by bicolor competitive fluorescent PCR.

Wang Yan Y Zhang Xiaofei X Ling Bo B He Changxiao C Xia Qingjie Q Chen Feng F Miyamori Isamu I Yang Zhao Z Fan Chunyuan C

Journal of clinical laboratory analysis 20130501 3

<h4>Objective</h4>To develop a reliable and specific method for rapid prenatal diagnosis of Trisomy 21 (Down syndrome).<h4>Methods</h4>We established a dual color competitive fluorescent Polymerase Chain Reaction (PCR) to measure the gene dosage of Down syndrome critical region (DSCR), a single copy sequence in chromosome 21. Another unique single copy sequence located on chromosome 2 (USC2) but not glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chose as reference gene.<h4>Results</h4>The ...[more]

PMID: 23686780

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Project description:BACKGROUND: Screening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA. METHODS AND FINDINGS: Highly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects. CONCLUSIONS: In summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma.

Project description:BackgroundDown syndrome (DS) is the most common known aneuploidy, caused by an extra copy of all or part of chromosome 21. Fetal-specific epigenetic markers have been investigated for non-invasive prenatal detection of fetal DS. The phosphodiesterases gene, PDE9A, located on chromosome 21q22.3, is completely methylated in blood (M-PDE9A) and unmethylated in the placenta (U-PDE9A). Therefore, we estimated the accuracy of non-invasive fetal DS detection during the first trimester of pregnancy using this tissue-specific epigenetic characteristic of PDE9A.Methodology/principal findingsA nested, case-control study was conducted using maternal plasma samples collected from 108 pregnant women carrying 18 DS and 90 normal fetuses (each case was matched with 5 controls according to gestational weeks at blood sampling). All pregnancies were singletons at or before 12 weeks of gestation between October 2008 and May 2009. The maternal plasma levels of M-PDE9A and U-PDE9A were measured by quantitative methylation-specific polymerase chain reaction. M-PDE9A and U-PDE9A levels were obtained in all samples and did not differ between male and female fetuses. M-PDE9A levels did not differ between the DS cases and controls (1854.3 vs 2004.5 copies/mL; P?=?0.928). U-PDE9A levels were significantly elevated in women with DS fetuses compared with controls (356.8 vs 194.7 copies/mL, P<0.001). The sensitivities of U-PDE9A level and the unmethylation index of PDE9A for non-invasive fetal DS detection were 77.8% and 83.3%, respectively, with a 5% false-positive rate. In the risk assessment for fetal DS, the adjusted odds ratios of U-PDE9A level and UI were 46.2 [95% confidence interval: 7.8-151.6] and 63.7 [95% confidence interval: 23.2-206.7], respectively.ConclusionsOur findings suggest that U-PDE9A level and the unmethylation index of PDE9A may be useful biomarkers for non-invasive fetal DS detection during the first trimester of pregnancy, regardless of fetal gender.

Dataset Information

Molecular detection of trisomy 21 by bicolor competitive fluorescent PCR.

Publications

Molecular detection of trisomy 21 by bicolor competitive fluorescent PCR.

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