Potential significance of methylation differential genes in clinical diagnosis of down’s syndrome

Abstract

Prenatal preventions of Down's syndrome include genetic counseling and prenatal diagnosis. The high cost of massive parallel sequencing prevents its routine use in clinics. Recent studies have found that certain genetic loci have opposite maternal-fetal methylation status, which suggests a new strategy to identify fetal DNA. The aim of this study is to investigate the feasibility of combining methylation markers together with MSRE-qPCR to achieve precise diagnostics of trisomy 21 with advantages of less time consuming and less costly, making it especially ideal for initial screening of 21-Trisomy syndrome. Theoretically after methylation sensitive restriction endonuclease digestion, only the fetal DNA would be undigested in peripheral cell-free DNA. We detected cffDNA in maternal peripheral blood. On the other hand we mixed the placental DNA with normal blood genomic DNA in order to mimic clinical samples in enriching cffDNA in total cell-free DNA. Normal human blood genomic DNA was diluted in serial proportion as a control group (Table 1). CffDNA, placenta tissue DNA and peripheral blood genomic DNA were extracted in accordance with the instruction respectively. First steps, placental DNA (considered as fetal DNA) and genomic DNA (considered as the maternal DNA) were digested with methylation sensitive restriction endonuclease BstUI. Second, real-time fluorescence quantitative PCR amplification of HLCS, C21orf25 and RASSF1A gene were performed for 0.1-1% placental DNA in the 9 sample groups with BstUIdigestion. Third, cffDNA samples were subjected to BstUIdigestion, and followed by qPCR quantification targeting the three genes. The quantification of each gene target is calculated according to 2-ΔΔCt. For cffDNA samples, the ratio of HLCS to RASSF1A in trisomy 21 to euploid was 1.442, which was close to theoretical chromosome dosage ratio1.5:1. The trisomy 21 mixture samples could be differentiated from euploid mixture samples even when the fraction of placental DNA was as low as 0.1%, indicating the high sensitivity of the method using employed in the experiment. The approach correctly identified 3 cases of trisomy 21 fetus from total 8 cases of clinical samples, which was 100% consistent with karatyping result. This study showed that HLCS gene and RASSF1A gene together with MSRE-qPCR, the approach was highly sensitive and highly specific for non-invasive prenatal diagnosis of 21-Trisomy syndrome. It is fast and cost efficient, making it especially ideal for initial screening of 21-Trisomy syndrome.Tabled 1Placental DNA mixed groups and normal blood genomic DNA diluted groupsExperimental groups121 trisomy placental DNA (1%) mixed with normal blood genomic DNA (99%)Experimental groups2Normal placental DNA (1%) mixed with normal blood genomic DNA (99%)Experimental groups321 trisomy placental DNA (0.33%) mixed with normal blood genomic DNA (99.67%)Experimental groups4Normal placental DNA (0.33%) mixed with normal blood genomic DNA (99.67%)Experimental groups521 trisomy placental DNA (0.1%) mixed with normal blood genomic DNA (99.9%)Experimental groups6Normal placental DNA (0.1%) mixed with normal blood genomic DNA (99.9%)control groups1Normal blood genomic DNA (diluted to 1%)control groups2Normal blood genomic DNA (diluted to 0.33%)control groups3Normal blood genomic DNA (diluted to 0.1%) Open table in a new tab