Novel methylation speciifc real-time PCR test for the diagnosis of Klinefelter syndrome

• 来源期刊: 亚洲男性学杂志（英文版）
• 年，卷（期）: 2014,（9）
• 所属期刊栏目: ORIGINAL ARTICLE
• 页码范围: 684-688
• 页数: 5页
• 语种: 英文
• DOI: 10.4103/1008-682X.125914

摘要

The aim of this study was to design a molecular assay for the diagnosis of Klinefelter syndrome(KS), based on the detection of supernumerary X?chromosomes(X?chs). DNA was extracted from peripheral blood samples of twenty?six47,XXY males; two 46,XY/47,XXY males; twenty?two46,XY males; and 15females; and deaminated. Methylation?speciifc quantitative polymerase chain reaction(MS?qPCR) was performed using primers for unmethylated and methylated copies of the X?ch inactive?speciifc transcript(XIST?U and XIST?M) gene. X?ch disomy was determined on the basis of XIST methylation status. Degree of mosaicism in the 46,XY/47,XXY males was compared with karyotype and lfuorescentinsituhybridization(FISH) results. Data analysis was performed using the Roche? LightCycler software V. 3.5.3., including determination of crossing points(CPs) by ift?point analysis and melting curve analysis. X?ch disomy was detected in all female controls and KS patients; male controls expressed XIST?M only. CPs ranged from 29.5 to 32.5(standard deviation(s.d.) 0.8) for XIST?U and from 29 to 31(s.d. 0.6) for XIST?M. Limit of detection of mosaicism was 1%. Based on XIST?U/XIST?M ratios for the two 47,XXY/46,XY patients, the calculated degree of mosaicism(1.8%and 17.8%) was comparable to FISH results(2.3% and 15%, respectively). Turnaround time from DNA deamination to ifnal data analysis was under 9h. We conclude that MS?qPCR is a sensitive, speciifc and rapid test for the detection of X?ch disomy, with applicability for the screening and diagnosis of KS, even in the setting of low grade47,XXY/46,XY mosaicism.