KIinefeIter综合征的诊断：新型甲基化特异性实时定量PCR

本文研究目的为设计一种基于额外X染色体（X-ch）检测的分子检测方法用于Klinefelter综合征的诊断。从26名47,XXY男性,2名46,XY／47,XXY男性,22名46,XY男性和15名46,XX的女性的外周血标本中提取DNA,并进行脱氨基处理。甲基化特异性的定量多聚酶链反应（MS-qpPCR）以非甲基化和甲基化的X染色体非活化特异性转录基因（XIST-U和XIST-M）拷贝为模板。X染色体二倍体通过XIST基因甲基化状态来判定。46,XY／47,XXY男性的嵌合程度通过核型分析和原位荧光杂交（FISH）的结果比较来判定。数据分析应用Roche LightCycler software v．3．5.3,包括通过拟合点分析和溶解曲线分析决定交叉点（CPs）。所有对照组女性和Ks患者均检测到X染色体二倍体,而对照组男性只表达XIST-M。XIST-U和IXIST-M的交叉点范围分别是（29．5-32．5,SD0．8）和（29-31,SD0．6）。嵌合度的检测极限为1％。根据2名47,XXY/46,XY患者的XIST-U／）（IST-M比值分析,计算出的嵌合率（1．8％和17．8％）与FISH结果（2．3％和15％）相当。从DNA脱氨基到最终数据分析的间隔小于9小时。本文得出结论：MS-qpPCR是一种敏感、特异及快速的X染色体二体检测方法,可以用于KS的筛查和诊断,甚至在低嵌合水平的47,XXY／46,XY患者也适用。

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

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-six 47,XXY males; two 46,XY/47,XXY males; twenty-two 46,XY males; and 15 females; and deaminated. Methylation-specific quantitative polymerase chain reaction （MS-qPCR） was performed using primers for unmethylated and methylated copies of the X-ch inactive-specific 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 fluorescent in situ hybridization （FISH） results. Data analysis was performed using the Roche LightCycler software V. 3.5.3, including determination of crossing points （CPs） by fit-point analysis and melting curve analysis. Xoch 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 XlST-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 final data analysis was under 9 h. We conclude that MS-qPCR is a sensitive, specific 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 grade 47,XXY/46,XY mosaicism.