Mehta Akanksha, Mielnik Anna, Schlegel Peter N, Paduch Darius A
Department of Urology, Weill Cornell Medical College, New York, NY; Department of Urology, Emory University School of Medicine, Atlanta, GA, USA.
Asian J Androl. 2014 Sep-Oct;16(5):684-8. doi: 10.4103/1008-682X.125914.
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. 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 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.
本研究的目的是基于对额外X染色体（X-chs）的检测，设计一种用于诊断克兰费尔特综合征（KS）的分子检测方法。从26名47,XXY男性、2名46,XY/47,XXY男性、22名46,XY男性和15名女性的外周血样本中提取DNA并进行脱氨基处理。使用针对X染色体失活特异性转录本（XIST-U和XIST-M）基因的未甲基化和甲基化拷贝的引物进行甲基化特异性定量聚合酶链反应（MS-qPCR）。根据XIST甲基化状态确定X-ch二体性。将46,XY/47,XXY男性的嵌合度与核型和荧光原位杂交（FISH）结果进行比较。使用罗氏®LightCycler软件V. 3.5.3进行数据分析，包括通过拟合点分析和熔解曲线分析确定交叉点（CPs）。在所有女性对照和KS患者中检测到X-ch二体性；男性对照仅表达XIST-M。XIST-U的CPs范围为29.5至32.5（标准差（s.d.）0.8），XIST-M的CPs范围为29至31（s.d. 0.6）。嵌合度的检测限为1%。基于两名47,XXY/46,XY患者的XIST-U/XIST-M比率，计算出的嵌合度（1.8%和17.8%）与FISH结果（分别为2.3%和15%）相当。从DNA脱氨基到最终数据分析的周转时间不到9小时。我们得出结论，MS-qPCR是一种用于检测X-ch二体性的灵敏、特异且快速的检测方法，适用于KS的筛查和诊断，即使在低级别47,XXY/46,XY嵌合的情况下也是如此。
