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 (R) 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.