Rapid Screening For Klinefelter Syndrome With A Simple High-Resolution Melting Assay:A Multicenter Study

Klinefelter syndrome(KS)is a group of syndromes manifested by the presence of an extra X chromosomes in males.KS is one of the most common sex chromosomal disorders and the most common reason that cause the infertility in males.The incidence of KS is 0.1-0.2%in all live male births,3%in infertile males,and 10%-12%in non-obstructive azoospermic patients.Increasing evidence has shown that a diverse range of treatments and interventions such as testosterone replacement treatment and testicular sperm extraction,if offered at the appropriate time,effectively improve the quality of life and alleviate later complications of KS patients.It also reduce the incidence of KS and improve the quality of the population.Unfortunately,most Klinefelter syndrome patients have not been diagnosed.Only 25%of KS patients were diagnosed.Of diagnosed cases,most are diagnosed in adulthood during infertility investigation.They have been missed the opportunity for the best fertility treatment,bearing both physical and psychological burdens.Therefore,to develop the postnatal population-based KS screening is an important measure to improve the diagnosis rate of KS.To date,several methods were developed for the clinical detection of KS including karyotyping,Fluorescence In Situ Hybridization(FISH),Multiplex Ligation-dependent Probe Amplification(MLPA)and Real-time Quantitative Polymerase Chain Reaction(qPCR).However,there are some limitations of these methods such as the operational simplicity,testing cost,clinical sensitivity and specificity.So these methods cannot be applied to clinical detection on a large scale.The High Resolution Melting curve(HRM)method,as a new technique in the field of molecular diagnosis,has been widely used in the screening of diseases.Therefore,the purpose of this study is to establish a simple HRM detection method that can be used for the screening of people with Klinefelter syndrome.The sensitivity and specificity of the method were evaluated by three medical centers using 1373 clinical blood samples.The HRM assay utilized a single primer pair to simultaneously amplify specific regions in ZFX gene and ZFY gene.The ratio of ZFX/ZFY is 1 in normal male,but in cases of KS,the ratio of ZFX/ZFY will change.As a result,the specific melting profiles differ,and can be differentiated in the data analysis process.We evaluated the detection limits,cut-off values,and clinical sensitivity and specificity of the HRM assay.At the same time,in order to explore the potential of HRM assay in neonatal screening for Klinefelter syndrome,60 neonatal dried blood spots were also collected for testing.The experimental results show that the HRM method for screening of Klinefelter syndrome established in this study displayed high analytical specificity over a wide range of template DNA amounts(5-50ng),high resolution for detecting KS mosaicism,and high clinical sensitivity(100%)and specificity(99.8%).It also has some advantages of reproducibility,high throughput,rapid and high efficiency(2h),low cost.In addition,HRM assay is easy to perform and automate,and can detect both whole blood samples and dried blood spots.Therefore,this HRM assay is a preferred postnatal population-based KS screening method that can be used for different age groups.