Detection Of Copy Number Variation In Spinal Muscular Atrophy (SMA) And Duchenne Muscular Dystrophies (DMD) By Multiplex Ligation-dependent Probe Amplification-based Real-time PCR

Spinal muscular atrophy(SMA)and Duchenne muscular dystrophy(DMD)are common genetic disorders which can cause severe and untreatable phenotypes.SMA is of high prevalence in the general population.In China,the carrier prevalence is as high as 1/42.Therefore,populational screening for SMA carrier is of clinical importance.DMD is the largest known human gene,spans a wide range of genomic DNA and thus it is susceptible to the high frequency of various mutations.Therefore,population-based carrier screening for these two diseases is an efficient way to reduce birth defects and improve the quality of population.To date,several techniques were developed for the clinical detection of these diseases,including denaturing high performance liquid chromatography(DHPLC),multiple ligation-dependent probes amplification(MLPA),real-time PCR(also known as Quantitative PCR),next-generation sequencing(NGS).However,some drawbacks of these techniques such as high cost,low throught,or low sensitivity,limit their clinical application in populational screening.Moreover,SMA and DMD were conventionally detected separately in these methods.Our study aimed to develop a new technique,called MLPA/qPCR,for the simultaneous screening of SMA and DMD,in order to meet the requirements of the clinical carriers screening and improve the efficiency of disease detection.The contents were mainly divided into four parts,including the exploration of MLPA/qPCR system principle,the establishment of MLPA/qPCR detection system,the evaluation of system performance and the screening of the populational carriers.To address the performance of the MLPA/qPCR detection system,the effects of multiple reaction factors on the hybridization system and PCR detection system were investigated.Finally,an optimal reaction system was established and the method of statistics analysis was improved.In order to evaluate the efficiency of the newly developed MLPA/qPCR detection system,107 cases of SMA and 24 cases of DMD that had already been diagnosed previously by the traditional MLPA,were tested by the MLPA/qPCR.The results showed very high diagnostic sensitivity(SMA:100.0%,DMD:100.0%),high diagnostic specificity(SMA:100.0%,DMD:99.4%)and good repeatability(CV=8.7%).In addition,the mothers of 185 patients with unclear muscle weakness or atrophy were detected by the MLPA/qPCR and MLPA,respectively.The results showed high clinical sensitivity(SMA:100.0%,DMD:92.9%)and high clinical specificity(SMA:100.0%,DMD:99.4%).We also concluded that 6 hot mutations in DMD gene in the mainland of China are accounted for 39.4%.To investigate the incidence of SMA and DMD carriers in Xiamen,the 2962 cases of clients with local medical card were recruited to screen by the MLPA/qPCR system.The results revealed that the incidence of SMA carriers in Xiamen area was 1/46,which corresponded with the document(1/42).Similarily,the incidence of DMD carriers in Xiamen(1/2022)was close to the literature record(1/2300).These observations demonstrated the feasibility of this technique can be used for the simultaneous screening of SMA and DMD.In conclusion,our study firstly integrated the real-time fluorescence PCR technique and MLPA technique together into a new method,called MLPA/qPCR.This novel method can be used to simultaneous detection of SMA and DMD.In comparison with former methods,this technique is proven to be rapid,inexpensive,easy to set up,of high clinical sensitivity and specificity.Our study will not only reduce the birth of diseased children and improve the population quality,but also it could be a competitive tool for the clinical population-based screening of these common genetic diseases.