A Study On The Genetic Diagnostic Platform For Duchenne/Becker Muscular Dystrophy Based On Sequence Capture Microarray And Next-Generation Sequencing AND Its Applications In Clinical Practice

Background:Duchenne muscular dystrophy and Becker muscular dystrophy are the most common types of muscular dystrophies. The pathogenic gene of DMD/BMD is DMD gene （or Dystrophin gene）, which is the largest human gene to date. The DMD gene contains79exons and has many different causative mutation types. There are about30percent DMD/BMD patients suffered from SNVs and INDELs in DMD gene. It is difficult to detect SNVs and INDELs in such a huge gene. With the development of the DNA sequencing assay and gene chip, we can establish a new method to detect SNVs and INDELs in DMD gene.Objective:To set up a new platform based on sequence capture microarray and next-generation sequencing to detect SNVs and INDELs in DMD gene and test its sensitivity and specificity.Material and Methods:We employ YH standard gDNA to build the filter principle for our targeted capture NGS platform. Two DMD patients （"XZX" and "YZY"） with complete loss of dystrophin by muscle biopsy and one apparent patient （"CDY"） excluded by pathologic diagnosis finally were enrolled in the study. The gDNA from the three samples were conducted Sanger sequencing of the79exons of DMD gene first, then sequenced on the targeted capture NGS platform. Investigators were blinded to the results from the other method in the whole processing until final unblinding.Results:①By Sanger sequencing, we detected c.2833delC in exon22of the patient "XZX" and c.6391₆392delCA in exon44of the patient "YZY". There was no causative mutation in the DMD gene of the patient "CDY".②Compared with YH gDNA, we verified that the filter,"Ratio of Reads"=26.6%, was the best one for our platform③By targeted capture NGS, the results of the three samples were exactly the same as the Sanger sequencing results. At the same time, we detected some non-causative SNPs.④Targeted capture NGS platform can be utilized in the SNV and INDEL detection in the DMD gene.Conclusion: 1. Targeted capture NGS platform can be applied in DMD/BMD diagnosis for SNVs and INDELs detection. Its sensitivity and specificity are good.2. Compared with Sanger sequencing, targeted capture NGS platform has the advantages of less turnaround time, less cost and stable processing. Background:The platform based on targeted capture and next-generation sequencing often aimed to detect SNVs and INDELs in the past. With the meticulous application of the platform on a certain gene, people were aware of the possibility that it can be used to detecting CNV. DMD is an X-linked recessive inheritary disease. The DMD gene locates in the X chromosome. The male patients have only one X chromosome, which makes detection of CNV in DMD gene easier. While we focused on this topic, another team had published a small-scale retrospective study on CNV in DMD patients by targeted capture NGS.Objective:To establish a standard bioinformative workflow for CNV detection by calculating and analyzing the reads from the targeted capture NGS. Then we applied it to a large-scale test to validate the practical feasibility and efficacy.Methods:A total of418individuals including124healthy controls （62males,62females）,41DMD/BMD patients （40males,1females）,8asymptomatic carrier （all females） and245non-DMD controls （144males,101females） were enrolled in the study and tested by the targeted capture NGS platform. All the enrolled patients and carriers were with CNVs. The normalized sequencing depths of the DMD gene from healthy controls were used to calculate the mean values and standard deviations of each exon. The patients, carriers and non-DMD controls were analyzed through a novel three-step computational framework. Lastly, we counted the sensitivity and specificity of CNV detection by our platform.Results:1. All the patients and carriers were detected large deletions or large duplications via MLPA assay.2. The results from healthy controls demonstrated the normalized sequencing depth of female was twice as that of male and the standard deviation was small.3. The screening results from the DMD patients/carriers and non-DMD controls showed excellent sensitivity （99.26%） and specificity （99.99%）.Conclusion1. Through our novel three-step computational framework, the targeted capture NGS platform could be utilized to detect CNVs in DMD gene. Its sensitivity and specificity are fairly good.2. The targeted capture NGS platform may distinguish the breakpoints of large deletions or duplications more accurate. We found a new subtype of mutation,partial exonic deletion. Background:It is more than180years when the first case report of Duchenne muscular dystrophy was published. At first, doctors diagnosed the disease by clinical manifestations. Now, the patients were diagnosed on gene analysis. Southern blotting was the first testing assay used in clinical gene diagnosis. From then on, many different platforms, such as multiplex PCR, MLPA, aCGH and Sanger sequencing, were applied for DMD detection. Each assay has its advantages and disadvantages. The common shortage is none of them could find all types of mutations. We developed a new platform based on targeted sequence capture and next-generation sequencing, which could comprehensively scan the DMD gene and discover almost all the mutations.Objective:To test and verify the practical feasibility and applicable field of our targeted capture NGS platform in clinical circumstances.Material and Methods:From October2011to August2012, a total of60individuals including50clinically diagnosed DMD/BMD patients （48males,2females）,4proband’s mothers and6asymptomatic female carriers with suspected family history were enrolled in the neurology department of PUMCH. All the participants without previous gene diagnosis were tested by the targeted capture NGS platform for CNV or SNV in the DMD gene.Results:①Among50participants,47patients were detected causative mutations. There are8CNVs （7males,1female） and39SNVs （38males,1female）.3participants with negative results were performed muscle biopsy. The immunohistochemistry staining of dystrophin demonstrated one of them was DMD and the other two weren’t DMD.②The four proband’s mothers were found to be heterozygous SNV carriers. Among the six asymptomatic female carriers with suspected family history, three persons were demonstrated as CNV carriers while the rest were not detected a causative mutation.③All the SNVs founded by targeted capture NGS were validated by the Sanger sequencing. Some of the CNVs were validated by MLPA.④We first systemically report a new subtype of deletion, partial exonic deletion. In our study, there were four pedigrees with this type of mutation.Conclusion:1. Our platform based on targeted sequence capture and next-generation sequencing could be a solid tool in clinical gene diagnosis of DMD/BMD for its ability of comprehensive detection.2. Partial exonic deletion is a new subtype of mutation, which might influence the patients’phenotype and the effect of gene therapy in the future.