The Genetic Analysis Of Muscular Dystrophy And Study Of Nevel Mutation

Part 1 The Analysis o F Genetic Diagnosis of Duchenne/Becker Muscular Dystrophy and Mutation Spectrum of Dystrophin GeneObjective Clinical data of patients suspecting with muscular Dystrophy(MD)in our hospital during 2011 to 2018 were collected.We aimed to make genetic diagnosis for DMD/BMD patients and extent the mutation spectrum of DMD gene in Guangxi province,and study the genotype/phenotype correlations.Methods 1.We collected the clinical and examination data of all the patients suspecting with MD.2.Multiplex ligation-dependent probe amplification(MLPA)was used to detect the exons of DMD gene in these patients.Genetic diagnosis can be made if MLPA found large rearrangement.When no mutation was identified by MLPA,next generation sequencing(NGS)was used to analyze muscle diseases related genes exons and the adjacent noncoding regions.3.Then large rearrangements frequency and breakpoints distribution were analyzed.4.The correlation of genotype and phenotype was made by checing the reading frame rule.5.Leiden database was used to to learn whether the point mutations were novel.SIFT,Poly Phen,Mutationtaster were used to predict the influence of the mutations.Results 1.198 cases were enrolled in our study.175 were finally diagonosed to be DMD/BMD.12 were other MD.11 did not found the pathogenetic mutation by genetic analysis.94.4% MD patients achieved genetic diagnosis.Slow progress in muscle weakness was found in DMD/BMD patients.Their CK level is significantly high.2.1 The positive rate of MLPA was 66.7%.113 cases were delection and 19 were duplication mutation.77 different mutations were found.24 cases were single exon delection.Whereas 89 were mutipul exons delection.The largest one was exon 1-60.2.2 The deletions primarily clustered at exons 45–55,followed by exons 2–19.The duplication locations were involved the 3‘ end of the gene.3 Large rearrangement breakpoints are clustered in hot spot intron 43-55.Intron 44 was the most common one.Delection breakpoints are clustered in intron 43-55.Intron 44-54 and 43-55 were the starting breakpoint and ending breakpoint which involved frequentliest respectively.No hot spot was found in duplication breakpoints.4 26 cases(19.7%)carried in frame mutations.106(80.3%)were out of frame.4 cases were too young to evaluat the phenotype.The reading frame rule holds true for 86.71% DMD/BMD.5 A total of 43 cases with point mutations were detected by NGS analysis.Furthermore,24 previously unreported mutations were detected.Point mutations including 23 nonsense,8f rameshift,1 missense and 11 splice site mutations.Conclusions 1.Most of the patients suspected with MD were diagnosed to be DMD/BMD.94.4% patients got the genetic diagnosis by our strategy.2.MLPA combined with NGS was effective for detection of the mutations in dystrophin gene exons.3.The mutation typies in Guangxi population were variable.The frequency of delection and duplication were different from previous studies in Hongkong,Taiwan and some other Asia coutries.4.The distribution of delection was similar with previous data.The duplication locations were in contrast to previous studies,which involved the 3‘ end of the gene.The distribution of delection breakpoints was similar with previous data.Whereas the distribution of duplication breakpoints was different.5.The reading frame rule holds true for 86.71% DMD/BMD.It is useful in the evaluation for the diseas.6.The constitusion of point mutations was similar with previous studies.Part 2 The Clinical,Pathological and Molecular Analysis of a Family with Bethlem Myopathy Caused by a Novel Splicing Mutation Identified in COL6A2Objective We aimed to study the pathogenicity of the novel mutation by analyzing the clinical phenotype,imaging,skeletal muscle biopsy pathology,and genetic result in a Family with Bethlem myopathy.The pathogenicity and pathogenesis of the mutation was identified from m RNA and protein levels.This study provides theoretical basis for accurate genetic counseling and even gene therapy in the future.Methods 1.The clinical manifestations,examinations,and imaging data of a family with Bethlem myopathy were collected,genetic pedigree was draw.2.Muscle diseases panel based on next generation sequencing was chosen according to the clinical characteristic.3.1 Sanger sequencing was used to indentifie the status of the probands and family members,then coseparation status were analyzed.3.2 In order to learn whether the mutation was novel,several databases were searched for the relevant information of the mutation.3.3 Retrieving the occurrence rate of the mutation among the normal population in the NCBI db SNP database,Hap Map database,Genome l000 database and Chinese population SNP database.4.Biological softwares were used to predict the influence of the mutation on protein function.5.Fibroblast was cultured from patients‘ skin.Vimentin immunohistochemistry was used to indentidied the fibroblast.6.RNA-sequencing was applied to learn m RNA changes.7.RT-PCR was used to clarify the sequence of the splicing variants.8.Differential gene exzpression analysis was used to compare the expression of COL6A2 in the m RNA for patient and normal control.9.Real-time PCR was used to indetified the expression of COL6A2 in the m RNA.10.Muscle biopsy pathological examination and Immunohistochemical staining were undergone to confirm the pathogenicity of the mutation.Results 1.1 This family has three patients and the hereditary mode is autosomal dominant inheritance.The clinical manifestations including slow progress in muscle weakness,experienced difficulty getting up from the floor and climbing stairs,walking on tiptoe,elbow and hip contracture,long finger flexors,slightly elevated CK level.The father has the most severe symptoms,his hands cannot be combined,can't stand up after squat,the second son performances the lighest symptoms,his hands can still folded,can get up from the floor.1.2 MRI manifestations of bilateral lower limb skeletal muscles reveals symmetric diffuse fatty infiltration on both sides of the lower extremities.1.3 Muscle biopsy showed that mild dystrophic features could be observed in muscular fibers.2 Splicing mutation c.736-1G>C in COL6A2 gene was found in three patients.2.1 This variants were absent in normal controls from exome sequencing of 1000 Genomes,db SNP,Ex AC database.3.2 Sanger sequencing revealed this mutation was absent in other family members except these three patients.Itwas co-segregated within the family.3.3 This mutation was not been report in database before.It was a novel mutation.3.4 Mutation Taster predicted it to be harm.4 Human Splicer Finder and Alternative Splice Site Predictor suggested that this mutation will result in the elimination of wild-type splice acceptor sites that would result in either skipping of the respective exon or the creation of new cryptic splice acceptor site or intron retention additionally.5 Fibroblastwas locally aligned in parallel clusters.Vimentin immunohistochemistry revealed vimentin was positive expressed.6 RNA-sequence found two types of splicing variants.One was part of intron 4 was retention(36%);another one was exon 5 skipping(14%).7 RT-PCR demonstrated an additional PCR product.It was confirmed to be the products of splicing variants by Sanger sequence,which would cause a truncated protein.8 Differential expression analysis revealed the expression of COL6A2 in m RNA of patients was less than the normal control.9 Real time PCR demonstrated the expression of COL6A2 in patient‘s fibroblast was 1/10 times of normal control.10 Collagen VI immunohistochemistry revealed Collagen VI is normal in basement membrane surrounding muscle fibres,wheras Collagen VI is virtually absent in fibroblast.Conclusions 1.The clinical phenotypes are consistent with Bethlem myopathy.The older,the more severe of the disease.2.We report a novel splicing mutation in COL6A2 which was responsible for a family with BM.The inheritance pattern is autosomal dominant.3.RNA-sequencing is a simple and economic approach in analyze the splicing influence on m RNA.What is more,the percentage of splicing variant can be determined.4.The pathogenic mechanism of the mutation c.736-1G>C is abnormal splicing in m RNA.The mutation activates a cryptic splice acceptor site and exon skipping,which result in a truncated protein.5.The expression of COL6A2 was lower in ptient‘s m RNA.6.MRI of lower limb skeletal muscles is beneficial for the diagnosis of BM and the location of the Muscle biopsy.And it can evaluate the severity of the affected muscles.The content of fatty is a way to evaluate the progression of the disease.7.The Collagen VI immunohistochemistry in BM patients‘ muscle and fibroblast might be different.We should make a diagonosis according to the phenotype,genetic analysis and immunohistochemistry.