Genetic Analysis And Prenatal Diagnosis Of DMD/BMD

Background and objectivePseudo-hypertrophic muscular dystrophy is the most common X-linked recessive muscular disease,including Duchenne muscular dystrophy(DMD)and Becker muscular dystrophy(BMD),with an incidence of about 0.02%in live births of male infants.Children with DMD usually develop the disease between 2 and 5 years old,with progressive muscular atrophy and gradual loss of motor ability.They often lose the ability to walk at about 12 years old and eventually die of heart and lung failure.The clinical symptoms of children with BMD are similar to those of children with DMD,but the onset time is later and the course of disease progresses slowly.Usually,the normal life span is not affected,but the quality of life of the patients is seriously affected.At present,there is no efective clinical treatment method,and gene therapy is the most promising and curable method.Accurate gene diagnosis and prenatal diagnosis technology are important ways for early detection,early prevention and early treatment.In this study,the combined application of MLPA,NGS(highthroughput sequencing),Sanger sequencing,fluorescence quantitative PCR technology and other methods for gene diagnosis.Besides,the application of STR linkage analysis method for prenatal diagnosis,and through the two ways of mutual authentication,analysis of DMD gene mutation spectrum,a new mutation rate heritage rate,the analysis of the data in our study will exert positive effects on the gene therapy of the disease.Materials and methods1.In this study,2042 unrelated DMD/BMD families who had been clinically and genetically diagnosed were selected from the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University between January 1,2005 and August 31,2019.2.MLPA analysis was performed on pedigree propositus using MLPA detection kits to identify deletion or duplication sites.3.For patients with negative MLPA results,79 exons of DMD were detected by NGS,and the mutation sites were verified by Sanger sequencing.4.For pregnant women who need prenatal diagnosis in their families,molecular diagnosis of the fetus can be carried out by extracting fetal villi or amniotic fluid from pregnant women and applying STR linkage analysis method.If healthy children are healthy,peripheral blood is extracted after birth for verification.If children with abortion were selected,flow products were collected for verification.Result1.Genetic analysis was performed on 2042 Chinese Han DMD/BMD family probs using MLPA,NGS and Sanger sequencing.Mutations were detected in 1986 families,and no mutation was detected in 56 families,with a detection rate of 97.26%(1986/2042).DMD and BMD accounted for 78.60%and 21.40%,respectively,including 33 female propositus.Large fragment deletion,duplication and small mutation accounted for 71.85%,8.76%and 19.39%,respectively.Among them,the most common types of exon deletion and duplication were E45-50 deletion and E2 duplication,respectively,and no hot spots were found in small mutations.The present study examined the family history of DMD in 1595 families.58.62%(935/1595)was inherited from the mother,and the rate of new mutation was 41.38%(660/1595).In the present study,34.28%(700/2042)of the patients whose symptoms could be alleviated by an existing gene therapy approach.2.In 872 pregnant women in the family,931 prenatal diagnoses were performed by linkage analysis(one pregnancy was counted as two pregnancies).Among them,20.73%(193/931)were male fetuses,16.33%(152/931)were female carriers,37.59%(350/931)were normal male fetuses,and 25.35%(236/931)were normal female fetuses.3.Genetic diagnosis of neonates or fluid products was performed,and the results were all matched.ConclusionIn this study,MLPA,second-generation sequencing and Sanger sequencing were combined to conduct gene analysis for DMD families,which could provide a clear diagnosis for DMD families.Since the new mutation rate(de novo)of DMD is as high as 41.38%,the fatality rate is high,the prognosis is poor,and there is no effective cure,so far,prenatal diagnosis and genetic counseling should be paid great attention.In prenatal diagnosis and genetic counseling,even families with no birth history or family history of DMD still need to pay attention to the possible risk of DMD.It is very important to carry out non-invasive prenatal diagnosis services for DMD,carry out neonatal screening,expand the scope of prenatal screening for DMD,and prevent the birth of children with newly developed mutations of DMD.