Study On Pathogenicity Of A Novel Splicing Mutation For LMNA Gene Causing Limb Girdle Muscular Dystrophy 1B

ObjectiveThis study was aimed to study the clinical manifestations,pathological biopsy and the pathogenicity gene detection of a suspected limb-girdle muscular dystrophy type 1B.The pathogenicity of the novel mutation was judged by retrieving Database,and the effect of the novel mutation on mRNA splicing was predicted by bioinformatics softwares.The pathogenicity of the novel mutation on mRNA level and protein level was finally confirmed by experimental methods,and preliminary explorating the pathogenesis to offer help to the clinical diagnosis and genetic counseling of limb-girdle muscular dystrophy.Method1.The clinical data,myocardial enzymes and other blood biochemical examinations,electromyography and cardiac ultrasound results of a suspected limb-girdle muscular dystrophy type 1B were collected.2.Next-generation sequencing of myopathy panel was done taking into consideration combined with the clinical features of the child.3.The gene mutation result was verified by Sanger sequencing and was also confirmed that his parents did not carry the mutation.The polymorphic loci was filtered out by retrieving the NCBI dbSNP database and the Genome l000 database.Human Gene Mutation Database and NHLBI Exome Sequencing Project were cross checked so as to confirm if the novel mutation is a new mutation.Bioinformatics softwares were used to predict the effect of the mutation on mRNA,as well as possible pathogenic mechanism.4.Biopsy of the child’s gastrocnemius muscle was taken for HE and IHC to understand muscle pathology and protein expression.5.The experiments were designed to verify the theory of bioinformatics combined with the transcriptome sequencing of the RNA of the gastrocnemius muscle to understand the effect of the mutation on mRNA splicing.The primary skin fibroblast from the skin of the child was cultured.The immunohistochemical study in Lamin A protein of skin fibroblasts was taken to understand the expression of Lamin A protein in the child.RT-PCR on the cDNA of skin fibroblasts of the child combined with normal controls was taken to understand the difference in mRNA of LMNA gene between the child and normal controls.Results1.The proband was walking weakly for two years with progressively increased muscle weakness,a waddling gait,difficulty in climbing stairs and squatting,CK 779 U/L,upper limb muscle strength 4 points,lower limb muscle strength 4-points,Gower’s sign(+).Electromyography does not suggest myogenic damage and heart color Doppler ultrasound detection was normal.2.The next-generation sequencing of myopathy panel found that the child had a c.810+2T>C splicing heterozygous mutation of the LMNA gene.3.Sanger sequencing verified the result and confirmed that his parents did not carry the same mutation.Both the NCBI dbSNP database and the Genome l000 database showed that the mutation was extremely low in the normal population and was not a polymorphic locus.There is no pathogenicity report of this mutation in HGMD databasea and ESP database,that is,this is a novel mutation.GERP++RS predicted that the mutation site is in a highly conserved region,both Human Splicer Finder and NetGene2 predicted that the mutation made splicing change in mRNA.4.Gastrocnemius muscle HE staining showed muscle fibers in different sizes,the number of nuclei in muscle fibers had increased significantly,with connective tissue hyperplasia and mild muscular dystrophy.Muscle immunohistochemistry showed that Lamin A protein expression was normal.5.The transcriptome sequencing showed that one part of the muscle mRNA of the child was normal,accounting for 92.2%;and the other part was 4 intron retentions,accounting for 7.8%.The primary fibroblasts of the skin were fusiform and irregular triangles.The cell bodies are long and narrow,translucent,and rich in cytoplasm.The immunohistochemistry study of Lamin A protein in skin fibroblasts was absent.RT-PCR of the cDNA in the child’s skin fibroblasts showed that the mRNA expression level of LMNA gene was significantly lower than the normal controls.Conclusion1.The c.810+2T>C splicing heterozygous mutation of LMNA gene was reported as a pathogenic mutation of LGMD1 B.2.The pathogenesis of the c.810+2T>C splicing heterozygous mutations of the LMNA gene is that the mutation leads to alternative splice donor site and intron retention to form an aberrant splice variant,thereby forming truncated proteins.3.When studying the effect of splice site mutations on mRNA splicing,transcriptome sequencing is simple and efficient,and the proportion of aberrant splice variants can be determined.