Pathogenicity Study Of Limb-girdle Muscular Dystrophy Type 2Q Caused By Plec Gene Novel Mutations

ObjectiveThis study conducted a pathogenic research on a patient with suspected limb-girdle muscular dystrophy type 2Q.The research clearly diagnoses at the genomic level,and preliminary explores the pathogenic mechanism of the disease.To provide corresponding helps for genetic counseling and clinical diagnosis of limb-girdle muscular dystrophy.Method1.Collecting clinical material,biochemical tests,electromyography and imaging data of proband with suspected limb-girdle muscular dystrophy.2.According to the clinical characteristics of the proband,next-generation sequencing(NGS)of myopathy panel was performed.3.Polymorphic loci were excluded by retrieving the Genome l000 database(http F://www.1000 genomes.org/)and the NCBI db SNP database(https://www.ncbi.nlm.nih.gov/snp/).According to the ACMG genetic variation classification criteria and guidelines,consult the relevant database to determine whether the mutation site and its pathogenicity are reported in the literature.Sanger sequencing was used to verify suspected pathogenic mutation sites and analyze whether patient’s parents carry the mutation,and draws a pedigree of family.Bioinformatics softwares were used to predict the effect of mutation on protein biological function.4.Extract RNA from proband muscle tissue and sequence the transcriptome to understand whether the suspected pathogenic mutation affects m RNA expression in muscle tissue.5.Analysis of the experimental results of hematoxylin-eosin staining(HE)and immunohistochemical staining(IHC)to understand the pathological changes of muscles and related protein expression in proband.6.Culturing the fibroblast from the skin of the proband,and the suspected lesion protein was immunohistochemically stained to understand the expression of plectin protein in the skin fibroblasts of the proband.Results1.Proband climbing hard was found at 3 years old,could’t jumping,squatting difficult,uncoordinated running,and normal intelligence.Slight atrophy can be seen in the shoulder and pelvic girdle muscles.Upper and lower proximal limb muscle strength were grade 4 on the MRC scale,upper and lower distal limb muscle strength were grade 4+ on the MRC scale,muscle tension was normal.Gower’s sign(-).The serum creatine kinase(CK)was up to 1121U/L.Electromyography suggest neurogenic damage.Muscle MRI indicated fatty infiltration of both lower limbs.2.The next-generation sequencing revealed that PLEC gene of proband carry complex heterozygous missense mutations in exons 31(c.6428C>T)and32(c.13192G>A),respectively.3.The Genome 1000 database and the single nucleotide polymorphism database indicated that mutation sites were non-polymorphic.c.6428C>T and c.13192G>A had not been reported and were novel mutations when referring to relevant databases.Sanger sequencing verified that PLEC gene complex heterozygous mutations of the proband were derived from c.13192G>A of the father and c.6428C>T of the mother,respectively.None of the parents had related clinical manifestations,and there was cosegregation within the family.Comprehensive analysis of the prediction results suggests that c.13192G>A and c.6428C>T complex heterozygous mutation may be pathogenic.4.Transcriptome sequencing results showed that there was no significant change in m RNA expression of muscle tissue in proband.5.Nucleus accumulation and nuclear chain in the muscle fibers can be seen by hematoxylin and eosin staining.Immunohistochemistry staining of plectin showed plectin protein aggregation in the sarcoplasm.6.The fibroblasts of proband’s skin are irregular triangle,the cell body is bright,narrow and long with abundant cytoplasm.Immunohistochemistry showed that plectin protein was aggregation in skin fibroblasts.Conclusion1.According to the clinical features and laboratory results,the proband was clinically diagnosed as conforming to limb-girdle muscular dystrophy type 2Q,which is an autosomal recessive inheritance disease.2.It is first reported that PLEC gene 31 exons(c.6428C>T)and 32 exons(c.13192G>A)complex heterozygous missense mutation can lead to limb-girdle muscular dystrophy type 2Q,expanded the mutation spectrum of PLEC gene and provide help for the clinical diagnosis of limb-girdle muscular dystrophy type 2Q.3.MRI can be used to understand the proportion of fat in the diseased muscle,affected muscle group and severity,and help to judge the condition of LGMD2 Q.4.Based on clinical manifestations,the accuracy of pathogenicity of new mutations can be improved by various bioinformatics software to predict the pathogenicity,RNA sequenclng to measure m RNA expression,pathological biopsy to detect the tissue.