Phenotype–genotype Analysis Of LMNA-related Muscular Dystrophy Patients And Functional Study Of LMNA Mutants

Objective:This study aims to analyza the clinical data of Chinese patients withinfantile-onsent LMNA related muscular dystrophy, discuss the clinical manifestations,muscle pathological features and characteristics of genotype with genetic analysis,analysis of the phenotype-genotype and the functional changes of some mutatedLMNA are investigated to understand the potential pathogenic mechanism.Methods:17cases of LMNA related muscular dystrophy were clinically diagnosed andcollected according to the diagnostic criteria. Skeletal muscle specimens werebiopspied from the probands for routine histological stains and observing the changesof the ultrastructure of muscle fibers under electron microscopy. Genomic DNA wasextracted from peripheral blood leukocytes of the patients and their parents,polymerase chain reaction（PCR） was performed to amplify the12exons of LMNA,PCR products were detected by agarosegel electrophoresis and then purified. TheLMNA gene mutations were analysed by DNA sequencing. RNA was extracted fromperipheral blood leukocytes of some patients, then reverse transcription-polymerasechain reaction（RT-PCR） was performed with cDNA which was produced throughreverse transcription and primers which were designed for the sequence containingthe splice site. RT-PCR was verify the changes by splice site mutation on RNA level.The functional study, the in vitro-expression and subcellular location of lamin A/C,was performed by immunofluorescence of cultured skin fibroblasts with specificantibody and constructions of eukaryotic expression mutant plasmid.Result:1、Phenotype features:17cases including11cases of L-CMD and6cases ofEDMD were consistent with the clinical diagnosis of LMNA related musculardystrophy. L-CMD cases presented neonatal-onsent or infantile-onsent rapidly progressing trunk and neck-axis muscle weakness, poor head control, jointcontractures with progression from distal joints to proximal joints, spinal deformity,hypotonia, hyporeflexia or abolition of tendon reflex; EDMD cases presented earlychildhood-onsent slowly progressing scapular and fibular muscle weakness andatrophy, joint contractures with progression from proximal joints to distal joints,spinal deformity, hypotonia, hyporeflexia or abolition of tendon reflex. Most caseshad mild or moderate elevation of CK levels, myogenic injury examined by EMG,disorders of cardiac conduction on the electrocardiogram and normal brain MRI..2、Pathological features: muscle biopsies were performed on12in17cases forroutine histological stains and observation of the ultrastructure of muscle fibers underelectron microscopy. Dystrophic changes or myopathic changes associated withinflammation on early-onset L-CMD were observed by HE staining. Theultrastructure of fibers under electron microscopy showed focal or extendeddisruption of filament, indistinct sarcomeres, many vacuoles in the muscle fibers,increased adipose and connective tissue, abnormal nuclear morphology withheterochromatin condensation, focal loss of nuclear membrane, accumulation ofmitochondria around the nucleus, nuclear bands, and nucleolar holes.3、Genotype features: the17cases were genetically identified by the detection ofLMNA mutations. The mutations including13missense mutations,3deletionmutations and1splicing mutation were all heterozygous.16kinds of mutationsincluding8novel mutations were found. The sources of mutations on14cases wereverified:1paternal mutation and13de novo mutations. Mutations（c.91G>A、c.94₉6delAAG、c.116A>G、c.745C>T、c.746G>A、c.832G>C、c.1124C>G、c.1580G>C） were correlated with LMNArelated muscular dystrophy.4、 Lamin A/C function changes affected by LMNA mutations: Nucleis ofunequal size and abnormal morphology were exhibited and lamin A/C expressionwith abnormal distribution was detected in the nucleus on dermal fibroblasts fromone case by immunofluorescence. After transfection with wild-type and mutants, thelamin A/C expression with abnormal distribution was detected in the mutants byusing fluorescence microscope. Conclusions:1、The phenotype of the cases corresponds with L-CMD or EDMD, the biopsiedspecimens from patients exhibited dystrophic changes or myophic changes withinflammatory changes on early-onset L-CMD, destruction of a large number ofmuscle fibers and abnormal nuclear morphology were observed under transmissionelectron microscopy.2、The mutations in our study which were almost de novo in the study were allheterozygous and included8novel mutations. Among them, c.91G>A、c.94₉6delAAG、c.116A>G、c.745C>T、c.746G>A、c.832G>C、c.1124C>G、c.1580G>C were correlated with LMNArelated muscular dystrophy.3、Lamin A/C expression with abnormal distribution in the nucleus because ofLMNA mutations resulted in the abnormal morphology of nucleus, and it might bethe pathogenic mechanism.