The Function And Mechanism Of The Novel Homozygous Mutation Of DNAJB6 In Myofibrillar Myopathy

Backgrounds: DNAJB6 is a newly discovered pathogenic gene of myofibrillar myopathies(MFMs)in recent years.The protein encoded by DNAJB6 exists in nucleated cells as two subtypes of DNAJB6 a and DNAJB6 b,which can eliminate abnormal proteins and maintain the normal function of cell homeostasis.Previous studies have shown that the DNAJB6 b subtype is the responsible protein of DNAJB6gene-related MFMs.Our study found a novel DNAJB6 homozygous mutation c.695?699del(p.V232 G fs*7)in the genetic test results of an MFM patient.This mutation is located in exon 9 and does not encode the DNAJB6 b subtype,but only leads to the C-terminal of DNAJB6 a subtype.However,the function of DNAJB6 a in the pathology of MFMs is unknown.Therefore,the hypothesis is that the novel mutation causes MFMs by affecting the function of DNAJB6 a subtype proteins.In order to prove this hypothesis,this study intends to combine bioinformatics,cell biology,molecular biology and genetic engineering techniques to carry out clinical,in vivo animal and in vitro cell research to clarify the role of the mutation in the development of MFMs,and explore its mechanism of action,to provide innovative scientific basis for precise diagnosis and treatment of DNAJB6 related MFMs.This research consists of three parts:Part 1: Clinical study of the homozygous mutation of DNAJB6 c.695?699del in a family of patients with myofibrillar myopathy Methods: Comprehensively screen the clinical data of family members,complete the sanger sequencing of the DNAJB6 gene of peripheral blood samples,predict the pathogenicity of the mutation site according to the ACMG guidelines and bioinformatics software;at the same time,use immunofluorescence staining,q-PCR and Western Blot and other methods detect the distribution and level of DNAJB6 and its related proteins(Desmin,TDP-43,LC3 B,p62 and Dysferlin protein)in the skeletal muscle tissue of the proband.Results: Only the proband had the clinical phenotype of MFMs and the homozygous mutation of DNAJB6 gene c.695?699del in the family of the proband.Compared with the normal controls,the expression of DNAJB6 protein in the muscle tissue cells of the proband is absent in the nucleus,and abnormally accumulates under the cell membrane and in the cytoplasm.At the same time,Desmin,TDP-43,LC3 B,p62 and Dysferlin proteins abnormally accumulate or express ectopically in the cytoplasm.Compared with the normal controls,the expression level of human(h)-DNAJB6 b m RNA in the muscle tissue of the proband had no significant difference,and the expression level of total h-DNAJB6 m RNA was significantly reduced.At the same time,the wild type(wt)h-DNAJB6 a protein expression in the muscle tissue of the proband is absent;the total h-DNAJB6(wt h-DNAJB6a+mut h-DNAJB6a+h-DNAJBb)expression level is about that of the 1/8 of the level of normal control;the expression levels of Desmin and TDP-43 were significantly increased,about twofold of the expression level of the normal control.According to ACMG guidelines,based on population frequency,family co-segregation,pathogenicity evaluation database prediction results and the experiment results,it is determined that h-DNAJB6 c.695?699del(p.Val232 Gly fs*7)homozygous mutations exist in PS4,PM2,PM4 and PP1 evidences.Conclusion: A novel homozygous mutation that h-DNAJB6 c.695?699del(p.Val232 Gly fs*7)was found in patients with MFM for the first time.According to the ACMG guidelines,we speculate that this new mutation is a possible pathogenic variant of MFMs.Part 2: The effect of homozygous mutation of DNAJB6 c.698?702del on motor function and skeletal muscle pathological phenotype of mouseMethod: We use the CRISPR/Cas9 gene strategy to construct mouse(m-)DNAJB6gene c.698?702del(p.V233 G fs*80)knock-in(KI)mice that are homologous to the mutation of the proband.We prove the effects of the mutation on m RNA and protein expression levels,and continuous observe multiple behavior indicators,skeletal muscle histopathology and molecular pathology changes at 3,6,9,12,18 month of age of KI mice.Results: Compared with littermate wild-type mice,the m RNA(P=0.0023)and protein(P=0.0018)expression levels of skeletal muscle m-DNAJB6 a of12-month-old homozygous mice were significantly lower;Compared with mice,the suspension time(P=0.023),swinging time(P=0.034)and running time(P=0.022)of both upper limbs of homozygous mice at 9 months of age were significantly reduced.Homozygous mice at 12 months of age The grip of the limbs continued to decrease significantly(P=0.0023);compared with the littermate wild-type mice,the activity of succinate dehydrogenase and cyclooxygenase in the muscle cells of the 12-month-old homozygous mice was partially reduced,and the type IIA muscle fibers were distributed The ratio was significantly reduced(P=0.025),the distribution ratio of type IIB muscle fibers was significantly increased(P=0.012),a large number of rimmed vacuoles appeared in the muscle cytoplasm of homozygous mice at 18 months of age,and DNAJB6 and Desmin proteins were under the cell membrane and cell Abnormal aggregation in the qualitative,and the expression of TDP-43,LC3 B and p62 proteins around the vacuole was significantly increased(P=0.002,0.004 and0.001).Conclusion: The homozygous mutation of m-DNAJB6 c.698?702del(homologous mutation of h-DNAJB6 c.695?699del)only reduces the expression of m-DNAJB6 a in mouse skeletal muscle,and can cause decreased motor behavior and similar MFMs in mice histopathology,molecular pathology phenotype.Part 3: The effect of DNAJB6 c.695?699del homozygous mutation on the anti-aggregation ability of poly Q protein and its mechanism Methods: We use plasmid construction and cell transfection methods,compare the expression level and position changes of wild-type(WT)and mutant(mut)h-DNAJB6 a plasmids in HEK293 T cells;we explore the mechanism of h-DNAJB6 c.695?699del homozygous mutation affecting the expression level of DNAJB6 a by m RNA and protein stability experiments.In addition,by constructing an anti-aggregation cell model of h-DNAJB6 a against poly glutamine(poly Q)protein,comparing WT and mut h-DNAJB6 a plasmids The anti-aggregation ability of poly Q and the change of the anti-aggregation ability of mut h-DNAJB6 a protein at different doses.Results: Cellular immunofluorescence results showed that WT h-DNAJB6 a protein was only distributed in the nucleus,while mut h-DNAJB6 a protein was scattered throughout the cell;compared with WT h-DNAJB6 a protein,the same gene dose of mut h-DNAJB6 a protein The expression in HEK293 T cells was significantly decreased(P=0.0012),and its m RNA stability was significantly decreased(P=0.0078);however,the effect of WT h-DNAJB6 a and mut h-DNAJB6 a at the same expression level on the anti-aggregation ability of poly Q protein There was no significant difference(P=0.12),but its anti-aggregation ability gradually decreased with the decrease of mut h-DNAJB6 a protein expression level.Conclusion: h-DNAJB6 c.695?699del affects the protein expression level by reducing the m RNA stability of h-DNAJB6 a,and therefore reduces its anti-aggregation function against abnormal proteins.Total conclusion:The new recessive mutation c.695?699del of h-DNAJB6 can lead to myofibrillar myopathys,and the possible mechanism is that it may reduce the expression level of DNAJB6 a protein to cause abnormal protein deposition in cells.The innovations of this research:1.For the first time,a new mutation site of DNAJB6 was discovered in a patient with myofibrillar myopathy.This site has not been reported in related databases.2.It was reported earlier that the inheritance mode of this gene mutation is autosomal dominant inheritance,but in this case,the inheritance mode of this patient is autosomal recessive inheritance,which is likely to be a new inheritance mode of skeletal muscle diseases caused by this gene mutation.3.Previous studies have suggested that DNAJB6 b carrying pathogenic mutations is the responsible protein for the occurrence of MFM,but the new mutations discovered in this study only affect the expression of DNAJB6 a and lead to the clinical myopathy phenotype.Therefore,we speculate that DNAJB6 a also plays an important role in the maintenance of skeletal muscle structure and function,and its loss of function is a new mechanism leading to the occurrence of MFMs.4.The function of the C terminal region of DNAJB6 a was previously unknown.This study found that the deletion of the C terminal region can significantly reduce the stability of DNAJB6 a m RNA and lead to a decrease in protein expression,which is a new knowledge of the C terminal region in the molecular structure of DNAJB6 a.