Sidt2 Lossinduces Mouse Skeletal Muscle Damage And The Study Of Diagnosis And Treatment Of Malonicaciduria

Objectives Analyze skeletal muscle phenotypes and autophagy mechanism of the muscle-specific Sidt2 knockout mice.Study phenotypes of malonicaciduria and analyze clinical treatment effects.Methods （1）Sidt2^f/fCre（-）mice and Sidt2^f/fCre（+/-）hybrid breeding obtained Sidt2^f/fCre（+/-）model mice(i.e.Sidt2^f/fCre)and Sidt2^f/fCre（-）control mice(i.e.Sidt2^f/f).DNA from mouse tails was amplified to confirm that Sidt2 was successfully deleted at DNA level.（2）The body weights of knockout mice and control mice were measured to analyze body weight changes and growth differences of knockout mice.The tibialis anterior muscle,gastrocnemius,and soleus were isolated to analyze skeletal muscle morphology and weights between the two groups.Serum creatine kinase（CK）changes of model mice were also examined and analyzed.Skeletal muscle tissue sections of 2-month-old model mice and control mice were subjected haematoxylin-eosin and immunohistochemistry staining.（3）Western blot and immunofluorescence detection were performed to investigate autophagy-related proteins p62,LC3,Lamp2 and Ubiquitin expression levels in skeletal muscle-specific Sidt2 knockout mice.The lysosome enzymes were analyzed.（4）The new mutation was confirmed by comparing with the patient’s biological parents and direct sequencing of the corresponding sites in normal controls.Amino acid homology analysis was done to exclude polymorphism.（5）The patient was advised to have a standard therapy.The clinical outcomes were followed up.Results Muscle-specific Sidt2 knockout mice have been successfully generated to perform the corresponding experiments.Body weights of skeletal muscle-specificSidt2knockout mice from two weeks old to twenty-four weeks old had no significant changes compared with the body weights of control littermates;The absolute weights of the tibialis anterior were smaller in Sidt2^f/fCre mice at 8 weeks of age,while there were no obvious differences in the absolute weights of gastrocnemius and soleus muscles as compared to Sidt2^f/f controls.However,relative muscle weights after normalization to body weight showed virtually no difference between Sidt2^f/fCre and Sidt2^f/fanimals in the soleus muscle,gastrocnemius and soleus muscles.The levels of serum creatine kinase were increased at one month old while there was no significant change at two months old.HE staining revealed myofiber structure and size disorder,along with the presence of centronucleated muscle fibers,basophilic inclusions in myofiber and inflammatory cell infiltration among myofibers.PAS and ORO stainings were negative.ACP,α-GPD,NADH andAMP stainings were positive.Western blotting analysis showed high levels of p62,LC3 and Ubiquitin proteins but a normal level of beclin1 protein in Sidt2^f/fCre mice at eight weeks old compared with the control littermates.Immunofluorescence showed that the p62fluorescent particles in model muscle sarcoplasmic co-localized with ubiquitin fluorescent particles,and partially co-localized with Lamp2 fluorescent particles.The lysosome enzyme of model mice was normal.The patient had a novel heterozygous mutation（c.911G>A,p.G304E）in exon4 of the MLYCD gene.Genome analysis of the patient revealed a57Kbp heterozygous deletion in 16q23.3 comprising the first three exons of MLYCD gene on the other chromosome.The missense mutation was located in the highly conserved region of the gene.After treatment for one and a half months,the patient’s urinary malonic acid became lower than pretreatment,while malonylcarnitine increased slightly after one and a half months.At the following-up examination in six months,the urinary malonic acid was undetectable while the blood malonylcarnitine did not change remarkably.Meanwhile,the parents observed an improvement in the patient’s mental health compared with the past.Conclusions Muscle-specific Sidt2 knockout mice had been successfully generated to accomplish the study.The model mice presented elevated serum CK,myofiber structure disorder,along with the presence of centronucleated muscle fibers,this muscle damage phenotype may be caused by the dysfunction of the autophagy pathway;We reported an affected Chinese female who carried a novel heterozygous mutation and a large deletion in the MLYCD gene.After a standard treatment,the patient had an improvement in the clinical symptoms.