Clinical Phenotype-Genotype Characteristics ,mtDNA Pathogenicity And Pathological Mechanism Of Pure Mitochondrial Myopathy

Part Ⅰ:Clinical Phenotype-Genotype Characteristics of Pure Mitochondrial MyopathyBackgroundMitochondria are organelles coated by double membranes,which exist in most cells of human body.Except for mature red blood cells,each cell contains hundreds of mitochondria.The respiratory chain of mitochondria is composed of five enzyme complexes,which is an important place for cells to carry out aerobic metabolism and oxidative phosphorylation(OXPHOS).Electron transmission through respiratory chain plays a significant role in cell energy production,intracellular signal transduction,cell growth,proliferation,differentiation,aging and withering.Mitochondrial disease is a heterogeneous disease caused by mitochondrial DNA(mtDNA)or nuclear gene(nDNA)mutation,leading to OXPHOS of mitochondrial respiratory chain,and insufficient cell energy source.According to different organs involved,mitochondrial diseases can be classified as mitochondrial encephalopathy,mitochondrial myopathy and mitochondrial peripheral neuropathy.The clinical phenotypes of mitochondrial disease are complex and diverse,and the symptoms vary with the age of onset.Although multiple organ involvement is an important feature of mitochondrial disease,there are still such subtypes as single organ involvement.Primary mitochondrial myopathy refers to mitochondrial disease with skeletal muscle involvement as the main manifestation caused by mtDNA or nDNA mutation.The skeletal muscle involvement here refers to but not limited to the involvement of skeletal muscle.Based on current understanding,primary mitochondrial myopathy can be roughly divided into two categories,one is ocular muscle involvement phenotypes with chronic progressive external ophthalmoplegia(CPEO)or Kearns-Sayre syndrome as the main manifestations and the other is subtypes with trunk muscle or limb band muscle involvement as the main manifestations.The latter has not been officially given a name,for which some scholars call it pure mitochondrial myopathy(PMM),also others call it limb girdle mitochondrial myopathy(LGMM)or isolated mitochondrial myopathy(LMM).PMM is rare to been seen in clinic.At present,most researches on PMM are case reports at home and abroad,lacking systematic clinical,pathological and genetic research,and no consensus has been reached.PMM may be an independent mitochondrial subtype.Its clinical features include muscle weakness,exercise intolerance,fatigue,elevated serum creatine kinase(CK),myalgia and,in rare cases,rhabdomyolysis.It was our team that first used this term in China in 2003,and reported typical cases found in clinical practice.By summarizing case reports,we found that PMM usually follows a benign course,but in some cases,the disease can also develop rapidly.We found that PMM is often misdiagnosed as myositis,muscular dystrophy,lipid deposition disease and so on,and its early diagnosis is still challenging in clinic.Therefore,this part aims to comprehensively analyze the clinical effects,muscle pathology and genetic characteristics of PMM cohort in our center in recent 20 years,and conduct long-term follow-up.We believe that PMM is a unique clinical subtype of mitochondrial disease,which should be recognized by more clinicians,so as to provide help for the early diagnosis and treatment of PMM patients.Methods1.This study included 36 patients with PMM diagnosed by clinical manifestations,pathological results or genetic testing from January 2000 to December 2020 for data collection and long-term follow-up.The study approved by the ethics committee of Qilu Hospital of Shandong University.2.Collected laboratory data of 36 PMM patients,including data of initial diagnosis,CK level,lactate level,brain MRI(magnetic resonance imaging),EEG,minimental state examination(MMSE),Montreal Cognitive Assessment(MoCA),electromyography,echocardiography,etc.3.Skeletal muscle biopsy and tissue staining were performed in 36 PMM patients,including standard hematoxylin eosin(HE)staining,modified Gomori trichrome(MGT)staining,succinate dehydrogenase(SDH)staining,cytochrome c oxidase(COX)staining,SDH/COX double staining,and Oil red O(Oro)staining.The pathological changes of muscle tissue were observed by electron microscope.4.The DNA of peripheral venous blood,oral mucosa,urinary sediment and biopsy skeletal muscle tissue of PMM patients were extracted.The full length of mtDNA and/or nDNA were sequenced with muscle tissue DNA to identify the pathogenic gene.Sanger sequencing technology was used to verify multiple tissues and summarize gene characteristics.Results1.Clinical and demographic information:male/female ratio was 58.3%vs 41.7%.The median age of symptoms was 18.5 years(range 0-58 years),the median age of PMM was 24.5 years(range 4-58 years),and the interval between symptoms and diagnosis was 8.0 ± 7.9 years.We found that myasthenia(n=32,88.9%)was the main clinical feature of PMM,followed by fatigue(n=25,69.4%),exercise intolerance(n=24,66.7%),myalgia(n=12,33.3%),muscle atrophy(n=11,30.6%),cardiomyopathy(n=4,11.1%)and respiratory failure(n=8,22.2%).Of all PMM patients,16 had a family history of mitochondrial disease.2.Laboratory examination results:Serum CK level of most patients(n=31,86.1%)was normal or moderately elevated,and about half of patients(n=19,52.8%)had hyperlactatemia.Electromyography was performed in 31 patients,of which 16 patients(51.6%)were myogenic lesions,10 patients(32.3%)were neurogenic lesions,and 5 patients(16.1%)had normal electromyographic manifestations.All patients were assessed by MMSE and MoC A during enrollment and follow-up,the scores of all the patients were normal.3.Pathological examination of muscle tissue:RRF can be seen on MGT staining in all patients,and the proportion of RRF was between 2.5%and 71.4%.31 patients(86.1%)had COX missing muscle fibers,and the missing proportion ranged from 3.0%to 93.0%.6 patients showed strongly SDH-positive vessels(SSV),and about half of the patients(n=20,55.6%)showed mild to moderate increase of lipid droplets on ORO staining.Electron microscopic examination showed that the morphology and quantity of mitochondria were abnormal.4.Gene detection results:21 patients(58.3%)carried mtDNA point mutations,including 11 patients with m.3243A>G mutation,3 patients with m.8344A>G mutation,2 patients with m.8356T>G mutation,1 patient with m.9176T>C mutation,2 patients with m.5708C>T mutation,1 patient with m.7453G>A mutation,and 1 patient with m.14502T>G mutation.We also found that 2 patients carried mtDNA deletion mutations.5.Follow-up situation:Among 36 PMM patients,we collected follow-up data of 33 patients.Our average follow-up time was 6.7±5.8 years.25 of the 33 patients were stable or improved.Most of these patients were treated with mitochondrial nutrients.Three patients died during follow-up,including two patients died of respiratory failure and 1 died of cardiomyopathy.Five patients had aggravated symptoms periodically during follow-up.Conclusion1.The clinical manifestation of PMM can be limited to muscle always or for a long time,mostly have good outcomes.2.PMM is often associated with subclinical peripheral neuropathy,most of which are axonal lesions.3.The primary genetic defect was mt-tRNA mutation.Part Ⅱ:Pathogenicity Analysis of Novel mtDNA Variation in Pure Mitochondrial MyopathyBackground:Mitochondria is the only organelle containing genetic material.The total length of mtDNA is 16569 bp.It does not bind to histones,a naked closed-loop double chain shape,and both chains can be encoded.There is no intron in the whole sequence of mtDNA,and its coding sequence accounts for 93%of all bases.mtDNA genes are closely arranged and lack the protection of histones,which is very vulnerable to reactive oxygen species(ROS)in oxidative respiration.Therefore,compared with nDNA,mtDNA is more prone to mutation.In mtDNA,tRNA is a hot spot mutation.The reasons why tRNA is prone to mutation include the following three aspects:first,compared with the typical clover structure of cytoplasmic tRNA,the structure of mitochondrial tRNA is loose and unstable.Second,the copy number of mitochondrial tRNA is low,and one amino acid corresponds to only one mitochondrial tRNA copy.Third,the secondary structure of some mitochondrial tRNAs is incomplete.For example,tRNASer(CCU)and tRNALys(UUU)lack D-ring in the secondary structure,which are the reasons why mtRNA is prone to mutation.In PMM cohort,we found three mitochondrial new mutation sites,the pathogenicity of which is not completely clear,including 2 patients with m.5708C>T mutation,1 patient with m.7453G>A mutation and 1 patient with m.14502T>G mutation.Those three types of mutations are located in tRNAAsn,tRNASer(UCN)and ND6 subunit of complex I respectively.We completely verified the function of the pathogenicity of three new mutation sites and explored its pathogenic mechanism.This part mainly introduces the pathogenicity verification of m.7453G>A variant ectopic point.MethodsThis part will verify the pathogenicity of m.7453G>A variation on mitochondrial function.See the following technical roadmap for specific ideas.Firstly,we compared muscle tissues of patients and normal people for pathological staining to analyze the damage of mitochondria in patients’ skeletal muscle,then selected COX positive muscle fibers and COX negative muscle fibers on the stained samples for single fiber DNA sequencing,and used the comparison of muscle tissues for mitochondrial protein synthesis analysis.In addition,we constructed the transmitochondrial cell lines from patients and normal controls of the same haplotype,carried out monoclonal screening,selected wild and mutant transmitochondrial cell models with the same nuclear background,and analyzed mitochondrial tRNA steady-state level,mitochondrial protein synthesis,cell oxygen consumption rate,ROS production,and mitochondrial membrane potentia.Thus,the pathogenicity of m.7453G>A mutation was verified in multiple dimensions.#12Technical roadmap figure:The verification method for the pathogenicity of m.7453G>A mutationResults1.In gene analysis of PMM cohort,we found a new missense mutation of m.7453G>A in tRNASer(UCN),which encodes MT-TS1 gene.Sanger sequencing was used to detect the heterogeneity of m.7453G>A mutation in different tissues,which was highly conserved among different species.Single fiber sequencing of muscle fibers showed that the mutation load of COX negative fibers was significantly higher than that of COX positive fibers.2.Muscle biopsy:It shows pathological characteristics of typical mitochondrial diseases.About 70%of COX negative muscle fibers were found by COX staining,most of which showed broken red fibers(RRF)on MGT staining,suggesting the dysfunction of skeletal muscle mitochondria.3.Activities of mitochondrial respiratory chain complexes:compared with wild cells,activities of respiratory chain complex Ⅰ of mutant cells decreased by 59.3%(P<0.01),and activities of complex Ⅳ decreased by 40.1%(P<0.05).4.RNA level:Northern Blot detection showed that cells carrying 7453G>Amutation presented a decrease in the total amount of tRNASer(UCN).Taking various mitochondrial tRNAs as a reference,the total amount of tRNATrp decreased by 73.6%,53.8%,53.9%,48.8%,48.6%,50.2%,51.9%,52.3%and 46.1%5.Protein level:Western Blot showed that MT-CYB,MT-COl,nDNA encoded NDUFB8 and UQCRC2 decreased significantly,while MT-ND1,MT-ND4 and MT-CO4 decreased slightly.The expression levels of MT-ATP6,SDHB and ATP5A remained unchanged.The expression of mtDNA-encoded subunit MT-ND1,MTND4,MT-ND5,MT-CYB and MT-CO1 in mutant cell lines decreased from 72.79%to 22.44%,and the expression levels of nDNA-encoded subunit NDFUB8,UQCRC2 and MT-CO4 were 20.4%,44.3%and 72.9%of control group respectively.6.Evaluation of mitochondrial respiration:We analyzed the respiration of mutant cells and control cells by Seahorse energy metabolism instrument.Compared with wild strain,mutant cell lines showed only 32.8%basal oxygen consumption rates(OCR)at baseline.ATP production,proton leakage,maximum respiration and reserve OCR were reduced to 31.5%,26.2%,27.2%and 9.3%respectively.Further evaluating the effect of mutation on activities of mitochondrial respiratory chain complexes,we found that complex Ⅰ,Ⅱ and Ⅳ mediated respiration showed significant decreases,which were 15.8%,21.4%and 14.8%respectively.7.Determination of ROS production:red fluorescence intensity of mutant cell lines increased to 263%of wild cell lines,indicating that the production of ROS in m.7453G>A mutant cells increased.8.Determination of mitochondrial membrane potential:the ratio of red/green fluorescence of mutant cell lines decreased significantly,which was 29.8%of that of wild cell lines,indicating that m.7453 G>A mutation reduced the cell membrane potential.Conclusion1.This part first reports the clinical phenotype of PMM caused by new m.7453G>A mutation,which expands the pathogenic gene spectrum of PMM.2.m.7453G>A mutation affects the experssion of tRNASer(UCN).The metabolic disorder of tRNA leads to mitochondrial translation abnormalities,respiratory defects,and increases ROS production,this further damages the mitochondrial intracellular environment,and finally results in the oxidative stress vicious cycle.Part Ⅲ:Expression of GDF15 in Muscle of Pure Mitochondrial Myopathy and its Characteristics of Intracellular DistributionB ackground:Growth differentiation factor 15(GDF15)is the cytokine of transforming growth factor beta(TGF-β)superfamily.Over the past two decades,GDF15 has attracted great attention in medical field due to its multiple roles in various diseases,including cancer,cardiovascular disease and obesity.GDF15 was initially identified as the member of TGF-β superfamily,which was based on the similarity of its amino acid sequences with other members of the protein family.GDF15 precursor protein consists of 167 amino acids and contains an N-linked glycosylation site at amino acid position 70.It dimerizes through a specific disulfide bond to form a pro-GDF15 dimer precursor.The dimer protein is cleaved at RXXR site to form a mature GDF15 containing 112 amino acids.Mature GDF15 dimer protein can be found in human serum,but whether GDF15 precursor and dimer are active in biology is still unknown.Under physiological conditions,GDF15 is highly expressed in placenta,while its expression in colon,kidney and prostate is low.The increase of age,body stress or environmental factors may increase the level of GDF15.Under pathological conditions,GDF15 is highly expressed in a variety of diseases,including tumor,cardiovascular disease,kidney disease,liver disease,mitochondrial disease,infectious diseases and so on.Our previous research found that the level of GDF15 in the serum of patients with mitochondrial disease was greatly increased,and the level of GDF15 in the serum was positively correlated with the severity of clinical manifestations of mitochondrial disease.We know that most patients with mitochondrial disease suffer from damaged skeletal muscle.But now there is no research on whether GDF15 will be expressed in skeletal muscle,the tissue source of up-regulated GDF15 in the circulatory system of patients with mitochondrial disease and its characteristic intracellular distribution.Therefore,this part studies the expression of GDF15 in skeletal muscle through PMM cohort,and defined its characteristics of intracellular distribution.Method:1.Routine staining and special immunohistochemical staining were performed on PMM patients.Routine staining included standard HE staining,MGT staining,SDH staining,COX staining and SDH/COX double staining.Special staining includes mitochondria staining labeled mitochondria and GDF15 staining.2.In order to determine whether skeletal muscle can express GDF15,we homogenized the muscle tissues of patients and healthy controls and extracted protein components.The expression of mitochondrial complex proteins and GDF15 proteins were analyzed by Western blot.3.To study the correlation between the expression of GDF15 and pathological damage,the correlation between the expression of GDF15 protein and the proportion of RRF in pathological staining was analyzed.At the same time,the correlation between GDF 15 positive muscle fibers and RRF in pathological staining was researched.Results:1.Western blot showed that in PMM patients,five mitochondrial subunit levels decreased in varying degrees.Among them,the protein levels of complex Ⅱ and Ⅲdecreased in 2 patients,the protein levels of complex Ⅰ,Ⅱ and Ⅳ decreased in 5 patients,the protein levels of complex Ⅰ,Ⅲ and Ⅳ decreased in 5 patients,and all complex protein levels decreased in 4 patients.Western Blot detection of GDF 15 protein indicated that the expression of GDF 15 was quite low in healthy controls,while the expression level of GDF 15 protein increased in PMM patients.2.By further analyzing the correlation between GDF 15 protein level and RRF in pathological staining,we found that there was an obvious positive correlation between them(r=0.8114,P<0.0001).Immunohistochemical analysis of serial sections of muscle samples showed that GDF 15 positive muscle fibers were highly consistent with RRF,and the correlation coefficient was 0.9711,P<0.0001.Conclusion1.The present study describes,for the first time,the level of GDF15 protein in skeletal muscle of PMM patients was significantly increased,suggesting that GDF15 can be expressed in skeletal muscle.2.For PMM,the GDF15 positive fibers were well colocalized with RRF.3.For PMM patients,GDF15 may play a role in targeting mitochondria or work through mitochondria.Part Ⅳ:Study on the Diagnostic Value of GDF15 in Mitochondrial DiseasesBackground:The clinical manifestations of mitochondrial diseases are heterogeneous.The diagnosis of mitochondrial diseases is still challenging even for doctors with rich clinical experience.At present,the diagnosis of the disease is mainly based on clinical manifestations,muscle biopsy and gene detection.Considered the invasive nature of muscle biopsy,expensive and time-consuming gene testing,they can not be widely used in clinical screening of mitochondrial diseases.Previous studies have also reported that the levels of lactic acid,creatine kinase(CK)and pyruvate in serum can be used for the screening and diagnosis of mitochondrial diseases,but these markers lack sensitivity and specificity,thus it can not be used either.Therefore,finding a reliable serum biomarker is of great importance for the diagnosis of mitochondrial diseases.GDF15 is one of the members of transforming growth factor β superfamily.Many tissues and organs of human body can secrete GDF15.When the body is in inflammation,hypoxia and oxidative stress,GDF15 secretion increases to maintain homeostasis.In recent years,studies at home and abroad have found that GDF15 may be a potential biological marker of mitochondrial disease.In 2014,GDF15 was considered as a marker for the diagnosis of TK2 gene deficient mitochondrial disease.In 2015,Yatsuga and his colleagues found in a cohort study that GDF15 was highly specific for the diagnosis of mitochondrial diseases.In 2016,Davis et al.showed that GDF15 had higher diagnostic efficiency than fibroblast growth factor 21(FGF21).In 2019,Poulsen and his colleagues further studied the role of GDF15 in the course and prognosis of mitochondrial disease.In this part,we analyzed GDF15 in the serum of patients with mitochondrial diseases in Qilu hospital since 2000,and divided it according to different clinical phenotypes and gene types.Further,we meta-analyzed all the reported studies on GDF15 as a diagnostic marker of mitochondrial diseases,so as to clarify the potential value of GDF15 as a biomarker of the disease.Method:1.This study included 122 patients with mitochondrial disease diagnosed by clinical manifestations,laboratory examination and muscle biopsy from January 2000 to December 2020.Blood samples were collected in the morning on an empty stomach.2.GDF15 in patients’ serum was analyzed by ELISA,and the kit used was Abcam GDF15.3.Diagnostic Meta data collection:data collection and diagnostic value analysis were carried out in accordance with the requirements of relevant guidelines for diagnostic Meta analysis.We conducted a comprehensive search of the database,including PubMed,EMBASE,MEDLINE,Web of Science and Cochrane.The deadline for literature searched is January 1,2020.The keywords used in the search were:("mitochondrial disease" or "mitochondrial myopathy" or "oxidative phosphorylation disorder" or "respiratory chain defect")and("fibroblast growth factor 21" or "FGF-21" or "FGF21")and("growth differentiation factor 15" or"GDF-15" or "GDF15").4.Diagnostic Meta data analysis:We used Quality Assessment of Studies of Diagnostic Accuracy included in Systematic Reviews 2(QUADAS-2)to assess data quality and bias risk.Heterogeneity between studies was analyzed by Cochran Q and I2.According to the available data,calculated the true positive value,false positive value,true negative value and false negative value of each study,diagnostic odds ratios(DORs),drew Summary Receiver Operating Characteristics(SROC),and calculated the area under curve(AUC).Sensitivity analysis and subgroup analysis were used to find the source of heterogeneity,and Deeks funnel chart was used to evaluate publication bias.Results:1.ELISA analysis:(1)The level of GDF15 in patients’ serum in mitochondrial disease group(2157.1 ± 1974.3pg/ml,range[109.8-12416.3]pg/ml)was obviously higher than that in healthy control group(156.4 ± 89.1pg/ml,range[46.1423.7]pg/ml).(2)Clinical phenotype classification:the level of GDF15 in patients’ serum with myopathy(2761.9±2148.5pg/ml,range[165.1-8716.5]pg/ml)was significantly higher than that in patients with encephalopathy or neuropathy(336.5 ± 301.3pg/ml,range[135.1-12416.3]pg/ml).(3)Genotype classification:the level of GDF15 in patients’ serum with MT tRNA mutation(2379.7±2280.4pg/ml,range[109.8-12416.3]pg/ml)was greatly higher than that in patients with MT nd mutation(487.3 ±557.9pg/ml,range[120.9-2273.7]pg/ml).2.The results of meta-analysis showed that among the 7 diagnostic studies on GDF15,the sensitivity of GDF15 was between 0.53 and 0.98(I2 92.93%,95%CI 89.1596.71),the specificity of GDF15 was between 0.63 and 0.97(I2 88.91%,95%CI 82.16-95.66),the total sensitivity of GDF15 was 0.83(95%CI 0.65-0.92),the total specificity was 0.92(95%CI 0.84-0.96),and DOR was 52(95%CI 13-205),AUC was 0.94(95%CI 0.92-0.96),positive likelihood ratio was 9.90(95%CI 4.6021.20),and negative likelihood ratio was 0.19(95%CI 0.08-0.42).The overall diagnostic efficiency of GDF15 in mitochondrial diseases was better than that of FGF21.Conclusion1.There are differences in GDF15 levels in patients’ serum with different clinical phenotypes and genotypes.The GDF15 level in patients’ serum with myopathy phenotype was significantly higher than patients with encephalopathy or neuropathy phenotype(p<0.0001).The GDF15 level in patients’ serum with MttRNA genotype was significantly higher than patients with Mt-ND genotype(p<0.0001).2.GDF 15 and FGF21 have high sensitivity and specificity as diagnostic markers of mitochondrial diseases.Compared with FGF21,GDF15 has better diagnostic efficiency and should be used as an important means to screen mitochondrial disease.