The Heterogeneity Of Genotype-Phenotype Of MELAS-non A3243G And Pathogenicity Of Novel Variant In MtDNA

Part Ⅰ:The heterogeneity of Genotype-Phenotype of MELAS-non A3243GBackgroundPrimary mitochondrial disease is a hereditary metabolic disorder caused by mutations in mitochondrial DNA(mtDNA)or nuclear DNA(nDNA),leading to mitochondrial oxidative phosphorylation dysfunction and insufficient production of adenosine triphosphate(ATP).Among them,mitochondrial encephalopathy,lactic acidosis,and stroke-like episodes(MEL AS)is the most common primary mitochondrial disease.MEL AS is difficult to diagnose due to its phenotype-genotype variability,and it has a serious impact on the health of patients with no effective treatment.MELAS mainly caused by mtDNA mutations,among which the m.3243A>G variant,which located on the mt-TL1 gene,accounts for approximately 80%.Previous studies mainly focus on the clinical characteristics,treatment,and prognosis of MELAS caused by the m.3243A>G variant(MELAS-A3243G).However,the clinical features of MELAS caused by other mtDNA variants(MELAS-non A3243G)are often overlooked.Objective1.To investigate the phenotype of MELAS-non A3 243G.2.To explore the clinical,myopathological and brian MRI features of MELAS-non A3243G.Methods1.This is a retrospective study which included 26 MELAS-non A3243G patients diagnosed at the Department of Neurology,Qilu Hospital of Shandong University between January 2012 and June 2022.Based on gene variants,MELAS-non A3243G was divided into four categories:MELAS caused by mutations in mtDNA which encodes mitochondrial complex I subunit(mt-ND)(MELAS-mtND),MELAS caused by mutations in mtDNA which encodes mt-tRNA(non-m.3243A>G)(MELAS-mt tRNA),MELAS caused by mutations in mtDNA wich encodes mitochondrial complex Ⅳ subunit(mt-COX)(MELAS-mtCOX),and MELAS caused by large-scale deletions in mtDNA(mtDNA del)(MELAS-mtDNA del).This study explored the clinical,myopathological and brian MRI features of different subtypes of MELAS-non A3243G.2.The most common subtype of MELAS-non A3243G(MELAS-mtND)was compared with MELAS-A3243G.Results1.This study included 26 MELAS-non A3243G patients from 23 unrelated pedigrees,accounting for 23.0%(26/113)of the mtDNA-related MELAS in our center.The male-to-female ratio was 13:13,and the median age of onset was 26 years(IQR:20.5).Seven patients exhibited maternal inheritance.Among the 26 MELAS-non A3243G patients,the most common mutation gene was mt-ND(16%,18/113),followed by mt-tRNA(non-m.3243A>G variant)(6.5%,6/113),mt-COX(1.1%,1/113),and mtDNA del(1.1%,1/113).The two most common variants were m.10191T>C(mt-ND3)(17.4%,4/23)and m.13513G>A(mt-ND5)(13.0%,3/23).Analysis of the heteroplasmy revealed that MELAS-non A3243G patients were more likely to have a variant that was absent in blood cells than that in MELAS-A3243G(36.4%vs 1.4%,p<0.001).2.This study enrolled 18 MELAS-mtND patients,accounting for 69.2%(18/26)of MELAS-non A3243G.Compared with MELAS-A3243G patients,MELAS-mtND patients had a higher body mass index(BMI)(24 ± 2.5 vs.17.8±2.7,P=0.003),less short stature(23.1%vs.60.8%,P=0.027)and fewer migraine(33.3%vs.62.1%,P=0.025).Furthermore,MELAS-mtND patients had less multisystem involvements including hearing loss(27.8%vs.54.0%,P=0.043)and diabetes(11.1%vs.37.9%,P=0.030).The brain MRI evaluated at the first stroke-like episode showed significantly more nonclassic brain MRI(small cortical lesions)in MELAS-mtND patients(66.7%vs.12.2%,P<0.001),with fewer involvement in temporal lobes(22.2%vs.65.3%,P=0.002)and occipital lobes(27.8%vs.55.1%,P=0.047).Compared with MELAS-A3243G patients,MELAS-mtND patients had more normal muscle pathology(31.3%vs.4.1%,P=0.004)and,fewer ragged red fibers/ragged blue fibers(RRFs/RBFs)(62.5%vs.91.9%,P=0.002),COX-deficient fibers/blue fibers(25.0%vs.85.1%,P<0.001)and strongly succinate dehydrogenase-reactive blood vessels(SSVs)(50.0%vs.81.1%,P=0.009).Furthermore,MELAS-mtND patients had a significantly lower MDC score(7.8 ± 2.7 vs.9.8 ± 1.9,P<0.001)with fewer definite MELAS(62.5%vs.90.5%,P=0.004).3.Six MELAS-mt tRNA(non-m.3243A>G variant)patients were included from three unrelated families.Muscle pathology of MELAS-mt tRNA patients showed that the proportion of COX deficient fibers>20%was much higher than that in MELAS-A3243G(83.3%vs.24.43%,P=0.007).4.The present study also reported a MELAS patient carrying a novel mt-COX3 gene mutation(m.9553G>A),as well as a MELAS patient with mtDNA del.Conclusion1.mt-ND is the most common pathogenic gene for MELAS-non A3243G.The two common variants were m.10191T>C(mt-ND3)and m.13513G>A(mt-ND5).2.MELAS-non A3243 G patients are more likely to have a variant that was absent in blood cells.3.Compared with MELAS-A3243G patients,MELAS-mtND patients presented with less short stature,lower proportion of migraine,lower multisystem involvements(hearing loss and diabetes),lower presentation of RRFs/RBFs,COX-deficient fibers/blue fibers and SSVs but higher probability of nonclassic brain MRI.Part Ⅱ:A novel nonsense variant in mt-COX3 causes MELAS syndromeBackgroundMitochondrial encephalopathy,lactic acidosis,and stroke-like episodes(MELAS)syndrome caused by mutation in the mitochondrial gene encoding complex Ⅳ subunit(mt-COX)(MELAS-mtCOX)is rare.To date,only three cases of MELAS caused by mutations in mt-COX3 have been reported according to the MITOMAP database.Our previously study identified a patient in the MELAS-non A3243G cohort,carrying a novel variant(m.9553G>A)in the mt-COX3 gene.However,the pathogenicity of m.9553G>A is not clear.ObjectiveTo clarify the pathogenicity of the m.9553G>A variant.MethodsIn the present study,the pathogenicity of the m.9553G>A variant was investigated from multiple perspectives,including clinical data analysis,mutation pathogenicity prediction,genetic heterogeneity analysis,mitochondrial protein expression,and mitochondrial respiratory chain enzyme activity.Furthermore,we also conducted a literature review on variants in the mt-COX3 and truncation mutations in mt-COX.Results1.The m.9553G>A vatiant was segregated in family members.This variant was heteroplasmic in multiple tissues,where the mutation load was 89%in muscle samples,13%in oral epithelial cells,and not detectable in the blood cells.2.The 9553 site is highly conserved among multiple species and the m.9553G>A vatiant changed a highly conserved amino acid to a stop codon(p.Trp116*).3.Single muscle fiber analysis showed clear segregation of the m.9553G>A vatiant with the mutation load was 90.9%in COX deficient fibers while 42.9%in COX positive fibers(P<0.001).4.Western blot analysis revealed a significantly decrease in several subunits of complexⅣ(mt-COX1,mt-COX2,mt-COX3,and COX4),along with the subunit UQCRC2 in complex Ⅲ in the muscle samples of the patient when compared to the controls.5.COX respiration activity was remarkably reduced(58.84%)relative to the controls according to spectrophotometric assays.6.The phenotype of mt-COX3 mutations and mt-COX trunctuate mutations is complex and variable.Nearly half of these patients have a variant that was absent in blood cells.Futhermore,muscle pathology of these patietns showed significantly high amount of COX deficient fibers.ConclusionOur results indicated that this m.9553G>A variant may be responsible for the MELAS syndrome in the proband by affecting the stability and function of COX.The study expands the clinical and molecular spectrum of mt-COX3 specific mitochondrial diseases.Part Ⅲ:Mitochondrial encephalopathy Due to a Novel Pathogenic Mitochondrial tRNAGln m.4349C>T VariantBackgroundPrimary mitochondrial disease is a genetic metabolic disorder caused by mutations in mitochondrial genes(mitochondrial DNA,mtDNA)or nuclear DNA(nDNA)that result in dysfunction of mitochondrial oxidative phosphorylation and inadequate production of adenosine triphosphate(ATP).Among which,the most common mutations are variants in genes encoding mitochondrial tRNA(mt-tRNA).To date,the MITOMAP database has recorded 380 variants in mt-RNA.However,only 49(12.9%)are pathogenic variants.With the advancements in the technology and the decreasing cost of next-generation sequencing(NGS)techniques,more and more mtDNA variants have been reported to be associated with mitochondrial diseases.However,the detailed molecular pathogenesis of these mt-tRNA gene mutations remains unclear.We previously identified a patient with mitochondrial encephalopathy,carrying a novel variant m.4349C>T,which encoding mitochondrial tRNAGln.ObjectiveTo clarify the pathogenicity of the m.4349C>T variant.MethodsWe performed the clinical emulation,muscle histochemistry,northern blotting analysis of tRNA levels,biochemical measurement of respiratory chain complex activities and mitochondrial respirations in muscle tissue and cybrid cells.Results1.The m.4349C>T variant was segregated in the family members.This variant was heteroplasmic in multiple tissues,where the mutation load was 94.8%in muscle,75.0%in urothelium and 55.0%in blood cells.2.The 4349 site is highly conserved among multiple species,the m.4349C>T variant changed G to A at the position 53 of mt-tRNAGln,which disrupted the highly conserved G53-61C base pairs.3.Northern blot analysis revealed a decrease in mt-tRNAGln expression in the muscle sample of the patient with the m.4349C>T variant.4.Western blot analysis showed a decrease in several mitochondrial proteins expression in the muscle sample of the patient with the m.4349C>T variant.5.By constructing cybrid cells carrying m.4349C>T and testing its effect on the stability of mt-tRNAGln and mitochondrial function,it was found that this mutation firstly disturbed the translation machinery of mitochondrial mt-tRNAGln and decreased several mitochondrial proteins,impaired mitochondrial respiratory chain complex activities,followed by remarkable mitochondrial dysfunction and ROS production.ConclusionThis study illustrated the pathogenicity of a novel m.4349C>T mutation and provided a better understanding of the phenotype associated with mutations in mitochondrial tRNAGln gene.