A Clinical And Basic Research On Mitochondrial Encephalomyopathy, Lactic Acidosis And Stroke-like Episodes

Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is a rare genetic disease. Its prevalence rate with mitochondrial DNA (mtDNA) defects in the West about12.48/100,000. MELAS results in defects of respiratory chain complex mainly due to the mtDNA mutations, causing the damage of organ or tissue which require energy. Clinical considerations is a syndrome characterized with stroke-like episodes, muscle weakness and/or lactic acidosis.In the present study, we explored the clinical features, imaging findings and muscle pathology; screened the whole mitochondrial DNA and respiratory chain complex, and analyzed the relationship among clinical type, genotype and biochemical characteristics in MELAS patients.METHOD:This study included25patients with MELAS diagnosed by clinical features and muscle biopsy. The imaging of brain CT scan and MRI was performed in all patients. The frozen sections of muscle biopsy were stained by Henatoxylin and eosin (HE), Modified Gomori Trisome (MGT), succinate dehydrogenase(SDH), cytochrome C oxidase (COX), Oil red O (ORO), Nicotimamide-adenine dinucleotid (NADH), anti-mitochondrial antibody. The paraffin-embeded muscles were observed by electron microscope.The total DNA was extracted from25muscle samples and13blood samples in the same sampling time.24pairs of primers were designed and synthesized based on the comparison of mitochondrial DNA (homo sapiens) retrieved from Genbank. DNA PCR, gel electrophoresis and sequencing were performed in25muscle samples. Data were analyzed by the blast software version3.25of Mutation Surveyor, revised Cambridge reference sequence (rCRS), MITOMAP database (update to March1st,2011) and Phylotree (update to March,30th,2011). The novel mutations were confirmed by100normal controls to exclude possibility of Single Nucleotide Polymorphisms. Prediction of protein secondary structure was performed using SOSUI.We analyzed the m.3243A>G heteroplasmy between muscle and blood in13patients with MELAS.The main clinical phenotypes, including muscle weakness, epilepsy, mental retardation and genotypes were analyzed.Mitochondria was isolated by differential centrifugation from25muscle samples with MELAS and28normal controls. The activities of respiratory chain complexes Ⅰ-Ⅴ and citrate synthase were determined by colorimetric assay, and the normal ranges of CI/CS, CII/CS, CIII/CS, CIV/CS, CV/CS were calculated, The correlation between the respiratory chain complexes deficiency and clinical type/genotype was analyzed.Results:(1) The average age of onset in MELAS patient was24.0±10.0years old. The median age at diagnosis was28.5±12.2years old. Initial presentations of MELAS were epilepsy (52%), headache (36%), blurred vision (32%). The main clinical manifestations were muscle weakness (64%), stroke-like seizures(60%), mental retardation(48%). Serum creatine kinase(CK) and lactate dehydrogenase(LDH) were normal or mildly elevated, some patients had CK-LDH inversion. Head CT in5cases showed the calcification in the basal ganglia and/or cerebellar. The MRI in25patients exhibited single or multiple intracranial lesions involving the cortex, showing low signal intensity on T1W, high signal intensity on T2W, FLAIR and DWI. Muscle pathology in MELAS patients showed ragged red fibers (RRF), some muscle fibers and blood vessels stainied SDH. The expression of COX was elevated surrounding muscle fibers and SSV in22cases (88%). Anti-mitochodrial antibody showed that part of the muscle fiber brown calm.(2)We found total692polymorphisms in25cases, including652single nucleotide polymorphisms (SNP),34reported mtDNA base substitution disease mutations and6novel mutations.The numbers of cases of mutant gene occurred in this study in turn were MT-TL1(76%), MT-ND3(68%), MT-CYB (64%), MT-ND2(36%), MT-ND6(32%), MT-DLOOP (28%). Others were MT-RNR1, MT-TT, MT-TC, MT-TK, MT-TW, MT-TH, MT-TE, MT-TA, MT-ND5, MT-ND4, MT-CO1, MT-C02, MT-ND1, MT-ATP6. The most common point mutation was m.3243A>G (76%).There were no significant differences between phenotype and genotype. Mutant genes in turn were MT-TL1, MT-ND3, MT-CYB in patients with muscle weakness, in turn were MT-TL1, MT-CYB, MT-ND3.in patients with seizure, and in turn were MT-TL1, MT-ND6, MT-ND3, MT-CYB.in patients with mental retardation.The main clinical manifestations were epilepsy, muscle weakness and endocrine disorders in patients with MT-TL1gene mutation. However, The main clinical manifestations were epilepsy, muscle weakness, mental retardation and endocrine disorders in patients with MT-ND3gene mutation.Haplotype analysis revealed that12/25cases (48%) were H line who belong to the Wast European and Asian origin. In addition,5/25cases (20%) were M line and8/25cases (32%) were D line who belong to the east European and Asian origin.The novel mutations found in this research were m.7513insT, m.8477T>C, m.3959G>A, m.1694T>C, m.3995A>G and m.1888G>C. The prediction of protein secondary structure showed that the mutation of m.7513insT, m.8477T>C, m.3959G>A and m.3995A>G may cause the changes of protein secondary structure in different degrees. The rate of m.3243A> G mutation in blood samples was53.8%of muscle tissue.(3) In the MEL AS group,11cases (44%) had respiratory chain complex Ⅰ deficiency;1case (4%) with respiratory chain complex Ⅱ deficiency;9cases (36%) with respiratory chain complex Ⅲ deficiency;10cases (40%) with respiratory chain complex IV deficiency; and7cases (28%) with respiratory chain complex V deficiency. Nine cases occurred combined deficiency of2respiratory chain complexes,4cases occured combined deficiency of3respiratory chain complexes.There were no significant differences between biochemistry and genotype/clinical type. MT-TL1and MT-ND were the most common mutation genes in respiratory chain complex I deficiency, while MT-TL1, MT-CYB and MT-ND3were the most common mutation genes in respiratory chain complex Ⅲ and Ⅳ deficiency.Affected systems in the patients with respiratory chain complex Ⅰ deficiency included skeletal muscle, brain and endocrine system. Affected systems in the patients with respiratory chain complex Ⅳ deficiency included brain, skeletal muscle and endocrine. Howerver, the involvement of skeletal muscle and brain was equivalent in MELAS patients with respiratory chain complex Ⅲ and Ⅴ deficiency. Conclusion:MELAS is Mitochondrial encephalomyopathy characterized with neurological manifestations and radiography as well as pathological features. It is most commonly caused by an A-to-G transition mutation at position3243of mitochondrial genome. We found six novel mutations, m.7513insT, m.8477T>C, m.3959G>A, m.1694T>C, m.3995A>G, in.1888G>C.The most common change in respiratory chain is complex Ⅰ deficiency. Muscle is the best selection in the genetic and biochemical test.