A case of overlap myoclonic epilepsy with ragged-red fibers-Leigh syndrome associated with mitochondrial DNA 8344A>G mutation / 中华神经科杂志

Overlap myoclonic epilepsy with ragged-red fibers (MERRF)-Leigh syndrome is a rare mitochondrial encephalomyopathy. A case of MERRF-Leigh syndrome associated with mitochondrial DNA 8344A>G (m.8344A>G) mutation was reported in this article. The patient has suffered from the disease since 15-year old with myoclonus, exercise intolerance, ataxia, limb weakness, dysphasia, dyspnea, blurred vision and hearing loss. Magnetic resonance imaging revealed lesions on right thalamus, bilateral medulla and lumbar spinal cord and atrophy of cervical spinal cord. Electromyography showed predominantly axonal damage of both sensory nerve and motor nerve. Histochemical analyses revealed ragged red fibers, ragged blue fibers, succinate dehydrogenase-stronghly reactive vessels and decreased cytochrome oxidase activity. Gene tests demonstrated a high level of m.8344A>G mutation and m. 14484T>C mutation. MERRF-Leigh overlap syndrome with m.8344A>G mutation was rare. Bulbar paralysis following myoclonus is the main clinical symptom.

Identification of a novel mutation in the BCS1L gene causing Bj(o)rnstad syndrome / 中华皮肤科杂志

Objective To identify gene mutations in the BCS1L gene in a patient with Bj(o)rnstad syndrome mainly manifesting as congenital pili torti and sensorineural hearing loss.Methods Clinical data were collected and DNA was extracted from the peripheral blood of the patient and her parents.All the exons and their flanking sequences of the BCS1L gene were amplified by PCR followed by Sanger sequencing,and the sequencing results were compared with the normal sequences.A few hairs were collected from the patient,and examined by the scanning electron microscope.Results There were two mutations in BCS1L gene in the patient,i.e.,a heterozygous nonsense mutation in exon 4 and a heterozygous missense mutation in exon 8.The nonsense mutation in exon 4,which caused a change from CGA to TGA at position 144 and resulted in the substitution of arginine by termination codon (p.R144*),was a novel mutation in the BCS1L gene causing Bj(o)rnstad syndrome,and had never been repotted in the literature.The missense mutation in exon 8 led to a change from CGC to CAC at position 306 and resulted in the substitution of arginine by histidine (p.R306H).The patient's mother only carried a heterozygous mutation c.430 C＞T (p.R144*) in exon 4 of the BCS1L gene,and her father only carried a heterozygous mutation c.917 G＞A (p.R306H) in exon 8 of the BCS1L gene.Scanning electron microscopy showed that flats,grooves and longitudinal twisting irregu larly appeared at intervals on the surface of hair shafts.Conclusions A novel mutation in the BCS1L gene,which causes a change from CGA to TGA at position 144 in the BCS1L gene and results in a premature termination codon,is firstly reported in a patient with Bj(o)rnstad syndrome,and the compound heterozygous mutations c.430 C＞T and c.917 G＞A in the BCS1L gene are associated with the clinical manifestations of the patient.Genetic analvsis is helpful for the diagnosis of Bj(o)rnstad syndrome.

Neurological mitochondrial diseases / 中华神经科杂志

Mitochondrial disease (MD) is a group of metabolic disorders,caused by mitochondrial DNA or nuclear DNA mutations.With the development of research in mitochondrial medicine,the phenotypic spectrum of MD has expanded significantly.The common phenotypes in Chinese population included mitochondrial encephalopathy with lactic acidosis and stroke like episodes,Leigh syndrome,Leber hereditary optic neuropathy and chronic progressive external ophthalmoplegia.The rare phenotype included myoclonic epilepsy with ragged-red fibers,Kearns-Sayre syndrome,sensory ataxic neuropathy,mitochondrial neurogastrointestinal encephalomyopathy,Alpers disease,limb girdle mitochondrial myopathy,neuropathy with ataxia and retinitis pigmentosa.However,considerable clinical variability exists and some individuals do not fit into the known disease category.The revolution in genetic technologies has dramatically improved the diagnosis strategy of MD.In this article the pathology,clinical symptoms,laboratory findings and therapy in MD are sumarrized so as to provide guidance for clinical practice.

Prenatal diagnosis of mitochondrial disease: analysis of 28 pedigrees with mitochondrial DNA mutation / 中华围产医学杂志

Objective To perform a prenatal diagnosis for the second fetuses from 28 pedigrees with proband of mitochondrial disease due to mitochondrial DNA (mtDNA) mutation.Methods From April 2011 to November 2015,peripheral blood samples of 28 probands and their parents,urine samples of these probands and their mothers as well as amniotic fluid samples of the second fetuses from the 28 pedigrees were collected in Peking University First Hospital.DNA sequencing was used to identify mtDNA mutations.Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to verify mutation sites,calculate mutation loads,and further confirm the diagnosis after birth.Microsatellite maker analysis was also performed on five short tandem repeats located in nuclear genes to exclude maternal contamination.Statistical analysis was carried out using independent t-test.Results In the 15 pedigrees carrying A3243G mutation,13 mothers and nine fetuses carried A3243G mutation.Neither the other two mothers nor their fetuses were positive for A3243G mutation.Among the 12 pedigrees with T8993G mutation,there were eight mothers carrying T8993G mutation and all of their fetuses carried the same mutation;and the other four mothers and their fetuses were negative for T8993G mutation.T10191C mutation was only found in one proband and the second fetus of that pedigree,but not in the mother.None of the fathers had mtDNA mutation.Results of PCR-RFLP were consistent with those of DNA sequencing.Short tandem repeat analysis demonstrated that amniocyte samples were from fetuses without maternal contamination.No mtDNA mutations were found in the six newborns who were negative for mtDNA mutations in prenatal diagnosis.The mean mutation load in urine samples of the six mothers without A3243G mutation in amniocytes was significantly lower than that of the nine mothers with A3243G mutation [(10.1 ±4.8) ％ vs (28.2 ± 15.1) ％,t=2.290,P=0.043].Conclusions The lower the mtDNA mutation load in maternal urine samples,the less the possibility she bears a child with mtDNA mutation.However,prenatal diagnosis of mitochondrial disease is necessary.

Effects and mechanism of overexpression of Mfn-2 gene on photodynamic therapy sensitivity of T47D cells in human breast cancer / 国际肿瘤学杂志

Objective To investigate the effects and possible mechanisms of Mfn-2 gene overexpression on photodynamic therapy (PDT) sensitivity of T47D cells in human breast cancer.Methods pEGFP and pEGFP-Mfn-2 were transfected into human breast cancer T47D cells,and then the mRNA and protein expression of Mfn-2 gene in T47D cells were detected by real-time PCR (RT-PCR) and Western blotting in pEGFP group and EGFP-Mfn-2 group,respectively.After pEGFP and pEGFP-Mfn-2 were transfected into human breast cancer T47D cells for 48 hours,methyl thiazolil tetracolium (MTT) assay was used to measure the PDT sensitivity of T47D cells in human breast cancer in pEGFP group and pEGFP-Mfn-2 group.pEGFP + PDT group and pEGFP-Mfn-2 + PDT group were obtained by PDT irradiating pEGFP and pEGFP-Mfn-2 which were transfected into human breast cancer T47D cells.Cell apoptosis and mitochondrial membrane potential of T47D cells were assayed by flow cytometry in pEGFP + PDT group and pEGFP-Mfn-2 + PDT group.Laser scanning confocal fluorescence microscope was applied to observe the morphological ultrastructure of mitochondria in pEGFP group,pEGFP-Mfn-2 group,pEGFP + PDT group,and pEGFP-Mfn-2 + PDT group.Results RT-PCR showed that after transfecting T47D cells with pEGFP,the expression of Mfn-2 mRNA was 1.01 ±0.12.After transfecting T47D cells with pEGFP-Mfn-2,the expression of Mfn-2 mRNA was 1 067.00 ±41.72.There was statistical significance (t =67.541,P ＜ 0.001).Western blotting revealed that compared with the pEGFP group,the pEGFP-Mfn-2 transfection group had higher expression of Mfn-2 gene in T47D cells.MTT assay showed that pEGFP-Mfn-2 transfection significantly enhanced the PDT sensitivity of T47D cells in hunan breast cancer compared with the pEGFP + PDT group.When the concentration of methylene blue was 5.00 μmol/ml,the survival rate of the pEGFP +PDT group and pEGFP-Mfn-2 + PDT group were (59.96％ ± 1.21％) vs.(46.50％ ± 1.72％),with significant difference (t =34.403,P ＜ 0.001).Flow cytometry assay showed that the cell apoptosis rates in pEGFP-Mfn-2 + PDT group was markedly higher compared with the pEGFP + PDT group [(81.21 ± 2.13)％ vs.(68.82 ±2.64)％,P=0.024],with statistical significance.Also,the mitochondrial membrane potential was obviously lower in pEGFP-Mfn-2 +PDT group compared with the pEGFP +PDT group [(1.37 ±0.12)％ vs.(23.33 ± 1.86)％,P＜0.001],with statistical significance.Laser scanning confocal fluorescence microscope showed that cells in pEGFP group showed network structure,both pEGFP-Mfn-2 group and pEGFP + PDT group could cause mitochondrial fusion,and the pEGFP-Mfn-2 + PDT group could induce mitochondrial disintegration and lose its normal morphology completely.Conclusion Mfn-2 may enhance the PDT sensitivity of T47D cells in human breast cancer,which is possibly related with the normal morphology alteration of mitochondria and the inducement of mitochondrial apoptosis.

Effect of mfn2 on mitochondrial function in steatosis hepatocytes / 中国病理生理杂志

AIM: To investigate the effect of mfn2 on mitochondrial function in steatosis hepatocytes. METHODS: Plasmid pEGFP-mfn2 was transfected into hepatocyte strain L02 by Lipofectamine 2000 in vitro, then the steatosis model of hepatocytes was establish by oleic acid induction. RT-PCR was used to evaluate mRNA expression and Western blotting was use to detect the protein expression. ATP level was determined by firefly luciferase bioluminescent. ROS production was measured by fluorescence probe DCFH-DA. Chondrosome transmembrane potential of L02 was observed by labeling of JC-1 and FCM. RESULTS: The stable expression of ectogenesis mitofusin2 in L02 cells was confirmed by RT-PCR and Western blotting. In the model of oleic acids induced lipid formation, Mfn2 obviously inhibited the descent of chondrosome transmembrane potential and ATP level, and increased ROS production in L02 cells. CONCLUSION: Up-regulated expression of mfn2 attenuates mitochondria dysfunction caused by oleic acids induced lipid formation.