The Pathogenicity Of Mitochondrial ND1 Gene M.4160T>C Variant

BackgroundMitochondria are involved in a variety of metabolic processes and signaling pathways in eukaryotic organisms,and their structure and function are regulated by both the mitochondrial genome and nuclear genome.The mitochondrial genome is a double-stranded closed-loop structure with a length of 16569bp,consisting of an inner light strand(L)and an outer heavy strand(H).The 37 genes of the mitochondrial genome are closely arranged on the heavy and light chains.Because of their exposed structure,mitochondrial genes are susceptible to damage by reactive oxygen species(ROS)generated in oxidative phosphorylation(OXPHOS)on the inner mitochondrial membrane.Therefore,mitochondrial genes are prone to mutation.There are seven mitochondrial DNA(mtDNA)involved in encoding mitochondrial complex I(NADH ubiquinone oxidoreductase)proteins in the mitochondrial genome,ND1-ND6 and ND4L genes,respectively.Mutations in MT-ND1,MT-ND4 and MT-ND6 genes are the most common causes of Leber’s hereditary optic neuropathy(LHON).Mitochondrial gene mutations can cause mitochondrial dysfunction,and insufficient ATP synthesis,leading to retinal ganglion cell(RGC)degeneration and optic nerve atrophy,triggering the LHON phenotype.More than 30 mtDNA mutations have been found to cause LHON,among which three mutation hotspots are m.3460G>A,m.11778G>A and m.14484T>C mutations.The clinical manifestations of LHON are mainly acute or subacute bilateral painless vision loss and central visual field deficiency.Genetic detection is essential to make a definite diagnosis when the clinical manifestations are atypical,or there is no clear maternal genetic history.However,variants of undetermined significance(VUS)cannot be used to determine or exclude the diagnosis of LHON.Therefore,the pathogenic mechanism studies of new variants or VUS are essential to supplement and improve the phenotypic and genetic spectrum of LHON and assist in clinical diagnosis and treatment.MethodsThe pathogenicity and pathogenic mechanism of mitochondrial ND1 gene m.4160T>C variant were investigated by performing molecular genetics and molecular biological analysis of a patient presenting a unique LHON plus dystonia.Firstly,we improved the clinical data of the patient,and performed genealogy verification and heterogeneity measurement.Then,the platelets of the family members were extracted as exogenous mitochondrial donors and human thymidine kinase-deficient osteosarcoma cell lines(143B TK-)were used as nuclear donor cells to construct cybrid cells by cytoplasmic hybrid technique.Cells carrying the m.4160T>C variant and wild-type cells were used as variants and controls,respectively.Finally,the mitochondrial-related protein expression levels,mitochondrial respiratory chain enzyme complex activity and its mediated respiratory oxygen consumption rate,as well as cellular respiratory oxygen consumption rate of cybrid cells were detected.The data were processed and analyzed.Results1.Clinical data:Ophthalmologic examination showed the best-corrected visual acuity(BCVA)of the patient were 1/20 in the eyes.The optical coherence tomography(OCT)and optical coherence tomography angiography(OCTA)demonstrated the retinal nerve fiber layer(RNFL)atrophy and a significant decrease in blood flow of the radial peripapillary capillaries(RPC).The head MRI manifested abnormal signals in the bilateral basal ganglia region and the area around the midbrain aqueduct.2.Genetic analysis:The mitochondrial genome sequencing using urine sediment showed that the patient carried the MT-ND1 gene m.4160T>C variant.Sanger sequencing revealed that the m.4160T>C variant was heteroplasmic in the blood(80.2%),urine sediment(90.8%),and oral mucosal(81.7%)samples of the patient.No variant was detected in the patient’s mother and sister.Conservation analysis of mtDNA and nucleotide sequences showed that the mitochondrial 4160 site was highly conserved among different species.The m.4160T>C variation changed the leucine to proline at amino acid position 285(p.Leu285Pro).3.Western blotting analysis showed the level of some proteins expression was markedly decreased in variant cybrid cells,such as mtDNA-encoded complex Ⅰ subunits MT-ND1,MT-ND4,MT-ND5,complex Ⅴ(ATP synthase)subunit MT-ATP6,and nDNA encoding subunit NDUFB8;while the levels of some protein expression,such as nDNA encoding complex Ⅳ(cytochrome C oxidase)subunit MT-CO1,complex Ⅲ subunit UQCRC2 and complex V subunit ATP5A remained unchanged between the mutant and controls cybrid cells.4.The mitochondrial biochemical activity assay showed that the enzyme activities of mitochondrial complex Ⅰ and complex Ⅱ(succinate dehydrogenase)of variant cybrid cells were significantly reduced,which were 31.8%and 74.4%of those in the control group,respectively(P values<0.05).5.The detection of mitochondrial respiration function showed that the basal respiratory oxygen consumption rate(OCR),ATP-linked respiration OCR,and maximal respiration OCR of variant cybrid cells were significantly decreased,which were 59.8%,57.7%,56.6%and 65.2%of those in the control group,respectively(P values<0.05).The mitochondrial complexⅠ and complex Ⅱ mediated respiration OCR in variant cybrid cells decreased significantly,which were 61.2%and 52.5%of those in the control group(P values<0.05).There was no significant difference in the respiratory OCR of complex Ⅳ between two groups(P values>0.05).ConclusionsThe mitochondrial ND1 gene m.4160T>C variant is pathogenic,which can decrease the expression of mitochondrial proteins,affect the enzyme activity of mitochondrial complex I and complex Ⅱ,and impair mitochondrial respiratory chain function,causing insufficient ATP production degeneration.These pathological processes lead to the loss of oxygen-consuming retinal ganglion cells and optic nerve atrophy,ultimately triggering the LHON phenotype.