Association Study Between Genetic Variation In Whole Mitochondrial Genome,Oxidative Stress And Ischemic Stroke

Background and Aims: Ischemic stroke?IS?is a neurological disease characterized by ischemia,hypoxia or necrosis of brain tissue in perfusion area due to the cerebral arterial stenosis or obstruction.IS has the characteristics of high morbidity,high disability and high mortality,which brings heavy burden to individuals,families and society.The pathological mechanism of IS is very complex.It is caused by cerebral ischemia,such as oxidative stress,excitatory amino acid toxicity,energy disorder,neuroinflammation and neuronal cell death,which interact to form a complex information network and eventually lead to cascade injury.Mitochondria,as an important organelle of energy metabolism,widely exist in neurons and glial cells.Mitochondria play an important role in different stages of IS and in different mechanisms of cascade reaction.Therefore,the association study between mitochondria and IS provides a new perspective for fully explaining the pathological mechanism of IS.The human mitochondrial DNA?mt DNA?molecule is a circular molecule consisting of 16,569 pairs of bases,encoding ribosomal RNA?r RNA?,transfer RNA?tRNA?,and the important component of mitochondrial oxidative phosphorylation,?OXPHOS?.mt DNA is maternal inheritance.Due to the lack of an effective mt DNA protection and repair system,the sequence mutation rate is high,more than 10 times that of the nuclear genome?n DNA?mutation.Some mutations affect mitochondrial oxidative phosphorylation,leading to mitochondrial dysfunction.Some mutations gradually accumulated in the population to form haplogroups and haplotypes.These haplotypes enriched in specific environments can affect the function of mitochondria,leading to human susceptibility to certain diseases.It has been reported that the relationship between mt DNA and IS was studied by partial mt DNA mutation,mt DNA haplotype and mt DNA large fragment deletion.Our team also found that mitochondrial haplotype D4 b may be a protective factor of IS in Henan Han population.However,the whole mitochondrial genome,including the D-Loop region,22 RNA genes,2 t RNA genes,and 13 protein-coding genes play important roles in maintaining normal mitochondrial function.Any change in the locus of mt DNA may lead to mitochondrial dysfunction,leading to the occurrence of related diseases.Whole mitochondrial genome sequencing technology plays a crucial role in the discovery of genetic factors in variety of diseases.At present,study on IS genetics by whole mitochondrial genome sequencing has not been reported.In addition,recent studies have shown that non-traditional risk factors,such as oxidative stress,have become the focus of IS field.Oxidative stress runs through vascular endothelial cell injury,atherosclerotic plaque formation,plaque rupture,stroke events,post-stroke ischemia-reperfusion injury,and even vascular restenosis after interventional therapy.Therefore,it is very important to find effective oxidative damage markers to study the pathogenesis,degree of oxidative damage,therapeutic efficacy and prognosis of IS.In summary,this study contained three parts.The first part was to screen the whole mitochondrial genetic variations in IS patients and normal controls by next-generation sequencing?NGS?,and obtained the data of mt DNA mutations of whole mitochondrial genome with IS.In the second part,we expanded the samples to verify the association between mt DNA variation and IS,and obtained susceptible mt DNA mutations as genetic markers of IS.In the third part,superoxide dismutase?SOD?,hydrogen peroxide?H₂O₂?,catalase?CAT?and malondialdehyde?MDA?were selected as indicators for assessing oxidative stress in vivo,and the effects of oxidative stress on the occurrence and development of IS were discussed.This study will reveal the pathogenesis and characteristics of IS from a new perspective of extranuclear inheritance,and provide a theoretical basis for the prevention and treatment of IS.Materials and MethodsPart I Screening of whole mitochondrial genome variation in ischemic strokeSubjects: The study protocol was authorized by the Ethics Committee of Zhengzhou University.All the subjects signed the informed consent.Case group: 52 IS patients with age?60 years old were chosen from the First Affiliated Hospital of Henan University of Traditional Chinese Medicine and the Department of Neurology of Zhengzhou First People's Hospital during May 2017 to November 2017.All patients were diagnosed according to the IS diagnostic criteria established by the WHO,and various tests?signs,history,biochemical tests,MRI or CT?were used to rule out atrial fibrillation,malignant tumors,and cerebral hemorrhage.Control group: 55 healthy individuals with age and gender matched with case group,and the volunteers with cardiovascular disease or diabetes were excluded.Methods: DNA was extracted using Beijing Tiangen's batch number DP332 Blood Genome Extraction Kit,and mitochondrial genomic DNA was enriched by PCR,followed by construction of a DNA library for high-throughput in a 2�150 bp double-end sequencing mode on the Illumina Hiseq platform.Sequencing and analysis.Results: 1.A total of 9 unreported mt DNA mutations were found in the IS and control groups.2.There were statistically significant differences in 54 mt DNA mutations between the IS and control groups,P< 0.05.3.Seven mt SNPs and eight low-frequency sites related to IS were selected for expanding sample validation.Part II Verification of IS-related mitochondrial gene variation using SNa Pshot techniqueSubjects: The study protocol was authorized by the Ethics Committee of Zhengzhou University.All the subjects signed the informed consent.The case group consisted of 200 IS patients who were hospitalized in the First Affiliated Hospital of Henan University of Traditional Chinese Medicine and the First Affiliated Hospital of Zhengzhou City from May 2017 to August 2018,with an average age of 56.3�8.84 years.The diagnosis of all patients was based on the IS diagnostic criteria established by the WHO,and various tests?history,physical signs,clinical biochemical tests,CT or MRI?were used to rule out atrial fibrillation,malignant tumors,cerebral hemorrhage and other diseases.The control group consisted of 200 healthy individuals with age and gender matched with case group and no cardiovascular and cerebrovascular diseases collected at the First Affiliated Hospital of Henan University of Traditional Chinese Medicine and Zhengzhou First People's Hospital at the same time.The average age was 55.69�3.41 years old.Methods: Seven mt SNPs and eight low-frequency sites screened in the first stage were detected by multiple SNa Pshot techniques,and the original data were analyzed by Genemapper 4.0 software.Statistical analysis and analysis of experimental data were performed using SPSS 21.0 statistical software.Results: 1.The frequency of m.T195 C and m.T12338 C loci were statistically different between the IS group and the control group,P<0.05.2.The frequency of m.T195 C,m.C311 T,m.C16355 T loci were statistically different between LAA subtype and control group,P<0.05.Part III Correlation between oxidative stress and ischemic strokeSubjects: The study protocol was authorized by the Ethics Committee of Zhengzhou University.All the subjects signed the informed consent.The case group consisted of 101 patients with IS who were hospitalized in the First People's Hospital of Zhengzhou City and the First Affiliated Hospital of Zhengzhou University from May 2017 to August 2018 with an average age of 53.02�0.91 years old.The diagnosis of all patients was based on the IS diagnostic criteria established by the WHO,and various tests?history,physical signs,clinical biochemical tests,CT or MRI?were used to rule out cerebral hemorrhage,aortitis,malignancy,atrial fibrillation,hyperthyroidism,tuberculosis,blood disease and serious liver and kidney dysfunction.The control group consisted of 101 healthy individuals with matched gender and age with case group and no cardiovascular and cerebrovascular diseases with an average age of 52.05�0.58 years.Methods: The Beyotime kits were used to detect oxidative stress indicators in the IS and control groups.The indicators include malondialdehyde?MDA?,superoxide dismutase?SOD?,hydrogen peroxide?H₂O₂?,and catalase?CAT?.Statistical analysis of experimental data were performed using SPSS 21.0 statistical software.Results: 1.The levels of CAT and H₂O₂ were statistically different between the IS group and the control group?P< 0.05?.2.H₂O₂ levels and CAT levels in male IS patients were statistically different from normal males?P<0.05?.The CAT level of female IS patients was significantly lower than that of normal women?P<0.05?.Conclusions: 1.Based on the whole mitochondrial sequencing technique,nine unreported mt DNA mutation loci are found for the first time in this study,which enrich the resource bank of mt DNA genetic variation.2.A total of 54 mt DNA variant sites which may be related to the pathogenesis of IS are found,which provide data and reference for the association study of IS.3.The m.T195 C and m.T12338 C are protective factors for IS.The m.T195 C and m.C311 T are protective factors for LAA subtype.The m.C16355 A is a risk factor for LAA subtype.4.Patients with IS have elevated H₂O₂ levels and decreased CAT levels,which are independent risk factors for IS,suggesting a significant oxidative stress response in IS.