The Role And Mechanism Of Mitochondrial DNA Haplogroup M9 In Diabetic Retinopathy

Diabetic retinopathy(DR),as the major microvascular complication of type 2 diabetes mellitus(T2DM),is the primary cause of blindness in working-age people and lacks of effective treatment.which is bound to cause a great economic burden on individuals,families as well as society.The popular trend of DR in China in recent y ears is not optimistic,with a tendency to catch up with developed countries.Moreover,the national prevalence of DR is higher in rural areas than that in urban areas and higher in northern areas than that in southern and eastern coastal areas.Yunnan Province is located in the southwest plateau of China,with a unique geographical and cultural structure.A previous survey of this study has found that compared with other regions,the prevalence of DR in patients with T2DM in Yunnan Province is increased.Therefore,Yunnan Province is facing a severe DR prevention and control task.Actively looking for the major risk factors of DR and making a clinical decision tree model of DR is urgent.The pathogenesis of DR is complex and has not been fully elucidated.The existing evidence shows that DR can be subject to double "pressure" from genetic and environmental factors.Therefore,in addition to revealing the clinical risk factors of DR,it is particularly necessary to further find the factors that can integrate the geographical environment and genetic characteristics of Yunnan Province,and explore the correlation between these factors and DR.Previous studies have shown that mitochondria is not only closely related to the retina,but also a "key link" in communicating the gene inheritance and geographical environment.As a special organ of the human body that integrates the vascular and nervous system,the retina needs to consume a large amount of adenosine triphosphate(ATP)to maintain its visual function.Mitochondria,in addition to act as the main place for the production of ATP,their normal function is particularly vital for maintaining normal retinal metabolism.Mitochondrial dysfunction caused by mitochondrial DNA(mtDNA)mutation has been confirmed to be related to DR.Moreover,due to the lack of histone protection,mtDNA is prone to mutation.During the process of migrating around the world and adapting to the natural environment,human ancestors gradually accumulated some ancient mtDNA mutations and finally formed a specific mtDNA genetic background,namely,mtDNA haplogroups.Recently,there are many lines of evidence indicating that mtDNA haplogroups have functional consequences,which are mostly related to energy metabolic diseases,and could be affected by geographical,ethnic and other factors.To date,the studies associated with mtDNA haplogroups and DR have mainly been limited in European and American countries,and the results are controversial.More importantly,there are few related studies in China.Therefore,combined with the unique geographical and human background of Yunnan Province,it is of great significance to further research on mtDNA haplogroup and DR,and elucidate its potential pathogenic mechanism.In general,this study is planned to be carried out from the following three stages.In the first stage,the associated risk factors of DR in the Han population with T2DM in Yunnan Province were revealed,and the clinical decision tree model was constructed.In the second stage,based on the samples in the first stage,screening of mtDNA haplogroup related to DR(namely candidate haplogroup)was performed.In the third stage,the molecular mechanism of candidate haplogroup in DR was revealed by comparing the mitochondrial function between the candidate and control haplogroup cybrids and utilizing the point mutation and transcriptome sequencing.In summary,from clinical research to in vitro research,this study was designed to enrich the pathogenesis of DR.Part 1 Risk factors analysis and clinical decision tree model of diabetic retinopathy in Yunnan ProvinceObjectives:To elucidate the associated risk factors of DR in patients with T2DM in Yunnan Province and further construct a clinical decision tree model.Methods:According to fundus photography,a total of 1654 Han population with T2DM were divided into the without DR(WDR)group(n=826),and the DR group(n=828).Furthermore,the DR group was divided into the non-proliferative diabetic retinopathy(NPDR)group(n=403),and the proliferative diabetic retinopathy(PDR)group(n=425).Based on the recorded clinical data,univariate analysis,logistic regression analysis,and clinical decision tree model were performed by SPSS 20.0.Results:1.Univariate analysis showed that compared with the WDR group,the proportion of female,duration of diabetes,standing systolic pressure(SBP),standing diastolic pressure(DBP),cholesterol(Chol),blood urea nitrogen(BUN),uric acid(UA),serum creatinine(Scr),fasting glucose and hemoglobin A 1c(HbA1c)in the DR group were all significantly increased(all P<0.05);body mass index(BMI),hip circumference and estimated glomerular filtration rate(eGFR)in the DR group were all significantly decreased(all P<0.05).Compared with the NPDR group,the proportion of female,supine SBP,supine DBP,Chol,low-density lipoprotein-cholesterol and BUN in the PDR group were significantly increased(all P<0.05),while age,hip circumference and eGFR were all significantly decreased(all P<0.05).2.Logistic regression analysis showed that female(odds ratio(OR)=1.520),diabetes duration?10 years(OR=2.118),standing or supine position SBP?140 mmHg(OR=1.417,OR=1.881),Chol ?6.22 mmol/L(OR=1.591)were risk factors for DR in patients with T2DM.In addition,the poorer the stage of chronic kidney disease(CKD)or the HbAlc,the risk of DR in T2DM patients increased gradually.Moreover,female(OR=2.161),diabetes duration? 10 years(OR=1.483),the progress in CKD stages were risk factors for PDR.3.In the decision tree model,the duration of diabetes was the primary risk factor that affected the occurrence of DR in patients with T2DM,namly,the root node.The extraction rules were interpreted as:For patients with diabetes duration<10 years,if they met:?CKD stage=G3/G4/G5;or ?CKD stage=G2 and BMI<24 kg/m2;or?CKD stage=G1 and standing SBP?140 mmHg,then DR was prone to occur.On the contrary,for patients with diabetes duration?10 years,if they met:?supine SBP<140 mmHg and CKD stage=G2/G3/G4;or ? supine SBP?140 mmHg and CKD stage=G3/G4/G5,then DR was prone to occur.Conclusions:Female,diabetes duration?10 years,standing or supine SBP?140 mmHg,Chol?6.22 mmol/L,deterioration of CKD stage and HbA1c are important risk factors for DR in the Han population with T2DM in Yunnan Province.In addition,the decision tree model developed in this study suggests that the duration of diabetes should be considered for the first time when assessing the risk of DR in patients with T2DM.If the diabetes duration?10 years,the standing SBP or BMI are further evaluated according to the CKD stage;if the diabetes duration ? 10 years,the CKD stage is further evaluated according to the supine SBP.Finally,the classification of DR is obtained.This model could help clinicians in Yunnan Province(especially primary medical staffs who lack related DR detection means such as ophthalmoscope)to make more effective clinical predictions of DR risk of patients with T2DM,with its simplicity and intuitiveness,making it worth promoting in future clinical practice.Part 2 Analysis of mtDNA haplogroups and mtDNA single nucleotide variants associated with diabetic retinopathy in Yunnan ProvinceObjectives:To identify the mtDNA haplogroups and mtDNA single nucleotide variants associated with DR in patients with T2DM in Yunnan Province.Methods:According to fundus photography,a total of 832 patients with T2DM(all from Part 1)were divided into the WDR group(n=403),the DR group(n=429).Furthermore,the DR group was divided into the NPDR group(n=227),and the PDR group(n=202)based on the severity of DR.Firstly,the genomic DNA was extracted from the peripheral blood of patients,and the mtDNA haplogroups were classified based on the sequenced mtDNA.Then,statistical analyses were performed by SPSS 20.0 as follows:?univariate analysis of the clinical baseline data of the enrolled patients was applied to make sure that these subjects of Part 2 can represent the whole subjects of Part 1.?a principal component analysis was conducted based on the mtDNA haplogroup distribution frequencies for all samples.?logistic regression analysis was used to identify the risk mtDNA haplogroup related to DR,namely candidate mtDNA haplogroup.?the differences in clinical parameters between candidate haplogroup and other haplogroups were compared.?associations between the single nucleotide variants of mtDNA-DLOOP and DR were also tested.Results:1.Univariate analysis showed that age,duration of diabetes,hip circumference,supine SBP,Glu0,HbA1c,Chol,BUN,UA,and Scr in the DR group were significantly different from those in the WDR group(all P<0.05),which was consistent with the results of Part 1 of this study,suggesting that the clinical data of these part of subjects have good stability.2.The 832 patients with T2DM could be classified into 40 mtDNA haplogroups.Among them,mtDNA haplogroups that were shared by at least 3%of the subjects in this study were mainly A,B,C,D,R9,G,M*,M7,M9,and N9.3.The principal component map revealed no apparent population stratification,although haplogroups closely clustered within the southwest province in China.And the south and southwest China had a more diverse haplogroup distribution than that in east and northeast China.4.Logistic-regression analysis indicated that haplogroup M9 was an independent protective factor for DR(P=0.004,OR=0.1).5.The Chol concentration in subjects with haplogroup M9 was significantly lower than that in other haplogroups(P=0.042).Even in WDR subjects,the Chol concentration was still significantly lower in subjects with haplogroup M9 than that in other haplogroups(P=0.038).6.Three single nucleotide variants of mtDNA D-LOOP,including mt.T146C,mt.T16298C,and mt.C16327T,were identified to be related to DR(all P<0.05).Conclusions:A more diverse haplogroup distribution exists in south and southwest China.This study finds for the first time that haplogroup M9 is an independent protective factor for DR in patients with T2DM in Yunnan Province.The Chol concentration in subjects with haplogroup M9 is significantly lower than that in other haplogroups.Part 3 Comparison of mitochondrial function and transcriptome analysis of M9 and non-M9 cybridsObjectives:To reveal the molecular mechanism of the haplogroup M9 as a DR protective factor by comparing the mitochondrial function in M9 and non-M9 cybrids,and by performing point mutation and transcriptome analysis.Methods:Firstly,according to previous reports,mtDNA haplogroups unrelated to glucose and lipid metabolism were used as the control of M9,namely non-M9.Then,the M9 and non-M9 cybrids were constructed to perform the mitochondrial function.Secondly,mt.T3394C and mt.G4491 A mutant plasmids were constructed and transfected into M7 cybrids respectively.According to the types of plasmids,cybrids were divided into normal control(NC)group,empty vector(Vector)group,wide type(WT)group,and mutant type(MUT)group to observe the effect of mt.T3394C and mt.G4491A mutation on mitochondrial function of M7 cybrids.Finally,RNA was extracted from M9 and non-M9 cybrids for transcriptome analysis.Results:1.Haplogroup M7 and D5,which were not related to glucose and lipid metabolism,were used as controls for haplogroup M9,namely non-M9.2.M9 and non-M9 cybrids were successfully constructed.The mitochondrial function analyses of cybrids showed that the mitochondrial membrane potential and mitochondrial mass of M9 cybrids were significantly higher than that of M7 cybrids(P<0.001),and there was no significant difference from D5 cybrids(P>0.05).There was no significant difference in ATP content among M9,M7,and D5 cybrids(all P>0.05).However,the reactive oxygen species(ROS)and mitochondrial superoxide levels of M9 cybrids were significantly lower than those of M7 and D5 cybrids(all P<0.01).3.The mt.T3394C mutant plasmid was successfully constructed for M7 cybrids transfection.The level of cytoplasmic ROS in the MUT group was significantly lower than that in the NC group,WT group,and Vector group(all P<0.05).The level of mitochondrial superoxide in the MUT group was significantly lower than that in the Vector group and WT group(all P<0.001),higher than that in the NC group(P<0.001).4.The mt.G4491 A mutant plasmid was successfully constructed for M7 cybrids transfection.The level of cytoplasmic ROS in the MUT group was significantly lower than that in the WT group and Vector group(all P<0.001),higher than that in the NC group(P<0.001).The level of mitochondrial superoxide in the MUT group were higher than that in the NC group and Vector group(all P<0.01),no significant difference was found between the MUT group and the WT group(P>0.05).5.The transcriptome analysis showed that there were 892 differentially expressed genes(DEGs)in M9 cybrids compared with non-M9(M7,D5)cybrids,of which 450 were up-regulated and 442 were down-regulated.The Gene Ontology(GO)analysis of DEGs mainly focused on cell components.The Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways analysis showed that the up-regulated DEGs were enriched in 5 pathways and the down-regulated DEGs were enriched in 3 pathways.Among them,four pathways,including PI3K-Akt signaling pathway,ECM-receptor interaction,HIF-1 signaling pathway,and NF-Kappa B signaling pathway were associated with DR.Cluster heatmaps of DEGs met with false discovery rate<0.05,and fold change?2 showed that 6 genes were up-regulated and 9 genes were down-regulated.Notably,NRCAM and ADAMTSL3 were expressed in the retina.Conclusions:Compared with non-M9(M7,D5)cybrids,the level of cytoplasmic ROS and mitochondrial superoxide in M9 cybrids are lower,and the expression of key signaling pathways and nuclear genes related to the pathogenesis of DR are also significantly down-regulated in M9 cybrids,suggesting haplogroup M9 exerts a vital role in the pathogenesis of DR.Moreover,the unique mutation mt.T3394C of the haplogroup M9 can effectively reduce the level of cytoplasmic ROS and mitochondrial superoxide in M7 cybrids,suggesting that this mutation partially mediate the protection of haplogroup M9 against DR.