The Impacts Of Mitochondrial DNA Haplogroups On The Morbidity And Mortality Of Severe Sepsis In Han Population

Severe sepsis is defined as sepsis plus evidence of organ dysfunction,and the development of sequential organ dysfunction is the most common cause of death in the intensive care unit.The pathophysiology of organ dysfunction associated with sepsis is not yet clear,but likely involves multiple factors,such as microcirculatory dysfunction,disturbed tissue oxygenation,deranged apoptosis,and direct cytotoxicity of cytokines and other sepsis-related compounds.Recently,lots of studies indicated that impaired cellular oxygen utilization,or 'cytopathic hypoxia',might play an important role in the development of multiple organ dysfunction in severe sepsis. Most of cellular oxygen delivered to tissues is used by the mitochondria,which produce ATP,the main intracellular energy source,needed for normal cellular function and metabolic homeostasis by oxidative phosphorylation,a process conducted by a series of five enzyme complexes located on the inner mitochondrial membrane. According to these findings,there is a strong likelihood that mitochondrial dysfunction contributes significantly to morbidity and mortality in the clinical setting during severe sepsis.The mitochondrion,contains multiple copies of a small circular genome of approximately 16,000 nucleotide base pairs,and this mitochondrial DNA(mtDNA) encodes for 13 peptides(subunits of complexesⅠ,Ⅲ,andⅣand the ATP synthase complex),2 ribosomal ribonucleic acids(RNAs),and 22 transfer RNAs.Human mtDNA is maternally inherited and the population can be divided into several mtDNA haplogroups on the basis of specific single nucleotide polymorphisms(SNPs) scattered throughout the mitochondrial genome.A body of evidences suggested that mtDNA haplogroups had functional importance,being associated with respiratory-chain activity and diseases susceptibility,such as Alzheimer disease, Parkinson's disease,breast and esophageal cancer.More recently,it has been reported that mtDNA haplogroup H,the most common subdivision of mtDNA in Europe,was a strong independent predictor of outcome during severe sepsis,conferring a 2.12-fold increased chance of survival at 180 days compared with individuals without the haplogroup H.Although different population has its unique mtDNA haplogroup types, we hypothesize the similar phenomena should occurred in other ethnic groups beside Europeans.So far,there is no relative data reported.The Han people constitute China's and the world's largest ethnic group,making up about 93%of the country's population and nearly 20%of all humankind.Most of the Han mtDNA can be allocated to specific subhaplogroups of Eurasian founder haplogroups M,N,R(which itself a subhaplogroup of N shared between Europe and East Asia).In the present study,we established the efficient and convenient strategy to determine the individuals' mtDNA haplogroups and prospectively studied a cohort of 181 patients with severe sepsis,to determine whether the main mtDNA haplogroups of Han people have impact on the morbidity and clinical outcome. PartⅠStrategy selection and method establishing for the mitochondrial DNA haplotypingObjective:To select an appropriate strategy for the mtDNA haplotyping and establish efficient methods suitable for large scale research.Material and methods:70 healthy volunteers were recruited at the First Affiliated Hospital,College of Medicine,Zhejiang University.We determined the mtDNA haplpgroups by comprehensive analyzing to the sequences of mtDNA hypervariable segmentⅠ(HVSⅠ)and haplotyping specific polymorphisms in the mtDNA coding region.The haplotyping diagnostic polymorphisms were accordance with the up-dated East Asia mtDNA haplogroups tree.HVSⅠfrom position 15996 to 16498 (relative to the revised Cambridge reference sequence(CRS)were amplified and sequenced.Fluorescence-labeled oligonucleotide probes were designed to examine the diagnostic polymorphisms in mtDNA coding region via real-time PCR allele discrimination.The mtDNA haplogroups determined by the established methods were confirmed by the typical haplotyping method-restriction fragment length polymorphism(RFLP).Results:70 healthy volunteers' mtDNA haplogroups were determined.RFLP analyzing were used to proved the haplotyping result of mtDNA haplogroup A,D,M7 and M9.For RFLP,the mtDNA haplogroup A generated 2 DNA bands(290b and 257bp)as well as mtDNA haplogroup D(312bp and 284bp)and M7(361bp and 47bp),while mtDNA haplogroup M9 produced numerous restriction bands and had the diagnostic bands(76bp and 36bp).The results of the RFLP were uniformly consisted with the sequencing and oligonucleotide probes' tests.Conclusion:mtDNA HVSⅠsequnces analysis combined with mtDNA haplotying diagnostic polymorphisms test was a good way to determine the individual's mtDNA haplogroup with perfect sensitivity as well as specificity.Fluorescence-labeled oligonucleotide probes' test was an efficient method to determine mtDNA polymorphisms and suitable for large scale investigation. PartⅡMitochondrial DNA haplotyping of the local Han population and the susceptibility to severe sepsisObjective:To investigate the susceptibility of mtDNA haplogroups to severe sepsis in Han population.Material and methods:181 patients with severe sepsis were recruited sequentially on admission to the intensive care unit at the First Affiliated Hospital,College of Medicine,Zhejiang University,as defined by the diagnostic criteria published by International Sepsis Definitions Conference.And,570 healthy volunteers were recruited as the control cohort.All the individuals' mtDNA haplogroups were determined by HVSⅠsequencing and specific coding region polymorphisms testing.Results:The frequencies of the major subhaplogroups of Eurasian founder haplogroups M,N,R in the healthy control cohorts were 47.7%,48.5%,37% respectively,while in the patient group were 47%,50.8%,36.5%respectively.Fisher' exact test indicated that the frequency of the main subhaplogroups of Han population in the study cohort did not differ significantly from the control group.The patients with mtDNA haplogroup R had higher maximum body temperature than those without haplogroup R(37.88±0.129℃VS 37.54±0.106℃,P=0.044)on the first 24 hours when recruited.Conclusion:The major mtDNA haplogroups of Han population M,N and R had not obvious association of the morbidity of severe sepsis.At the acute phase of the severe sepsis,the patients with mtDNA haplogroup R had higher body temperature than those without the haplogroup. PartⅢThe impact of mitochondrial DNA haplogroups on the clinical outcome of severe sepsisObjective:To assess the impact of the mtDNA haplogroup R on the clinical outcome of the severe sepsis.Material and method:The study cohort and control cohort were same to the PartⅡ. Acute physiology and chronic health evaluation(APACHE)Ⅱscores were documented at study entry,and recorded daily sepsis-related organ failure assessment (SOFA)scores until discharge from the intensive care unit or death,along with clinical outcome throughout 180 days.We used death during the 180-day period or survival at 180 days as endpoints.Follow-up was complete for all study patients. Auunivariate comparisons followed by mltivariate logistic regression analysis were performed to test the independent contribution of mtDNA haplogroup R to the prediction of the long term outcome of the disease.The odds ratios with 95% confidence intervals(CI)were used to estimate the association between the independent variables and the dependent variable.Results:Auunivariate analysis indicated that survivors were significantly different from non-survivors from some demographic and clinical characteristics,including younger age,lower APACHEⅡand SOFA scores,less likely to have chronic ill health (as defined in the APACHEⅡsystem),a lower occurrence of chest-related sepsis,and a higher occurrence of abdominal-related sepsis,while with respect to sex and sources of admission(whether surgical,medical,elective,or emergency),there was no significance between the survivor and non survivor cohorts.On admission to intensive care unit,the frequency of the main subhaplogroups of Han population in the study cohort did not differ significantly from the control group.Fisher' exact test indicated a higher 180-day survival rate in patients with mtDNA haplogroup R(p=0.006) compared with individuals without haplogroup R,and Kaplan-Meier analysis showed significantly higher survival over 180 days in patients with mtDNA haplogroup R than those without the haplogroup(p=0.0063).A comparison between demographic and clinical characteristics of the R and non-R haplogroup cohorts showed no significant differences.To avoid the potential influence of some confounding risk factors,we subsequently did logistic regression analysis using 180-day survival as the independent variable and the following dependent variables:age,sex,APACHEⅡscore and first 24h SOFA score,main source of sepsis,presence of chronic ill health, and the presence or absence of mtDNA haplogroup R.Higher APACHEⅡscore(odds ratio 1.130,95%CI 1.009-1.266,p=0.035)and SOFA score(odds ratio 1.186,95%CI 1.003-1.403,p=0.046),and the presence of chronic ill health(odds ratio 3.499,95% CI 1.581-7.741,p=0.002)were independently associated with poor outcome.MtDNA haplogroup R was a strong independent predictor of outcome,cofferfing 4.543-fold (95%CI 1.903-10.844,p=0.001)increased chance of survival at 180 days compared with those without the haplogroup.Age(p=0.140),sex(p=0.418),and main source of sepsis(p=0.115)were not independently associated with long-term outcome. Furthermore,our data also showed that mtDNA haplogroup R had independent associations of the patients' de-ventilation rate,indicating higher mechanical support discharge rate(OR 9.315,95%CI 3.014-27.684,p＜0.001).Conclusion:In Han population,mtDNA haplogroup R was a strong independent predictor for the outcome of severe sepsis,conferring increased chance of long-term survival compared with individuals without the haplogroup.Furthermore,mtDNA haplogroup R indicated higher de-ventilation rate of the patients needing respiratory support.