Relationship Between Single Nucleotide Polymorphisms In Mitochondrial Displacement-loop And The Risk Of Chronic Kidney Disease

Objective: Chronic kidney diseases(CKD) is one of the important diseases affecting human health, its clinical manifestations are different in every stage,After entering the kidney failure stage, can appear acute heart failure, severe hyperkalemia, gastrointestinal bleeding, central nervous system disorders and so on,even have dangerous that threaten the life. The incidence of the disease is very high on a global scale. Recent epidemiological survey show that the incidence of chronic kidney disease in the adult population in China is as high as 10.8%. The occurrence and development of chronic kidney disease(CKD) is a multifactor and multistage comprehensive process, the susceptible factors mainly include age, diabetes, obesity, metabolic syndrome, a high protein diet, hyperlipidemia, high uric acid hematic disease, etc., but thefactors above can not fully explain the high incidence of chronic kidney disease. Research has shown that genetic factors plays a vital role in the process of development and incidence of chronic kidney disease(CKD). DNA in human cells are susceptible to oxidative stress damage, DNA combined with histone in the cell nucleus are less susceptible to oxidative stress damage, even if by damaged, the presence of certain nuclear enzyme can effectively repair the damaged DNA. While the Mt DNA located in the cytoplasm is lack of histone protection and effective repair mechanism, and thus is more likely to be damaged and cause mutations. Mt DNA mutations can occur in the coding region of each gene locus, but mainly are concentrated in the non-coding regions including D-loop. The study found that the gene polymorphism in the Mt DNA D-loop region is associated with aging, and the occurrence of various diseases, such as hemodialysis, coronary artery disease and cancer, etc., but it is rarely reported the relationship between chronic kidney disease(CKD) and the gene polymorphism in the Mt DNA D-loop region. Therefore, this experiment plans to detect the sequence of Mt DNA D- loop region for CKD patients and healthy people,to discuss and the relationship between the Mt DNA D-loop region gene polymorphism and the risk of chronic kidney disease.Methods:1 We selected 98 CKD5 patients who are in maintenance hemodialysis in hebei medicaluniversity hospital hemodialysis center in case group, including 53 men and 45 women. The inclusion criteria are above this(1) the age of dialysis is six months or more;(2) 2-3 times a week, 4-4.5 hours every time;(3) without acute infection, trauma, and other active disease in nearly one month;(4) without the use of lipid-lowering drugs and antioxidant in nearly two weeks; the same usage and dosage of recombinant human erythropoietin in nearly three months of; 6 volunteered for this topic research. The exclusion criteria are above this(1) serious liver damage;(2) autoimmune disease in active;(3) severe malnutrition. We selected 159 healthy people who receive physical examination in the fourth hospital of hebei medical university medical center as control group, including 96 men and 63 women. All the people don’t have symptoms and medical history of chronic kidney disease, don’t have serious heart and liver disease, don’t have family history of chronic kidney disease. This experiment has been approved by the fourth hospital of hebei medical university ethics committee, all the patients have signed the informed consent.2 Collected the clinical data of the two groups, including age, gender, kidney disease onset time and the serological indexes for the first time. Acquisition the venous blood samples of the two groups after overnight fasting, to extract the DNA of blood leukocyte. The Mt DNA D-loop area is amplified By PCR(Polymerase chain reaction, PCR,and is electrophoresed on a 1% agarose gel electrophoresis, The fragments are successful amplified and then inspect the sequences. Using the Snapshot gene sequencing method to determine the D-loop sequence, due to the D-loop area is very long, the same specimen should be sequenced in both directions. D-loop Sequence that be measured using DNAman software should be compared with the standard Revised Cambridge Reference Sequence(r CRS) in the mitochondria library, and to determine the frequency of Mt DNA D- loop polymorphism loci within the case group and control group, and the data should be in statistical analysis.3 Using SPSS 17.0 software package to analysis the data, The measurement data are expressed with mean±standard deviation, the comparison of Mt DNA D-loop polymorphic loci rate between the two groups is analysed by chi-square test, the difference was statistically significant in P<0.05.Results:1 there was no statistically significant difference between the case group and the control group in the distribution of age, gender(P value was 0.166 and 0.166, respectively), two group matches with each other.2 we found 103 polymorphic loci in the D-loop area of Mt DNA between the two groups.The frequence of 27 polymorphism loci is greater than 5% in healthy controls and(or) in the case group.3 we disscuse the relationship between gene polymorphism in mt DNA D-loop and the risk of chronic kidney disease.Compared with healthy controls, the frequency of A mutated to G(99% vs0 %, P < 0.001) in 73 loci significantly increased in patients with chronic kidney disease, the frequency of T mutated to C(16.3% vs0 %, P < 0.001) in 146 loci significantly increased, the frequency of T mutated to C(15.3% vs0 %, P < 0.001) in 150 loci significantly increased, the frequency of T mutated to C(0% vs 7.1%, P = 0.003) in 195 loci significantly increased, the frequency of C mutated to CA(0% vs 6.1%, P = 0.006) in 16266 loci significantly increased, The mutation rate in the rest of the site of the two groups have no obvious difference(P > 0.05).Conclusions: A mutated to G in 73 loci, T mutated to A in 146 loci, T mutated to C in 150 loci, T mutated to C in 195 loci, C mutated to CA in 16266 loci in D-loop region are associated with the risk of chronic kidney disease. Analysising Mt DNA D-loop sequence in the general population help to identify people who are at higher risk of chronic kidney disease, in order to intervent and prevent the disease in early stage.