Identification Of Sequence Polymorphisms In The ND Region Of Mitochondrial DNA As A Risk Factor For Hepatocellular Carcinoma

Objective: Hepatocellular carcinoma is one of the most frequent cancer in the world and it is the third leading cause of cancer deaths worldwide.In China,the incidence of HCC is increasing and now accounts for 55% of all HCC cases in the world.Single nucleotide polymorphisms(SNPs)accumulated in the mitochondrial DNA(mtDNA)links to the tumor formation.Our previous researches discovered that SNPs in the mitochondrial non-code region of displacement loop(D-loop)raised the risk of hepatocellular carcinoma(HCC).In this study,cancer risk-associated SNPs in the ND genes of mtDNA coding region were assessed in HCC patients and health controls.Methods:1 Samples: Blood samples were collected from 80 HCC patients at Fourth Hospital of Hebei Medical University,who underwent tumor resection in the Department of Hepatobiliary Surgery from September 2007 to March2008.Normal blood samples were collected from age-and number-matched healthy controls.2 DNA extraction: Genomic DNA was extracted immediately with a Wizard Genomic DNA extraction kit(Promega)from HCC patient's blood samples and normal blood samples.Then the DNA was frozen immediately in refrigerator until used.3 PCR amplification and sequence: The sequence of 13 paired primers were designed according to NCBI database(http://www.ncbi.nlm.nih.gov/snp/).Polymerase chain reaction(PCR)was performed using a PCR Master Mix Kit according to manufactor's instructions(Promega),and purified before sequencing.Sequencing was done by Sangon Biotech(Shanghai)Company.4 Statistical analysis: The entire mitochondrial ND gene was checked for meaningful SNPs,then enlarged the sample size of these valuable sites for further study.The student's t-test was used to analyse differences of clinical features between HCC patients and age-matched controls.The ?2 test was used to analyse the genotype frequencies of SNPs.All analyses were performed using SPSS software(version 21.0).A P-value<0.05 was considered statistically significant,and all statistical tests were two sides.Results:1 As for clinical characteristics,there was no statistical difference in age and gender between HCC group and heathy controls(P>0.05).2 The SNPs analysis of the 13 fragments were performed in 30 HCC patients and number-matched controls.The nucleotide position at sites4820G/A(P=0.237),4824A/G(P=0.112),5301A/G(P=0.237)were identified for their potential association with HCC.3 After expanded the genetic sample size of 4820G/A,4824A/G,5301A/G in ND and for further statistical analysis,the genotype distribution frequency of 5301 A and 5301 G was different between normal and HCC patients(? 2=4.783 P=0.029).The 5301A/G inducing the amino acid of I repalced by L at the 278 th resudial of MT-ND2 protein may influence the function of respiration chain,resulting in the development of hepatocellular carcinoma.4 The relationship between the HCC risk-associated SNPs and clinical features of HCC was then analyzed.The result showed: there was no association of 5301A/G SNPs with clinical features,including gender,age,TNM classification,size of the tumor,portal vein thrombosis and Child classification.Conclusion:1 For the SNP 5301 A/G targeting in ND,the frequency distribution was significant difference between HCC patients and healthy controls.The SNPs for 5301 A/G was associated with the cancer risk for hepatocellular carcinoma development.2 No association exist for the genotype distribution of 5301 A/G with clinical characteristics(gender,age,TNM classification,size of the tumor,portal vein thrombosis and Child-Pugh classification)in HCC(P>0.05).