Association Of Hepatitis B Virus Genotype, Gene Subtype And Basic Core Promoter Region / Pre - C Region Gene Mutation With Liver Cancer Family Aggregation In

Objective:To explore the relationship among the distribution and characteristics of hepatitis B virus (HBV) genotype and subgenotype in the high hepatocellular carcinoma (HCC) incidence area of Guangxi and HCC aggregation of families.Methods:103 pairs of samples from the members matching for same sex, age (±5) and same living environment in 39 HCC-aggregation families (the experimental group) and 59 carcinoma-free families (the control group) were collected from July 2007 to July 2012, all of the subjects were infected with HBV and HBV DNA≥100IU/ml. Nested polymerase chain reaction (PCR) and sequencing methods were applied for analyzing HBV genotype, and then nested PCR and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for analyzing subgenotype.Results:(1)The baseline HBV DNA level of HCC-aggregation families members was significantly higher than that of non-cancer control families members (P=0.008),the proportion of HBV DNA≥105copies/ml viral load high members of HCC-aggregation families and carcinoma-free families members was 35.9% and 17.5%, the difference was significant (P=0.003)(2)Four HBV genotypes B, C, B/C, D were detected, occupying 30.6%、59.2%、4.4%、2.4%, respectively. B, C genotype were major genotypes, genotype C was prevalent. The proportions genotype B, genotype C, combined genotype B/C and genotype D in HCC aggregation families and carcinoma-free families members were 31.1%, 63.1%,1.9%,1.9% and 30.1%,55.3%,6.8%,2.9%, respectively, no significantly differences were found (P>0.05). HBeAg positive rates of genotype C andgenotype B were significant difference (P=0.000).In genotype B and genotype C, no significantly differences were showed in such factors as gender, age,ethnic groupand HBV DNA level (P> 0.05).The distributions for genotype B and genotype C of all level relatives in HCC-aggregation families were not significant differences. The genotype C rates of male and≥30 years old members in HCC aggregation families were significantly higher compared with the members in carcinoma-free families (P<0.01), but they were not significantly different in the members of female,<30 years old, ethnic group, HBeAg status between two groups (P>0.05). No significant differences were found in terms of gender, age, ethnic group or HBeAg status stratified in the genotype B membersin the two groups (P>0.05)(3)The case proportions of subgenotypes in all subjects were B2 (30.6%), Cl (38.8%),C2 (20.4%), B2C1 (2.9%), B2C2 (1.5%), noother subgenotype was detected, B2 and C1 weremajor subgenotypes.The proportions of subgenotype B2, subgenotype C1, subgenotype C2, combined subgenotype B2C1 and combined subgenotype B2C2 in HCC-aggregation families and carcinoma-free families members were 31.1%,63.1%,1.9%,1.9% and 30.1%,55.3%, 6.8%,2.9%, respectively, no significantly differences were found (P>0.05). HBeAg positive rate of subgenotype C1 was significantly higher than that of subgenotype B2 (P=0.000). No significantly differences were showed in such factors as gender, age,ethnic group, and HBV DNA level among subgenotype B1, subgenotype C1 and subgenotype C2 (P> 0.05). Differences were not showedin the distributions for subgenotype B1, subgenotype C1 and subgenotype C2 of all level relatives in HCC-aggregation families. The subgenotype C1 rates of male and≥30 years old members in HCC-aggregation families were significantly higher compared with the members in carcinoma-free families (P<0.05), but they were not significantly different in the members of female,<30 years old, ethnic group, HBeAg status between two groups (P>0.05).No significant differences were foundin terms of gender, age, ethnic group or HBeAg status stratified in the subgenotype B2 and subgenotype C2 membersin the two groups (P>0.05)Conclusions:HBV replication level and the high incidence of HCC for familial clustering in Guangxiwere closely relationship.Genotype B and genotype C were mainly, genotype C was prevalent, there are a small number of combined genotype B/C and genotype D. The subgenotype of genotype B all were subgenotype B2, there were two kinds of types (subgenotype C1 and subgenotype C2) in genotype C, subgenotype C1 was dominant, subgenotypesB2 and C1 were majority. No significant differences were found about the distribution of HBV genotypes and subgenotypes in HCC-aggregation families and carcinoma-free families. But the risk of HCC for the members who were men and ≥30 years old with genotype C and subgenotype C1 may be able to increase.Objective:To study the effect of hepatitis B virus (HBV) gene mutations in basal core promoter (BCP) region and precore region in the high hepatocellular carcinoma (HCC) incidence area of Guangxi on familial clustering of HCC.Methods:In order to avoid the influence of confounding factors for the objects of studying,103 pairs of subjects were selected from 39 HCC-clustering families (experimental group) and 59 non-cancer control families (control group) matching for gender, age and living environment in the high HCC incidence area of Guangxi from July 2007 to July 2012. All members were divided into Zhang (96 cases), Yao (52 cases) and Han (58 cases) three main ethnic groups based on ethnic distribution. Based on the HBeAg status, all members were divided into negative group (140 cases) and positive group (66cases). Based on mutation, all members were divided into mutations group and no mutations group. According to the classification of relatives, the HCC-clustering families members were divided into first-level relatives group (48 cases), second-level relatives group (32 cases) and third-level and above relatives group (23 cases).All of the subjects were infected with HBVand HBV DNA≥100IU/ml. The fragments of BCP region and the precore region genes were amplified by PCR, then the PCR products were sequenced and analyzed for detecting genetic mutations in BCP region and precore region of HBV DNA genome, and then to analyze the relationship between genemutations in BCP region and precore region of HBV DNA genome and the HBV genotypes.Results:(1)The baseline HBV DNA level and the number of HBV DNA≥ 105copies/mlviral load high members of HCC-clustering families were significantly higher than those of non-cancer control families members (P<0.01)(2)The sites of the previous ten frequency genetic mutations in BCP region and precore region of HBV were A1762T, G1764A, T1858C, A1775G, C1856T, G1896A, T1753C, A1846T, T1803Q G1898A, they were observed in 74.8%,70.9%,52.4%,46.6%,41.7%,34.0%,22.3%, 18.4%,14.6%,6.8%HCC high-incidence family members and in 53.4%,46.6%,42.7%,35.0%,22.3%,15.5%,9.7%,9.7%,7.8%, 3.9% non-cancer control families members, respectively. They have a variety of combinations of mutations, combined mutations were major, A 1762T/G1764A double mutations was the most common form of combined mutation. In ten mutations, the incidence rates of A1762T, G1764A, T1753C three sites in BCP region and C1856T, G1896Atwo sites in precore region of HBV were statistically differences in HCC high-incidence family members and non-cancer control families members (P<0.05). In addition, the differences in incidence rates of combined mutationsand A1762T/G1764A double mutations between two groups were significant (P<0.01). The indicators such as HBV DNA≥ 105copies/ml, A1762T/G1764A double mutations,A1762T, G1764A, T1753C, C1856T and G1896A had significant differences in univariate were included in the multiple logistic analysis showed that for HCC-clustering families HBV DNA≥105copies/ml (P=0.000, OR=18.637, 95%CI:6.014-57.757), A1762T/G1764A double mutations (P=0.009, OR=3.388,95%CI：1.360-8.441), C1856T (P=0.000, OR=10.270,95% CI:3.638-28.996) and G1896A (P=0.000, OR=7.597,95%CI： 2.720-21.213) mutations were independent risk factors.(3)The significantly different for the mutations rates of A1762T, G 1764A, T1753C, C1856T, G1896A were not found among Zhang, Yao and Han three main ethnic groups (P>0.05).The mutations rates of A 1762 T, G1764A, C1856T and G1896A sites in HBeAg negative group were higher than those in HBeAg positive group (P<0.01), but they were not significantly different in T1753C site between two groups (P> 0.05). The level of HBV DNA of A1762T, G1764Aand G1896Ain the groups with mutations were significantly lower compared with the groups with no mutations (P<0.01), no differences were found in T1753C and G1896A between two groups (P>0.05).The first-level relatives group, second-level relatives group and third-level and above relatives group showed statistically differences in the mutation rates of A1762T, G1764 Awith the first-level relatives group having significantly higher rates than second-level relatives group(P<0.05), the same results were not showed in T1753C, C1856T, G1896A among all level relatives in HCC-clustering families (P> 0.05)(4)In BCP region,genotype C had significantly higher prevalence of A1762T, G1764Aand T1753Cin comparison with genotype B (P< 0.05), no differences were found in A1775 G and T1803G between genotype B and genotype C (P>0.05)In precore region, genotype B had a significantly higher prevalence of C1856Tin comparison with genotype C (P=0.000), no differences were found in A1846T, T1858C, G1896A andG1898A between genotype B and genotype C (P>0.05)(5)In the members with genotype B, the mutation rate of C1856T in precore region in HCC high-incidence family was higher than that of in non-cancer control families family (P=0.032), in the two groups, no statistically differences forthe mutation rates of A1846T、T1858C、 G1896A、G1898A in precore region and A 1762T、G1764A、A1775G、 T1753C、T1803G in BCP region were found.In the members with genotype C, the mutation rates of A1762T、 G1764A、T1753C、T1803Gin BCP region and A1846T、G1896Ain precore region in HCC high-incidence family were higher than those of in non-cancer control families (P<0.05), no significant differences in the two groups about the mutation rates of A1775G in BCP region and C1856T、T1858C、G1898A in precore region (P>0.05) were found.(6) In BCP region mutations,subgenotype C1 had significantly higher prevalence of A1762T, G1764A and T1753C, and subgenotype C 2 had significantly higher prevalence of A1762T and G1764A in comparison with genotype B2 (P<0.05). No statistical differences in the mutations occurrence of A1762T, G1764A and T1753C between subgenotype C1 and C2, the results also were found in A1775G and T1803 G among subgenotype C 1, C1 and C2 (P>0.05)In precore region, the mutation rates of C1856T increased sequentially from subgenotype C1,C2 to B2, statistically differencesfor pairwise comparisons were found (P<0.05), subgenotype C2 had a significantly higher prevalence of G1898A than that of subgenotype C1 (P<0.05). No statistical differences were found in the mutations occurrence of A1846T, T1858C and G1896A among subgenotype B2, C1 and C2 (P>0.05)(7)In the members with genotype B2,the mutation rate of C1856T in precore region in HCC high-incidence family was higher than that of in non-cancer control families (P=0.032), the mutation rates of A1846T、 T1858C、G1896A、G1898A in precore region and A1762T、G1764A、 A1775G、T1753、T1803G in BCP region between the two groups had no significant statistically differences (P>0.05)In the members with genotype C1, the mutation rates of A1762T, G1764A, T1753C, T1803G in BCP region in HCC high-incidence family were higher than those of in non-cancer control families (P<0.05), no statistically differences were found about A1775G in BCP region and A1846T, G1896A, C1856T, T1858C and G1898A in precore region.In the members with genotype C2, the mutation rates of all the ten sites in BCP region and in precore region between the two groups had no significant statistically differences (P>0.05)Conclusions:(1)HBV DNA level, the genetic mutations of A1762T, G1764A, T1753C, C1856T and G1896A in BCP region and precore region of HBV DNA in the high hepatocellular carcinoma incidence area of Guangxi showed significant relationship with familial clustering of HCC. Combined mutation is a mutated form of the main, a synergistic role was complex mutations, A1762T/G1764A double mutations may have important predictive value for the incidence of HCC.(2)The mutations in BCP region and precore regionof HBV DNA may make HBeAg decreased or disappeared. The cases with mutations had a significantly lower level of HBV DNA compared with the cases without mutations, the high incidence of familial clustering of HCC in Guangxi may not be the mechanism by improving HBV DNA levels and working through mutations.(3)The mutations in BCP region were predominant in the members with genotype C and subgenotype C1, but genotype B and subgenotype B2 were associated with higher tendency in precore region. The risk of HCC for the members of genotype C and subgenotype C1 with mutations and genotype B and subgenotype B2 with mutations in BCP region may increase.