Particular HBV genotypes are associated with the outcome of chronic HBV infection and the response to antiviral therapies [8, 18–20]. The prevalence of HBV genotypes varies geographically, where genotypes B and C account for more than 90% of chronic HBV infection in East Asia . Furthermore, HBV strains circulating in an area can reflect the ethnic mix of its population [13, 14, 21]. In the western part of Indonesia (Sumatra, Nias, Mentawai, Kalimantan, Java and Lombok islands), HBV/B was predominant . Interestingly, in Minangkabau population of West Sumatra the predominant HBV genotype is HBV/C with subgenotype C1 [13, 14]. HBV/C was found to be predominant in the eastern part of Indonesia (Papua and Papua-influenced populations of Moluccas) but of different subgenotypes than those found in the western Indonesia (C6 and C7) [13, 14, 22].
HBV/C was highly prevalent in the Minangkabau ethnic group, whilst in non-Minangkabau ethnic groups HBV/B was more prevalent. In the western Indonesian population, the HBV/C was exclusively high in Minangkabau population . While our data supports the data from previous studies, it also reveals the distinct HBV genotypes that are conserved in those of Minangkabau ethnicity residing in Padang, as well as those of who live in western Java (Figure 3). Minangkabau ethnic people have strong traditions, and usually marry within their own group. Studies of Minangkabau associations in different regions indicate that they often develop out of pre-existing kin or locality-based networks that promote social interactions among their members . The present data illustrate a conserved transmission route of HBV infection which was most likely vertical, as it was obtained in high prevalence areas where HBV infection usually occurs perinatally or during infancy and early childhood . Thus, distinct HBV genotypes can be kept in societies living closely together even after migration over long distances .
The HBV genotype C is independently associated with a higher risk of HCC and associated with more rapid progression to cirrhosis than genotype B [8, 20, 23]. A prospective study from Hong Kong of 1,006 patients with chronic HBV infection followed up for a median of 7.7 years showed that the highest risk of developing HCC was in persons infected with HBV genotype C2, with the next highest being C1, followed by those infected with genotype B (presumably Ba) . Differences between the Asian genotypes B and C appears to be influenced by the subgroup of genotype B, because in Japan where the genotype B1 (Bj) is prevalent, no differences in the development of HCC between genotypes B and C was observed . The samples in this study were mostly from blood donors, however the data demonstrated a higher proportion of patients with liver disease among those of Minangkabau ethnicity than in the non-Minangkabau ethnic group (P=0.010) (Table 1).
Different HBV genotypes display distinct patterns of mutations at the Pre-S region and at the EnhII, BCP, and precore (EnhII/BCP/Precore) region in the HBV genome . The prevalence of pre-S mutants among different HBV genotypes was significantly higher in patients with genotypes B (25.0%) and C (24.5%) than the other genotypes (P<0.05), but there was no significant difference between genotypes B and C . The A1762T/G1764A mutation is more commonly found in patients with genotype C than those with genotype B . In Padang population, the data demonstrated significantly higher prevalence of pre-S mutant, T1753V, and A1762T/G1764A in the Minangkabau compared to the non-Minangkabau ethnic group. This was due to the higher prevalence of HBV/C and liver disease patients who provided samples from the Minangkabau ethnic group. The pre-S mutation was mostly found in HBV/C and in samples from liver disease patients of the Padang population (data not shown).
In contrast to the results in Padang population, pre-S mutations in samples from the Minangkabau ethnic group (from Padang and western Java) were less prevalent compared to those from the Javanese ethnic group, although this difference was not statistically significant. In particular, pre-S2 start codon mutation was more common in samples from the Javanese ethnic group than in those from the Minangkabau ethnic group (Table 2). This is most likely due to the higher percentage of liver disease patients in the Javanese ethnic group. The frequencies of mutations at the pre-S2 promoters were significantly higher in the patients with HCC than in the patients without HCC (pre-S2 promoter mutation: 15.3% vs 8.9%, P=0.032) . Furthermore, pre-S2 start codon mutation was associated with advanced liver disease and was the more common type of pre-S mutation in Indonesian patients regardless of the HBV genotype [15, 16].
The prevalence of the A1762T/G1764A mutation was significantly higher in the Minangkabau compared to the Javanese ethnic group. This result demonstrated the high prevalence of the A1762T/G1764A mutation in HBV/C regardless of the stage of infection. A previous meta-analysis study, evaluating 43 studies with a total of 11582 HBV-infected participants, found that the frequency of C1653T, T1753V, and TA mutations increased successively from asymptomatic carrier to cirrhosis, and were independent factors associated with HCC. The C1653T mutation in HBV subgenotype C2 and T1753V and A1762T/G1764A in HBV subgenotypes C1 and C2 were statistically significantly associated with an increased risk of HCC. Furthermore, the study mentioned that pre-S mutations C1653T, T1753V, and A1762T/G1764A accumulated during the progression of chronic HBV infection from the asymptomatic carrier state to HCC (P
<0.001 for each mutation) .
Random errors and variations in the HBV genome that occur over long periods while immune selection pressures operate at the population level have led to the emergence of distinct genotypes and subgenotypes in specific geo-ethnic populations. Since these variants are transmission competent, they can stably circulate within the given geo-ethnic population . HBV genotypes may add additional support to anthropological data on ancient migration events . The distribution of HBV genotypes/subgenotypes in the Indonesian archipelago is related to the ethnic origin of its populations and suggests that the HBV distribution is associated with the ancient migratory events in the peopling of the archipelago .
HBV/C1 was commonly found in Southeast Asia (the western part of Indonesia, Malaysia, Thailand, Vietnam and Bangladesh) and Southern China [12, 13, 20, 21, 27]. The Malay populations in the western (Melayu Minangkabau) and southern parts (Melayu Jawa and Melayu Bugis) of the Peninsular Malaysia were believed to have had more historical and cultural links with the populations from the Indonesian archipelago . The Neighbor-Joining tree based on the genetic distance measure of Fst (a method to show population genetic structure by partitioning genetic variance within populations relative to between populations), demonstrated that the Malay Minangkabau are grouped with the Indonesian Melayu. This topology may reflect the migrations of Malay Minangkabau to Malay Peninsula from Sumatra, the geographic origins of Indonesian Melayu . Genotypes B and C were equally frequent in ethnic Malays, where most genotype C strains were subgenotype C1 .
The multiethnic origin of the populations is reflected in the HBV genotype distribution in different parts of the country . The successfully subgenotyped samples from the non-Minangkabau ethnic group classified in this study was comprised of half Minangkabau descendants, Javanese and Sundanese (originating from Java island), and ethnic groups from other parts of Sumatra island (41.0%, 23.1%, 35.9%, respectively). Most of the HBV/C1 samples from the non-Minangkabau ethnic group belong to the half Minangkabau (72.2%) and Bengkulu people (11.1%). Bengkulu is a province near West Sumatera province. On the other hand, most of the HBV/B3 and HBV/B7 were found in the Javanese (45.5% and 66.7%, respectively). Three samples with intragenotypic recombinant HBV were detected in the non-Minangkabau ethnic group, two with B8/B3 and one with B9/B7. The samples that contained B8/B3 recombinant were detected in blood donors originated from Mentawai (11.101.135), an island near West Sumatra province, and from Simeuleu (11.101.118), an island near Aceh province, whilst the B9/B7 genotype was detected in a blood donor sample that originated from Bengkulu (11.101.121). Co-infection with more than one HBV genotype may also be a more common occurrence than what was previously thought. Among the Asian patients from Vietnam, significantly higher associations were found between mixed genotype infections and acute hepatitis B, liver cirrhosis, and HCC although the specific combination was impossible to identify due to the different mixtures reported . There were five samples from Padang population (1.9%) that were found to be co-infected with HBV/B and HBV/C, and all five were from blood donors. Two out of five were successfully cloned for subgenotyping (11.101.057 and 11.101.072). From sample 11.101.057, the S region was amplified and cloned. Five clones were obtained, four clones were HBV/B3 and one was HBV/C1. Whilst from sample 11.101.072, the pre-S region was successfully amplified and cloned. Interestingly, a mixture of subgenotypes variants was found. From eight clones obtained, five clones were HBV/B7, one clone was a putative intergenotypic recombinant strain of B/A, one clone was HBV/C1, and one clone was a putative intergenotypic recombinant strain of C/A.
In conclusion, the prevalence of HBV in blood donors in Padang was relatively low. The predominant HBV genotype and subgenotype in HBV carriers of Minangkabau ethnic group is HBV/C with subgenotype C1, which is different from the Indonesian population in general. The transmission route of HBV infection is most likely vertical, which allowed circulation of a conserved HBV genotype within the Minangkabau ethnic population. The prevalence of pre-S, A1762T/G1764A, and T1753V mutations were higher among HBV carriers of Minangkabau ethnicity. However, the association of the high prevalence of HBV/C1 and mutant variants with increased risk of advance liver disease among HBV carriers of Minangkabau ethnicity needs further investigation.