Hepatocyte Nuclear Factor-4α P2 Promoter Haplotypes Are Associated With Type 2 Diabetes in the Japanese Population

The inclusion criteria used for diabetic and nondiabetic subjects in this study have been previously described (17). Diabetes was diagnosed according to the criteria of the World Health Organization (WHO) (18). All subjects enrolled in this study were of full Japanese ancestry. SNPs were genotyped in 192 nondiabetic subjects (mean age 69.1 ± 6.93 years and mean BMI 23.9 ± 2.77 kg/m²) and type 2 diabetic subjects (mean age 63.1 ± 4.16 years and mean BMI 24.6 ± 2.78 kg/m²) (first case-control cohort). We confirmed the results derived from our first case-control cohort in a second cohort (384 type 2 diabetic subjects with a mean age of 62.5 ± 9.78 years and a mean BMI of 24.4 ± 3.92 kg/m² and 384 nondiabetic subjects with a mean age of 68.4 ± 9.80 years and a mean BMI of 23.4 ± 5.87 kg/m²) and a third cohort (192 type 2 diabetic subjects with a mean age of 63.7 ± 8.31 years and mean BMI of 23.8 ± 1.39 kg/m² and 192 nondiabetic subjects with a mean age of 69.1 ± 8.32 years and a mean BMI of 22.9 ± 2.7 kg/m²). The three cohorts of subjects (first, second, and third cohorts) were recruited during different times (in the order of first, second, and third cohorts). The type 2 diabetic subjects of the first and second cohorts were recruited from the outpatient clinic of University of Tokyo Hospital; they were all residents of the Tokyo metropolitan area. The type 2 diabetic subjects of the third cohort and the nondiabetic subjects of all the three cohorts were recruited from the Hiroshima Atomic Bomb Casualty Council Health Management Center; they were all residents of Hiroshima, a city 500 miles west of Tokyo (note that all of the nondiabetic subjects were recruited from Hiroshima). Informed consent was obtained from all of the subjects enrolled in the study, and the study was approved by the Ethics Committee of the University of Tokyo.

To establish an SNP map encompassing HNF4A, SNPs were ascertained by both direct sequencing and a search of databases. We conducted a screening for SNPs by direct sequencing at all the exons of HNF4A and their exon-intron boundaries and along a 1,500-bp region 5′ upstream of exon D, which has been reported to be a β-cell–specific promoter region. The conditions and sequences of the primers used in the PCR shall be made available upon request. The SNPs were identified based on the sequences reported in GenBank that contain the HNF4A gene (accession no. NT_086910). From the public database, rs6065723, rs1884612, rs4812822, rs4810424, rs1884613, rs1884614, rs2144908, rs2273618, and rs3818247 were selected and validated in 30 type 2 diabetic subjects. The SNPs were genotyped in type 2 diabetic subjects and nondiabetic subjects by direct sequencing. PCR was performed under standard conditions, and the sequencing reactions were performed using the BigDye terminator kit (Applied Biosystems, Foster City, CA) and resolved using an ABI 3700 automated DNA sequencer (Applied Biosystems). The results were integrated using a Sequencher (Gene Codes Corporation, Ann Arbor, MI) and individual SNPs were manually genotyped. Ambiguous base callings were eliminated from further analysis. There was no genotyping error based on blind replicates for two SNPs in 192 samples.

The characteristics of the populations were described as means ± SD. The proportions of specific genotypes or alleles in subjects with and without type 2 diabetes were compared using the χ² test. The differences among subjects with different SNP genotypes were statistically tested using ANOVA. The statistical analyses, except for the haplotype estimation, were performed using JMP for Windows, Version 4.00 (SAS Institute, Cary, NC).

The frequencies of each haplotype were estimated, and the differences in the haplotype frequencies between nondiabetic and diabetic subjects were assessed using a software based on the expectation-maximization (EM) algorithm, SNPAlyze (Dynacom, Tokyo, Japan), and PHASE, Version 2 (http://www.stat.washington.edu/stephens/software.html). The differences in the haplotype frequencies were then analyzed using the χ² and permutation tests. The linkage disequilibrium (LD) structure in the HNF4A region was plotted using GOLD software.

Genomic DNAs from 192 unrelated type 2 diabetic and nondiabetic Japanese individuals were genotyped at an average of one SNP every 4.1 kb across an ∼78-kb region harboring the HNF4A gene and its alternative upstream promoter, P2. All the genotypic distributions of the SNPs that were identified were in Hardy-Weinberg equilibrium in both the nondiabetic and type 2 diabetic subjects (P > 0.05). Among those identified, SNPs with a minor allele frequency >0.10 were investigated for LD in HNF4A and for their association with type 2 diabetes. As shown in Fig. 1A, SNPs were used to determine the pattern of LD in the chromosomal region of HNF4A. The LD plot illustrates that the chromosomal region beginning with rs6065723, −27 kb upstream of the P2 promoter, and rs6073418, 17kb downstream of the P2 promoter, represents one block of strong LD, which is consistent with previous reports (15,16,20). No difference in the distributions of either the genotypes or the SNPs in HNF4A were observed between the nondiabetic and type 2 diabetic subjects (Table 1). We then tested the association of haplotypes with susceptibility to type 2 diabetes and found very strong evidence of an association between type 2 diabetes and a certain haplotype in the promoter region of the HNF4A gene. This highly significant association was seen with the haplotype consisting of two SNPs, namely rs1884614 and rs2144908, in the P2 promoter region of the HFN4A gene (overall P value = 3.82 × 10⁻⁴) (Table 2). In contrast, there was no significant association between type 2 diabetes and haplotypes consisting of polymorphisms not located in the P2 promoter region. Indeed, the extent of association between haplotypes and type 2 diabetes decreased with increasing distance from the P2 promoter region (Fig. 1B). This result supports the idea that a type 2 diabetes susceptibility polymorphism might be located in the P2 promoter region but not in any other region of the HNF4A gene. Martin et al. (19) reported that haplotype analysis allowed more accurate mapping of the Alzheimer’s disease susceptibility locus to the APOE gene than analysis of individual SNPs, similar to our findings. The association of type 2 diabetes with the rs1884614-rs2144908 haplotype was replicated in two other case-control cohorts. The second cohort (overall P value = 1.51 × 10⁻⁴) consisted of 384 diabetic and 384 nondiabetic subjects. The third cohort (overall P value = 0.019) (Table 2) included both type 2 diabetic and nondiabetic subjects recruited from the same area of Japan to exclude the possibility of false haplotype associations as a result of population stratification between samples enrolled from different areas of Japan (Tokyo and Hiroshima). The T-G rs1884614-rs2144908 haplotype was found consistently more frequently in the type 2 diabetic subjects than in the nondiabetic subjects of the first (0.052 vs. 0.024, haplotype-specific P value = 0.04, odds ratio [OR] 2.23 [95% CI 1.06–5.35]), second (0.042 vs. 0.012, haplotype-specific P value = 3.6 × 10⁻⁴, OR 3.50 [95% CI 1.67–7.11]), and third cohorts (0.039 vs. 0.007, haplotype-specific P value = 0.003, OR 5.83 [95% CI 1.69–22.4]) (Table 2).

We found no association between individual SNPs in the P2 promoter region and type 2 diabetes, which differs from the results reported by Silander et al. (15) in a Finnish population and by Love-Gregory et al. (16) in Ashkenazi Jews. However, our result was consistent with that reported in two recent articles published by Bagwell et al. (20) and Winckler et al. (21), who reported no evidence of any association of single SNPs with type 2 diabetes in Scandinavians and American Caucasians. These discrepant results among the studies may be attributable to the different genetic backgrounds of the subjects. In contrast, we found significant and consistent association between certain haplotypes and type 2 diabetes, which is consistent with the report of Bagwell et al. in which haplotypes consisting of SNPs in the P2 promoter region, but not any individual SNPs, were significantly associated with type 2 diabetes. The haplotype found to be associated with type 2 diabetes in the present study is relatively uncommon. However, it is noteworthy that the association between this haplotype and type 2 diabetes was observed consistently in all the three cohorts, suggesting the existence of an as-yet-unidentified polymorphism that might be relatively uncommon but associated with susceptibility to type 2 diabetes.

The results of the present study revealed that the P2 region of the HNF4A gene might be a susceptibility region for type 2 diabetes common to different ethnic groups, including the Japanese. Given that specific haplotypes, but not individual SNPs, in the P2 promoter region were significantly associated with type 2 diabetes, there might exist an as-yet-unidentified but functional polymorphism in the P2 promoter region of the HNF4A gene directly influencing the susceptibility to type 2 diabetes. It is also possible that SNPs constituting the haplotype in the P2 promoter region of the HNF4A gene associated with type 2 diabetes may coordinately affect the susceptibility to type 2 diabetes.

This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (to T.K.); Grant-in-Aid for Scientific Research on Priority Areas “Applied Genomics” from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and Grant-in-Aid from the 21st Century Program (to R.N.).

We thank Naoko Miyama and Yuko Okada for technical assistance.