SNPs associated with lipid levels
Lipid association data for 21 previously established HDL-C SNPs in full-heritage Pima Indian samples are shown in ESM Table 3. Twelve SNPs were significantly (p < 0.05) associated with HDL-C in 2,675 full-heritage Pima Indians and all the associations were directionally consistent with those reported in individuals with European ancestry . These 12 SNPs were further genotyped in the remaining participants (n = 5,035 were informative for type 2 diabetes of which 2,819 also had lipid measures). Re-analysis of the 12 HDL-C-associated SNPs in all participants with lipid measures (n = 5,494, representing 2,717 full and 2,777 non-full-heritage Pima Indians) identified the strongest HDL-C associations for SNPs rs17231506 and rs12720922 at the CETP locus, which reduced HDL-C by approximately 6.5% per risk allele (Table 2, ESM Table 4).
Analysis of HDL-C-associated SNPs for association with type 2 diabetes
The 12 HDL-C-associated SNPs were further analysed in 7,710 participants informative for type 2 diabetes. SNPs at HNF4A, CETP, KLF14, MMAB and GALNT2 loci had nominal associations with type 2 diabetes (Table 3). Three of these SNPs (in HNF4A, CETP and ABCA1) had significant genotype–sex interaction (p
_int_sex < 0.05) for type 2 diabetes association and were re-analysed in male and female participants separately (no genotype–sex interaction observed for the HDL-C associations, p
_int_sex > 0.05). Rs4731702 in KLF14 had marginal evidence for genotype–sex interaction (p
_int_sex = 0.06) for type 2 diabetes association and was also re-analysed separately in male and female participants. For three of these SNPs (CETP, KLF14 and HNF4A) the HDL-C-lowering allele significantly (Bonferroni correction requires p < 4.2 × 10−3 based on 0.05/12 SNPs) associated with increased risk of type 2 diabetes in female but not male participants (Table 3). This sex-specific effect was directionally consistent in full and non-full-heritage Pima Indian samples when analysed separately, although the association for the CETP SNP in non-full Pima female participants did not reach statistical significance (p = 0.10, ESM Table 5). In contrast, the HDL-C-lowering allele for the ABCA1 SNP associated with decreased risk for type 2 diabetes in female participants, but did not achieve Bonferroni-corrected significance (Table 3). SNPs in GALNT2 and MMAB did not achieve a Bonferroni-corrected significant association with type 2 diabetes in the combined dataset and showed no evidence for a genotype–sex interaction; thus they were not further analysed separately in male and female participants (Table 3). Similar results were obtained when the 12 HDL-C-associated SNPs were analysed prospectively using a Cox proportional hazard model; the HDL-C-lowering allele of SNPs in CETP, KLF14 and HNF4A were associated with increased risk for type 2 diabetes in female participants and the SNP in ABCA1 was protective for type 2 diabetes in female participants (ESM Table 6).
To evaluate whether the sex-specific associations with type 2 diabetes for SNPs in CETP, KLF14 and HNF4A are mediated by HDL-C levels, we analysed the SNPs in the subset of samples informative for both type 2 diabetes and HDL-C levels (n = 5,494) and additionally adjusted for HDL-C (Table 4). In male participants, the type 2 diabetes associations were not significant with or without adjustment for HDL-C; however, in female participants all three SNPs remained nominally significant even after adjustment, although the effects were attenuated (Table 4). When additionally adjusted for other lipid measures (LDL-C, TG and TC levels) the female sex-specific type 2 diabetes association of SNPs in KLF14 and HNF4A remained nominally significant (n = 2,977; p = 0.03, OR 1.16 and p = 0.02, OR 1.54, respectively) although this adjustment rendered the association of the CETP SNP non-significant (p = 0.14).
Additional analysis at CETP
SNPs in the CETP locus provided the strongest evidence for association with HDL-C. Therefore, an additional 12 tag SNPs in CETP were analysed, along with the previously analysed rs12720922 and rs17231506 variants, for association with HDL-C and type 2 diabetes in full-heritage Pima Indians (Table 5). Rs6499863, which strongly associated with HDL-C (p = 5.6 × 10−11, effect = −5.4%; Table 5, Fig. 1a), had the strongest evidence for association with type 2 diabetes (p = 4.6 × 10−4, OR 1.26 [1.11, 1.43]; Table 5, Fig. 1b). Rs6499863 had a significant genotype–sex interaction for type 2 diabetes such that this SNP was significantly associated with type 2 diabetes in female (p = 2.8 × 10−5, OR 1.44 [1.21, 1.71]; Table 5, Fig. 1d) but not male participants (Table 5, Fig. 1c). This SNP was further genotyped and analysed in the non-full-heritage Pima Indian sample where the association with HDL-C (p = 1.3 × 10−6, effect = −4.9%; Fig. 1a) and type 2 diabetes in female participants (p = 0.04, OR 1.26 [1.01, 1.55]; Fig. 1d) but not in male participants (p = 0.70, OR 1.05 [0.82, 1.34]; Fig. 1c) was replicated. In the full-heritage and non-full-heritage Pima Indian combined sample, rs6499863 robustly associated with HDL-C (p = 3.6 × 10−15, effect = −4.9%; Fig. 1a) and significantly increased the risk for type 2 diabetes (p = 1.8 × 10−4, OR 1.22 [1.10, 1.35]; Fig. 1b). The genotype–sex interaction remained significant (p = 0.007) such that this SNP provided evidence for type 2 diabetes association in the combined analysis in female (p = 5.0 × 10−6, OR 1.36 [1.19, 1.55]; Fig. 1d) but not male participants (Fig. 1c).
Among female participants informative for both HDL-C levels and type 2 diabetes, the association of rs6499863 with type 2 diabetes was significant before and after adjustment for HDL-C levels (n = 3,044; p
before adjustment = 9.9 × 10−8, OR 1.49 [1.29, 1.72] and p
after adjustment = 9.9 × 10−6, OR 1.40 [1.20, 1.62]), and remained significant when adjusted for other lipid measures (TC, LDL-C and TG) (n = 2,936, p
after adjustment = 6.7 × 10−4, OR 1.31 [1.12, 1.53]). Rs6499863 also associated with estimates of insulin resistance (HOMA-IR) (p = 0.02, β [SD units] = 0.04; Fig. 1e), where the association was restricted to female participants (p = 0.005, β [SD units] = 0.06; Fig. 1e). Rs6499863 further associated with insulin sensitivity as assessed by the euglycaemic–hyperinsulinaemic clamp technique in 561 non-diabetic individuals (p = 0.02, effect = −4% per allele; Fig. 1f), where the association was only found in female participants (p = 0.03, effect = −4% per allele; Fig. 1f).
GRS analysis of HDL-C SNPs in Pima Indians
A multi-allelic GRS, which reflected the aggregate effect of all 21 established SNPs analysed (GRS21), associated with HDL-C such that there was an average decrease of HDL-C by 1.2% per risk allele (p = 1.9 × 10−14). However GRS21 was not associated with HOMA-IR, HOMA-B or type 2 diabetes. This analysis was restricted to only full-heritage Pima Indians since we did not genotype all 21 variants in the entire sample. A refined GRS (GRS12) created from the 12 SNPs significantly associated with HDL-C in full-heritage Pima Indians, and therefore genotyped in the entire sample, strongly associated with HDL-C in the combined analysis (p = 1.4 × 10−47, effect = −2.2%; Fig. 2a). GRS12 also positively associated with type 2 diabetes and HOMA-IR when analysed in the non-full-heritage Pima samples (n = 4,004, p = 0.002, OR 1.06 [1.02, 1.10] and n = 2,728, p = 0.03, β [SD units] = 0.012 per risk allele, respectively) and in the entire dataset (p = 0.04, OR 1.03 [1.00, 1.05] and p = 0.003, β [SD units] = 0.01, respectively, Fig. 2c, d), but not with HOMA-B (Fig. 2b). However, the GRS did not have a significant interaction with sex for the traits analysed. When rs4731702 (KLF14) and rs1800961 (HNF4A) (established loci for type 2 diabetes) were removed from the GRS12 analysis, the association with HOMA-IR remained significant (p = 0.02) but the association with type 2 diabetes was rendered non-significant (p = 0.60).