Bayesian clustering implemented in the program STRUCTURE revealed that both self-reported EA cases (n = 620) and controls (n = 443) were ‘genetic’ EAs. The average EA ancestry proportions for cases and controls were 0.977 (s.d. = 0.047) and 0.979 (s.d. = 0.046), respectively. In contrast, the mean EA ancestry proportion for the 214 AA reference subjects was 0.048 (s.d = 0.103). Moreover, the program STRAT revealed no significant difference in ancestry proportions between EA cases and controls (Σχ2 = 291.99, d.f. = 292, P = NS). Therefore, population admixture in our sample can be neglected, and additional SA analyses are unnecessary.
LD analysis results are shown in . The majority of OPRD1 SNPs (OPRD1^1–OPRD1^9) were distributed in a single haplotype block. The two remaining SNPs (OPRD1^10 and OPRD1^11), located 793 bp apart in the 3′ region, are in another haplotype block. Two pairs of SNPs were in high LD (OPRD1^1 and OPRD1^2: D′ = 0.95, r2 = 0.833; OPRD1^5 and OPRD1^6: D′ = 0.97, r2 = 0.656). Among seven OPRK1 SNPs, five (OPRK1^3–OPRK1^7) were in one haplotype block. However, OPRK1^1 showed LD with both OPRK1^3 and OPRK1^4, and OPRK1^2 also showed LD with both OPRK1^3 and OPRK1^4. In addition, OPRK1^5 and OPRK1^6 were in close LD (D′ = 0.97, r2 = 0.663).
Linkage disequilibrium (LD) analysis of OPRD1 and OPRK1 markers in European Americans (EAs) (cases and controls together) ((a) 11 OPRD1 SNPs; (b) 7 OPRK1 SNPs). The numbers in the squares are D′ ×100.
No significant deviations from HWE were detected in controls for any OPRD1 SNPs, nor were there deviations from HWE in cases or controls for any OPRK1 SNPs. However, two OPRD1 SNPs showed departure from HWE in AD subjects (OPRD1^6, P = 0.01; OPRD1^9, P = 0.02) or in CD subjects (OPRD1^6, P = 0.02). None of these were significant after correction for multiple comparisons. In addition, the genotype distributions for these two SNPs were consistent with genetic models that best fit these data. The OPRD1^6 genotype data fit either an additive model for AD (χ2 = 1.43, d.f. = 2, P = 0.489) and DD (CD and/or OD) (χ2 = 0.56, d.f. = 2, P = 0.755) or a recessive model for DD (χ2 = 2.91, d.f. = 2, P = 0.234). The OPRD1^9 genotype data fit an additive model for both AD (χ2 = 4.55, d.f. = 2, P = 0.103) and DD (χ2 = 2.27, d.f. = 2, P = 0.322). In other words, the deviation from HWE of genotype distributions for OPRD1^6 and -^9 may reflect either random effects (particularly a problem with multiple testing of many SNPs) or a real disease association of these two markers. Additionally, the OPRD1 SNP, rs419335 (not listed in ), which is located between OPRD1^3 and OPRD1^4 (6656 bp from OPRD1^3 and 9765 bp from OPRD1^4), was also genotyped by the TaqMan technique in all our case and control subjects. However, its genotype distribution was neither in HWE nor did it fit any of the disease models. Rechecking the allelic discrimination plot for this marker revealed that the three clusters of genotypes of this marker were not well separated, leading to the conclusion that the deviation was attributable to genotyping error, so it was excluded from analyses.
As shown in , nominally significant associations (P
< 0.05) were obtained with allelic and genotypic association analyses: OPRD1^1 (in the 5′ region) and OD (allelic); OPRD1^2 (or G80T in exon 1) and OD (both allelic and genotypic); OPRD1^6 (in intron 1) and AD (genotypic), CD (genotypic) and OD (allelic); OPRD1^7 (in intron 1) and AD (genotypic) and CD (genotypic); and OPRD1^11 with OD (genotypic). OPRD1^2 (or G80T) showed a strong association with OD (allelic, P
= 0.005; genotypic, P
= 0.008). Its minor (G) allele was significantly more frequent in OD subjects (21.0%) than in controls (13.2%). In addition, the G80T heterozygote (C/T) showed a significantly higher frequency in OD subjects (32.4%) than in controls (24.2%) (Supplementary Table S2 in Supplementary Materials
). To correct for multiple testing, we calculated the effective number of independent markers and the experiment-wide significance threshold required to keep the Type I error rate at 5%. The effective numbers of independent markers were 8.67, 8.71 and 8.74, respectively, for AD, CD and OD association analyses. Correspondingly, the threshold significance levels were 5.78×10−3
. Only the association between G80T and OD remained significant after correcting for multiple comparisons
P-values for comparisons of genotype and allele frequency distributions between cases and controls
Using a moving window analysis of three SNPs, an association of a haplotype of alleles of SNPs OPRD1^3, -^4, and -^5 and OD was detected (global P
-value after adjustment for multiple comparison or aP
= 0.03). Moreover, moving window analyses indicated that two OPRK1
haplotypes were associated with AD (haplotype OPRK1^2-^3-^4: aP
= 0.02; haplotype OPRK1^3-^4-^5: aP
= 0.004). To analyze the possible association of OPRD1
variants and SD using a different approach, six OPRD1
tag SNPs (selected by Tagger in Haploview) and all seven OPRK1
SNPs were included in haplotype analyses, results of which are summarized in . A significant difference in haplotype frequency distributions was found between OD subjects and controls (aP
= 0.005). This global P
-value might be attributable to rare haplotypes, because the global P
-value computed from the haplotypes abundant enough to be listed (frequency > 2%) did not reach statistical significance (OD vs Con: χ2
= 0.38, P
= 0.53). However, the frequency in cases of a specific OPRD1
T, which harbors the G-allele of G80T (in exon 1) and the C-allele of C921T (in exon 3), was more than double in controls (AD, 9.3%; CD, 9.1%; OD, 13.4%; controls, 4.7%). For OPRK1
, no significant difference in haplotype frequencies was observed between cases and controls. Nevertheless, one specific OPRK1
haplotype GGCTTCT occurred significantly more frequently in AD subjects (25.4%) than in controls (18.6%) (χ2
= 8.12, d.f. = 1, P
= 0.004). Furthermore, the positive results obtained from these two specific haplotypes (OPRD1 G
T and OPRK1
GGCTTCT) were confirmed by HTR analyses. As shown in Supplementary Table S3 in Supplementary Materials
T was the deleterious OPRD1
haplotype for AD (P
< 0.005, β
= 2.68, odds ratio (OR) = 14.55), CD (P
= 0.01, β
= 3.25, OR= 25.90) and OD (P
< 0.001, β
= 4.98, OR= 145.37), and GGCTTCT was the OPRK1
risk haplotype for AD only (P
= 0.02, β
= 0.52, OR= 1.68).
Comparison of haplotype frequency distributions between cases and controls
There were significantly more males among cases (P < 0.01) and cases were significantly older than control subjects (P < 0.01) ( and ). When potential confounding effects of sex and age were considered in backward logistic regression analyses in three genetic models (additive, dominant and recessive), four OPRD1 markers (OPRD1^2, -^4, -^7 and -^9) showed association with SD (). Specifically, the minor (G) allele of OPRD1^2 (or G80T), which was significantly more frequent in OD subjects than in controls, was a risk factor for OD and it exerted its effect on susceptibility to OD via an additive mode of action (P = 0.04, β = 1.27, OR= 3.56). In contrast, the minor (C) allele of OPRD1^7 was a protective allele for AD, consistent with a dominant mode of action (P = 0.04, β =−0.83, OR= 0.44). Moreover, logistic regression analyses with the recessive model demonstrated that the minor allele (the G-allele) of OPRD1^4 might play a protective role for CD (P = 0.04, β = −1.80, OR= 0.16), whereas the minor allele (the G-allele) of OPRD1^9 might exert a risk effect on CD (P = 0.03, β = 2.17, OR= 8.79). Additionally, haplotype logistic regression analyses, which also took sex and age into consideration, confirmed the haplotype association results, that is, the OPRD1 haplotype GCAACT might have a risk effect on AD (P = 0.03, β = 1.86, OR= 6.43) and has a significant risk effect on OD (P < 0.001, β = 3.92, OR= 50.57). The logistic regression analysis results for OPRK1 are summarized in . Three OPRK1 SNPs (OPRK1^2, OPRK1^3 and OPRK1^4) were shown to be associated with AD or CD. The minor (T) allele of OPRK1^3 showed a protective effect on CD through an additive mode of action (P = 0.02, β =−1.07, OR= 0.34); the minor (T) allele of OPRK1^2 might exert a risk effect on CD (P = 0.03, β = 2.61, OR= 13.65), and the minor (C) allele of OPRK1^4 might play a protective role for AD (P = 0.005, β =−1.08, OR= 0.33) and CD (P = 0.02, β =−1.00, OR= 0.37), both consistent with a recessive mode of action. Furthermore, OPRK1 haplotype GGCTTCT might contribute to risk for AD (P = 0.009, β = 1.06, OR= 2.90) and possibly for CD (P = 0.03, β = 1.02, OR= 2.76) ( and ).
Backward stepwise logistic regression (LR) analysis of OPRD1 SNPs in cases and controls
Backward stepwise logistic regression (LR) analysis of OPRK1 SNPs in cases and controls