GWA results in CLHNS mothers
In 1786 CLHNS mothers with complete genotype and phenotype data (Supplementary Material, Table S1
), we observed 22 SNPs significantly associated with natural log-transformed Hcy (log-Hcy) levels (P
< 5 × 10−8
), all located within or near the carbamoyl phosphate synthetase 1 (CPS1
) gene on chromosome 2 (Fig. ). The most strongly associated SNP (rs7422339, P
= 4.7 × 10−13
) was imputed, and encodes a Thr1405Asn substitution. Tests of association for the other CPS1
SNPs conditional on rs7422339 suggested a single CPS1
signal. The most significant SNP outside the CPS1
region was rs6722617 (P
= 1.9 × 10−7
), an imputed SNP located on chromosome 2q11.2 near lysozyme G-like 1 (LYG1
) and thioredoxin domain containing 9 (TXNDC9
), neither of which has been previously implicated as having effects on Hcy or other CVD-related traits. Several MTHFR
SNPs showed modest evidence of association (best P
= 2.7 × 10−4
for rs13306561). However, the widely studied MTHFR
C677T variant (rs1801133) was not directly tested as part of the genome-wide panel, and the imputation quality score did not meet the minimum requirement for inclusion in analysis.
GWA results for log-Hcy levels in 1786 CLHNS mothers. Result for directly genotyped SNP rs1801133 (MTHFR) is included. Loci described in the text are annotated with gene symbols.
Four analyses showed little-to-no evidence for genetic heterogeneity among CLHNS subjects: principal component (PC) analysis of population substructure (Supplementary Material, Fig. S1
), a minimal effect of adjusting for PCs (data not shown), the observed genomic control inflation factor (λGC
) of 1.0 and a quantile–quantile plot of GWA P
-values (Fig. ).
Figure 2. Quantile–quantile plot of SNP association with log-Hcy in CLHNS mothers. Black closed circles indicate all SNPs and dark-gray closed circles exclude all SNPs in 1 Mb windows centered on each previously reported SNP associated with log-Hcy (Table (more ...)
The most significant variants in the CPS1
regions were both imputed SNPs. To confirm association in the mothers and to test these variants for association with log-Hcy in the offspring (n
= 1679; 54.8% male, Supplementary Material, Table S2
), we directly genotyped rs7422339 and rs6722617. We also genotyped the MTHFR
SNP rs1801133 in both the mothers and the offspring.
The test of association between log-Hcy and the directly genotyped CPS1 SNP rs7422339 remained significant in the CLHNS mothers (P = 5.3 × 10−12, Table ). The estimated increase in log-Hcy for each additional A allele was 0.071 [standard error (SE) = 0.009], and the proportion of variability in the log-Hcy level explained by genotype was 3.0%. This SNP was not significantly associated with log-Hcy levels (P = 0.11) in the pooled CLHNS offspring sample (Table ). However, evidence for association was present in the female-only subset (n = 759; P = 0.0087), with the direction of effect the same as observed in the mothers (increase of 0.040 in mean log-Hcy for each additional A allele). A formal test for a gender-by-genotype interaction in the offspring-only sample was not significant (P = 0.095), but the statistical power to detect such an interaction was only 54% for the effect size observed. The test for interaction between rs7422339 genotype and generation (mothers versus offspring) was significant (P = 0.0004). There was no significant evidence for an interaction between age and genotype in the mothers (P > 0.10).
Evidence of log-Hcy association with CPS1 rs7422339 and MTHFR C677T
Evidence of log-Hcy association for the directly genotyped LYG1-TXNDC9 SNP rs6722617 (P = 1.2 × 10−7) was similar to the result based on its imputed genotype. The estimated increase for log-Hcy was 0.62 (SE = 0.23) for each additional C allele. No evidence for association between rs6722617 and log-Hcy was observed in the offspring-only sample (P = 0.58).
The directly genotyped MTHFR C677T SNP showed convincing evidence of association (P = 8.7 × 10−10) and explained 1.6% of the proportion of variability in log-Hcy in the CLHNS mothers (Table ). The estimated increase in log-Hcy for each additional copy of the T allele was 0.083 (SE = 0.015). This SNP was even more strongly associated with log-Hcy levels in the gender-combined offspring sample (P = 1.9 × 10−26, Table ). Each additional copy of the T allele was associated with an increase in log-Hcy of 0.171 (SE = 0.016), and the proportion of variability in the log-Hcy level explained by this locus was 5.1%. The gender-stratified results in the CLHNS offspring indicated that the MTHFR C677T association appeared slightly stronger in males (P = 1.1 × 10−16; β = 0.199; R2 = 7.3%) than females (P = 3.6 × 10−10; β = 0.136; R2 = 5.1%), and there was marginal evidence for a gender-by-genotype interaction (P = 0.053). The combined study of mothers and offspring for MTHFR C677T resulted in a smaller P-value (P = 1.3 × 10−29) than when analyses were restricted to the offspring alone. Evidence for an interaction between genotype and generation (P = 1.2 × 10−5) suggests that the increase in mean log-Hcy levels associated with each additional T allele is greater in the offspring cohort than in the mothers, although this result may be somewhat confounded by the marginal evidence for a gender interaction in the offspring. We did not observe evidence for an interaction between age and genotype in the mothers (P > 0.10). The MTHFR C677T genotypic means in the CLHNS mothers and offspring are most consistent with a recessive model for the T allele. The sample sizes for the 677T homozygotes (n = 7 for mothers and n = 16 for offspring), however, were too small to test this model.
Additional covariate adjustment for estimated folate, vitamin B6 and vitamin B12 intake levels in our linear models did not appreciably change any tests of genotype association or genetic effect size estimates for CPS1 rs7422339 or MTHFR C677T in either the CLHNS mother or offspring cohorts (data not shown). Furthermore, we found no evidence of interaction between MTHFR C677T genotype and dietary intake levels of folate (data not shown).
We observed strong evidence for association between CPS1
variation and log-Hcy level in this GWA study of 1786 Filipino mothers from the CLHNS cohort. The most significantly associated SNP (P
= 4.7 × 10−13
) encodes Thr1405Asn in CPS1 and explains ~3% of the variability in log-Hcy levels in this cohort. This finding confirms a recent report by the Women's Genome Health Study (WGHS), which observed significant evidence for an association between Hcy levels and rs7422339 (P
= 1.9 × 10−11
) in a sample of 13 974 Caucasian women. The protein encoded by CPS1
catalyzes the first committed step of the hepatic urea cycle, and rs7422339 has been previously reported to be associated with nitric oxide level and vascular smooth muscle activity, necrotizing enterocolitis, pulmonary hypertension and, also in the WGHS, fibrinogen level (15
). Consistent with our evidence of association in the female but not male CLHNS offspring, the WGHS did not observe replication evidence for CPS1
in samples from the Precocious Coronary Artery Disease Study (PROCARDIS), a subset of 840 post-myocardial infarction patients from several European countries, until they performed a gender-stratified analysis (male n
= 658, P
> 0.05; female n
= 161, P
= 0.0009). Although the P
-value for gender-by-genotype interaction in the CLHNS offspring was not statistically significant (P
= 0.095), together these results suggest that the effects of this gene may be stronger in women than in men.
The CLHNS mothers also showed evidence for association with the widely studied MTHFR
C677T SNP (rs1801133), which explained an estimated 1.6% of the variation in log-Hcy levels. Follow-up analysis of the CLHNS young adult offspring (age 20–22 years) suggests that MTHFR
C677T has a much stronger effect in this younger generation, with an estimated proportion of variance explained by genotype of 6.3%, which is very large for a single SNP for a complex phenotype. We also observed marginal evidence (P
= 0.053) for a gender-by-genotype interaction in the offspring, suggesting that, in contrast to CPS1
, there may be a stronger effect of this SNP in males than in females. These findings are consistent with several reports that have suggested that the effect of MTHFR
C677T is stronger in males than in females (10
) and that the importance of MTHFR
C677T genotype decreases with increasing age (22
), although no evidence for gender differences was reported in the PROCARDIS replication sample (13
). Hcy levels have been shown to be consistently higher in males than in females after childhood (23
), and estrogen lowers plasma Hcy (25
). In our study, CHLNS male offspring had significantly higher mean Hcy levels (P
< 0.0001) than female offspring despite higher mean dietary intake of folate, vitamin B6
and vitamin B12
(Supplementary Material, Table S2
), suggesting that any increase in the effect of MTHFR
C677T genotype in CLHNS male offspring compared with female offspring is not due to lower folate intake.
Evidence for differences in genotype effects between the CLHNS mothers and offspring was also detected at CPS1. In contrast to MTHFR, the observed effect of CPS1 was stronger in the mothers than in the offspring (Table ). The difference in effects for CPS1 could be due to bias based on the identification of this locus in the mothers and only following up the most significant results in the offspring, although this bias is mitigated by the report of the CPS1 association in the WGHS. The differences in genetic effect strength between CLHNS mothers and their offspring could also be attributed to age or other factors. We observed no evidence for a CPS1–age interaction in the mothers, where there is an age range of 34 years (data not shown). Nonetheless, there remains the possibility that the difference may not be a linear effect with age, but rather a consequence of different life stages or environmental exposures between younger and older adults.
Effect sizes appear substantially different between CLHNS and WGHS women at both the CPS1
loci. Overall mean levels of Hcy were the same (CLHNS mean = 11.4, WGHS mean = 11.4), but CLHNS mothers’ Hcy levels had less variability [CLHNS standard deviation (SD) = 3.0, WGHS SD = 4.7]. CPS1
SNP rs7422339 was estimated to explain a greater proportion of the variability in log-Hcy levels in the CLHNS mothers (R2
= 3.0%) than in the WGHS samples (R2
= 0.3%) (13
) despite a lower frequency (0.24 versus 0.31) of the minor allele in CLHNS mothers. Similarly, MTHFR
C677T explained a greater proportion of the variability in log-Hcy levels in CLHNS mothers than in WGHS women (CLHNS R2
= 1.6%; WGHS R2
= 0.95%) despite a lower estimated frequency of the minor allele (0.22 versus 0.33). At both loci, a significantly higher estimated increase of log-Hcy levels per each additional copy of the minor allele was observed in CLHNS mothers compared with WGHS women (see Table for effect size estimates and their associated SEs for both studies). The estimated proportions of variability explained by CPS1
SNP rs7422339 (R2
= 0.9%) and MTHFR
= 5.1%) in CLHNS female offspring were also higher than in WGHS women, although the estimated increase in log-Hcy levels per minor allele for rs7422339 in CLHNS female offspring was less than the estimated increase observed in WGHS women. The higher estimated proportion of variation explained by rs7422339 in CLHNS female offspring can be attributed to the lower overall variability of Hcy levels in CLHNS female offspring (SD = 3.0) compared with WGHS.
Because environmental factors, such as dietary intake of folate, vitamin B6 and vitamin B12, and indicators of estrogen levels in women are predictive of the Hcy level, it is possible that differences across populations could play a role in how genes influence Hcy. Both CLHNS and WGHS Hcy measurements were made in environments in which folate and vitamin B12 fortification was not widely available (WGHS measurements were made prior to 1998, when fortification of refined wheat products became mainstream in the USA). Adjustment for folate, vitamin B6 and vitamin B12 did not appreciably change genetic association test results in either CLHNS sample. We also found no evidence for any interactions between either CPS1 rs7422339 or MTHFR C677T genotype and folate levels to suggest their genotypic effects are stronger in those with lower reported intake levels of folate. Other important differences to consider when comparing results from CLHNS mothers with other studies include substantially lower use of hormone replacement therapy (<1%), which was widely reported in the WGHS (44.9%), and low oral contraceptive use in CLHNS (<2%).
Three of the four other loci identified in the WGHS for which SNP genotypes were available in the CLHNS also showed evidence of association (P
< 0.05, Table ). Evidence for the NOX4
locus (rs11018628) was relatively strong in the CLHNS mothers (P
= 9.8 × 10−5
), and we observed modest evidence for rs460879 (P
= 0.0039) at CHMP1A-DPEP1
and rs4267943 (P
= 0.050) at CENPQ-MUT
, but not for rs2061821 at AKAP13
= 0.79). The direction of estimated effects in the CLHNS mothers was consistent with those observed in the WGHS for the NOX4
SNPs. We did not observe evidence for the replication of rs10986018 at GPR51
reported in a meta-analysis by Hazra et al
). The GPR51
C allele frequency, however, was much smaller in the CLHNS (<0.01) than in the European-ancestry based sample examined in that study.
Finally, we did not replicate an observed association for rs1999594 near MTHFR
), for which a joint analysis including MTHFR
SNPs rs1801133 and rs1999594 showed that both SNPs were significant (14
). The frequency of the rs1999594 minor allele was lower in CLHNS mothers compared with the previous reports; however, there was no evidence of a main effect for this locus (β = −0.002) in CLHNS mothers. The different results for rs1999594 in CLHNS may be influenced by weaker linkage disequilibrium (LD) between rs1801133 and rs1999594 (HapMap CHB + JPT R2
= 0.00, D
′ = 0.23; HapMap CEU R2
= 0.26, D′ = 0.91).
This study represents the first GWA results for Hcy in an Asian population. Our findings suggest that at least some of the main genetic factors influencing plasma Hcy levels are likely shared across populations of European and Asian descent, despite different genetic backgrounds and environmental and dietary exposures. These results also suggest that the effects of genotype on plasma Hcy levels may differ across gender and life stages and should motivate future studies to take these factors into consideration.