In this study, we resequenced PON cluster genes PON1, PON2, and PON3 in 16 children and 14 mothers of Mexican ancestry living in Salinas Valley, California, and identified over 90 genetic variants, including indels, which have not been previously explored, and also some novel SNPs. Many of these variants had significantly different allele frequencies in comparison to Caucasions despite an overall similar haplotype structure within the PON gene cluster. Several PON variants in CHAMACOS mothers and children, not in strong LD with main PON1 SNPs (R2<0.20), including a newly identified deletion in the 5’ UTR, were found to be associated with AREase and POase activity that are predictive of susceptibility to OPs and oxidative stress. Multiple polymorphisms provided a modest improvement of the assessment of functional significance for PON levels and substrate-specific activities in comparison to the haplotype tagSNPs. Functional effects of PON genetic variation also differed by age, and should be considered in protecting vulnerable subpopulations.
Overall, the distribution and location of haplotype blocks was similar in CHAMACOS Mexican-Americans from Salinas Valley, CA and Caucasians (HapMap data) with several regions of strong LD separated by regions of high recombination. In the CEPH population, Jarvik et al (2003)
described four nonrecombinant regions – one at the promoter region, one near the PON155
SNP, one encompassing the PON1192
SNP, and one at the 3’ UTR region. Likewise, in Salinas Mexicans, we observed high LD in the promoter region and identified haplotype blocks surrounding the two nonsynonomous coding SNPs (blocks 1 and 2) and the 3’ UTR region (block 3). While the LD structure in Salinas mothers and children appears similar to that found in Caucasians, we did observe one important difference between the two; in Mexicans, many SNPs in haplotype blocks 2 and 3 were highly correlated with each other (across blocks) whereas these two blocks were more distinct with low LD between SNPs in CEPH subjects.
Although differences in LD structure between ethnic groups were modest, allele frequencies for some SNPs varied more noticeably. Several studies have previously demonstrated that the genotype distributions of the four main functional PON1 SNPs (192, 55, -108, -162) vary widely across ethnic groups (Chen et al. 2003
; Rojas-Garcia et al. 2005
). Here, we found allele frequencies of other PON1
polymorphisms in our population were also quite different from other studied groups. For instance, 12 SNPs with MAF less than 5% in Caucasians had much higher frequencies (20% or higher) in Mexicans. These potentially important and common SNPs could easily be overlooked in other studies if they are rare in well-characterized populations. Our data may aid study design in future PON
genetic association studies in Mexican and other Latino populations, providing more detailed data on LD structure and further informing tagSNP selection.
Indels are the second most frequent type of polymorphism in the genome, and have been associated with human diseases (Kondrashov and Rogozin 2004
; Sun et al. 2007
; Zoghbi and Orr 2000
). Despite their potential relevance, few PON1
indels have been functionally characterized. We observed four insertions and eight deletions in the PON1
gene, four of which were novel. Furthermore, four deletions and one insertion were significantly associated with either AREase or POase activity. Since several of them were also correlated with the coding SNP PON155
, it is not clear whether these associations are due to the indels themselves or to their LD with PON155
. Future studies should include additional types of sequence variations like indels because they are common genetic variants with likely functional significance.
Genetic association studies often focus on coding SNPs, particularly nonsynonomous SNPs resulting in amino acid changes because they are likely to be functional. However, recent studies suggest that SNPs in other regions of the genome, including introns and 3’UTR regions may also have functional consequences. Intronic SNPs can affect splicing elements thereby influencing transcription and gene expression(Le Hir et al. 2003
). In our study, we identified several intronic SNPs which were associated with AREase and POase activity. Splicing is one potential mechanism explaining their impact on PON1
phenotype. MicroRNA, which is involved in post translational regulation of gene expression, binds to sequences in the 3’UTR region(Borel and Antonarakis 2008
). Several studies have shown SNPs in these miRNA binding sites can mediate regulation of gene expression(Abelson et al. 2005
; Sethupathy et al. 2007
). In our study, we identified several SNPs in the 3’UTR that are predicted miRNA binding sites and were associated with AREase activity. Future studies employing reporter silencing assays may help to establish whether these SNPs affect gene regulation through miRNA-mediated mechanisms.
Since the LD structure of PON1
comprises 4 haplotype blocks in Caucasians, Jarvik et al.(2003)
previously suggested that most of PON1 variation could be captured using just the two promoter SNPs PON1-108
and the two coding SNPs PON155
. Indeed many studies, including our own, have examined effects of these SNPs (and also PON1-909
). In this study, we extended our analyses to include SNPs and indels spanning the entire PON
gene cluster and identified several additional genetic variants associated with AREase and POase activity. However, in combination with the four known functional SNPs, they still only explain a small percentage of additional phenotypic variation. Particularly with AREase activity, other factors including genetic variants in other genes, environmental exposure or epigenetic modification may also influence PON1
expression. We also found that similar to previous studies showing differences in the percent variation explained by the same SNPs in newborns and mothers(Chen et al. 2003
; Holland et al. 2006
), the relative contribution of some SNPs to PON1 phenotype was different in these age groups. For instance, the 3’ UTR SNP (rs854551) was not significantly associated to POase activity in newborns, yet was strongly associated with POase activity in mothers (p=1.14 × 10-3
). These data suggest that the role of specific genetic variants is not static and at different ages or physiological conditions (at the time of delivery), their relative influence on PON1
phenotype may change.
In conclusion, we sequenced PON family genes in a cohort of Mexican-American children and mothers from Salinas Valley, CA, and identified several novel SNPs, and indels including several SNPs in introns and the 3’UTR, some of which were independently associated with PON1 phenotype. Allele frequencies of many of the SNPs were quite different in our Mexican-Americans in comparison to Caucasians. Additionally, the relative genetic contribution of PON1 SNPs toward molecular phenotype (enzyme activity) differed between mothers and their children. These functional effects of PON variation should be considered in protecting vulnerable subpopulations from OPs and other inducers of oxidative stress. Ethnic differences in the frequency and distribution of susceptible genotypes should also be taken into account in genetic association studies of PON.