-acting regulatory variants affecting gene expression are predicted to regulate expression of a large proportion of human genes (9
) and are likely to account for many of the SNP-trait associations at susceptibility loci identified through GWAS. Our recent GWAS identified a region on chromosome 12 containing SNPs strongly associated with plasma HDL-C level (3
). In the current study, we selected the strongly HDL-C-associated SNP rs7298565 to represent these SNPs and asked whether rs7298565 was associated with cis
-acting regulatory activity in five positional candidate genes. We demonstrated that the G-allele associated with lower HDL-C level is associated both with higher levels of MMAB
transcript based on AEI and gene expression and with higher MMAB protein levels.
AEI has been used to identify genes influenced by cis
-acting regulatory SNPs (rSNPs; 15
), and our results suggest that measuring AEI has good potential to help prioritize functional gene candidates in association regions containing more than one candidate gene. However, successful AEI experiments require that each candidate gene contain at least one common transcribed SNP such that sufficient informative heterozygous samples are available. In addition, the genes in which AEI is to be tested must be expressed in an available tissue relevant to the associated trait. In this study, we investigated common transcribed SNPs (MAF > 0.01) in each gene of interest in human hepatocytes.
We describe a statistical strategy that allowed us to evaluate AEI for transcribed SNPs that exhibit any observed level of LD with an HDL-C-associated SNP (Fig. and Table ) in the absence of information regarding haplotype phase. This strategy allowed us to evaluate all genes of interest. In each test, we compared the AEI in cDNA from samples heterozygous for the HDL-C-associated SNP to a reference, either gDNA from the entire set of heterozygous samples or cDNA from samples homozygous for the HDL-C-associated SNP. Pairs of SNPs with r2 = 1.0, D′ = 1.0 have the highest power to detect association because the gDNA reference group has the largest sample size. However, this comparison may also be subject to bias if, in the absence of AEI, there is a technical difference in the percent allele detected in gDNA compared with cDNA. For example, MMAB transcribed SNP rs11067233 shows ~47% C allele in the gDNA but ~51% C allele in the cDNA of HDL-C SNP homozygotes, and this difference could cause an over (or under) estimation of AEI. For SNPs in which r2 < 1 between the transcribed SNP and the HDL-C associated SNP, using the cDNA reference group reduces power but also the risk of bias because the compared values are all based on cDNA. Testing transcribed SNPs with varying degrees of LD with the trait-associated SNP should allow evaluation of any possible bias in the more powerful test. For studies in which phase can be determined across markers with high accuracy, testing the phased samples for allelic expression differences could provide greater power.
AEI may have more power to detect allelic effects on mRNA level compared with stratifying mRNA level by genotype. AEI offers increased sensitivity because each sample acts as its own internal control for sample-to-sample differences in cellular environment. In contrast, total mRNA levels can be influenced by inter-individual variation that arises from differences in environmental factors, physiological states, trans-acting factors or overall gene expression between individuals of different ancestry. We observed stronger evidence for association of rs7298565 allele with transcript level using AEI (PgDNA = 1.4 × 10−13, n = 45) than by evaluating total MMAB mRNA level stratified by genotype (P = 0.0081, n = 89). The observation of increased MMAB protein with each copy of the G allele of rs7298565 is consistent with observations at the mRNA level and provides further support for a regulatory variant being responsible for the association with HDL-C level at this locus.
These results provide evidence of AEI in human hepatocytes and suggest a cis
-acting SNP affects expression of MMAB. MMAB
is expressed in many tissues, with high expression in the liver, which may be most relevant for processes influencing cholesterol level. Tests for the presence of AEI in other cell types important in regulating HDL-C level may produce different results. Expression quantitative trait locus (eQTL) studies also detected association of rs7298565 with MMAB
expression in 400 human liver samples (P
= 5.5 × 10−15
), a result confirmed in 950 liver samples (P
= 4 × 10−23
), but not all genome-wide eQTL analyses of primary or transformed lymphocytes reported SNP association with MMAB
). In addition, a recent genome-wide study in lymphoblastoid cell lines detected association between SNPs in moderate LD with rs7298565 (r2
= 0.11–0.67; D
′ = 1) and allelic expression in unspecified genes located in a 167 kb region that includes MMAB
). Further study is necessary to determine the strength of association between HDL-C associated SNPs and MMAB expression in other cell types.
The role of MMAB
in HDL-C biology is unclear. MMAB
is a mitochondrial enzyme that catalyzes conversion of vitamin B12 into its active form, adenosylcobalamin (4
is an essential participant in the catabolism of certain amino acids, short chain fatty acids and the side chain of cholesterol to the citric acid cycle intermediate succinyl-CoA (20
). An increase in expression of MMAB
might therefore be expected to lower levels of total cholesterol, including levels of HDL-C. Given the severity of methylmalonic acidemia, a disorder resulting from deleterious mutations leading to impaired MMAB
), and the evidence presented here of cis
-acting variation in MMAB
expression, we hypothesize that a variant in high LD with the HDL-C-associated SNP rs7298565 is likely to be a rSNP that has a modest effect on synthesis or stability of mRNA.
Our data cannot completely exclude the biological candidate MVK
, nor the other nearby genes KCTD10
, as functional targets of an HDL-C-associated SNP. Of the transcribed SNPs tested in our study, rs7957619 in MVK
had the lowest MAF and only 16 heterozygote samples were available. We had 100% power to detect AEI in MVK
based on the amount of AEI observed in MMAB
. A recent study in mice reports that Mmab
share a conserved promoter region and are both regulated by binding of sterol regulatory element binding protein 2, suggesting a potential novel link between the functions of these genes (22
). In humans, MVK
are also arranged in a head-to-head orientation on chromosome 12 and are predicted to share a 258 bp bidirectional promoter (L. Elnitski, Ph.D, unpublished data, 2008). Although neither our data nor existing liver eQTL data (2
) suggest evidence of cis
-acting regulation in MVK
, chromosome 12 HDL-C-associated variants could regulate levels of MVK
in untested tissues or through functional effects unrelated to total mRNA levels.
A primary challenge remains to identify the functional SNP(s) that affect MMAB
transcript levels at this HDL-C locus. Associated SNPs include a non-synonymous SNP in MMAB
, rs9593. Although both alleles of rs9593, encoding lysine and methionine, are predicted to have normal physiological activity (20
), we cannot exclude that this variant may also affect HDL-C level.
In summary, these data show that increased expression of MMAB is associated with SNPs associated with decreased HDL-C level, strengthening the evidence that MMAB is a target of an HDL-C-associated SNP and providing evidence of an effect on protein levels. Further studies are necessary to identify the functional SNP(s) affecting MMAB, and to determine how variation in MMAB gene expression may affect HDL-C level.