In this study, we identified several variants in the LPA gene that are strongly associated with Lp(a) levels in a diverse epidemiologic study. More specifically, three SNPs in non-Hispanic whites, twelve SNPs in non-Hispanic blacks, and six SNPs in Mexican Americans were strongly associated at p<0.0001. While no single LPA variant was significantly associated in all three racial/ethnic groups, six SNPs were significantly associated in two subpopulations and the directions of effects were consistent.
Most previously published studies characterizing the relationship between Lp(a) and LPA
have focused on the effects of the kringle IV-2 copy number polymorphism. More recently, a genome-wide association study in Hutterites identified one SNP in LPA
(rs6919346) that associated with Lp(a) levels, independent of kringle IV-2 copy number
. Subsequent studies have found this variant to be independently associated with increased Lp(a) levels in European Caucasians
and South Asians and Chinese
. In our study, the same allele (G) was also strongly associated with increased trait levels not only in non-Hispanic whites (β
) but also in non-Hispanic blacks (β
). In contrast, the association in Mexican Americans was much less robust (p
0.02), but the effect trended in the same direction (β
0.18). This intronic tagSNP is not in linkage disequilibrium (LD) with any genotyped SNP (Supplementary Figure S1
), nor with the kringle IV repeat
. As others have suggested
, rs6919346 may be tagging the causal variant or, due to the fact that it resides in a CRE-binding site, may play a role in gene expression.
It is interesting to note that while we did replicate the association between Lp(a) levels and rs6919346, we did not necessarily replicate the associations reported recently for rs10945682 and rs7765803
rs10945682 was not associated with Lp(a) levels in NHANES III at the significant threshold of p<0.0001 (). Furthermore, while the direction of effect in non-Hispanic whites was consistent with that observed for Europeans and Asians studied by Lanktree et al (taking into account the coded allele), the direction of effect was opposite in the non-Hispanic black and Mexican American subpopulations in NHANES III. LPA
rs7765803 was not associated with Lp(a) levels in non-Hispanic whites (p
0.2471) while it was strongly associated in European and Asian populations in Lanktree et al. Finally, the data reported here are not consistent with the linkage disequilibrium data reported by Lanktree et al. LPA
rs10945682 and rs6919346 are reported to be in the same linkage disequilibrium block
but the LD calculated in our non-Hispanic white samples and in HapMap CEU suggests there is little LD (r2
0.06 and 0.03 in non-Hispanic whites and CEU, respectively) between the two SNPs. It is possible that this discrepancy can be explained by unidentified population substructure or by the use of different LD measures, but this is unclear from the literature and requires further investigation.
As alluded to above, the relationship between LPA
tagSNPs and Lp(a) levels may represent a direct (i.e. causal) or indirect (i.e. proxy for true causal variant) relationship. The latter situation most likely applies to the majority of SNPs genotyped in this study. Of the 19 LPA
SNPs, 17 are located in introns, and the two nonsynonymous SNPs (rs7765803 and rs41265936, Supplementary Table S1
) are not predicted to alter protein function using SIFT
. Additional studies are needed to determine if these variants regulate LPA
expression in vivo
. However, since apo(a) is present only in humans, Old World primates, and the hedgehog, resources for these studies are limited to transgenic mice and rabbits as models
In an attempt to evaluate the joint effect of significantly associated variants, a genetic risk score (GRS) was calculated. Based on this GRS, these variants together explained 7%, 9%, and 11% of the variance in Lp(a) levels in non-Hispanic whites, non-Hispanic blacks, and Mexican Americans, respectively. In comparison to the effect attributed to the kringle repeat region based on previous studies
, the effect of these SNPs is considerably small.
This study has several strengths and limitations. The greatest strength is the use of a large and diverse population. While there have been several studies of LPA
SNPs and its association with Lp(a) that have included both European and African descent populations, no single study, to our knowledge, has also included Mexican Americans genotyped for the same LPA
SNPs. This latter point cannot be under emphasized as the Hispanic or Latino population is the fastest growing minority population in the United States yet remains relatively underrepresented in genetic association studies
A limitation is that the method of measuring serum Lp(a) levels in NHANES III does not account for apo(a) isoform size. While accurate measurement of apo(a) isoform is ideal, the reliability of the Lp(a) measurement used here has been adequately demonstrated
. Furthermore, there is no generally accepted laboratory procedure or national standardization program for Lp(a) measurement, which may help to explain the lack of generalizabilty across studies
A second major limitation is that NHANES III does not have data on kringle repeat size for each participant. Several methods are used to measure kringle repeat size such as Southern blot
and quantitative PCR
, neither of which can be used in NHANES III DNA samples given investigators are aliquoted limited amounts of DNA from crude cell lysates. Without these data, it is unclear if the associations between LPA
SNPs and Lp(a) levels reported here are independent of the KIV-2 copy number variant, which has a well-established, large effect on Lp(a) levels.
The amount of linkage disequilibrium, or lack thereof, between the KIV-2 region and other LPA
variants is a controversial issue. Previous studies have reported strong LD between the KIV-2 alleles and SNPs in or around LPA
. In contrast, additional studies indicate the lack of strong LD
. More specifically, the tagSNPs genotyped in this study had been selected from a previous study
that provided data on kringle IV-2 repeat size, and no strong LD (r2
>0.80) was found for any of the SNPs tested
. However, there was moderate LD (r2
0.45 in European American and r2
0.57 in African American samples) between kringle repeat sizes 10 and 14 and LPA
SNPs 74970 and rs41271028, respectively
74970 was not genotyped here. LPA
rs41271028 was genotyped here but was not significantly associated with Lp(a) levels in any of the three subpopulations after correction for multiple testing (). Thus, the tagSNPs genotyped here and significantly associated with Lp(a) levels after correction for multiple testing are not in high or moderate LD with specific kringle repeat sizes examined in the original dataset reported by Crawford et al
. Further studies are needed in NHANES and other large datasets to characterize the full spectrum of LPA
genetic variation and its impact on Lp(a) levels in diverse populations.
Another limitation of this study is that only approximately 30–35% of the LDSelect “bins” for European Americans and African Americans are represented by tagSNPs as many LPA SNPs failed assay design or genotyping in NHANES III. And, tagSNPs selection was limited to common variation, leaving rarer variation such as LPA rs10455872 (<5% MAF) untested. Thus, much of the genetic variation in LPA and its association with Lp(a) levels in these populations remains to be explored. Furthermore, tagSNPs were not selected specifically for the Mexican American subpopulation. At the time of tagSNPs selection, HapMap 3 Mexican American samples were not available at the time, and it was unclear which populations should be used for tagSNPs selection to adequately represent this admixed population. It is important to note that, however, that while our tagSNPs selection process may have been biased for populations of European- and African-descent, the allele frequencies observed in NHANES III Mexican Americans were very similar to that of non-Hispanic whites. Furthermore, our lack of Mexican American specific tagSNPs does not undermine the observation that there is an excess of significant variants associated only in non-Hispanic blacks compared to non-Hispanic whites.
Because of these strengths, and despite these limitations, we have taken an important step in understanding how LPA
genetic variants contribute to Lp(a) levels in a diverse population. One of the major findings of our study was that there were notably more significant associations between Lp(a) and LPA
SNPs in non-Hispanic blacks compared to non-Hispanic whites and Mexican Americans. Moreover, nearly half of these associations were exclusive to non-Hispanic blacks. Our results suggest that between-population differences in Lp(a) levels can be explained, in part, by multiple population-specific cis
-acting variants in LPA
. While the role of multiple trans
-acting factors in Lp(a) trait distribution has been disputed
and cannot be ruled out, our results reaffirm the need for more comprehensive studies of the effects of LPA
variants in large, diverse populations.