The surprising finding from this study was the identification of the R3500Q
mutation in APOB
as a major determinant of LDL-C levels and CAC in the Old Order Amish. This mutation accounted for approximately 13% of extensive CAC in the population, a substantial effect given that CAC is strongly associated with the risk of future cardiovascular events. The present sample included 181 carriers of the R3500Q
allele (ie, approximately 1 of every 8–9 participants), a much higher frequency for this mutation than in any other single population previously reported.13
Because the Old Order Amish in Lancaster County are descendents of approximately 300 founders who emigrated to Pennsylvania from Switzerland in the latter part of the 1700s,14
we speculate that the R3500Q
mutation was introduced into the population by a single founding ancestor and has been maintained in the population at relatively high frequency through genetic drift.
mutation, which is responsible for familial defective apolipoprotein B-100,5
is believed to prevent the proper folding of apolipoprotein B by altering the interactions between 2 amino acids, thereby reducing the ability of the LDL-C particle to bind to the LDL receptor.15
The precise effects of this mutation on CVD risk have been hard to quantify because previous studies have either included few participants sampled from the general population or have focused on samples enriched with prevalent ischemic heart disease. In the largest published population sample, the APOB R3500Q
mutation was associated with total cholesterol levels 100 mg/dL higher and LDL-C levels 82 mg/dL higher based on only 7 carriers identified through a population screen of 9255 Danes16
compared with the present estimated effect sizes of 55 mg/dL for total cholesterol and 58 mg/dL for LDL-C based on a sample of 181 carriers. Previous studies16–18
based on identification of individuals with hypercholesterolemia or ischemic heart disease have reported even larger effect sizes. R3500Q
has been associated with ischemic heart disease, although a clear effect of this mutation on early coronary artery atherosclerosis has not been established. These findings reinforce the idea that the APOB R3500Q
mutation may increase ischemic heart disease risk by increasing LDL-C levels and CAC. This view is supported by evidence that the APOB R3500Q
mutation is a direct cause of elevated LDL-C levels, the known associations of APOB R3500Q
and LDL-C levels with ischemic heart disease, and previous studies,8,19–21
including from this population,8
showing that LDL-C concentrations are associated with the degree and presence of CAC.
The association between the R3500Q mutation and CAC could not be accounted for entirely by a 1-time measure of LDL-C levels because the R3500Q mutation remained strongly associated with CAC even after adjusting for or stratifying by LDL-C. A possible explanation is that R3500Q carriers have been exposed to a lifelong increase in LDL-C levels, which cannot be captured by a single cross-sectional LDL-C measurement. Alternatively, the mutation may be associated with features related to LDL-C metabolism that affect CAC development independently of circulating LDL-C levels. However, through this analysis of LDL sub particle distributions, we found no evidence to suggest that the mutation was associated with more atherogenic LDL.
Controlling for R3500Q
in the GWAS abolished almost all other associations with SNPs in the chromosome 2 region (data not shown). However, many SNPs in this region are in high-linkage disequilibrium, and carriers of the R3500Q
mutation share a common extended haplotype. We, thus, cannot exclude the possibility that there might be other variants in linkage disequilibrium with R3500Q
that are functional and contribute to elevated LDL-C levels and increased CAC levels. However, given what is known about the R3500Q
there does not seem to be a strong rationale for positing the presence of additional functional variants in the region that co-segregate with the R3500Q
mutation. Even if such variants did exist, it would be difficult to detect and attribute functional consequences to them because of the limited recombination along the at-risk haplotype.
mutation is relatively uncommon in non-Amish populations. Population-based surveys have reported carrier frequencies of 1 per 500 to 1 per 1250,23
and a carrier frequency of 1 per 209 has been reported in a Swiss population.13
Of 1840 individuals genotyped in the Baltimore-Washington Stroke Prevention in Young Women Study,24
we identified 2 heterozygotes, for a carrier rate of 0.11% (range, 0.02%–0.44%), in line with prior estimates of 0.08% to 0.41% reported from population-based studies.23
Importantly, even this low rate corresponds to a large number of carriers at the population level. For example, a carrier rate of 0.1% in the United States (2008 estimated population size of 305 million25
) would translate into an estimated 305 000 R3500Q
carriers. The markedly increased level of LDL-C and degree of CAC due to this single mutation have potentially important implications for personalized medicine in the full US population and in the Amish population, in whom this mutation is enriched. Because R3500Q
carriers are more likely than noncarriers to have an increased burden of sub clinical coronary atherosclerosis even at the same LDL-C levels, such individuals may benefit from earlier and more aggressive treatment with lipid-lowering medications. Is there value in screening for R3500Q
carriers at the population level? If so, how would this be most effectively accomplished, for example, by screening for the mutation or by screening for LDL-C levels? Prospective clinical trials are necessary to address these questions and to determine the most efficacious treatment strategies for carriers of this mutation, including at what age to start treatment.
In summary, we demonstrated that carriers of the R3500Q mutation in APOB are frequent in the Old Order Amish. In this population, the R3500Q mutation is a major determinant of LDL-C levels and CAC.