Common Low-Density Lipoprotein Receptor p.G116S Variant Has a Large Effect on Plasma Low-Density Lipoprotein Cholesterol in Circumpolar Inuit PopulationsCLINICAL PERSPECTIVE
Background—Inuit are considered to be vulnerable to cardiovascular disease because their lifestyles are becoming more Westernized. During sequence analysis of Inuit individuals at extremes of lipid traits, we identified 2 nonsynonymous variants in low-density lipoprotein receptor (LDLR), namely p.G116S and p.R730W.
Methods and Results—Genotyping these variants in 3324 Inuit from Alaska, Canada, and Greenland showed they were common, with allele frequencies 10% to 15%. Only p.G116S was associated with dyslipidemia: the increase in LDL cholesterol was 0.54 mmol/L (20.9 mg/dL) per allele (P=5.6×10−49), which was >3× larger than the largest effect sizes seen with other common variants in other populations. Carriers of p.G116S had a 3.02-fold increased risk of hypercholesterolemia (95% confidence interval, 2.34–3.90; P=1.7×10−17), but did not have classical familial hypercholesterolemia. In vitro, p.G116S showed 60% reduced ligand-binding activity compared with wild-type receptor. In contrast, p.R730W was associated with neither LDL cholesterol level nor altered in vitro activity.
Conclusions—LDLR p.G116S is thus unique: a common dysfunctional variant in Inuit whose large effect on LDL cholesterol may have public health implications.
Inuit were long believed to have lower cardiovascular disease (CVD) risk than nonindigenous populations.1–3 However, re-evaluation of population studies indicates that ischemic heart disease rates are similar between Inuit and nonindigenous people.4 Furthermore, ongoing Westernization in many Inuit communities has intensified their exposure to CVD risk factors such as smoking, calorie-dense processed foods, and a more comfortable but also sedentary lifestyle, all of which affect CVD risk and prevalence.4–10 Among classical CVD risk factors, Inuit adults tend to have higher plasma concentrations of low-density lipoprotein (LDL) cholesterol (LDL-C) than nonindigenous populations.11–15
Clinical Perspective on p 105
The predominant monogenic cause of elevated LDL-C concentration in most global populations is familial hypercholesterolemia (FH; Online Mendelian Inheritance in Man, 143890).16 Heterozygous FH prevalence may be as high as 1:200 in certain European populations, and it is a potent predisposition state for early CVD.11–13 To date, DNA sequencing and biochemical studies have identified >1600 rare loss-of-function mutations in the gene encoding the LDL receptor (LDLR), which can increase LDL-C levels by ≥100%, and underlie >95% of cases of molecularly diagnosed FH.16 But despite the relatively high levels of LDL-C observed in some Inuit, the role of LDLR gene variation has not been studied systematically.13–15 We thus investigated the LDLR locus in Inuit and tested for association of variants therein with plasma lipids. Through Sanger sequencing and targeted genotyping, we found 2 new LDLR variants common to 5 Inuit subgroups from across North America and Greenland: (1) p.G116S was both dysfunctional in vitro and associated with a relatively large increase in plasma LDL-C levels, whereas (2) p.R730W had minimal dysfunction and impact on the lipid profile.
For the purposes of this study, we referred to all participants as Inuit; however, we acknowledge that the circumpolar north is inhabited by a spectrum of diverse indigenous people.
Participants included Inuit >18 years of age (n=3324) residing in arctic communities across North America and Greenland. North American population-based samples were collected as part of regional health surveys that included (1) The Center for Alaska Native Health Research study of 2007, which covered 11 Southwest Alaska Yup’ik communities (n=1222),17 (2) the Qanuippitaa Health Survey of 2004, which covered 14 coastal communities in Nunavik, Quebec (n=429),6 (3) the Keewatin Health Assessment Study of 1990 to 1991, which surveyed the Keewatin (Kivalliq) region of Nunavut (n=210),18 and (4) the Adult Inuit Health Survey of 2008, which surveyed the Inuvialuit region of the Northwest Territories (n=281).19 Inuit living in West Greenland and Denmark (n=1182) were also included as part of our study cohort from a regional survey conducted in 1993 to 1994.20
The study was approved by the appropriate institutional research ethics boards including the Laval University Ethical Board and Comité Provincial de Santé Publique for use of Nunavik, Quebec samples; the University of Manitoba for use of Kivalliq samples; and McGill University for use of Inuvialuit samples. Yup’ik participants provided written informed consent using protocols approved by the University of Alaska Review Board, the National and Alaska Area Indian Health Service Institutional Review Boards, and the Yukon Kuskokwim Human Studies Committee. The Greenland population study was approved ethically by the Commission for Scientific Research in Greenland. Participants gave their written consent after being informed about the study both orally and in writing.
The LDLR promoter region and exons were Sanger sequenced within a discovery subset of 10 healthy Greenland Inuit with extreme plasma LDL-C concentrations >6.0 mmol/L (>95th percentile for non-Inuit adults). Two novel variants, p.G116S and p.R730W, were identified in LDLR exons 4 and 15, respectively (Figure I in the Data Supplement). Both variants were then genotyped in independent Inuit samples from 5 different regions (Table 1) with custom TaqMan single-nucleotide polymorphism (SNP) genotyping assays (Applied Biosystems; Foster City, CA). As a comparator, we genotyped a common polymorphism with a relatively large effect on LDL-C levels, namely the apoE gene (APOE) protein isoforms using TaqMan SNP genotyping assays for SNPs rs429358 and rs7412 (Applied Biosystems; Foster City, CA).21 Genotypes were tested for association with blood lipid traits, including total cholesterol, LDL-C, high-density lipoprotein (HDL) cholesterol, non-HDL cholesterol and triglyceride, as well as apoB concentration, where available.
Detailed descriptions of methods are provided in the Methods in the Data Supplement.
LDLR p.G116S and p.R730W Are Common and Exclusive to Inuit
To evaluate the genetic basis for elevated LDL-C in Inuit, we used candidate sequencing of the LDLR gene to screen 10 Inuit with plasma LDL-C concentrations >6.0 mmol/L (>95th percentile for non-Inuit adults). We found 2 heterozygous LDLR gene variants, namely p.G116S and p.R730W, in 3 and 4 Inuit with high LDL-C, respectively (Figure I in the Data Supplement). We then genotyped these variants in 3324 Inuit samples from Southwest Alaska, Northern Canada (Inuvialuit, Kivalliq, and Nunavik), and Greenland (Table 1). The p.G116S variant frequency ranged from 2% in Kivalliq to 13% in Greenland, with an overall frequency of 10% across all regions. The p.R730W variant frequency ranged from 11% in Greenland to 17% in Kivalliq, with an overall frequency of 14% across all regions. The variants were not in linkage disequilibrium (r2=0.017; P=NS). Both variants were absent from other indigenous population samples and neither was observed in 4281 European samples and 2193 black samples from the National Heart, Lung, and Blood Institut Exome Sequencing Project database. However, p.G116S was reported previously in a single hypercholesterolemic subject of unspecified ethnic background ascertained in a lipid clinic in Denmark.22
LDLR p.G116S Is Robustly Associated With Higher Plasma LDL-C Concentration
We stratified plasma lipoprotein profiles according to LDLR p.G116S or p.R730W genotype (Table 2). In each sample, p.G116S carriers had significantly higher total, non-HDL, and LDL-C concentrations compared with noncarriers (Tables IA and IIB in the Data Supplement). In the overall sample, p.G116S was associated with a ≈0.54 mmol/L (20.9 mg/dL) increase in LDL-C per copy (Table 3; P=5.6×10−49); mean plasma apoB and non-HDL cholesterol concentrations were also proportionately higher per copy of p.G116S. In contrast, p.R730W was not significantly associated with LDL-C overall (P=0.13). In the combined Inuit samples, LDLR p.G116S genotype had an additive (codominant) effect on LDL-C concentration (Figure 1): mean LDL-C concentration was significantly higher in p.G116S heterozygotes than in p.G116 homozygotes (P=2.0×10−34) and tended to be higher still in p.G116S homozygotes compared with heterozygotes (P=0.058). In contrast, the relationship between p.R730W and plasma LDL-C concentrations was not significant overall. Each copy of p.G116S was associated with increased risk of hypercholesterolemia, defined as a plasma LDL-C >5.0 mmol/L, which Canadian dyslipidemia guidelines23 suggest as the cutpoint for prescription of lipid-lowering treatment (Figure 2; odds ratio, 3.02; 95% confidence interval, 2.34–3.90; P=1.7×10–17). In contrast, p.R730W was not associated with increased risk of clinically actionable hypercholesterolemia.
LDLR p.G116S Has a Larger Effect Size on LDL-C Than the APOE E4 Isoform
We compared the effect size of p.G116S to that of the APOE E4 isoform, a well-established common variant associated with increased LDL-C.24,25 In Inuit, APOE E4 allele frequencies ranged from 21% to 27% (Table II in the Data Supplement) and each copy of E4 increased LDL-C by 0.18 mmol/L (7.0 mg/dL; P=9.0×10–11). Furthermore, the top LDL-C–associated variants from genome-wide association studies had effect sizes per allele ranging from 0.05 to 0.18 mmol/L.26 Thus, LDLR p.G116S in Inuit is unique, with >3-fold larger effect on LDL-C than any other common variant.
LDLR p.G116S Impairs LDLR Ligand-Binding Activity In Vitro
Finally, we investigated the function of both variants in vitro, using cell-based models transfected with plasmid constructs encoding wild-type, p.G116S, or p.R730W LDLR variants. Overall, p.G116S tended to show increased mean mature LDLR expression by 31%, whereas p.R730W had reduced mean mature LDLR expression by 63% relative to the wild-type LDLR constructs. In vitro LDL-binding assays adjusted for total LDLR expression linked p.G116S with a significant 61% reduction in LDL-binding ability, whereas p.R730W had a nonsignificant 12% reduction in binding ability (Figure 3).
The LDLR p.G116S variant in exon 4 resides within the ligand-binding domain.27 Of missense or nonsense mutations in LDLR that cause monogenic FH, ≈20% reside within exon 4, which is considered to be a mutational hot-spot.28 The pathogenic relevance of p.G116 in receptor function was supported by identification of the p.G116C variant in a Polish patient with hypercholesterolemia.29 In contrast, p.R730W is within in exon 15, which encodes an attachment site for O-linked carbohydrate chains; this domain has no clear functional role.27 Less than 1% of disease-causing LDLR mutations reside within exon 15.28 Sequence conservation analysis suggested stronger evolutionary conservation at p.G116 compared with p.R730 (Figure II in the Data Supplement), whereas multiple algorithms predicted a more deleterious effect for p.G116S than p.R730 on LDLR function (Table III in the Data Supplement). The role of p.R730 in LDLR function remained unclear as a different mutation at p.R730, namely p.R730Q, was found in a sample from a Dutch FH cohort but was predicted to be benign and was reported as likely not disease causing.30
LDLR p.G116S thus seems to be an example of the hypothesized but to date elusive entity in lipoprotein genetics, namely a common genetic variant whose LDL-C raising effect, is intermediate between the modest effects attributable to genome-wide association study alleles and the large effects of rare LDLR mutations causing monogenic disease (FH). Although bioinformatic predictions further supported a functional consequence of p.G116S, the functional studies ultimately corroborate the observed phenotypic effect of p.G116S. The ≈ 60% reduced ligand-binding ability of cells expressing the p.G116S is intermediate between that of wild-type LDLR and of rare FH-causing mutations, which show ≤100% reductions of ligand-binding ability.27
Our discovery of the association between p.G116S and LDL-C concentration is of particular interest from a public health perspective, as Inuit communities may currently be at the tipping point of environment-related increased risk of CVD and metabolic disorders. In other populations, every 1-mmol/L increase in LDL-C corresponds to a ≈20% increase in CVD and ≈15% increase in all-cause mortality.31 Thus, the ≈0.5-mmol/L increase in LDL-C per p.G116S allele could potentially lead to ≈10% and ≈7.5% increased risk of CVD and all-cause mortality, respectively. Our analyses indicated that p.G116S carriers were at a ≈3-fold increased risk of high LDL-C (>5 mmol/L), which suggested that p.G116S carriers were also more likely to be candidates for pharmacological intervention than noncarriers (odds ratios, 3.02; 95% confidence interval, 2.34–3.90; P=1.7×10−17). Unfortunately, data on CVD end points were not systematically collected in the surveys that comprised this study, so the possible impact of p.G116S on metabolic and CVD risk among the Inuit cannot be directly inferred at this time. A link between this genetic variant and CVD risk would need to be formally evaluated, for instance, using Mendelian randomization or another appropriate prospective study design. Furthermore, it would be of interest to detect possible interactions between lifestyle factors, other risk factors, and the phenotypic impact of LDLR p.G116S. Although baseline between-population differences in lipid profiles might be consistent with environmental effects (Table 1), we have not systematically collected comprehensive diet and lifestyle data; although we would like to do this in the future, such an analysis is beyond the scope of the present report.
As with all association studies, a potential risk of population stratification artifacts exists. However, there are several reasons why we think that this is not a major issue here. First, we adjusted for geographic location in the association and correlation analyses for the combined Inuit cohort; the association of G116S with LDL-C was highly significant with this adjustment variable included. Second, although the minor allele of G116S varies by geographic region, the directionality of the association by genotype is the same and is individually significant, in each of the 5 subpopulations for LDL-C and the related traits of total cholesterol, non–HDL cholesterol, and apoB (see Tables IA and IB in the Data Supplement). Third, we have functionally evaluated the variants in vitro in 2 different cell lines and show a significant loss of binding function for the variant that is significantly associated with LDL-C levels, but no functional impact of the variant that is not associated with LDL-C levels. The findings for dysfunction of G116S are similar in quality, although smaller in magnitude, than those that we have seen for our patients with clinical FH with mutations in the LDLR gene. Finally, principal component analysis performed using genome-wide markers from the exome array on 3 of the 5 Inuit subpopulations shows a distinctive clustering, with no overlap at all with white or black clusters (data not shown). However, some small stratification artifacts are still possible.
Although we studied LDLR variation, we did not screen the additional FH genes APOB and PCSK9, so we cannot rule out similar additional effects on this quantitative trait. Furthermore, genome-wide association studies in other global populations have recently implicated >30 genes that modulate LDL-C concentration; cumulatively these might have a larger impact than the 0.54-mmol/L per allele effect of p.G116S.26 A comprehensive genetic screen for LDL-C–related variants using multilocus high-density genotyping strategies or microarrays, while of potential interest in these samples, is far beyond the scope of the studies reported here. Also, the reason that these distinct variants arose in circumpolar people in the first place cannot be determined or even reasonably speculated on at this time. The effect size on LDL-C is not consistent with any known or obvious survival advantage nor does there seem to be any potential for negative selection because CVD onset typically follows decades after the onset of the reproductive years. Finally, although the p.R730W variant seemed to have minimal impact on LDL-C at the population level, a possible impact on other pathways or networks cannot be ruled out from the studies performed here.
Thus, our screen for FH-related variation in the Inuit uncovered a unique genetic variant among global populations: LDLR p.G116S is a common, dysfunctional variant that is strongly associated with a large LDL-C–raising effect, although not causing classical FH. It seems to embody the type of variant that has been long sought-after in the post–genome-wide association study era and warrants consideration in evaluating clinical and public health implications as part of the fabric of CVD risk in the circumpolar north.
We are grateful to the participants in this research project. We thank Cynthia G. Sawyez for technical support and advice. We dedicate this article in memory of Dr Dewailly.
Sources of Funding
Dr Hegele holds the Edith Schulich Vinet Canada Research Chair (Tier I) in Human Genetics, the Martha G. Blackburn Chair in Cardiovascular Research, and the Jacob J. Wolfe Distinguished Medical Research Chair at the University of Western Ontario. This work was supported by the Canadian Institutes for Health Research (MOP-13430 and MOP-79533), the Heart and Stroke Foundation of Ontario (T-6066 and 000353), and Genome Canada through Genome Quebec.
The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.114.000646/-/DC1.
- Received April 4, 2014.
- Accepted October 2, 2014.
- © 2014 American Heart Association, Inc.
- Bjerregaard P,
- Dyerberg J
- Bjerregaard P,
- Mulvad G,
- Pedersen HS
- Goldstein JL,
- Brown MS
- Mohatt GV,
- Plaetke R,
- Klejka J,
- Luick B,
- Lardon C,
- Bersamin A,
- et al
- Saudny H,
- Leggee D,
- Egeland G
- Anderson TJ,
- Grégoire J,
- Hegele RA,
- Couture P,
- Mancini GB,
- McPherson R,
- et al
- Khan TA,
- Shah T,
- Prieto D,
- Zhang W,
- Price J,
- Fowkes GR,
- et al
- Cholesterol Treatment Trialists’ (CTT) Collaborators,
- Mihaylova B,
- Emberson J,
- Blackwell L,
- Keech A,
- Simes J,
- et al
The low-density lipoprotein (LDL) receptor (LDLR) gene has taught us much about lipoprotein metabolism and atherosclerosis risk. Severe loss-of-function mutations in LDLR occur in ≈1:250 people and can double plasma LDL cholesterol, causing familial hypercholesterolemia, an autosomal codominant condition that underlies ≈4% of early coronary heart disease cases. In contrast, common SNPs in LDLR have minor allele frequencies of ≈0.2, with LDL cholesterol raising effects of ≈0.12 mmol/L (5 mg/dL), which contributes to polygenic coronary heart disease risk in the general population. We report an LDLR variant, namely p.G116S, that is intermediate between these 2 extremes. It is common among Inuit people (Eskimos) living in Alaska, Northern Canada, and Greenland, with an overall frequency of ≈10%. It raises LDL cholesterol substantially by 0.54 mmol/L (21 mg/dL) per allele. In vitro studies show compromised ability of the variant receptor to bind LDL cholesterol. Although p.G116S heterozygotes are 3× more likely than noncarriers to have LDL cholesterol >5 mmol/L (194 mg/dL), they do not express classical heterozygous familial hypercholesterolemia. Similarly, p.G116S homozygotes have LDL cholesterol levels far below those observed in classical homozygous familial hypercholesterolemia. The p.G116S variant is thus unique: it is relatively common among Inuit, is associated with moderate loss-of-function, and raises LDL cholesterol by a moderately large amount. Carriers have a phenotype that is less severe than classical familial hypercholesterolemia; nonetheless, p.G116S is predicted to increase coronary heart disease risk among the Inuit, who are considered to be a vulnerable population in transition.