Common Low-Density Lipoprotein Receptor p.G116S Variant Has a Large Effect on Plasma Low-Density Lipoprotein Cholesterol in Circumpolar Inuit Populations

DOI: 10.1161/CIRCGENETICS.114.000646

The Common LDLR p.G116S Variant Has a Large Effect on Plasma LDL Cholesterol in Circumpolar Inuit Populations Running title: Dubé et al.; LDLR p.G116S elevates Inuit LDL cholesterol Joseph B. Dubé, MSc1; Jian Wang, MD1; Henian Cao, MD1; Adam McIntyre, BSc1; Christopher T. Johansen, PhD1,2; Scarlett E. Hopkins, RN3; Randa Stringer, BSc1; Siyavash Hosseinzadeh, BSc1; Brooke A. Kennedy, BSc1; Matthew R. Ban, BSc1; T. Kue Young, MD, PhD4; Philip W. Connelly, PhD5; Eric Dewailly, y, MD,, PhD6†;; 11,2 2 Hege He gele ge le,, MD1, le Peter Bjerregaard, MD7; Bert B. Boyer, PhD3; Robert A. Hegele,

1

Robarts Research s ch IInstitute, search nsti ns t tute ti tu ut , 2De Department D partment of Medicine, Schu Schulich uli lich ch School of Medicine Medicin inee and Dentistry, The University in Uni Alaska Nativee Health Health Research, Institute of Arctic Arr of Westernn Ontario, Ont n ario, London, Lo ondon, ON,, Canada;; 3The Center for Alaska he Dalla Daall lla La L Lana na S School ch choo hooll ooff P Public ublic He ub Heal Health, alth lth th,, Un University niv iverss of niverrsity of Alaska ni Alaask skaa Fairbanks, Faairba bank nk ks, Fairbanks, Faiirb r an a ks k , AK; AK K; 4The Biology, University he Keenan he Keenan Research Researc rcch Centre Cenntre for for Biomedical Biomedi dical Science Sciencce of St. St. t. M Michael's ichaael's H Hospital osspita tall & U ta University niiverssity ooff To Toronto, o Toronto; 5The Axee S A Santé annté des dees populations po opu p la lati tiion tion o s et et pratiques pra ratiqu quess optimales qu opttim i alles enn santé, saantté, é Centre Centr trre de Recherche Rech herche duu CHU C de Toronto, ON,, Canada; Cana Ca nada; 6Ax na The he National N ti Na tion i all IInstitute nstt of Québec & Département préventive, Université Canada; é épartement t de d médecine méd édeciine sociale éd socialle ett préventi tiive, Un U iversiitéé Laval, QC QC, C C anada; 7T Public H Health, University Southern Denmark, Copenhagen, Denmark ealtth, U niive v rssit ity y ooff So S uthe ut hern he rn D en nma m rk r , Co C penh pe n ag nh agen e , De en Denm nmar nm ak ††deceased dece de c as ce ased ed

Correspondence: Robert A. Hegele, MD Robarts Research Institute The University of Western Ontario 4288A-1151 Richmond Street North London, Ontario, N6A 5K8 Canada Tel: 519-931-5271 Fax: 519.931-5218 E-mail: [email protected]

Journal Subject Codes: [89] Genetics of cardiovascular disease, [90] Lipid and lipoprotein metabolism, [8] Epidemiology, [137] Cell biology/structural biology

1 Copyright by American Heart Association, Inc. All rights reserved.

DOI: 10.1161/CIRCGENETICS.114.000646

Abstract: Background - Inuit are considered to be vulnerable to cardiovascular disease (CVD) as their lifestyles become more Westernized. During sequence analysis of Inuit individuals at extremes of lipid traits, we identified two nonsynonymous variants in LDLR encoding the low-density lipoprotein (LDL) receptor, namely p.G116S and p.R730W. Methods and Results - Genotyping these variants in 3,324 Inuit from Alaska, Canada and Greenland showed they were common, with allele frequencies 10-15%. 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 x 10-49), which was >3-times larger than the largest effect sizes seen with 22--fo fold ld increased inc ncre reas re ased as ed ris iskk of is common variants in other populations. Carriers of p.G116S had a 3.02-fold risk classical ssic ss ical ic al ffamilial amil am i ia il iall hypercholesterolemia (CI 2.34 – 3.90, P = 1.7 x 10-17), but did not havee class hypercholesterolemia. s rol ster o em emia ia. In ia n vvitro, itro, p.G116S showed 60% 60% reduced liga ligand g ndd bbinding inding activity compared com m reeceptor. In n ccontrast, onntras astt, p. pp.R730W R7730 30W W was waas asso sociat ated ted w itth neither neit i he it her LDL LD DL ch chol ollesste tero rol nnor ro to wild-typee receptor. associated with cholesterol altered in vitro itrro ac aactivity. tivity ty y. n - LDLR p.G116S ns p.G11 116S 11 6S iss thus thu unique: uniqu q e:: a common qu com ommo on dysfunctional dyysf s unct ctiona nall va vvariant arian a t in Inuit who o Conclusions whose large effectt on LDL cholesteroll mayy ha hhave ve ppublic ubbliic hhealth eallth h iimplications. mp pli l cations. i

Key words: low-density lipoprotein cholesterol, cardiovascular disease, genetic variation, population genetics cholesterol, candidate gene resequencing

2 Copyright by American Heart Association, Inc. All rights reserved.

DOI: 10.1161/CIRCGENETICS.114.000646

Inuit were long-believed to have lower CVD risk than non-indigenous populations.1-3 However, re-evaluation of population studies indicates that ischemic heart disease rates are similar between Inuit and non-Indigenous 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 LDL cholesterol than non-indigenous populations.11-15 The predominant monogenic cause of elevated LDL cholesterol ol concentration inn mo m most global populations is familial hypercholesterolemia (FH, Online Mendelian Man delian In IInheritance heriitance in M [OMIM] 143890). 4 89 4389 890) 0).16 H 0) Heterozygous eter eroozygous FH (HeFH) prev prevalence val a ence may be as hhigh i h as 1:200 in certa ig certain a 133 European po ppopulations, opulations, and an nd it is is a potent pottentt predisposition po preedisppossitio on state sttatee for fo or early early CVD. ea CVD. D 11-13 Too date, T datte, DNA D

ioch io c em ch mic i all sstudies tud es hhave tudi ave id av den enttifi tifi f ed d >1,600 >1,60 6000 rare raare lloss-of-function ossos ss off-ffunct ctio ct ion mu io mut mutations tation tati tiions in the sequencing andd bbiochemical identified gene encoding d g the LDL receptor ding recept ptor ((LDLR), pt LDLR LD LR)),, whi which hich hi h can increase increase LD LDL L ch cholesterol holesterol levels byy 100% 10 or more, and underlie nderlie derli lie >95% >95 95% % off cases off molec molecularly le llarl arll di diagnosed d FH FH.16 B Butt despite de it the th h relatively relati latii e high levels of LDL cholesterol observed in some Inuit, the role of LDLR gene variation has not been systematically studied.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 two new LDLR variants common to five 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 cholesterol levels; while 2) p.R730W had minimal dysfunction and impact on the lipid profile.

Methods For the purposes of this study, we referred to all participants as “Inuit”; however, acknowledge 3 Copyright by American Heart Association, Inc. All rights reserved.

DOI: 10.1161/CIRCGENETICS.114.000646

that the circumpolar north is inhabited by a spectrum of diverse indigenous people. Participants included Inuit >18 years of age (N=3,324) 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 (SW) Alaska Yup’ik communities (N=1,222);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 1990-1991 which surveyed the Keewatin (Kivalliq) region of Nunavut (N=210);18 and 4) the Adult Inuit nuit Health Surveyy of o 2008 which surveyed the Inuvialuit region of the Northwest Territories (N=281). 281). ) 19 IInuit nuiit li living ngg iin n West a d De Denm nmar nm ark (N ar (N=1,182) were also includ uded ud e as part of our st study t cohort from a Greenland and Denmark included regional survey rvey conducted in 19 rv 1993-1994. 99399 3-1 -1994 4.20 dy was dy was approved app ppro rove vedd by tthe hee appro appropriate opria pria iatte iinstitutional nsti ns t tu uti tionnall research res esea es s arc rchh ethics ethi hics hi cs boards boa oard rds ds inclu i clu in Thee study including the Laval University y Ethical Ethicall B Board oard d andd C Comité omiité P Provincial rovin i cial i de de Santé S ntéé Publique Sa Publ bliq bl ique for use of Nunavik, Nun Quebec samples; mples; pll th the h U University Uni nii ersit sit i off Manitoba Manitob i ba for f use se off Kivalliq Ki alliq lliiq samples; ll pll andd McGill McGi Gill ll University U Uni nii 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 ethically approved 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 cholesterol concentrations >6.0 mmol/L (>95th percentile for non-Inuit adults). Two novel variants, p.G116S and p.R730W, were

4 Copyright by American Heart Association, Inc. All rights reserved.

DOI: 10.1161/CIRCGENETICS.114.000646

identified in LDLR exons 4 and 15 respectively (Supplemental Figure 1). Both variants were then genotyped in independent Inuit samples from five different regions (Table 1) with custom TaqMan SNP genotyping assays (Applied Biosystems; Foster City, CA). As a comparator, we genotyped a common polymorphism with a relatively large effect on LDL cholesterol levels, namely the apolipoprotein (apo) E 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 (TC), LDL cholesterol, HDL cholesterol, non-HDL cholesterol and triglyceride ride as well as apoo B concentration, where available. Detailed a ed de ailed desc descriptions scri sc ripptio ri ons n of methods are provid provided ded e in the Supp Supplemental u pleme menntal Materials section me section. n

Results LDLR p.G116S and p.R730W G G116S p.R R73 730W 0W W are are r common com ommo mon mo n an and d exclusive excl ex clus cl u iv ve to IInuit nu uit it To evaluatee tthe he ggenetic enet en etic i bbasis ic asis as is for for elevated ellevat elev atted L LDL DL cholesterol ch chol holles este t ro te roll in IInuit, nuit nu it,, we used used d candidate cand ca ndid didat atee sequencing of the LDL receptor gene (LDLR) to screen 10 Inuit with plasma LDL cholesterol concentrations >6.0 mmol/L (>95th percentile for non-Inuit adults). We found two heterozygous LDLR gene variants, namely p.G116S and p.R730W, in three and four Inuit with high LDL cholesterol, respectively (Supplemental Figure 1). We then genotyped these variants in 3,324 Inuit samples from Southwest (SW) 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 5 Copyright by American Heart Association, Inc. All rights reserved.

DOI: 10.1161/CIRCGENETICS.114.000646

European samples and 2193 African American samples from the NHLBI Exome Sequencing Project database. However, p.G116S was previously reported 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 cholesterol 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 cholesterol concentrations compared to non-carriers (Supplemental Tables 1A and 1B). In the overall sample p.G116S was associated with a ~0.54 mmol/L (20.9 mg/dL) g/dL) increase in LDL LD cholesterol per copy (Table 3; P = 5.6 x 10-49); mean plasma apolipoprotein rotein (a ((apo) po)) B andd nnono s rol ster o concentrations conc co nceentr nc traations were also proporti ion onaately higher per co copy of p.G116S. In HDL cholesterol proportionately contrast, p.R730W R730W was nnot ott significantly sig gnifi fica c ntly ca y associated assoociaateed with w th wi h LDL LDL cholesterol cho holestterroll overall overal alll (P al P = 00.13). .13) In e IInuit ed nuit nu it ssamples, ampl plles es, LD LDLR L p. pp.G116S .G1 G116S G1 S genotype gen ty geno ype had had an n add dit iti tive ive (co-dominant) (c dom (c (co-d o inan ant) t)) ef ffect fect on the combined additive effect LDL cholesterol s sterol concentration concentratiion (Figure (Fi Figu g re 1): 1)): mean L LDL DL ch DL cholesterol holesteroll concentrati concentration ion was sign significantly g ifii 34 higher in p.G116S G116S G116 G1 16S S heterozygotes hetero hete gotes te th than h iin p.G116 p G1 G116 16 hhomozygotes homo gotes ot (P=2.0x10 ((P P 2 0 10--34 )), and ndd tended t dedd to be

higher still in p.G116S homozygotes compared to heterozygotes (P=0.058). In contrast, the relationship between p.R730W and plasma LDL cholesterol concentrations was not significant overall. Each copy of p.G116S was associated with increased risk of hypercholesterolemia, defined as a plasma LDL cholesterol >5.0 mmol/L, which Canadian dyslipidemia guidelines23 suggest as the cutpoint for prescription of lipid-lowering treatment (Figure 2; OR 3.02, 95% CI 2.34 – 3.90, P = 1.7 x 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 cholesterol than the APOE E4 isoform We compared the effect size of p.G116S to that of the APOE E4 isoform, a well-established

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DOI: 10.1161/CIRCGENETICS.114.000646

common variant associated with increased LDL cholesterol.24, 25 In Inuit, APOE E4 allele frequencies ranged from 21% to 27% (Supplemental Table 2) and each copy of E4 increased LDL cholesterol by 0.18 mmol/L (7.0 mg/dL; P = 9.0 X 10-11). Furthermore, the top LDL cholesterol-associated variants from genome-wide association (GWA) 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 >3fold larger effect on LDL cholesterol 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 ell-basedd models transfected with plasmid constructs encoding wild-type, p.G116S, or p.R730 p.R730W 30W 30 W LDLR LDLR variants. var ariia G 16S ttended G11 ende en dedd to de to show increased mean m ature LDLR at R expre resssion by 31%, while re Overall, p.G116S mature expression had reduced me ha ean m aturre LD DLR R eexpression xpre ressionn by 663% 3% rrelative ellativ ve to o the the wild-type wil ild--ty ypee LDLR LD p.R730W had mean mature LDLR itro LD itro LDL L-b bindi indi ding ng assays assay ay ys adjusted adju ust sted ted ffor or ttotal otal ot al LDLR LDL DLR R expression expr ex xpres essi s onn llinked inkedd p. in pp.G116S G116 G1 16 with constructs. In vit vitro LDL-binding n 61% reduction in nt i L DL bi ndi ding ability, ab bil iliity, while whi hille p.R730W hi p.R R73 730W 0W W had had d a non-significant non-sig gnificant 112% a significant LDL binding n binding bindi di ability abilit bili bi lit (Figure ((Fig Fi re 3). 33)) reduction in 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 Fewer than 1% of disease-causing LDLR mutations reside within exon 15.28 Sequence conservation analysis suggested stronger evolutionary conservation at p.G116 compared to p.R730 (Supplemental Figure 2) while multiple algorithms predicted a more deleterious effect for p.G116S than p.R730

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DOI: 10.1161/CIRCGENETICS.114.000646

on LDL receptor function (Supplemental Table 3). 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 diseasecausing.30

Discussion LDLR p.G116S thus appears to be an example of the hypothesized but so far elusive entity in lipoprotein genetics, namely a common genetic variant whose LDL cholesterol raisingg effect is thee la larg rgee ef rg effe fect fe ctss of ct intermediate between the modest effects attributable to GWAS alleles and th large effects rare LDLR mutati mutations tion ons causing on c ussin ca i g monogenic disease (FH). (FH) H . While bioinformatic bioinfo form r atic predictions further fu ffunctional unctional consequence conse connseequuence ence en ce ooff p.G pp.G116S, .G G116S, 1166S, S thee functional funnctio cttio ona nal st sstudies uddie udie i s ultimately ulti tiimate tely ely corroborate corrobora cor orro ro obo bora the supported a fun h no heno noty ty ypi picc ef eeffect fectt ooff p. fe pp.G116S. G116 G1 16S. 16 S. Th he ~ 60 60% % re edu duce cedd li ce iga g nd nd--bind ndin in ng ab bil i itty of o ccells ellls ls observed phenotypic The reduced ligand-binding ability inte in teerm r ed edia iate ia te between bet etwe w en tthat we h t of w ha illdd ty ype pe L DLR DL R an andd of rare FH-causing FH-caa expressing the p.G116S iss intermediate wild-type LDLR whi hich ch show sho how w up to to 100% 100% % reductions red duc ucti tion ionss of lligandigandigan d-bi biindin nddingg ab bil ilit ity. yy.27 mutations, which ligand-binding ability. Our discovery of the association between p.G116S and LDL cholesterol 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 cholesterol corresponds to a ~20% increase in CVD and ~15% increase in all-cause mortality.31 Thus, the ~0.5 mmol/L increase in LDL cholesterol 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 ~3fold increased risk of high LDL cholesterol (>5 mmol/L) which suggested that p.G116S carriers were also more likely to be candidates for pharmacologic intervention than non-carriers (OR 3.02, 95% CI 2.34 – 3.90, p = 1.7 x 10-17). Unfortunately, data on CVD end points were not 8 Copyright by American Heart Association, Inc. All rights reserved.

DOI: 10.1161/CIRCGENETICS.114.000646

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. While 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; while we would like to do this th in the future, such an analysis is beyond the scope of the present report. As wit with studies, a potential risk w th all alll association asso as sociiat so ation t risk s of population stratification str tratification artefacts exists. However, there reasons why major hhere eree are several al rea asonss w hyy w wee bbelieve eliiev ve th that tthis hiss iss not hi ot a m ajo or or issuee here. heree. First, First Fi we adjusted forr geographic association combined geog ogra og raph ra p ic location ph loc ocat attio i n in tthe hee assoc cia iati tioon aand ti ndd correlation cor orrrela latti la ti n analyses tion ana naly lysses for ly for thee co comb mb bine ine Inuit cohort; G116S with LDL-C highly significant with adjustment t; the association off G1 t; G116 16S 16 S wi ith hL DL L-C C was high ghhly l sig i niificant wi ig ith this adj justm m variable included. Second, cll dded edd S Second e ndd while hhile il the h mi minor i all allele llelle off G116S G116 G1 16S S varies aries rii by b geographic phi hic region, region io the thh 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 TC, non-HDL-C and apo B (see Supplemental Tables 1A and 1B). 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 cholesterol levels, but no functional impact of the variant that is not associated with LDL cholesterol 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 three of the five Inuit subpopulations show

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DOI: 10.1161/CIRCGENETICS.114.000646

a distinctive clustering, with no overlap at all with Caucasian or African American clusters (data not shown). However, some small stratification artefacts are still possible. While 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, GWA studies in other global populations have recently implicated >30 genes that modulate LDL cholesterol 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 cholesterol-related variants using multi-locus high-density genotyping strategies or microarrays, arrays, while of ppotential oten ot e interest in these samples, is far beyond the scope of the studies reported ed here. Al Also, the t e reason th rea re that these distinct d inc disti nctt variants vari va riiants ts arose in circumpolar peoples peop ople op les in the first place plaace cannot be determin determined n or even reasonably upon The size LDL nablly speculated na ed upo poon at tthis his ttime. ime me. T he eeffect fffectt si siz ze onn L DL L ccholesterol hol ollesteero r l iiss nott consistent with w h any an ny kn know known wn or oobvious bv us survival bvious surviiva vall ad aadvantage, vant nta tage, e nnor or ddoes oess th oe tthere eree se er seem eem e to be b aany ny potential for o nega or negative g tive selection, selectiion,, since siince CV C CVD D onset ty typi typically pical i lly fo ffollows llows de ddecades caddes after the onset oof the reproductivee years. ears F Finall Finally, inall ll while hhile il th the h pp.R730W R730W R730 R7 30W W variant ariant rii t appeared ed d tto hha have a e minimal iniimall iimpactt oon LDL cholesterol 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 cholesterol-raising effect, although not causing classical FH. It seems to embody the type of variant that has been long sought-after in the post-GWAS era and warrants consideration in evaluating clinical and public health implications as part of the fabric of CVD risk in the circumpolar north.

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DOI: 10.1161/CIRCGENETICS.114.000646

Acknowledgments: We thank Cynthia G. Sawyez for technical support and advice. We dedicate this paper in memory of Dr. Eric Dewailly. Funding Sources: 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 CIHR (MOP-13430 and MOP-79533), the Heart and Stroke Foundation of Ontario (T-6066 and 000353) and Genome Canada through Genome Quebec. Conflict of Interest Disclosures: None. References: 1. Bjerregaard P, Dyerberg J. Mortality from ischaemic heart disease aand nd ccerebrovascular ereb er ebro eb rova ro vasc va scul sc ular ul a ar disease. Greenland Int J Epidemiol. 1988;17:514-519. 2. Middaugh 1980-86. Health. g JP. gh JP. Cardiovascular Card Ca rd dio i va vasscular deaths among Alaskan Alas aska as k n Natives. 1980ka 0-86. Am J Public Hea 0a 1990;80:282-285. 2--28 285. 3. Young TK, ME, O'Neil Cardiovascular population. K, Moffatt Moffattt M E O'N E, Neil JD JJD.. Ca Card diovaascullarr diseases dis isease ses in in a C Canadian an nad adia i n Ar ia Arctic ic po popula opuula Am J Publicc Hea Health. alt lth. h 11993;83:881-887. 993 93;8 ;8 83: 3:88 8881-8887. 4. Bjerregaard Hegele RA.. Lo Low cardiovascular a P,, Young ard g TK, K, H eg gele l RA R L w in iincidence ciide d nce of cardi d ovascullar ddisease di isease amongg the t Inuit--what is the evid evidence? iden den ence cee? Atherosclerosis. ce? Athe At hero he rosc ro scle scle lero rosiis. 2003;166:351-357. ro 2003 20 033;1 03;1 ;166 666:3 66:3 : 51-3 511-3 -357 57. 57 7. 5. Bjerregaard P, Mulvad G, Pedersen HS. Cardiovascular risk factors in Inuit of Greenland. Int J Epidemiol. 1997;26:1182-1190. 6. Chateau-Degat ML, Dewailly E, Louchini R, Counil E, Noel M, Ferland A, et al. Cardiovascular burden and related risk factors among Nunavik (Quebec) Inuit: insights from baseline findings in the circumpolar Inuit health in transition cohort study. Can J Cardiol. 2010;26:190-196. 7. Ebbesson SO, Adler AI, Risica PM, Ebbesson LO, Yeh JL, Go OT, et al. Cardiovascular disease and risk factors in three Alaskan Eskimo populations: the Alaska-Siberia project. Int J Circumpolar Health. 2005; 64:365-386. 8. Howard BV, Comuzzie A, Devereux RB, Ebbesson SO, Fabsitz RR, Howard WJ, et al. Cardiovascular disease prevalence and its relation to risk factors in Alaska Eskimos. Nutr Metab Cardiovasc Dis. 2010;20:350-358. 9. Jernigan VB, Duran B, Ahn D, Winkleby M. Changing patterns in health behaviors and risk factors related to cardiovascular disease among American Indians and Alaska Natives. Am J Public Health. 2010;100:677-683.

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DOI: 10.1161/CIRCGENETICS.114.000646

10. Kellett S, Poirier P, Dewailly E, Sampasa H, Chateau-Degat ML. Is severe obesity a cardiovascular health concern in the Inuit population? Am J Hum Biol. 2012;24:441-445. 11. Haase A, Goldberg AC. Identification of people with heterozygous familial hypercholesterolemia. Curr Opin Lipidol. 2012;23:282-289. 12. Jorgensen ME, Bjerregaard P, Kjaergaard JJ, Borch-Johnsen K. High prevalence of markers of coronary heart disease among Greenland Inuit. Atherosclerosis. 2008;196:772-778. 13. Redwood DG, Lanier AP, Johnston JM, Asay ED, Slattery ML. Chronic disease risk factors among Alaska Native and American Indian people, Alaska, 2004-2006. Prev Chronic Dis. 2010;7:A85. 14. Bjerregaard P, Jorgensen ME, Borch-Johnsen K. Serum lipids of Greenland Inuit in rrelation ea el to Inuit genetic heritage, westernisation and migration. Atherosclerosis. 2004;174:391-398. s. 20 2004 04;1 04 ;174 ;1 74:3 74 :391 :3 91-3 91 398. 98. 15. Ebbesson SO, Schraer C, Nobmann ED, Ebbesson LO. Lipoprotein profiles Alaskan n pro ofi file less in A le lask la skaan sk Siberian Yupik Eskimos. Arctic 1996;55:165-173. u kE upik skim sk im mos. Ar A ctic Med. Res. 1996;55: 5:16 5: 165-173. 16 16. Goldstein Arterioscler Thromb Vasc Biol. 2009;29:431e n JL, ein JL, Brown MS. MS. The The LDL L L receptor. LD reccepptorr. Ar rteerios oscl cler cl e Thr er hrombb Va asc Bio oll.. 20 2009;2 ;29:44 ;2 438. 17. Mohatt GV, Plaetke R R,, Klejka Luick Lardon Kllej ejka kaa JJ,, Lu Luic ickk B, ic B L a do ar donn C, C Bersamin Ber ersa er saami m n A, A et et al. all. The T e Center for Th Alaska Native Study: community-based i Health Researchh S ive tudy: dy a commu niity ty-ba b sed pa pparticipatory rtiicip i atoryy research study y of obesity andd chronic disease-related Circumpolar diise dise seas aseas e-re ereelate la ed protective la prot pr otec ot ecti ec tive ti ive ve and and nd risk ris iskk factors. f ctor fa cttors. ors. Int Int J Ci In Circ rcum rc um umpo mpo pola larr Health. la H alth. He 2007;66:8-18. 18 18. Moffatt ME, Young TK, O'Neil JD, Eidelheit S, Fish I, Mollins J. The Keewatin Health Assessment Study, NWT, Canada. Arctic Med Res. 1993;52:18-21. 19. Saudny H, Leggee D, Egeland G. Design and methods of the Adult Inuit Health Survey 2007-2008. Int J Circumpolar Health. 2012;71. 20. Bjerregaard P, Curtis T, Borch-Johnsen K, Mulvad G, Becker U, Andersen S, et al. Inuit health in Greenland: a population survey of life style and disease in Greenland and among Inuit living in Denmark. Int J Circumpolar Health. 2003;62 Suppl 1:3-79. 21. Fullerton SM, Clark AG, Weiss KM, Nickerson DA, Taylor SL, Stengard JH, et al. Apolipoprotein E variation at the sequence haplotype level: implications for the origin and maintenance of a major human polymorphism. Am J Hum Genet. 2000;67:881-900. 22. Damgaard D, Larsen ML, Nissen PH, Jensen JM, Jensen HK, Soerensen VR, et al. The relationship of molecular genetic to clinical diagnosis of familial hypercholesterolemia in a Danish population. Atherosclerosis. 2005;180:155-160.

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23. Anderson TJ, Gregoire J, Hegele RA, Couture P, Mancini GB, McPherson R, et al. 2012 update of the Canadian Cardiovascular Society guidelines for the diagnosis and treatment of dyslipidemia for the prevention of cardiovascular disease in the adult. Can J Cardiol. 2013; 29:151-167. 24. Khan TA, Shah T, Prieto D, Zhang W, Price J, Fowkes GR, et al. Apolipoprotein E genotype, cardiovascular biomarkers and risk of stroke: systematic review and meta-analysis of 14,015 stroke cases and pooled analysis of primary biomarker data from up to 60,883 individuals. Int J Epidemiol. 2013;42:475-492. 25. Ward H, Mitrou PN, Bowman R, Luben R, Wareham NJ, Khaw KT, et al. APOE genotype, lipids, and coronary heart disease risk: a prospective population study. Arch Intern Med. 2009;169:1424-1429. 26. Teslovich TM, Musunuru K, Smith AV, Edmondson AC, Stylianouu IM, Koseki M, et aal. Biological, clinical and population relevance of 95 loci for blood lipids. Nature. 2010;466:707s N s. atur at ure. ur e 20 2010 10;4 10 ;466 ;4 66:7 66 7 713. 27. Hobbs HH HH, Russell H H, Ru Russ ssel ss elll DW, el DW W, Brown MS, Goldstein n JJL. L The LDL receptor L. recep eptor locus in familial ep hypercholesterolemia: sterrolemia:: mutational st mu muta ut tion onal on al analysis ana naly na lysi ly s s of a membrane mem mbrranee protein. prot pr o ei ein. n. An Annu n Rev nu Reev Genet. Geneet. Ge 1990;24:133-170. 3-170. 28. Stensonn PD, PD D, Ball Ball EV, V Howells Howel ells l K, K, Phillips Philliips AD, Ph AD, Mort Mor ort M, M, Cooper Coope peer DN. DN N. The The Human H ma Hu mann Gene G ne Ge n Mutation Database: providing central mutation D providi d ngg a comprehensive di comp co mpre mp rehe re heens nsiv iv ve ce cen ntraal mu ntra m tati ta tion ti on ddatabase atab at a as ab a e fo forr molecular diagnostics and pe Hum Genomics. 2009;4:69-72. ppersonalized rsonalized d ggenomics. enomiics. Hu H m Ge G nomiics. 2009 0 ;4 09 4:6 699 72 72. 29. Chmaraa M M, Wasag B, Z Zuk M, Kubalska Wegrzyn A, Bednarska Bednarska-Makaruk M, ett al al. Wa B kM K balska ballskka JJ, Wegr We nA Bed d ka M Makar akka k M l Molecular characterization of Polish patients with familial hypercholesterolemia: novel and recurrent LDLR mutations. J Appl Genet. 2010;51:95-106. 30. Fouchier SW, Kastelein JJ, Defesche JC. Update of the molecular basis of familial hypercholesterolemia in The Netherlands. Hum Mutat. 2005;26:550-556. 31. Cholesterol Treatment Trialists' (CTT) Collaborators, Mihaylova B, Emberson J, Blackwell L, Keech A, Simes J. et al. The effects of lowering LDL cholesterol with statin therapy in people at low risk of vascular disease: meta-analysis of individual data from 27 randomised trials. Lancet. 2012;380;9841:581-90.

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DOI: 10.1161/CIRCGENETICS.114.000646

Table 1: Demographics and LDLR variant frequencies for select circumpolar populations

MAF (%) n

Age

Female (%)

BMI (kg/m2)

TC

LDL-C

HDL-C

Non-HDL-C

apo B

TG p.G116S

p.R730W

Greenland

1182

44±14

56

26±5

5.91±1.13

3.82±1.04

1.57±0.44

4.33±1.14

0.92±0.23

1.16±0.67

0.13

0.11

Kivalliq

210

37±16

54

27±4

5.00±1.03

3.09±0.92

1.45±0.41

55±1 1.0 .01 1 3.55±1.01

0.98 0. 9 ± 98 0.98±0.26

1.03±0.57

0.02

0.17

Inuvialuit

281

45±16

67

30±7 30 30±7

5.055±0.99 9 5.05±0.99

2.911±0 ± .889 2.91±0.89

11.37±0.42 .3 37±0 ±0.4 ±0 .42 .4 2

33.68±1.03 .6 68± ±1.03 3

0.9 91± ± 0.91±0.25

1.74±1.27

0.05

0.13

Nunavik

429

37±14

56

2 27±6

4.999±0 ±0.9 . 9 .9 4.99±0.99

2.79 2. 79±0 79 ±0.8 ±0 .86 .8 6 2.79±0.86

1. .63 63± ±0.4 .43 3 1.63±0.43

3.33 3. 3 ±1 33 ±1.0 .02 .0 3.33±1.02

0.96± ± 0.96±0.24

1.23±0.72

0.09

0.13

SW Alaska

1222

38±16

53

28±6

5.20±1.15

3.20±0.98

1.64±0.44

3.61±1.08

n.d.

0.94±0.56

0.10

0.16

Combined

3324

40±16

56

27±6

5.40±1.16

3.30±1.05

1.58±0.44

3.83±1.15

0.93±0.24

1.13±0.74

0.10

0.14

All demographics are reported ± standard deviation. Lipid-related traits are all reported in mmol/L except apo B which is in g/L. BMI, body mass index; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; MAF, minor allele frequency; n.d., no data; TC, total cholesterol concentration; TG, triglyceride concentration.

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DOI: 10.1161/CIRCGENETICS.114.000646

Table 2: Mean lipid traits based on p.G116S and p.R730W genotype in a combined Inuit cohort

p.G116S genotype (A)

p.R730W genotype (T)

Lipid traits

GG

GA

AA

CC

CT

TT

TC

5.29±1.11

5.94±1.24

6.20±1.25**

5.43±1.17 43±1 43 ±1.1 ±1 .177 .1

5.37±0.17 5

5.47±1.07

LDL-C

3.22±0.98

3.88±1.14 3.88 88±1 88 ±1.114 ±1

4.21±1.14** 4 21 4. 2 ±1 ±1.14* 4**

3.37±1.06 3 37 3. 3 ±1 ±1.0 .06 .0

3.28±1.04 3

3.30±0.82

apo B

0.90±0.23

1.03±0.24 1.03±0 0.2 .244

1.11±0.20** 1.11 1. 11±0 11 ±0.2 ±0 .20* .2 0***

0.93±0.23 0 93 0. 93±0 ±0.23 ±0

0.91±0.24 0

0.97±0.27

HDL-C

1.57±0.44

1.59±0.43

1.58±0.49

1.56±0.44

1.61±0.46

1.68±0.45*

TG

1.13±0.75

1.09±0.62

0.97±0.38

1.12±0.76

1.11±0.67

1.11±0.68

3.71±1.09

4.35±1.24

4.62±1.20**

3.86±1.15

3.76±0.16

3.80±1.10*

Non-HDL-C

* indicates P5.0 mmol/L was based on the current Canadian Cardiovascular Society recommendations for severe dyslipidemia requiring pharmacologic lipid-lowering treatment.15 Logistic regression was additionally adjusted based on geographic location for the combined Inuit cohort. Pairwise linkage disequilibrium and haplotype phase were assessed using PLINK (http://pngu.mgh.harvard.edu/purcell/plink/).16 Differences in LDLR expression and activity in cell culture between wild-type and mutant LDLR were compared using two-tailed unpaired t-tests.

4 Copyright by American Heart Association, Inc. All rights reserved.

The 839-amino acid mature protein is shown here with corresponding exon and domain annotations. Modified from Hobbs et al.17

5 Copyright by American Heart Association, Inc. All rights reserved.

Multiple amino acid residue sequence alignments from divergent species show conservation at amino acids 116 and 730 in LDLR (outlined in red). Amino acid residues conserved between homologs are highlighted in blue and indicate local conservation. LDLR gene representation was made using the UCSC Genome Browser.

6 Copyright by American Heart Association, Inc. All rights reserved.

Supplemental Table 1A: Plasma lipid traits in Inuit based on LDLR p.G116S or p.R730W genotype. total cholesterol (mmol/L) p.G116S genotype Greenland Kivalliq Inuvialuit Nunavik SW Alaska Combined

LDL-C (mmol/L)

GG

GA

AA

GG

GA

AA

5.74±1.09 4.93±0.97 5.00±0.98 4.90±0.94 5.16±1.10 5.29±1.11

6.40±1.07 6.02±0.97* 5.58±1.07 5.38±1.13 5.62±1.32 5.94±1.24

6.70±1.32** n.d. 5.05±0.11* 6.27±0.58** 5.57±0.96** 6.20±1.25**

3.63±0.99 3.02±0.87 2.88±0.86 2.72±0.82 3.12±0.91 3.22±0.98

4.34±0.98 4.11±0.90* 3.43±1.03 3.11±0.97 3.58±1.17 3.88±1.14

4.70±1.12** n.d. 3.27±0.20* 3.90±0.77** 3.61±0.96** 4.21±1.14**

total cholesterol (mmol/L) R730W genotype Greenland Kivalliq Inuvialuit Nunavik SW Alaska Combined

apolipoprotein B (g/L) GG

GA

0.89±0.22 1.02±0.24 0.95±0.24 1.36±0.12** 0.90±0.25 1.03±0.25 0.94±0.24 1.02±0.24 n.d. n.d. 0.90±0.23 1.03±0.24

LDL-C (mmol/L)

AA 1.10±0.20** n.d. 0.95±0.04* 1.24±0.19* n.d. 1.11±0.20**

apolipoprotein B (g/L)

CC

CT

TT

CC

CT

TT

CC

CT

TT

5.91±1.12 4.98±1.00 5.10±1.01 4.94±0.96 5.23±1.16 5.43±1.17

5.90±1.17 4.87±1.00 4.86±0.97 4.93±0.90 5.29±1.14 5.37±0.17

6.1±1.09 5.18±0.91 5.15±0.81 5.52±0.93 5.18±1.08 5.47±1.07

3.83±1.03 3.08±0.90 2.97±0.90 2.80±0.84 3.21±1.00 3.37±1.06

3.78±1.08 2.96±0.92 2.78±0.86 2.71±0.85 3.22±0.94 3.28±1.04

3.73±0.72 3.19±0.76 2.98±0.40 3.14±1.04 3.19±0.85 3.20±0.82*

0.92±0.22 0.97±0.25 0.92±0.26 0.95±0.23 n.d. 0.93±0.23

0.91±0.24 0.95±0.26 0.87±0.24 0.93±0.24 n.d. 0.91±0.24

0.95±0.31 0.98±0.22 0.91±0.18 1.04±0.28 n.d. 0.97±0.27

* indicates P