Nture Pulishing Group http://www.nture.com/nturegenetics Genetic vrition in PNPLA3 confers susceptiility to nonlcoholic ftty liver disese Stefno Romeo,, Juli Kozlitin,3,, Cho Xing,, Alexnder Pertsemlidis, Dvid Cox, Len A Penncchio 5, Eric Boerwinkle, Jonthn C Cohen & Helen H Hos,7 Nonlcoholic ftty liver disese (NAFLD) is urgeoning helth prolem of unknown etiology tht vries in prevlence mong ncestry groups. To identify genetic vrints contriuting to differences in heptic ft content, we crried out genome-wide ssocition scn of nonsynonymous sequence vritions (n = 9,9) in popultion comprising Hispnic, Africn Americn nd Europen Americn individuls. An llele in PNPLA3 (rs739[g], encoding IM) ws strongly ssocited with incresed heptic ft levels (P = 5.9 ) nd with heptic inflmmtion (P = 3.7 ). The llele ws most common in Hispnics, the group most susceptile to NAFLD; heptic ft content ws more thn twofold higher in PNPLA3 rs739[g] homozygotes thn in noncrriers. Resequencing reveled nother llele of PNPLA3 (rs[t], encoding S53I) tht ws ssocited with lower heptic ft content in Africn Americns, the group t lowest risk of NAFLD. Thus, vrition in PNPLA3 contriutes to ncestry-relted nd inter-individul differences in heptic ft content nd susceptiility to NAFLD. In humns, dipose tissue serves s reservoir to limit the deposition of triglyceride in the liver nd other metoliclly ctive tissues. The effectiveness of this uffer in protecting ginst the ccumultion of ft in the liver vries widely mong individuls: heptic ft content rnges from less thn % to more thn 5% of liver weight in the generl popultion. The ccumultion of excess triglyceride in the liver, condition known s heptic stetosis (or ftty liver), is ssocited with dverse metolic consequences, including insulin resistnce nd dyslipidemi 3,. In suset of individuls, heptic stetosis promotes n inflmmtory response in the liver, referred to s stetoheptitis, which cn progress to cirrhosis nd liver cncer 3,5. Nonlcoholic ftty liver disese (NAFLD) is the most common form of liver disese in Western countries. Approximtely % of liver trnsplnts done in the United Sttes re for cirrhosis relted to NAFLD. Fctors promoting deposition of ft in the liver include oesity, dietes, insulin resistnce nd lcohol ingestion 3,. The propensity to develop heptic stetosis differs mong ncestry groups, with Africn Americns hving lower (%) nd Hispnics higher (5%) frequency of the disorder thn Europen Americns (33%) in lrge US urn popultion. Hispnics lso hve higher prevlence of stetoheptitis nd cirrhosis, wheres Africn Americns re less prone to develop liver filure,7 9. The fctors responsile for these ncestryrelted differences in prevlence of heptic stetosis nd liver injury re not known. To identify DNA sequence vritions tht contriute to inter-individul differences in NAFLD, we crried out genome-wide survey of nonsynonymous sequence vritions in popultion-sed study comprising individuls of vrious ncestries, the Dlls Hert Study. We limited our nlysis to nonsynonymous sequence vritions to focus on vrints with higher likelihood of ffecting gene function. Heptic ft content ws mesured in the Dlls Hert Study using proton mgnetic resonnce spectroscopy ( H-MRS), the most ccurte, quntittive, noninvsive method ville,,. Of the,3 nonsynonymous vrints ssyed using chip-sed oligonucleotide hyridiztion 3, 9,9 exceeded the qulity control threshold for the study (see Methods) nd were included in the nlysis. We tested ech vrint for ssocition with heptic ft content in,3 Africn Americn, 9 Europen Americn nd 33 Hispnic study prticipnts in the Dlls Hert Study who otined H-MRS of the liver. To mximize sttisticl power, we pooled the three ncestry groups, nd included glol ncestry score (clculted using pnel of,7 ncestry-informtive SNPs) in the model to control for popultion strtifiction (see Methods). The quntile-quntile plot of P vlues showed no systemtic devition from the null distriution (Fig. ). A single vrint in PNPLA3 (rs739) ws strongly ssocited with heptic ft content (P = 5.9 ; Fig. ). The vrint is cytosine to gunine sustitution tht chnges codon from isoleucine to methionine; this residue is highly conserved in vertertes (Fig. ). Donld W. Reynolds Crdiovsculr Clinicl Reserch Center, Eugene McDermott Center for Humn Growth nd Development nd the Deprtment of Clinicl Sciences, University of Texs Southwestern Medicl Center, Hrry Hines Boulevrd Dlls, Texs 7539, USA. 3 Deprtment of Sttisticl Science, Southern Methodist University, Dlls, Texs 7575, USA. Perlegen Sciences, Mountin View, Cliforni 93, USA. 5 Genomics Division, Lwrence Berkeley Ntionl Lortory, Berkeley, Cliforni 97 nd US Deprtment of Energy Joint Genome Institute, Wlnut Creek, Cliforni 959, USA. Humn Genetics Center nd Institute for Moleculr Medicine, University of Texs Helth Science Center, Houston, Texs 773, USA. 7 Howrd Hughes Medicl Institute t the University of Texs Southwestern Medicl Center, Hrry Hines Boulevrd Dlls, Texs 7539, USA. These uthors contriuted eqully to this work. Correspondence should e ddressed to H.H.H. (helen.hos@utsouthwestern.edu) or J.C.C. (jonthn.cohen@utsouthwestern.edu). Received August; ccepted Septemer; pulished online 5 Septemer ; doi:.3/ng.57 nture genetics volume numer decemer
Nture Pulishing Group http://www.nture.com/nturegenetics log P oserved 3 log P expected log P PNPLA3 encodes mino cid protein of unknown function tht elongs to the pttin-like phospholipse fmily. The progenitor of this fmily, pttin, is mjor protein of potto tuers nd hs nonspecific lipid cyl hydrolse ctivity 5,. None of the other nonsynonymous sequence vrints tested in the genome-wide scn exceeded the Bonferroni-corrected threshold for significnce (P = 5. ; Fig. ). The ssocition etween PNPLA3 rs739 nd heptic ft content remined highly significnt (P = 7. ) fter djustment for ody mss index (BMI), dietes sttus, ethnol use, s well s glol nd locl ncestry (Fig. ), nd ws pprent in ll three ncestry groups (Fig. c nd Supplementry Tle online). Thus, the ssocition etween rs739 nd heptic ft content ws not ttriutle either to the effect of known risk fctors for liver ft ccumultion or to popultion strtifiction. The frequencies of the PNPLA3 rs739[g] llele were concordnt with the reltive prevlence of NAFLD in the three ncestry groups ; the highest frequency of the llele ws in Hispnics (.9), with lower frequencies oserved in Europen Americns (.3) nd Africn Americns (.7). Accordingly, we exmined the reltionship etween IM Pttin- like domin 3 5 7 9 3 5 7 9 X Chromosome Figure Genome-wide scn of liver triglyceride content mesured y proton mgnetic resonnce imging in the Dlls Hert Study (n =,). () Quntile-quntile plot of P vlues. () Sctter plot of P vlues. The dshed line denotes the Bonferroni-corrected significnce threshold (P = 5. ). 7 GASAG 7 P = 7. BMI (kg/m ) 3 DGGV Humn - ALVCSCFIPFYSGLI -55 Mouse ALVCSCFIPLFSGLI Rt ALVCSCFIPLFSGLI Horse ALMCSCFIPFLSGLM Pig ALLCSSFIPLVSGFI Dog ALLCSSFIPFFSGII Frog ALICSAFVPIYCGLI Zerfish ALICSCFIPVYCGLI HOMA-IR(U) - n:,359 77 5,33 953,9,99 35,9,99 35 3 Plsm TG (mg/dl) 75 5 5 PNPLA3 rs739 nd evidence of heptic inflmmtion, s indicted y relese of liver enzymes into the circultion, nd found significnt elevtion in serum concentrtions of lnine minotrnsferse (ALT) in ssocition with the PNPLA3 rs739[g] llele (P = 3.7 ). Anlysis of the three ncestry groups reveled tht the ssocition with ALT ws limited to Hispnics (P =.3 5 ), the group with the gretest prevlence of heptic stetosis nd susceptiility to cirrhosis (Supplementry Tle ),7. The PNPLA3 rs739[g] llele ws lso ssocited with serum sprtte minotrnsferse concentrtions in Hispnics (P =.). These findings re consistent with our prior oservtion tht higher proportion of Hispnics with heptic stetosis hve evidence of heptic inflmmtion 7, nd suggests tht the PNPLA3 rs739[g] llele dversely ffects liver function. Incresed heptic ft content is ssocited with insulin resistnce nd dyslipidemi (incresed plsm levels of triglyceride nd lower levels of high-density-lipoprotein cholesterol), ut the cusl nture of these reltionships remins poorly defined 3. We did not find ny ssocition etween the PNPLA3 rs739[g] llele nd BMI or indices of insulin sensitivity, including fsting glucose nd insulin concentrtions or homeosttic model ssessment of insulin resistnce (HOMA-IR), in the entire Dlls Hert Study (Fig. nd Supplementry Tle ). No ssocitions were oserved etween PNPLA3 genotype nd plsm concentrtions of triglyceride (Fig. ), totl cholesterol, highdensity-lipoprotein cholesterol or low-density-lipoprotein cholesterol (Supplementry Tle ). A corresponding nlysis in lrger smple (n =,), the Atherosclerosis Risk in Communities Study (ARIC) 7, lso showed no ssocition of PNPLA3 rs739 with BMI, indices of insulin sensitivity, or plsm concentrtions of triglyceride or high- Figure Assocition etween sequence vrint in PNPLA3 (rs739) nd heptic triglyceride content. () PNPLA3 is -residue protein tht contins pttin-like domin t the N terminus with consensus sequences for Ser-Asp ctlytic dyd (Gly-X-Ser-X-Gly nd Asp-X-Gly/Al). The IM sustitution (rs739) is locted etween the consensus sequences for the ctlytic dyd nd is highly conserved. () Medin heptic triglyceride (TG) content, ody mss index (BMI), homeosttic model ssessment of insulin resistnce (HOMA-IR) nd plsm TG levels for individuls y PNPLA3 genotype in the Dlls Hert Study. Vlues for heptic ft content were compred using ANOVA. Age, sex, BMI, dietes sttus, ethnol use nd glol ncestry were included s covrites in the model. (c) Medin heptic ft contents nd PNPLA3 rs739 genotypes in Europen Americns, Africn Americns nd Hispnics in the Dlls Hert Study. Associtions etween heptic ft content nd PNPLA3 rs739 genotypes were tested using ANOVA with ge, locl ncestry, sex, dietes sttus, ethnol intke nd BMI s covrites. CC CG GG P =. c n: P = 3. 7 75 Europen Americns P = 7.5 9 3 Africn Americns 79 99 Hispnics volume numer decemer nture genetics
Nture Pulishing Group http://www.nture.com/nturegenetics FS-Y() C99R 3 Pttin-like domin V7I C99G G5C VM IM I7T GG GT+TT P =. n = 77 77 Africn Americns V7I(3) E7D E7D c E33G S53I EK K3E S53R Numer of sujects with PNPLA3-rs[T] P = 7. density-lipoprotein cholesterol (Supplementry Tle online). On the sis of the oserved ssocitions etween the SNP nd heptic ft nd etween heptic ft nd HOMA-IR in the Dlls Hert Study, we clculted the power to detect n ssocition with HOMA-IR to e >9% in the Africn Americns nd 9% in the Europen Americns in ARIC. The dt from these studies indicte tht the PNPLA3 rs739[g] llele is ssocited with systemtic increse in heptic ft content ut not with mjor ltertions in glucose homeostsis or lipoprotein metolism. Thus, incresed heptic ft content does not inevitly led to insulin resistnce, which is consistent with recent oservtions in some niml models,9. To determine whether other sequence vritions in PNPLA3 contriute to differences in heptic ft content, we resequenced the coding region of PNPLA3 in the men (3 Africn Americns, 3 Europen Americns nd Hispnics) nd women who hd the highest levels of heptic ft in the Dlls Hert Study ( high group ), nd in sex- nd ncestry-mtched group with the lowest levels of heptic ft ( low group ). The numer of individuls with nonsynonymous vrints found only in the high group (n = ) ws similr to the numer of individuls with nonsynonymous vrints specific to the low group (n = 9), ut the three individuls with likely null muttions (FS-Y (p.tyrfs, delc) nd IVS7+ (c.+g>t)) were ll in the high group (Fig. 3), which is consistent with the hypothesis tht loss of function of PNPLA3 cuses n increse in heptic triglyceride content. Eight vrints were present in oth the low nd the high heptic ft groups (Fig. 3 nd Supplementry Tle 3 online), nd the six most common of these sequence vritions were genotyped in the entire smple. One vrint, PNPLA3 rs[t], which sustitutes n isoleucine for serine in codon 53, ws common in Africn Americns (MAF =.) ut rre in Europen Americns (.3) nd Hispnics (.) (Supplementry Tle 3) nd ws ssocited with significntly lower liver ft content. Medin heptic triglyceride content ws % lower in Africn Americns with the PNPLA3 rs[t] when compred to Africn Americns homozygous for the wild-type llele (3.3% versus.7%, P =. ; Fig. 3). Further evidence tht the vrint ws ssocited with lower heptic ft content ws provided y the finding tht, of individuls with the PNPLA3 rs[t] llele, significntly greter numer hd heptic ft content in the lowest decile of the popultion thn in the highest decile (Fig. 3). We did not find ny significnt difference in the numer of individuls identified in the extremes for ny of the other SNPs (dt not shown). The effect of PNPLA3 rs[t] on heptic ft content ws independent of the PNPLA3 rs739 polymorphism. Both vrints were sttisticlly significnt when included in multiple regression model, nd the PNPLA3 rs[t] llele ws significntly ssocited with heptic ft in Africn Americns homozygous for the PNPLA3 rs739[c] llele (dt not shown). The identifiction of second llele of PNPLA3 tht ws independently ssocited with heptic ft content further supports role for PNPLA3 in determining heptic triglyceride levels, nd indictes the presence of oth loss-of-function nd gin-of-function lleles t this locus. The mechnisms y which these lleles ffect heptic ft content re not known. The frequencies of oth PNPLA3 rs739[g] nd rs[t] in the three ncestry groups represented in the Dlls Hert Study re concordnt with ncestry-relted differences in heptic ft content. Exclusion of the individuls crrying either of these two lleles sustntilly ttenuted the differences in heptic ft content etween the ncestry groups; regression nlysis indicted tht these two sequence vritions ccounted for 7% of the oserved ncestry-relted differences in heptic ft content in the Dlls Hert Study. Thus, genetic vrition in PNPLA3 ccounts for lrge frction of the ncestryrelted differences in the propensity to ccumulte excess ft in the liver in this study popultion. The physiologicl sustrte(s) of PNPLA3 hs not een defined. Expression of PNPLA3 is under metolic control in dipose tissue nd liver; mrna levels re low in the fsted stte nd increse mrkedly with crohydrte feeding,. PNPLA3 structurlly resemles clcium-independent phospholipse A, ut the recominnt protein hs low phospholipse ctivity when expressed in insect (Sf9) cells. PNPLA3 hs more roust ctivity ginst triglyceride in vitro nd cn lso trnsfer ftty cids to nd from mono- nd dicylglycerol. It is not known whether the primry effect of PNPLA3 in the liver is to hydrolyze triglyceride or to trnsfer ftty cids etween lipids (trnscetyltion). Studies re in progress to determine the specific effects on lipid metolism of the nonsynonymous vrints in PNPLA3 identified in this study. Currently, we cnnot ccurtely predict which individuls with ftty liver will develop stetoheptitis nd progress to cirrhosis nd liver filure. The finding tht mrkers of liver inflmmtion (serum levels of liver-derived enzymes) were elevted in PNPLA3 rs739[g] crriers, which ws lso oserved in n independent genome-wide ssocition study 3, suggests tht this genetic vrint my confer incresed susceptiility to heptic injury. Pttin-like phospholipse fmily mem- 5 5 5 IVS7+ QK K3D G35S GT+TT F57() Heptic TG content <% >9% Liver TG High Low High + Low Figure 3 Identifiction of PNPLA3 llele (rs, encoding S53I) ssocited with lower heptic ft content in Africn Americns in the Dlls Hert Study. () Exons nd flnking introns of PNPLA3 were sequenced in the 3 Europen Americn nd 3 Africn Americn men nd women nd in the Hispnic men nd women with the lowest nd highest heptic triglyceride (TG) content determined using proton mgnetic resonnce imging. The sustitutions encoded y the nonsynonymous vritions identified in individuls in only the high, only the low nd in oth the high nd low groups re shown. All the vrints not found in oth groups were present in only single suject unless otherwise indicted y numer in prentheses.the rs numers for polymorphisms nd the oligonucleotides used for PCR-sequencing of the coding regions re provided in Supplementry Tles 3 nd. () Medin heptic triglyceride content in Africn Americns homozygous for the wild-type PNPLA3 llele (rs[g], GG) or crrying the rs[t] llele (GT + TT). (c) Numer of individuls with PNPLA3 rs[t] in the upper nd lower deciles of heptic ft content. nture genetics volume numer decemer 3
Nture Pulishing Group http://www.nture.com/nturegenetics ers in other orgnisms re upregulted in response to environmentl insults. The sequence vritions in PNPLA3 reported here my provide predictive informtion regrding the risk of developing heptic stetosis nd liver injury in response to environmentl stresses such s cloric excess, infections or drugs. METHODS Study popultions. The Dlls Hert Study is popultion-sed proility smple of Dlls County. The smpling frme nd the study design hve een descried in detil. Africn Americns were oversmpled (5% Africn Americn, self-identified s lck ; 9% Europen Americn, self-identified s white ; 7% Hispnic self-identified s hispnic ; nd % other ncestries). The institutionl review ord of University of Texs Southwestern Medicl Center pproved the study, nd ll study sujects provided written informed consent. We determined lcohol consumption ccording to nswers to previously vlidted questions. Blood pressure, height, weight nd BMI nd clculted vriles were mesured s descried. We otined fsting lood smples from 3,55 sujects (ges 3 5), nd,97 of these individuls completed clinic visit; we mesured heptic triglyceride content using H-MRS in, Africn Americns, Europen Americns nd Hispnics 7,. The ssocition etween PNPLA3 rs739 nd metolic phenotypes ws lso exmined in the Atherosclerosis Risk in Communities Study (ARIC), lrge prospective study tht focuses on crdiovsculr disese in Europen Americns nd Africn Americns. Detils of the ARIC study design nd the methods used to mesure plsm lipid levels hve een pulished previously 7,5. The dt used in this nlysis were collected from the seline exmintion. Genome-wide ssocition nd other sttisticl methods. Genome-wide ssocition nlysis ws done using,3 nonsynonymous sequence vritions from dsnp nd the Perlegen SNP dtse (ville on request). We ssyed SNPs in 3,33 Africns Americn, Europen Americn nd Hispnic prticipnts of the Dlls Hert Study using high-density oligonucleotide rrys (Perlegen Sciences). SNPs tht met ny of the following criteri were excluded (n =,3): error proility >%, genotype cll rte <%, or significnt devition from Hrdy- Weinerg equilirium (P <.). Of the 9,55 SNPs tht were successfully ssyed, were monomorphic in the Dlls Hert Study smple. We tested the remining 9,9 vrints for ssocition with heptic ft content in the, Africn Americn, Europen Americn nd Hispnic sujects in the Dlls Hert Study who underwent H-MRS of the liver nd in whom ncestry-informtive SNPs hd een ssyed previously; glol nd locl ncestries were inferred for ech individul using STRUCTURE under linkge model with,7 ncestry-informtive SNPs 7. We pooled ll prticipnts together nd inferred glol ncestry (the proility of n individul elonging to given cluster), setting the numer of clusters, K, equl to 3. Although the ncestry-informtive SNP pnel ws primrily designed for Africn Americns (men multipoint informtion content ( IC ) =.), it ws dequtely informtive in Europen Americns ( IC =.3) nd Hispnics ( IC =.). We lso inferred locl ncestry s the proility tht prticulr genomic region elonged to given cluster. The results were lmost identicl when ncestry djustment ws done with the sme SNPs using principl-component nlysis (dt not shown). The sttisticl significnce of 9,9 SNPs in the genome-wide ssocition study ws ssessed using nlysis of vrince (ANOVA). To ccommodte confounding fctors, we included ge, sex nd glol ncestry s covrites in the model. The dditive effect of ech vrint ws tested y encoding the genotype vrile s, or. Becuse the distriution of heptic ft levels is highly skewed, we pplied power trnsformtion (λ = /) to the trit efore the nlysis. To ccount for multiple testing, we djusted the significnce threshold for the numer of tests done using the Bonferroni method. SNPs with nominl P < 5. were considered significnt on genome-wide scle. We tested the ssocition etween PNPLA3 vrints nd heptic ft content within ech ncestry group using ANOVA, including ge, sex, BMI, dietes sttus, ethnol use nd locl ncestry s covrites. Individuls whose genetic ncestry ws not consistent with their self-reported ncestry (n =, 5 nd for Africn Americns, Europen Americns nd Hispnics, respectively) nd who hd frctionl ncestry tht ws more thn three times the interqurtile rnge elow the 5th percentile for their reference group were excluded from the nlysis. Becuse the distriution of heptic triglyceride content is skewed, we reported medins nd interqurtile rnges. We nlyzed the ssocition of PNPLA3 rs739 with BMI, HOMA-IR nd plsm triglyceride levels in the Africn Americns, Europens Americns nd Hispnics together using ANOVA including ge, sex nd locl ncestry s covrites. HOMA-IR ws djusted for BMI, nd plsm triglyceride levels were djusted for BMI nd dietes. To determine the contriution of the sequence vritions we identified in PNPLA3 to the ncestry-relted differences in heptic ft content, we exmined the proportion of vrince explined y ncestry (R) using liner model. We then determined the proportion of vrince explined y ncestry fter djustment for the PNPLA3 lleles (rs739[g] nd rs[t]) (R). The proportion of vrince due to ncestry nd explined y these two lleles ws determined from (R R)/R. Resequencing PNPLA3. The exons nd flnking introns of PNPLA3 were sequenced s descried previously 9 in the Africn Americn, Europen Americn nd Hispnic men nd women in the Dlls Hert Study with the highest nd lowest heptic triglyceride content. Oligonucleotide primers used for sequencing re shown in Supplementry Tle online. All sequence vrints identified were verified y mnul inspection of the chromtogrms, nd missense chnges were confirmed y n independent resequencing rection. Genotyping ssys. We developed fluorogenic 5 -nucleotidse ssys for PNPLA3 rs739 nd for the sequence vrints identified in oth the high nd low heptic triglyceride groups in the resequencing experiments. Sequence vritions in PNPLA3 were ssyed using the TqMn ssy system (Applied Biosystems) on 79HT Fst Rel-Time PCR instrument. Proes nd regents were purchsed from Applied Biosystems. Accession codes. GenBnk: PNPLA3, NM_55; PNPLA3, NP_795. Note: Supplementry informtion is ville on the Nture Genetics wesite. ACKNOWLEDGMENTS We thnk T. Hytt, J. Mrtin, W. Schckwitz, A. Ustszewsk, C. Wright nd the tem t Perlegen Sciences for technicl ssistnce. We thnk K. Lwson for the sttisticl nlysis of the dt from the ARIC study. We thnk J. Horton nd D. Hinds for helpful discussions. We re grteful to the stff nd prticipnts of the Dlls Hert Study nd the Atherosclerosis Risk in Communities Study for their contriutions. This work ws supported y grnts from the Donld W. Reynolds Foundtion, the US Ntionl Institutes of Helth (RLHL-955, PLDK nd HL-9), the US Ntionl Hert, Lung, nd Blood Institute (NHLBI) Progrm for Genomic Applictions (HL-) nd the US Deprtment of Energy (Contrct DE-AC-5CH3). AUTHOR CONTRIBUTIONS H.H.H., J.C.C., E.B., L.A.P. nd D.C. conceived, designed nd directed the study; J.K., S.R., C.X. nd A.P. performed nd interpreted the genetic nlysis. All the uthors pproved the finl mnuscript nd contriuted criticl revisions to its intellectul content. Pulished online t http://www.nture.com/nturegenetics/ Reprints nd permissions informtion is ville online t http://npg.nture.com/ reprintsndpermissions/. Wng, M.Y. et l. Adipogenic cpcity nd the susceptiility to type dietes nd metolic syndrome. Proc. Ntl. Acd. Sci. USA 5, 39 ().. Browning, J.D. et l. Prevlence of heptic stetosis in n urn popultion in the United Sttes: impct of ethnicity. Heptology, 37 395 (). 3. Browning, J.D. & Horton, J.D. Moleculr meditors of heptic stetosis nd liver injury. J. Clin. Invest., 7 5 ().. McCullough, A.J. The clinicl fetures, dignosis nd nturl history of nonlcoholic ftty liver disese. Clin. Liver Dis., 5 533 (). 5. Adms, L.A. et l. The nturl history of nonlcoholic ftty liver disese: popultion-sed cohort study. Gstroenterology 9, 3 (5).. de Alwis, N.M. & Dy, C.P. Non-lcoholic ftty liver disese: the mist grdully clers. J. Heptol. (Suppl. ), S S (). 7. Browning, J.D., Kumr, K.S., Soorin, M.H. & Thiele, D.L. Ethnic differences in the prevlence of cryptogenic cirrhosis. Am. J. Gstroenterol. 99, 9 9 ().. Clrk, J.M., Brncti, F.L. & Diehl, A.M. The prevlence nd etiology of elevted minotrnsferse levels in the United Sttes. Am. J. Gstroenterol. 9, 9 97 (3). volume numer decemer nture genetics
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