ARTICLE The Strength of Selection on Ultraconserved Elements in the Human Genome

Transcription

1 ARTICLE The Strength of Selection on Ultrconserved Elements in the Humn Genome Christin T. L. Chen, Jen C. Wng, nd Brk A. Cohen Ultrconserved elements re stretches of consecutive nucleotides tht re perfectly conserved in multiple mmmlin genomes. Although these sequences re identicl in the reference humn, mouse, nd rt genomes, we identified numerous polymorphisms within these regions in the humn popultion. To determine whether polymorphisms in ultrconserved elements ffect fitness, we genotyped unrelted humn DNA smples t loci within these sequences. For ll single-nucleotide polymorphisms tested in ultrconserved regions, individuls homozygous for derived lleles (lleles tht differ from the rodent reference genomes) were present, vile, nd helthy. The distriution of llele frequencies in these smples rgues ginst strong, ongoing selection s the force mintining the conservtion of these sequences. We then used two methods to determine the minimum level of selection required to generte these sequences. Despite the lck of fixed differences in these sequences etween humns nd rodents, the verge level of selection on ultrconserved elements is less thn tht on essentil genes. The strength of selection ssocited with ultrconserved elements suggests tht muttions in these regions my hve sutle phenotypic consequences tht re not esily detected in the lortory. Five percent of the humn genome is estimted to e under purifying selection. 1,2 However, only 1.5% of the genome encodes protein, leving twice s much conserved noncoding DNA s coding DNA. Consistent with this estimte, thousnds of conserved noncoding sequences hve een discovered in studies tht sought to identify mmmlin sequences with unusully slow rtes of sustitution. 3 5 At the extreme end of the sequences identified in these studies re the ultrconserved elements, 6 sequences in which runs of 200 consecutive nucleotides re identicl in lignments from the humn, mouse, nd rt reference genomes. The underlying ssumption of comprtive genomics is tht sequences tht contriute to the fitness of n orgnism will evolve slowly, reltive to selectively neutrl sequences. Thus, the ultrconserved elements, which evolve exceptionlly slowly, might encode importnt functions. An lternte hypothesis is tht these sequences re situted in regions of the genome with low muttion rtes, resulting in fewer thn expected nucleotide sustitutions over time. Drke et l. 7 suggested tht conserved noncoding sequences re likely to e functionl nd not muttion cold spots, ecuse the derived lleles in these regions show is towrd eing minor-frequency lleles. On the sis of this oservtion, Drke et l. 7 concluded tht purifying selection mintins these sequences in the genome. Kryukov et l. 8 lso concluded tht purifying selection, rther thn decrese in muttion rte, drives the conservtion of these sequences. Despite the high levels of conservtion these sequences exhiit, oth Kryukov et l. 8 nd Keightley et l. 9 suggested tht muttions in conserved noncoding regions re only slightly deleterious. However, the strength of selection required to mintin the sequence conservtion of ultrconserved elements, the most extreme representtives of conserved noncoding sequences, hs yet to e determined. In this study, we estimted the mgnitude of selection consistent with the mintennce of ultrconserved elements, y nlyzing the distriution of polymorphisms within these elements nd the nucleotide differences in these sequences in the chimpnzee reference genome. We lso compred the estimted selection coefficients with those ssocited with essentil genes, to pprecite their significnce. By determining the mgnitude of selection tht constrins the evolution of ultrconserved elements, we will e etter le to devise pproprite experiments tht revel their potentil functions. Mteril nd Methods Polymorphisms in Ultrconserved Elements The coordintes of the ultrconserved elements were converted to the My 2004 version (hg17) of the University of Cliforni Snt Cruz (UCSC) Genome Browser. 10 The coordintes of ll recorded SNPs in the humn SNP dtse (dsnp) 11 were checked to see whether they fll within the coordintes of ech ultrconserved element. Ech SNP tht ws found in n ultrconserved element ws checked to see whether frequency informtion ws recorded, whether it ws found using two different methodologies, nd whether it ws withdrwn fter sumission. We clculted the P vlue of oserving, t most, 24 vlidted From the Deprtment of Genetics, Center for Genome Sciences (C.T.L.C.; B.A.C.), nd Deprtment of Psychitry (J.C.W.), Wshington University School of Medicine, St. Louis Received Septemer 26, 2006; ccepted for puliction Jnury 25, 2007; electroniclly pulished Ferury 20, Address for correspondence nd reprints: Dr. Brk A. Cohen, Deprtment of Genetics, Center for Genome Sciences, Cmpus Box 8510, Wshington University School of Medicine, 4444 Forest Prk Prkwy, St. Louis, MO E-mil: cohen@genetics.wustl.edu Am. J. Hum. Genet. 2007;80: y The Americn Society of Humn Genetics. All rights reserved /2007/ $15.00 DOI: / The Americn Journl of Humn Genetics Volume 80 April

2 SNPs in these ultrconserved regions, using two pproches. First, we used cumultive Poisson sttistics with genome-verge SNP density of 1.84 SNPs per 1 k of sequence. The genome-verge SNP density ws derived y dividing the numer of ll verified SNPs in the dtse y the size of the humn genome. l in the Poisson eqution ws computed y multiplying the genome verge y the numer of ses in ultrconserved elements (126,007 p). The second pproch ws to use the empiricl frequency distriution of SNPs in the genome. We otined this distriution y rndomly smpling 100,000 different sets of genomic regions tht mtched the length distriution of the ultrconserved elements nd counting the numer of vlidted SNPs in ech set. Selection of SNPs Locted Within nd Outside Ultrconserved Elements The two genotyping experiments were pproved y the pproprite institutionl review ords, nd ll humn DNA smples were deidentified. For the first experiment, we selected 24 SNPs. Hlf of the SNPs re locted within the ultrconserved elements; 9 of 12 lie in intergenic regions, nd the remining 3 re in introns or UTRs. The other 12 SNPs, selected to e controls, were locted in regions with low proility of eing under selection. We determined whether 50-p window in the humn-mouse-rt (HMR) lignment ws under selection y clculting the percentge of identity in tht window. A mtch occurred when ll three species hd the sme nucleotide in the sme position. Otherwise, mismtch ws recorded for tht position. The percentge of identity for given window ws the totl numer of mtch positions divided y the window size. A score ws then derived from the percentge of identity y using the scoring system tht ws modified from previous studies 2 nd ws sed on the cumultive inomil distriution. We modified the scoring scheme y using HMR ncient repets to estimte the expected frequency of neutrl positions with three-wy mtches, insted of humn-mouse ncient repets. Any window in which 191% of the positions were identicl in ll three species hd 95% proility of eing under selection (C. T. L. Chen nd B. A. Cohen, unpulished dt). To ensure tht the control SNPs we picked were not locted in regions of high selection, we scnned 50-p flnking sequences surrounding ech SNP of interest, using 50-p overlpping windows, nd clculted the score of ech window. A SNP ws selected only if!91% of the ses were threewy mtches in ll the windows. The distnces etween ech of the pired SNPs rnge from 28 p to just over 1 k. Aout hlf of the nery SNPs hd een vlidted y multiple ls, ccording to dsnp, s of July We genotyped these SNPs in 752 cse-control humn DNA smples provided y the Collortive Study on the Genetics of Alcoholism (COGA) Consortium. One SNP in the first experiment (rs in ultrconserved region [UC] 51) hd low frequency of derived lleles, s documented in dsnp. To ensure tht the oservtion ssocited with this SNP ws not due to indequte smple size, we genotyped it in dditionl smples, long with one SNP upstrem nd one SNP downstrem from it. These two SNPs were locted outside regions of high conservtion. We lso chose two SNPs locted within one ultrconserved region (UC 268) to genotype in dditionl smples, since there were no frequency dt ssocited with them in dsnp. In ddition, we selected two more pirs of SNPs to genotype in the second experiment. Ech pir included one SNP locted within the ultrconserved regions (UC 140 nd UC 353) nd nother SNP locted outside regions of high conservtion, defined s detiled ove. We genotyped these nine SNPs in 721 control humn smples provided y the genetic core t Alzheimer Disese Reserch Center t Wshington University. The MssARRAY system ws employed in genotyping humn DNA smples. 12 SpectroDESIGNER softwre ws used to select primers for ech SNP (Sequenom). Stndrd Sequenom PCR protocols were used, followed y shrimp lkline phosphtse tretment nd the homogenous MssEXTEND rection, s detiled in the Mss- ARRAY ppliction notes (Sequenom). The SpectroACQUIRE nd SpectroAnlyzer modules in the Typer softwre were used to nlyze the SNP dt (Sequenom). We compred the llele frequencies of SNPs etween the COGA cse nd control smples nd found no significnt differences etween these two groups of smples. Therefore, we comined the smples in ll lter investigtions. To determine whether SNP ws in Hrdy-Weinerg equilirium (HWE), we used two different implementtions of Fisher s exct test. Generl goodness-of-fit tests, such s the x 2 nd likelihood (G test) tests, were not suitle, since some of the expected genotype numers were!10, mking the smpling distriution of the test sttistics only pproximtely equl to the theoreticl x 2 distriution. 13 The symptotic ssumption did not hold in these cses. The first implementtion used the Mrkov chin Monte Crlo (MCMC) method to estimte the P vlues nd ws developed for multiple lleles. 14 The progrm ws run using the prmeters: 2,000 initil steps, 500 chunks, nd 5,000 s the size of ech chunk. The second implementtion (EXACT) ws derived from the first implementtion, with specific ppliction to illelic SNPs. 15 An level of.05 ws selected s threshold. Any SNP with P!.05 in either test ws deemed to e out of HWE. To clculte linkge disequilirium etween pirs of SNPs, the genotype dt were compiled to construct hplotype informtion for ech individul. Individuls with miguous hplotypes were removed efore clcultion of linkge disequilirium. Fisher s exct test ws pplied to ech set of genotype dt. An level of.05 ws selected s threshold. Any pir of SNPs with P vlue!.05 ws considered to e in linkge disequilirium. Clcultion of Selection Coefficients with the Use of Fixed Differences To clculte the strength of selection cting on ultrconserved elements, we mde two ssumptions. Since the proility of oserving long run of consecutive nucleotides ws so low, 6 we ssumed there were no neutrlly evolving positions in ultrconserved elements. Also, since we were interested in the verge selection on ultrconserved elements nd not in the selection on individul ses, we ssumed tht ech nucleotide ws under the sme mgnitude of selection in these regions. We employed equtions developed y Kimur 16,17 to relte the mount of sequence divergence etween humn nd chimpnzee with the strength of the selection coefficient. The sustitution rte per nucleotide etween two species cn e modeled s k p 8Nemp(p,s) 4Nemtp(p,s), (1) where N e is the effective popultion size; is the fixtion p(p,s) proility s function of s, the selection coefficient, nd of p, the initil frequency of the mutnt llele; t is the time, in genertions, since divergence of the two species; nd m is the muttion rte per se per genertion. The Americn Journl of Humn Genetics Volume 80 April

3 Tle 1. Definition of Reltive Fitness for Possile Genotypes in Kimur s Equtions Genotype A 1 A 1 1 s A 1 A 2 1 hs A 2 A 2 1 Reltive Fitness A 1 refers to the derived llele, nd A 2 refers to the ncestrl llele. s is the selection coefficient, nd h is the dominnce fctor. Nucleotide differences etween the humn nd chimpnzee genomes could hve risen in two wys. Humns nd chimpnzees my hve inherited different lleles from their common ncestors, s modeled y the first prt of eqution (1). Muttions could lso hve occurred fter the specition event nd could hve susequently reched fixtion, s modeled y the second prt of eqution (1). The fixtion proility of ny muttion p(p,s) cn e clculted in terms of the dominnce prmeter 17 h: p 2cDx(1 x) 2cx e dx 0 p(p,s) p 1, (2) 2cDx(1 x) 2cx e dx 0 where p is the initil mutnt-llele frequency, c p Ns e, nd Dp 2h 1. We solved for s, given h, k, N e, m, t, nd p, y rerrnging equtions (1) nd (2): p 2cDx(1 x) 2cx e dx 0 k p(s) p 1 p. ( 3) 4Nem ( t 2Ne) 2cDx(1 x) 2cx e dx 0 Assuming tht the effective popultion size ws the sme for oth chimpnzee nd humn, we set N e to e 10, Assuming tht ny muttion occurring in the ultrconserved region ws deleterious, we set m to e the verge muttion rte in the humn genome, which ws 2.5# The numer of genertions since the divergence of humn nd chimpnzee ws estimted to e 250,000, with n verge life spn of 20 yers. 20 Assuming tht the muttion leding to new llele ws rre, we set p to e 1/(2N e) p 1/20,000. We used mouse s n outgroup, to determine which llele in chimpnzee nd humn ws the ncestrl llele. The fitness scheme for possile genotypes A 1 A 1,A 1 A 2, nd A 2 A 2 cn e found in tle 1, where A 1 refers to the derived llele nd A 2 refers to the ncestrl llele. Since the rel dominnce fctor, h, ws not known, we smpled different vlues of h tht represent different selection models (tle 2). To determine the numer of fixed differences in the ultrconserved elements etween humn nd chimpnzee genomes, we compred ech of the 481 ultrconserved sequences with the chimpnzee genome (Novemer 2003 version) y using the BLAT tool (UCSC Genome Browser). 2,10 When BLAT did not yield hits, we checked whether the queried sequences were situted in gps etween contigs or supercontigs. We found one ultrconserved element (UC 294) to e completely missing in the chimpnzee ssemly. There ws one contig (contig 36351) spnning the entire region round UC 294, ut BLAT could not identify ny sequence on tht contig tht ws homologous to UC 294. We looked for UC 294 in 10 chimpnzee DNA smples, using PCR with primers internl to this element, nd showed tht this element ws present in the chimpnzee even though the sequence ws not identified in the Novemer 2003 ssemly. We did not include ny ses tht were deleted or inserted in the humn genome reltive to the chimpnzee genome, since mny insertions nd deletions were likely to e errors in the genome ssemlies, such s the UC 294 ssemly error descried ove. The sustitution rte ws clculted y dividing the numer of fixed differences y the totl numer of ligned ses etween the two genomes; it ws 1.16 # Assuming tht ll ultrconserved elements evolved s one llele nd tht the sme selection force ws cting on ech se in these regions, we solved 3 for ŝ, using eqution (3) with 1.16 # 10 s the sustitution rte. We then relxed these ssumptions y clculting selection coefficients for ech of 481 ultrconserved elements, using the sustitution rtes derived for ech element. We performed the clcultions in Mthemtic. To interpret the vlues of selection coefficients, we clculted gˆ s gˆ p Ns e ˆ nd estimted the rtio of expected numers of replcement differences etween the two species under the selected model nd neutrl model to e 2g/(1 ˆ e 2gˆ ) t the present time. 21 Clcultion of Selection Coefficients with the Use of Humn Polymorphisms We employed the Poisson rndom field frmework to model polymorphisms in these sequences nd to clculte the mximumlikelihood estimte of selection coefficients. 22 To ccommodte different smple sizes for ech SNP tht we genotyped nd to estimte selection coefficients given the dominnce fctor, severl modifictions were mde. Let Tle 2. Dominnce Fctor (h) Definition of Dominnce Models Positive Selection Coefficient Negtive 1 Underdominnce Overdominnce 0 A 1 recessive, A 2 dominnt A 1 dominnt, A 2 recessive.5 Incomplete dominnce Incomplete dominnce 1 A 1 dominnt, A 2 recessive A 1 recessive, A 2 dominnt 2 Overdominnce Underdominnce n 1 n 1 i i n 1 ip1 ip1 [ ] F(n,i; g,h) l(g,hfx) p ln (n!) ln (x!) x ln, F(n,j; g,h) s defined in the work of Willimson et l., 22 where g p 2Ns e for diploid orgnisms, h is the dominnce fctor, n is the totl numer of lleles (constnt for ech SNP), i is the numer of lleles with ncestrl SNPs (different for ech SNP), nd x i is the numer of SNP hving i copies in n lleles. A 1 refers to the derived llele, nd A 2 refers to the ncestrl llele. jp1 694 The Americn Journl of Humn Genetics Volume 80 April

4 To optimize l(g,hfx), we needed to optimize n 1 ip1 [ ] F(n,i; g,h) xi ln n 1. F(n,j; g,h) jp1 To ccommodte different smple sizes for ech SNP, we rewrote the ove s S n 1 kp1 [ ] F(n k,i k;g,h) ln, F(n k,j k;g,h) jp1 where S is the totl numer of SNPs, n k is the totl numer of lleles for SNP k, nd i k is the numer of ncestrl lleles tht SNP k hs. Insted of optimizing l(g,hfx), we optimized l(gfh,x). This ws equivlent to optimizing S n 1 kp1 [ ] F(n k,i k;g,h) ln, F(n k,j k;g,h) jp1 since l(g,hfx) l(gfh,x). The optimiztion ws performed in C progrm with the use of different h vlues, s listed in tle 2. Similr modifictions were done to the equtions to clculte the 95% CI for gˆ. We first clculted gˆ for the ultrconserved elements s single llele nd then clculted ĝ only for those elements tht hrored SNPs. In this nlysis, mouse ws not n pproprite outgroup to use to determine which llele in the humn popultion ws the ncestrl llele. Muttions in these elements could hve occurred nd fixed on the linege leding to humns, fter humns nd rodents diverged. Insted, we used the chimpnzee s n outgroup. The fitness scheme cn e found in tle 3. Clcultions of Proilities of Oserving t Lest 12 Frequent SNPs under Wek nd Strong Selection We compred the proilities of oserving t lest 12 frequently derived SNPs under wek nd strong selection, given rnge of the totl numer of SNPs within the ultrconserved elements in the popultion, using ll possile llele-frequency distriutions of SNPs nd rnge of dominnce models. First, we estimted the proility of SNP found t ech frequency in popultion of 1,000, using F(n,i; g,h) n 1, F(n,j; g,h) jp1 Tle 3. Definition of Reltive Fitness for Possile Genotypes Employed in the Poisson Rndom Field Model Genotype Reltive Fitness A 1 A 1 1 2s A 1 A 2 1 2hs A 2 A 2 1 A 1 refers to the derived llele, nd A 2 refers to the ncestrl llele. s is the selection coefficient, nd h is the dominnce fctor. s defined in the work of Willimson et l., 22 where n ws the smple size, i ws the frequency of the derived SNP in the smple, g ws the selection coefficient, nd h ws the dominnce fctor. A popultion size of 1,000 ws pproprite, since ll SNPs in our smple were genotyped in 11,000 individuls. Since very wek selection hs een defined s FgF 1, nd strong selection hs een defined s FgF k 1, 23,24 we chose g p 1 nd 5 to represent wek nd strong purifying selection, respectively, in our nlysis. As efore, we used rnge of dominnce models: h p 1, 0, 0.5, 1, nd 2. We defined SNP s frequent if its frequency in the popultion is 5%. The comintoril lowup of considering ll possile rrngements of SNPs with frequencies from 1% to 99% in 12 different positions necessitted this simplifiction. Then, the proility of SNP eing rre ws computed s the sum of proilities of SNP eing found in!5% of the individuls. Since the totl numer of SNPs in the ultrconserved regions ws not known, we used totl SNPs in our clcultions. The proility of oserving t lest 12 frequent SNPs ws clculted using the cumultive inomil distriution. Comprison of Essentil Genes with the Ultrconserved Elements We used the mmmlin phenotype rowser t Jckson Lortory to select exons tht, when replced with null lleles, led to emryonic lethlity during fetl growth or development. The humn homologues of these exonic sequences were identified, nd the nucleotide sequences were used s queries to identify the homologous sequences in the chimpnzee genome with the use of the BLAT tool (UCSC Genome Browser). 10,25 We removed ny se tht ws ligned to gp in either of the two genomes efore counting the numer of different ses etween the genomes. Since we ssumed tht there were no neutrl ses in the ultrconserved elements, we removed the third position of every codon in the essentil genes, to mke certin tht ech se in the essentil genes ws under selection. For ech ultrconserved element nd essentil gene tht ws not polymorphic, we clculted the selection coefficient, using eqution (3), s detiled erlier with h p 0.5, nd plotted the distriutions of selection coefficients in diffusion time scle ( ĝ p Ns e ). We used the Mnn- Whitney test to compre the distriutions of selection coefficients etween ultrconserved elements nd essentil genes. Lists of essentil genes nd ultrconserved elements cn e found in tles A3 nd A4. To ssess the overrepresenttion of nonexonic ultrconserved elements with high frequencies of derived lleles, we used the hypergeometric distriution. Results We investigted whether chnges in the nucleotide sequences of ultrconserved elements re tolerted y exmining the distriution of verified SNPs in these regions in the humn genome. At the time when ultrconserved elements were found, only six vlidted SNPs were re- The Americn Journl of Humn Genetics Volume 80 April

5 Tle 4. SNP Frequencies of SNPs in the Ultrconserved Regions Ultrconserved Element In Totl Smple With Homozygous Ancestrl Alleles No. of People With Heterozygous Alleles P for HWE With Homozygous Derived Alleles MCMC (SD) EXACT c rs ,361 1, (.080).0015 (6.45#10 5 ).0015 rs (.746).28 (1.79#10 3 ).28 rs (.053).14 (6.14#10 4 ).14 rs (.161).49 (1.25#10 3 ).49 rs (.402).75 (1.36#10 3 ).81 rs (.341).73 (1.32#10 3 ).80 rs (.144).54 (1.08#10 3 ).65 rs (.140).54 (1.09#10 3 ).64 rs NP NP NP NP NP rs NP NP NP NP NP rs (.417).11 (1.59#10 3 ).11 rs NP NP NP NP NP rs (.167).35 (1.33#10 3 ).35 rs (.325).13 (1.60#10 3 ).15 rs (.296).37 (2.05#10 3 ).42 rs NP NP NP NP NP NOTE. NP p not polymorphic. Vlues in prentheses re derived-llele frequencies in the smple. Uses the MCMC method to estimte the P vlue for the null hypothesis tht two lleles re in HWE. c Uses implementtions to estimte the P vlues specificlly for the illelic SNPs. ported in these sequences. 6 In our study, we found 102 SNPs recorded in dsnp, of which were verified y two or more reserch groups. Two pproches were used to determine the significnce of oserving, t most, 24 SNPs in the ultrconserved elements. With ckground density of 1.84 vlidted SNPs per 1,000 nucleotides in the humn genome, we clculted the proility of oserving, t most, 24 SNPs in the ultrconserved elements 68 to e 1.14 # 10, using cumultive Poisson sttistics. With the ssumption tht ll 102 SNPs re vlidted, the P vlue increses to 2.74 # 10 22, which remins significntly lower thn the genome verge. We lso generted the empiricl frequency distriution of vlidted SNPs in the genome y rndomly smpling 100,000 sets of genomic regions tht mtched the length distriution of ultrconserved elements nd counting the numer of vlidted SNPs in ech set. The numer of SNPs in ech set rnged from 140 to 341, with n verge of 203. This suggests tht the proility of oserving, t most, 24 SNPs in the ultrconserved elements is!10 5. With the ssumption tht ll 102 SNPs re vlidted, the P vlue remins!10 5. The pucity of SNPs in ultrconserved regions is consistent with the high conservtion of these sequences etween humns nd rodents. Ancestrl Alleles of Ultrconserved Elements Are Not Required for Norml Development in Humns If the perfect conservtion of ultrconserved elements cross species is due to purifying selection, nd if muttions in these sequences re deleterious, then some of the polymorphisms in these sequences in humn popultions my e deleterious recessive muttions. This hypothesis predicts tht, given the frequency of SNP in n ultrconserved region, there should e n excess of heterozygotes nd corresponding shortge of derived-llele homozygotes. Ancestrl lleles of ultrconserved elements re defined s lleles tht re found in the rodent reference genomes, wheres the derived lleles re lleles tht re different from those in the rodents. To determine whether strong purifying selection cts on the derived lleles, we genotyped rndom smple of 1600 phenotypiclly norml, unrelted humns from two sets of SNPs: one composed of 16 SNPs in ultrconserved elements nd nother set of 16 SNPs locted in the neutrl regions flnking the ultrconserved elements (tle A1). The distriutions of heterozygotes etween these distinct sets of SNPs were compred. Four of the 16 SNPs in the ultrconserved elements were not polymorphic in the smpled popultion (tle 4). For ech of the 12 polymorphic SNPs tht lie in n ultrconserved element, we found t lest one individul homozygous for the derived llele. Derived-llele homozygotes therefore do not cuse emryonic lethlity nd do not necessrily show gross oservle phenotypic normlities. We hypothesized tht, if ultrconserved elements re currently under strong selection, we might e le to detect it y testing whether the ncestrl nd derived lleles re in HWE. If homozygous derived lleles cuse emryonic lethlity or confer survivl disdvntges compred with homozygous ncestrl lleles, then the frequencies of the lleles in the smpled popultion would devite from HWE. Using Fisher s exct test, we oserved only one SNP (rs ) out of HWE (tle 4). For this SNP, the numer of individuls with the derived lleles ws fewer thn expected, which implies tht purifying selection my e currently cting on this locus. 696 The Americn Journl of Humn Genetics Volume 80 April

6 To exmine whether the SNPs in ultrconserved elements re more likely to e out of HWE, we exmined the genotype distriutions of SNPs djcent to the ultrconserved regions. Only 12 of the 16 SNPs genotyped were included in the finl nlysis. We identified individuls with two copies of the derived llele for ll 12 SNPs except one (rs ) (tle A2). However, this SNP, rs , ws determined to e in HWE, suggesting tht the oserved lck of individuls with homozygous derived lleles is due to the low frequency of the derived llele. Of 12 SNPs tested, only 1 (rs471578) ws not in HWE. This is pproximtely the sme rte of occurrence s the SNPs within the ultrconserved elements. Thus, SNPs in ultrconserved elements re not more likely to e out of HWE thn re SNPs outside these regions. Our dt rgue ginst strong, ongoing selection on ultrconserved regions ut do not rule out the possiility tht wek selection cts on these elements nd mintins their high conservtion. Fixed Differences Are Found in Ultrconserved Regions etween the Humn nd Chimpnzee Genomes We next investigted whether ultrconserved elements tolerte chnges y exmining their homologues in the chimpnzee genome. With the ssumption tht the ultrconserved elements re mintined y purifying selection, ny functions of these elements will likely e the sme in chimpnzees nd humns. We therefore expected tht these sequences would e perfectly conserved in the chimpnzee genome, s they re in the humn, mouse, nd rt genomes. Using BLAT 2 to identify homologues of ll 481 ultrconserved elements in the reference chimpnzee genome, we found there were 141 se differences mong the 121,830 ligned ses. The verge sustitution rte ws 1.16 # 10 sustitutions per se, 10-fold 3 lower thn the verge rte of sustitution etween humn nd chimpnzee. Even this low rte of sustitution ws unexpected, given tht ultrconserved elements were identified ecuse they lcked ny sustitutions mong the reference humn, mouse, nd rt genomes. Ultrconserved Elements Are under Negtive Selection We sought to determine wht level of selection is consistent with the oservtions tht ultrconserved elements exhiit oth polymorphisms within the humn popultion nd fixed differences etween humns nd chimpnzees. Becuse our ojective ws to clculte the verge mgnitude of selection on ultrconserved elements nd not on individul nucleotides, we treted ech nucleotide in the elements s eing under the sme level of selection. We clculted the strength of selection, using two methods. First, we estimted selection coefficients, using the numer of fixed differences etween humn nd chimpnzee genomes cross the entire set of ultrconserved elements. Using mouse s n outgroup, we defined the ncestrl llele s the one identicl to the llele in the mouse reference genome. We employed Kimur s equtions, 16,17 mking the ssumption tht ny muttion in these elements tht occurred fter specition of the humn nd chimpnzee hd sufficient time to either ecome fixed or dispper. Since the mgnitude of the selection coefficient ws confounded with the mode of interctions etween two different lleles, we chose five dominnce models representing different modes of interctions nd clculted the selection coefficient for ech model (tle 5). Under ll dominnce models, the selection coefficients (g) re negtive nd rnge from 2.72 to 1.11, which grees with the hypothesis tht the derived lleles of the ultrconserved elements re deleterious. These estimtes of selection coefficients represent the minimum mount of selection required on ech site in every genertion to mintin the oserved sequence conservtion. To pprecite the strength of selection on the ultrconserved elements, we compred the numer of muttions tht re expected to ecome fixed under ech estimted selection coefficient with tht under the neutrl model ( g p 0). If the derived llele is recessive nd the ncestrl llele is dominnt ( h p 0), then the numer of muttions tht would ecome fixed is 20-fold lower thn if the sequences re evolving under the neutrl model (tle 5). Alterntively, if the ncestrl llele is recessive nd the derived llele is dominnt ( h p 1), then this rtio ecomes sevenfold. We lso estimted selection coefficients y using the Poisson rndom field frmework 22 tht incorportes the frequencies of the SNPs in the ultrconserved elements. Mouse ws not n pproprite outgroup to use to determine which llele in the humn popultion ws the ncestrl llele, since muttions in these elements could hve occurred nd fixed on the linege leding to humns fter the split with rodents. Insted, we used chimpnzee s the outgroup for this nlysis. However, in ll positions tht re polymorphic in humns, the chimpnzee lleles were the sme s the rodent lleles. With use of this frmework, g rnged from 3.53 to 0.74 (tle 5). The vrinces of these estimtes re lrge, ecuse of the smll numer of ville SNPs in these regions. Thus, two independent clcultions oth suggest tht, under most dominnce models, the derived lleles re slightly deleterious. Our clcultions, sed on the oserved numer of frequent SNPs in ultrconserved elements, suggest tht these sequences re under wek selection. Becuse our estimtes re sed on genotyping known SNPs nd not on exhustive resequencing of ultrconserved elements, it is possile tht n unknown numer of SNPs hve een missed in our smple. The numer nd frequency distriution of these missed SNPs could ffect our estimtes of the implied selection coefficients in these regions. We therefore computed the proility of oserving t lest 12 frequent SNPs (the numer of frequent SNPs we oserved in our genotyping experiments) under model of either wek or strong selection, ssuming tht there my e 112 ctul SNPs in our smple. The result of these cl- The Americn Journl of Humn Genetics Volume 80 April

7 Figure 1. Comprison of wek nd strong selection, with the ssumption of n intermedite dominnce model ( h p 0.5). Wek nd strong selections were defined s g p 1 nd g p 5, re- spectively. Ech point represents the proility of oserving t lest 12 frequent derived-llele SNPs, with the ssumption of different numers of totl (oserved nd unoserved) SNPs. cultions suggests tht wek selection, rther thn strong selection, is the correct model over very rod rnge of totl possile SNPs (fig. 1). For exmple, we oserved 12 frequent SNPs in our genotyping experiments. dsnp contins n dditionl seven vlidted SNPs t known frequencies nd five more vlidted SNPs t unknown frequencies in these sequences. The totl numer of SNPs is, therefore, likely to e t lest 24. In this rnge, strong selection ( g p 5) is incomptile with our oservtions, nd the likelihood rtio etween the two models suggests tht wek selection ( g p 1) is t lest 20 times more likely to e the correct model thn strong selection, with the ssumption of n intermedite dominnce model. Overll, we tke this nlysis s evidence tht wek selection, rther thn strong, is operting on ultrconserved elements. The nlysis does not rule out strong selection completely, especilly if the totl numer of unoserved SNPs is very lrge. We think tht is unlikely to e the cse ecuse the oserved numer of SNPs in ultrconserved sequences is sixfold lower thn the verge cross the genome. Wek selection is lso consistent with our estimtes from the nlysis of sustitutions in these regions etween humns nd chimpnzees. Genes whose products re essentil for the proper development of n orgnism re ssumed to e under strong purifying selection. We compred the strength of selection cting on the ultrconserved elements with tht cting on essentil genes (tles A3 nd A4). If the strength of selection cting on ultrconserved elements is similr to tht cting on essentil genes, then this would support the notion tht the exceptionlly high conservtion of these sequences reflects importnt, ut currently unknown, functions in the mmmlin lineges. We clculted the strength of selection cting on ech essentil gene nd on ech individul ultrconserved element, using n dditive model ( h p 0.5), nd compred the two distriutions of selection coefficients (fig. 2). As expected, purifying selection ppers to e cting on ll essentil genes. The distriutions of selection coefficients for ultrconserved elements nd essentil genes re significntly different ( P!.0001; Mnn-Whitney s rnk sum test). The results suggest tht, on verge, the mgnitude of puri- Tle 5. Models Dominnce Fctor (h) Strength of Selection Coefficients on the Ultrconserved Elements Clculted for Different Dominnce Selection Coefficient ( ĝ) Kimur Model (Fixtion under g p 0)/ (Fixtion under ĝ) c Selection Coefficient ( ĝ) Poisson Rndom Field Model d 95% Confidence Limits for ĝ d (Fixtion under g p 0)/ (Fixtion under ĝ) c , , , , , Let A 1 e the derived llele, A 2 e the ncestrl llele, nd w(x) e the fitness of individuls with genotype x. When gˆ! 0, h p 1 implies tht w(aa) 1 2 is the highest mong the three genotypes, h p 0 implies tht w(aa)! 1 1 w(aa)p 1 2 w(aa) 2 2, h p 0.5 implies tht w(aa)! 1 1 w(aa)! 1 2 w(aa) 2 2, h p 1 implies tht w(aa)p 1 1 w(aa)1 1 2 w(aa) 2 2, nd h p 2 implies tht w(aa) 1 2 is the lowest mong the three genotypes. When gˆ 1 0, h p 1 implies tht w(aa) 1 2 is the lowest mong the three genotypes, h p 0 implies tht w(aa)1 1 1 w(aa)p 1 2 w(aa) 2 2, h p 0.5 implies tht w(aa)1 1 1 w(aa)1 1 2 w(aa) 2 2, h p 1 implies tht w(aa)p 1 1 w(aa)1 1 2 w(aa) 2 2, nd h p 2 implies tht w(aa) 1 2 is the highest mong the three genotypes. Clculted using fixed differences etween chimpnzee nd humn ultrconserved elements. c Fixtion under g p 0 1 e 2ĝ p, Fixtion under gˆ 2gˆ with the ssumption tht the muttion rtes re the sme under oth the neutrl nd the selected model. d Confidence limits clculted using 19 vlidted SNPs in humn ultrconserved elements. 698 The Americn Journl of Humn Genetics Volume 80 April

8 Figure 2. Distriutions of selection coefficients for ultrconserved elements nd essentil genes. fying selection cting on the ultrconserved elements is weker thn tht cting on the essentil genes. We lso oserved tht most of the fixed differences etween humns nd chimpnzees re concentrted in smll set of ultrconserved elements. Their selection coefficients (g) rnge from 1.45 to 1.35, with medin of We were unle to distinguish sttisticlly whether this smll group of elements represents distinct set of sequences evolving under different selective constrints or whether they re simply the til end of single distriution of selection coefficients encompssing ll ultrconserved sequences (dt not shown). For ultrconserved elements tht re polymorphic in the humn popultion ut show no fixed differences with their chimpnzee counterprts, we clculted selection coefficients, using the Poisson rndom field model (tle 6). Of the 13 elements, 10 contin SNPs with derived lleles t high frequencies. Of those 10 elements, 8 do not overlp exons in humns. 6 We exmined the remining two elements nd found no evidence of overlpping known exons in humns. This result suggests n overrepresenttion of nonexonic ultrconserved elements with derived lleles t high frequencies ( P!.002; hypergeometric distriution). Discussion The sence of solute sequence conservtion etween ultrconserved elements nd their homologues in chimpnzees is unexpected. Since the evolutionry distnce etween chimpnzees nd humns is much shorter thn tht etween rodents nd humns, one might expect tht the sequences of these elements would e preserved in the chimpnzee genome. However, not ll ultrconserved elements re conserved in the reference chimpnzee genome. If purifying selection preserves these sequences through evolution, then the functions of these elements my differ significntly in the chimpnzee. Alterntively, the existence of fixed nucleotide differences etween chimpnzees nd humns in these ultrconserved elements could e product of pst popultion fluctutions. Studies hve suggested tht oth chimpnzee nd humn popultions experienced ottleneck in which the popultion sizes decresed significntly nd then rpidly expnded. 26,27 The decrese in popultion size in oth species would llow severl slightly deleterious muttions to ecome fixed, producing high numer of fixed differences etween the two species. 8 The susequent rpid expnsion in popultion size would likely result in few polymorphic lleles within ech species. The most striking feture of the ultrconserved elements is the presence of so mny consecutive conserved nucleotides. There re no known functionl sequence elements tht require such long stretches of specific sequence. Nucleotide lignments of ORFs, noncoding RNAs, nd trnscription-fctor inding sites ll show chrcteristic ptterns of sustitutions The ultrconserved elements my therefore represent new clss of slowly evolving Tle 6. Selection Coefficients for Ultrconserved Elements Tht Are Polymorphic in Humn Popultions ut Show No Sustitutions with Chimpnzee Homologues Ultrconserved Element Type Selection Coefficient ( gˆ t h p.5) (Fixtion under g p 0)/ (Fixtion under ĝ) c 140 n n d p n n n n n n d p n n e n! As defined y Bejerno et l. 6 Type e suggests the element overlps the mrna of known humn protein-coding gene (including the UTR regions). Type n indictes there is no evidence of trnscription of this element from ny mtching EST or mrna from ny species. Type p refers to elements where evidence of trnscription is inconclusive. Scled s y effective popultion size. Let A 1 e the derived llele, A 2 e the ncestrl llele, nd w(x) e the fitness of individuls with genotype x. When gˆ! 0, h p 0.5 implies tht w(aa)! 1 1 w(aa)! 1 2 w(aa). When gˆ 1 0, h p 0.5 implies tht w(aa)1 w(aa)1w(aa) c Fixtion under g p 0 1 e 2ĝ p, Fixtion under gˆ 2gˆ with the ssumption tht the muttion rtes re the sme under oth the neutrl nd the selected model. d Although these elements re clssified s prtilly exonic y Bejerno et l., 6 we did not find ny evidence of their overlpping known exons in humns. e Progrm does not converge. The Americn Journl of Humn Genetics Volume 80 April

9 sequences tht re constrined t every position. However, since we clculted verge selection coefficients, our dt do not rule out the possiility tht different positions in these elements re under different levels of selection nd tht some positions my e neutrl with respect to fitness. If ultrconserved elements do contin some neutrl positions tht re conserved solely y chnce, then it is likely tht these elements will fll into known functionl sequence clsses. Indeed, one ultrconserved element ws recently shown to e trnscriptionl enhncer. 31 The estimtion of the numer of neutrl ses within ultrconserved elements is n ongoing effort tht involves compring the ptterns of sustitution in multiple mmmlin lineges. Our methods for estimting the verge selection cting on ultrconserved elements my e ffected y ises within dsnp. There my e more SNPs locted in these elements tht hve yet to e found nd documented. In ddition, our pproch of counting only fixed nucleotide differences etween humn nd chimpnzee reference genomes does not tke into considertion the possile existence of insertions or deletions in ultrconserved regions. It is lso possile tht some of these fixed differences my, in fct, e polymorphisms in the chimpnzee popultion. Tken together, these cvets suggest tht our estimtes of selection my e sed on underestimtes of polymorphism in these regions. We therefore rgue tht our estimtes re conservtive upper ounds of the strength of selection on these elements, since incorporting dditionl sequence chnges into our clcultions would likely lower our estimtes even further. Moreover, some frction of the rre polymorphism we oserved could result from pst popultion ottleneck nd susequent expnsion. If this scenrio were true, then the ctul levels of selection on ultrconserved elements re likely to e even weker thn we estimted ecuse some of the polymorphism we oserved is result of rpid popultion expnsion rther thn of purifying selection. The estimtes of selection we hve clculted refer to the verge level of selection cross ech ultrconserved element. The estimtion of selection coefficients for individul ses within ultrconserved elements would tke lignments drwn from hundreds of mmmlin genomes. 32 To circumvent this prolem, popultion genetic studies often ssume n priori distriution of selection coefficients, usully drwn from the gmm distriution. Since the functions of these elements re unknown, we did not know whether gmm would e n pproprite priori distriution. We hve therefore limited our conclusions to the verge levels of selection on ultrconserved elements. Our results show tht selection coefficients s low s % per genertion cn drive nucleotide differences to fixtion on the lineges leding to humns nd chimpnzees nd cn mintin the sequence conservtion of ultrconserved elements in humns. This mgnitude of selection is in greement with recent finding tht very wek selection ppers to e cting on the conserved noncoding regions, defined y the comprison of humn nd mouse reference genomes. 8 This reltively low level of selection is not enough to drive the llele frequencies of polymorphisms out of HWE in surviving dult popultions. Although this estimte of selection coefficients is dependent on the mode of interction etween the ncestrl nd derived lleles, the detection of phenotypes of individuls with derived lleles my e difficult in lortory setting. The fesiility of detecting phenotypic chnges relies on dequte smple size nd the numer of genertions in the study. For most studies in the lortory, selection of 0.1% is lredy elow the current level of detection. 33 In the cse where the minimum selection we clculted in this study cts constntly in every genertion, we should not necessrily expect to oserve ovious phenotypic chnges in individuls with muttions in ultrconserved elements. Acknowledgments We thnk Alison Gote, the COGA Consortium (funded y Ntionl Institute on Alcohol Ause nd Alcoholism nd Ntionl Institute on Drug Ause grnt U10AA0840), the Alzheimer Disese Reserch Center t Wshington University (funded y Ntionl Institutes of Helth grnt P50AG05681 to Dr. John Morris), nd Anne Bowcock, for providing humn nd chimpnzee DNA smples. We lso thnk Quo-Shin Chi, for dvice on solving Kimur s equtions; Scott Willimson, for ccess to his computer progrms nd suggestions on how to modify them; Stn Swyer, for dvice on sttisticl nlysis; Ro Mitr, Gil Bejerno, nd memers of the Cohen L, for helpful discussions; nd Ed Esprz, for proofreding the mnuscript. 700 The Americn Journl of Humn Genetics Volume 80 April

10 Appendix A Tle A1. Linkge Reltionship etween Ech Pir of SNPs inside nd outside the Ultrconserved Region Ultrconserved Region nd SNP Inside SNP outside Ultrconserved Region Loction of Outside SNP Reltive to Inside SNP Two-Tiled P 51: rs rs p downstrem 3.98#10 11 rs rs ,268 p upstrem 1.78#10 11 rs rs p downstrem 3.27# : rs rs p upstrem NA 67: rs rs p upstrem 1.68# : rs rs p downstrem 2.50# : rs rs p upstrem 1.53#10 5 rs rs p upstrem 5.34# : rs rs p downstrem 1.53# : rs12981 rs c 211 p upstrem 1.63# : rs rs p downstrem 1.28# : rs rs p upstrem 5.97#10 5 rs rs p downstrem NA 374: rs rs c 1,159 p downstrem 1.18# : rs rs p downstrem 4.79# : rs rs p upstrem NA Null hypothesis: two SNPs re t linkge equilirium. NA p not ville. One or oth SNPs in the pir re not polymorphic, nd the linkge reltionship cnnot e determined. c These pirs of SNPs re not in linkge disequilirium, nd the SNPs outside the conserved regions in these pirs re excluded from the nlysis. Tle A2. SNP Frequency of SNPs outside the Ultrconserved Elements Nery Ultrconserved Element In Totl Smple With Homozygous Ancestrl Alleles No. of People With Heterozygous Alleles P for HWE With Homozygous Derived Alleles MCMC (SD) EXACT c rs (.349).50 (2.01#10 3 ).56 rs (.653).44 (2.07#10 3 ).44 rs (.628).87 (7.52#10 4 ).94 rs NP NP NP NP NP rs (.903).39 (8.96#10 4 ).52 rs (.173).90 (3.70#10 4 ).90 rs (.0928).38 (9.68#10 4 ).38 rs (.0674).76 (3.71#10 4 ).76 rs (.104) 1 (0) 1 rs d (.0230) (7.55#10 5 ).0043 rs (.544).079 (1.38#10 3 ).094 rs (.165).0023 (1.04#10 3 ).0040 rs (.0511).25 (5.29#10 4 ).25 rs d (.0123).1 (3.22#10 4 ).10 rs (.904).82 (3.89#10 4 ) 1 rs NP NP NP NP NP NOTE. NP p not polymorphic. Vlues in prentheses re derived-llele frequencies in the smple. Uses the MCMC method to estimte the P vlue for the null hypothesis tht two lleles re in HWE. c Uses implementtions to estimte the P vlues specificlly for the illelic SNPs. d These SNPs re excluded from nlysis since they re not in linkge disequilirium with the SNPs locted within the ultrconserved regions. 701

11 Tle A3. Gene Essentil Genes nd Their Selection Coefficients GenBnk Accession Numer Gene Description Loction Selection Coefficient ( gˆ t h p.5) APAF1 NM_ Apoptotic peptidse ctivting fctor 1 Chr12: C1GALT1 NM_ Core 1 synthse, glycoprotein-n-cetylglctosmine Chr7: et-glctosyltrnsferse, 1 CITIED2 NM_ Cp/p300-intercting trnsctivtor, with Chr6: Glu/Asp-rich croxy-terminl domin, 2 EDG1 NM_ Endothelil differentition, sphingolipid G- Chr1: protein-coupled receptor EPO NM_ Erythropoietin Chr7: IKBKB NM_ Inhiitor of kpp light polypeptide gene Chr8: LIG4 NM_ DNA ligse IV Chr13: MEN1 NM_ Menin isoform 1 Chr11: MGAT2 NM_ Mnnosyl (lph-1,6-)-glycoprotein Chr14: MTF1 NM_ Metl-regultory trnscription fctor 1 Chr1: MYB NM_ V-my myelolstosis virl oncogene homolog Chr6: NCOR1 NM_ Nucler receptor corepressor 1 Chr17: NDST1 NM_ N-decetylse/N-sulfotrnsferse 1 Chr5: NF1 NM_ Neurofiromin 1 Chr17: NFAT5 NM_ Nucler fctor of ctivted T-cells 5 Chr16: PTHR1 NM_ Prthyroid hormone receptor 1 Chr3: RCE1 NM_ Prenyl protein peptidse Chr11: SERPINC1 NM_ Serpin peptidse inhiitor, clde C, memer 1 Chr1: TULP3 NM_ Tuy like protein 3 Chr12: VEGFC NM_ Vsculr endothelil growth fctor C Chr4: NOTE. Let A 1 e the derived llele, A 2 e the ncestrl llele, nd w(x) e the fitness of individuls with genotype x. When ĝ! 0, h p 0.5 implies tht w(a 1 A 1 )! w(a 1 A 2 )! w(a 2 A 2 ). When gˆ 1 0, h p 0.5 implies tht w(a 1 A 1 ) 1 w(a 1 A 2 ) 1 w(a 2 A 2 ). These coordintes refer to the gene positions in the Humn My 2004 (hg17) ssemly on the UCSC Genome Browser. Chr p chromosome. Scled s y effective popultion size. 702

12 Tle A4. Ultrconserved Elements Tht Show Sustitutions with Chimpnzee Homologues Ultrconserved Element Type Selection Coefficient ( gˆ t h p.5) 7 p n p n n n p n n n n n n n n n n p n n e p n p n e e e n n n n n n n n n e p p n p n n n e p p n e p n n e e p n n n 1.09 (continued) Tle A4. We Resources Ultrconserved Element (continued) Type Selection Coefficient ( gˆ t h p.5) 388 n n n e n e n p n n e p n n e e 1.35 NOTE. Let A 1 e the derived llele, A 2 e the ncestrl llele, nd w(x) e the fitness of individuls with genotype x. When gˆ! 0, h p 0.5 implies tht w(a 1 A 1 )! w(a 1 A 2 )! w(a 2 A 2 ). When gˆ 1 0, h p 0.5 implies tht w(a 1 A 1 ) 1 w(a 1 A 2 ) 1 w(a 2 A 2 ). As defined y Bejerno et l. 6 Type e suggests the element overlps the mrna of known humn protein-coding gene (including the UTR regions). Type n indictes there is no evidence of trnscription of this element from ny mtching EST or mrna from ny species. Type p refers to elements where evidence of trnscription is inconclusive. Scled s y effective popultion size. The URLs for dt presented herein re s follows: BLAT, dsnp, GenBnk, (for genes in tle A3) Sequenom Inc., The Jckson Lortory, UCSC Genome Browser, References 1. Chiromonte F, Weer RJ, Roskin KM, Diekhns M, Kent WJ, Hussler D (2003) The shre of humn genomic DNA under selection estimted from humn-mouse genomic lignments. Cold Spring Hr Symp Qunt Biol 68: Wterston RH, Lindld-Toh K, Birney E, Rogers J, Aril JF, Agrwl P, Agrwl R, Ainscough R, Alexndersson M, An P, et l (2002) Initil sequencing nd comprtive nlysis of the mouse genome. Nture 420: Bejerno G, Hussler D, Blnchette M (2004) Into the hert of drkness: lrge-scle clustering of humn non-coding DNA. Bioinformtics Suppl 1 20:I40 I48 The Americn Journl of Humn Genetics Volume 80 April