Recessive inheritance of gene disrupting alleles, either through homozygosity at a specific site or compound heterozygosity, have been demonstrated to underlie many Mendelian diseases and some complex psychiatric disorders. On the basis of exome sequencing data, an increased burden of complete knockout (homozygous or compound heterozygous) variants has been identified in autism. In addition, using single-nucleotide polymorphism microarray data, an increased rate of homozygosity by descent, or autozygosity, has been linked to the risk of schizophrenia (SCZ). Here, in a large Swedish case-control SCZ sample (11 244 individuals, 5079 of whom have exome sequence data available), we survey the contribution of both autozygosity and complete knockouts to disease risk. We do not find evidence for association with SCZ, either genome wide or at specific loci. However, we note the possible impact of sample size and population genetic factors on the power to detect and quantify any burden that may exist

Genovese, Giulio

Holmans, Peter

Hultman, Christina M

Lim, Elaine T

McCarroll, Steven A

Moran, Jennifer L

Purcell, Shaun M

Ruderfer, Douglas M

Sklar, Pamela

Sullivan, Patrick F

PubMed

Carolina Digital Repository

SHORT REPORTNo evidence for rare recessive and compoundheterozygous disruptive variants in schizophreniaDouglas M Ruderfer1,2,3, Elaine T Lim4, Giulio Genovese3, Jennifer L Moran3, Christina M Hultman5,Patrick F Sullivan6, Steven A McCarroll3,7, Peter Holmans8, Pamela Sklar1,9 and Shaun M Purcell*,1,2,3,4Recessive inheritance of gene disrupting alleles, either through homozygosity at a specific site or compound heterozygosity,have been demonstrated to underlie many Mendelian diseases and some complex psychiatric disorders. On the basis of exomesequencing data, an increased burden of complete knockout (homozygous or compound heterozygous) variants has beenidentified in autism. In addition, using single-nucleotide polymorphism microarray data, an increased rate of homozygosity bydescent, or autozygosity, has been linked to the risk of schizophrenia (SCZ). Here, in a large Swedish case-control SCZ sample(11 244 individuals, 5079 of whom have exome sequence data available), we survey the contribution of both autozygosity andcomplete knockouts to disease risk. We do not find evidence for association with SCZ, either genome wide or at specific loci.However, we note the possible impact of sample size and population genetic factors on the power to detect and quantify anyburden that may exist.European Journal of Human Genetics (2015) 23, 555–557; doi:10.1038/ejhg.2014.228; published online 5 November 2014INTRODUCTIONSchizophrenia (SCZ) is a highly heritable (h2 ~ 0.6–0.8), debilitatingdisorder with an increasing number of implicated genetic loci1–7 fromgenome-wide association studies (GWAS) of common single-nucleotide polymorphisms (SNPs) and rare copy number variants(CNVs). Recently, large-scale exome-sequencing studies have searchedfor rare variants that strongly increase risk,8,9 although the relativelyhigh baseline rate of rare neutral coding variation makes thischallenging.10 To focus on rare variants that are a priori more likelyto be highly penetrant for disease, one approach is to study de novomutations, as these are effectively uncensored by selection pressures.11A second strategy, adopted here, is to focus on the rare gene-disruptivevariants, defined as nonsense, essential splice site or frameshift, whereboth copies of a gene are affected through homozygosity at a single siteor compound heterozygosity (if an individual carries differentdisruptive variants on both the maternally and paternally derivedcopies of the gene). With respect to any one gene, we refer to these as‘complete knockout’ variants. A recent study in autism reportedcomplete knockout variants at a rate of 1 for every 20 healthyindividuals (0.05 per person) with a twofold higher rate in cases.12Whereas sequencing allows for direct discovery of rare completeknockout genotypes, rates of autozygosity (homozygosity by descent)can also be informative under recessive models. Previous studies haveused SNP microarrays to identify multi-megabase runs of homo-zygosity (ROH), that likely reflect autozygosity arising from a degree ofrecent inbreeding.13,14 A study of 9388, predominantly European, SCZcases and 12 456 controls demonstrated a significant positivecorrelation between the extent of ROH per individual and the diseaserisk.15 However, they did not identify any significant individual loci.Here we apply both approaches in a large Swedish population-basedsample8,16 of SCZ cases and controls: we first test the recessive modelindirectly by comparing the SNP-derived ROH burden in cases versuscontrols, and second, we use exome sequencing to assess directly theburden of complete knockout variants.METHODSSample ascertainment and data generationSamples were collected from national registers in Sweden, cases were identifiedthrough the Swedish Hospital Discharge Register and controls were randomlyselected from Swedish population registers. All subjects were appropriatelyconsented and the Institutional Human Subject Committees approved theresearch. Sequencing was performed at the Broad Institute using Illumina (SanDiego, CA, USA) GAII or HiSeq2000 machines. Exome capture was done usingeither version 1 or version 2 of the Agilent (Santa Clara, CA, USA) SureSelectHuman All Exon Kit. Additional detail on sample ascertainment and sequencedata generation has been previously published.8 BAM and VCF files areavailable in the dbGaP study phs000473.v1 (http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000473.v1.p1).Runs of homozygosity callingWe utilized previously published16 SNP imputation data of the entire Swedishdata set representing a superset of the individuals that have been sequenced. Weselected ~ 55 k SNPs in approximate linkage equilibrium from high-qualitygenotypes called from imputed dosages in 5001 cases and 6243 controls. Wesought to detect ROH using PLINK17 with parameter settings ROH length41Mb, at least one SNP per 100 kb and allowance of a single heterozygousevent within a window of 50 SNPs, splitting calls if spanning 500 kb with no1Division of Psychiatric Genomics, Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, USA; 2Institute for Genomics and Multiscale Biology, IcahnSchool of Medicine at Mount Sinai, New York, NY, USA; 3Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA; 4Analytic andTranslational Genetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Massachusetts General Hospital, Boston, MA, USA; 5Department of Medical Epidemiology andBiostatistics, Karolinska Institutet, Stockholm, Sweden; 6Department of Genetics, University of North Carolina, Chapel Hill, NC, USA; 7Department of Genetics, Harvard MedicalSchool, Boston, MA, USA; 8Medical Research Council Centre for Neuropsychiatric Genetics and Genomics, Institute of Psychological Medicine and Clinical Neurosciences, CardiffUniversity, Cardiff, UK; 9Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA*Correspondence: Dr SM Purcell, Department of Psychiatry, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy Pl, New York, NY 10029, USA. E-mail: shaun.purcell@mssm.eduReceived 10 March 2014; revised 16 July 2014; accepted 19 September 2014; published online 5 November 2014European Journal of Human Genetics (2015) 23, 555–557& 2015 Macmillan Publishers Limited All rights reserved 1018-4813/15www.nature.com/ejhgmarker. This method and parameter selection represents those suggested in theliterature after a careful comparison18 and is very similar to the approach usedpreviously on a subset of these data.15Compound heterozygous identificationTo evaluate the role of complete knockout variants in SCZ we implementeda method to identify these variants within the framework of the statisticalpackage Plink/Seq (http://research.mssm.edu/statgen/software.html). Themethod analyzes each transcript independently, ensuring that both thedisruptive variants are affecting the same protein. As most pairs of variantswill not be within the distance necessary to phase (length of sequencing read),we employed a previously used statistical phasing approach to identifycompound heterozygous events.12 We defined compound heterozygosity asan individual carrying a heterozygous genotype on each chromosome atdifferent locations within the same gene.Assessing significance using permutationSignificance of complete knockout variants was assessed by permutingindividual alleles independently assuming Hardy–Weinberg equilibrium(HWE). Permutation was performed within 296 groups of individuals matchedon IBS from GWAS data to control for subtle population substructure (averagenumber of individuals per group= 17). We assessed the significance of gene setsby summing across multiple variants assuming independence.RESULTSAmong 5001 cases and 6243 controls, we identified 4117 ROH in 2001individuals; 16% of individuals carried at least one ROH. The averagelength of ROH was 8Mb (median length of 6.8Mb) and ranged from1.6 to 55.2Mb. We also quantified each individual’s genome-widedegree of homozygosity using Wright’s inbreeding coefficient (F).After including the first 10 multidimensional scaling (MDS) compo-nents to account for ancestry, homozygosity was similar in cases andcontrols as assessed by the number of ROH (0.38 cases, 0.32 controls,P= 0.60), total kilobases of ROH (3123 kb cases, 2544 kb controls,P= 0.88) or F (0.0026 cases, 0.0029 controls, P= 0.09, Table 1).We next looked for complete knockout variants in a subset of 2477cases and 2481 controls for whom exome sequence data wereavailable.8 We identified 462 complete knockout rare (minor allelefrequency o5%) genotypes at 193 genomic locations, a rate of 0.09per person with no increased burden in cases (229 case; 233 control,Table 2). Of these, 405 were homozygous and 57 were compoundheterozygous, with no significant enrichment in either class. Analysisof nonsynonymous and silent complete knockout variants yieldedsimilar null results (data not shown).Testing each site individually for association with increased risk(one-sided), 30 (16%) had Po0.05 but none surpassed the Bonferronicorrection for multiple testing (Supplementary Table S1). This excessof nominally significant tests is a product of testing only those sitescarrying at least one complete knockout genotype: simulating nullgenotype data under HWE using the observed allele frequencies, wefound a similar proportion of nominally significant sites whenfollowing a similar analytic procedure (data not shown).For the X chromosome, males and females were analyzed separately.A single hemizygous disruptive variant in males results in a completeloss of gene function as in the autosomal complete knockout scenario.Using a minor allele frequency cutoff of 0.25%, we observed 49disruptive sites outside of the pseudoautosomal regions, a burden of41 in cases and 41 in controls (one sided Fisher’s exact test P= 0.5).No individual site achieved nominal significance (SupplementaryTable S2).Finally, we selected gene sets previously demonstrated to have asignificant polygenic burden of rare disruptive alleles (primarilyobserved as a single heterozygous genotype) in the same set ofindividuals.8 Across 2546 genes in seven sets, we identified 35complete knockout or male X chromosome disruptive genotypes, 19in cases and 16 in controls (P= 0.38; Supplementary Table S3).Although the set of genes carrying disruptive de novo alleles in SCZdisplayed nominal significance (4:0, one-sided P= 0.027), this doesnot survive correction for the seven tests. We also considered geneswith known syndromic autosomal recessive causes of autism orintellectual disability.19 Three case genotypes and five control geno-types were found across 119 intellectual disability genes and one casegenotype and zero control genotypes were found across 28 autismgenes; neither test reached nominal significance (P intellectualdisability= 0.72, P autism= 0.57).DISCUSSIONOverall, we did not observe a significant enrichment of ROH in SCZcases. In a larger sample (21 844), Keller et al.15 reported a significantlyhigher burden of ROH in SCZ cases, suggesting that the current studymay be under-powered. We note that the Keller et al. study included asmall proportion of the Swedish individuals reported here, althoughthe authors did not find evidence for increased ROH in the Swedishsubset, consistent with our results. This suggests that populationgenetic and ancestry characteristics of a sample could be a furtherdeterminant of the relative role of rare recessive variants in the riskfor SCZ.The amount of recent inbreeding will contribute to the levels ofhomozygosity present in a population. Our sample was ascertainedfrom Sweden, but includes a proportion of individuals of Finnishdescent. Utilizing populations with recent inbreeding will increase thefrequency of these events and the power to detect enrichment, anapproach that has been used successfully for CNVs.20In conclusion, this sample does not lend support for a major role ofdisruptive recessive or compound heterozygous variants in the riskof SCZ. There was no overall burden of ROH genome wide or ofcomplete knockout variants exome wide, no specific genomic site wassignificantly associated and no gene achieved significance aftercorrection. These results largely mirror those of de novo SCZ triostudies (Fromer et al.11), in which there is no significantly increasedTable 1 Summary of ROH and inbreeding coefficient (F) in fullSwedish GWAS sample, P-values are from a logistic regression with10 MDS components and wave as covariatesCases Controls P-valueN 5001 6243ROH rate 0.38 0.32 0.60ROH (kb) 3123 2544 0.88F 0.0026 0.0029 0.09Table 2 Summary of two-hit disruptive genotypesCase ControlN 2477 2481Homozygous 200 205Compound heterozygous 29 28All two-hit 229 233Rate 0.09 0.09Recessive and compound heterozygous variants in schizophreniaDM Ruderfer et al556European Journal of Human Geneticsburden overall and very few individual genes are unambiguouslyimplicated, although for de novo mutations, broader effects can beseen across subsets of functionally-related genes. Rare recessive andcompound heterozygous sites may still have a role in disease riskalthough larger samples or different analytic approaches may berequired.CONFLICT OF INTERESTThe authors declare no conflict of interest.1 Bassett AS, Chow EW, AbdelMalik P, Gheorghiu M, Husted J, Weksberg R: Theschizophrenia phenotype in 22q11 deletion syndrome. Am J Psychiatry 2003; 160:1580–1586.2 International Schizophrenia Consortium: Rare chromosomal deletions and duplicationsincrease risk of schizophrenia. Nature 2008; 455: 237–241.3 Ripke S, Sanders AR, Kendler KS et al: Genome-wide association study identifies fivenew schizophrenia loci. Nature genetics 2011; 43: 969–976.4 O'Donovan MC, Craddock N, Norton N et al: Identification of loci associated withschizophrenia by genome-wide association and follow-up. Nat Genet 2008; 40:1053–1055.5 Walsh T, McClellan JM, McCarthy SE et al: Rare structural variants disrupt multiplegenes in neurodevelopmental pathways in schizophrenia. Science 2008; 320:539–543.6 McCarthy SE, Makarov V, Kirov G et al: Microduplications of 16p11.2 are associatedwith schizophrenia. Nat Genet 2009; 41: 1223–1227.7 Lichtenstein P, Yip BH, Bjork C et al: Common genetic determinants of schizophreniaand bipolar disorder in Swedish families: a population-based study. Lancet 2009; 373:234–239.8 Purcell SM, Moran JL, Fromer M et al: A polygenic burden of rare disruptive mutationsin schizophrenia. Nature 2014; 506: 185–190.9 Kiezun A, Garimella K, Do R et al: Exome sequencing and the genetic basis ofcomplex traits. Nat Genet 2012; 44: 623–630.10 Zuk O, Schaffner SF, Samocha K et al: Searching for missing heritability: Designingrare variant association studies. Proc Natl Acad Sci USA 2014; 111: E455–E464.11 Fromer M, Pocklington AJ, Kavanagh DH et al: De novo mutations in schizophreniaimplicate synaptic networks. Nature 2014; 506: 179–184.12 Lim ET, Raychaudhuri S, Sanders SJ et al: Rare complete knockouts in humans:population distribution and significant role in autism spectrum disorders. Neuron2013; 77: 235–242.13 Lencz T, Lambert C, DeRosse P et al: Runs of homozygosity reveal highly penetrantrecessive loci in schizophrenia. Proc Natl Acad Sci USA 2007; 104: 19942–19947.14 Power RA, Keller MC, Ripke S et al: A recessive genetic model and runs ofhomozygosity in major depressive disorder. Am J Med Genet B Neuropsychiatr Genet2014; 165: 157–166.15 Keller MC, Simonson MA, Ripke S et al: Runs of homozygosity implicate autozygosity asa schizophrenia risk factor. PLoS Genet 2012; 8: e1002656.16 Ripke S, O'Dushlaine C, Chambert K et al: Genome-wide association analysis identifies13 new risk loci for schizophrenia. Nat Genet 2013; 45: 1150–1159.17 Purcell S, Neale B, Todd-Brown K et al: PLINK: a tool set for whole-genome associationand population-based linkage analyses. Am J Hum Genet 2007; 81: 559–575.18 Howrigan DP, Simonson MA, Keller MC: Detecting autozygosity through runs ofhomozygosity: a comparison of three autozygosity detection algorithms. BMC Genomics2011; 12: 460.19 Betancur C: Etiological heterogeneity in autism spectrum disorders: more than 100genetic and genomic disorders and still counting. Brain Res 2011; 1380: 42–77.20 Morrow EM, Yoo SY, Flavell SW et al: Identifying autism loci and genes by tracingrecent shared ancestry. Science 2008; 321: 218–223.Supplementary Information accompanies this paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)Recessive and compound heterozygous variants in schizophreniaDM Ruderfer et al557European Journal of Human Genetics

No evidence for rare recessive and compound heterozygous disruptive variants in schizophrenia

Ruderfer, Doug

Holmans, Peter Alan

Online Research @ Cardiff

https://cdr.lib.unc.edu/downloads/q237j0236

No evidence for rare recessive and compound heterozygous disruptive variants in schizophrenia

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