16 August 2011. The recent rise of whole-exome methods, which allow rapid and fairly inexpensive “deep sequencing” of all protein-coding regions in search of previously undetected genetic variants, promises to powerfully broaden the scope of psychiatric genomics beyond the common single-nucleotide variants sought in genomewide association studies (GWAS), and the large and rare structural anomalies targeted in copy number variation (CNV) studies. Indeed, based on their newly published exome-sequencing study, Maria Karayiorgou and Joseph Gogos of Columbia University in New York City and colleagues make the remarkable claim that rare, de novo single-point mutations and small indels (insertions and deletions) of the sort identified in their work may “account for more than half of the cases of sporadic schizophrenia.”
Whether or not this will prove accurate, the “mixed economy” of schizophrenia genomics (see SRF related news story) continues to thrive, as reflected in the variety of approaches and theoretical perspectives of several other recent studies.
A common cause
GWAS and the search for common genetic variants—generally defined as present in at least 5 percent of a population—are alive and kicking, as evidenced by two European studies published from overlapping consortia.
Some shortcomings of GWAS done so far are that sample sizes have been too small to assign statistical significance to many identified single-nucleotide polymorphisms (SNPs), or that potentially interesting SNP signals may have been “washed out” by the ethnic or diagnostic heterogeneity of many samples studied so far.
In a study published in Molecular Psychiatry, Marcella Riestchel of the University of Heidelberg, Mannheim; Sven Cichon of the University of Bonn; and colleagues throughout Germany and elsewhere in Europe aimed to address the latter concern by conducting a GWAS of well-characterized schizophrenia patients exclusively from Germany and The Netherlands (464 and 705, respectively) and 3,714 ethnically matched controls from the same countries.
No SNP reached genomewide significance in the first GWAS, which the authors ascribe to “insufficient power.” The group analyzed the top 43 SNP results in a separate sample of 2,569 patients and 4,088 controls from Germany, Holland, and Denmark, and found “nominal significance” for nine. The strongest associations were found for four closely associated SNPs located on chromosome 11 in introns near AMBRA1 (activating molecule in beclin-1-regulated), an important gene in early neural development. Another intriguing gene nearby is CHRM4, which codes for the muscarinic acetylcholine receptor M4, an attractive potential drug target that modulates dopaminergic transmission. Other candidate genes in the region are DGKZ (diacylglycerol kinase zeta) and Midkine (MDK). One other SNP, on chromosome 18 between CCDC68 (coiled-coil domain containing 68) and http://www.szgene.org/geneoverview.asp?geneid=161 TCF4 (transcription factor 4), survived Bonferroni correction for multiple testing.
In the combined GWAS and replication samples, one chromosome 11 T/C SNP identified in the previous analyses—rs11819869—surpassed genomewide statistical significance (3.89 x 10-9), a finding that was confirmed for the T risk allele of the SNP in 15 other European samples.
The researchers then looked for evidence that this variant marks a genetic influence on brain function, using data from normal subjects performing tests of cognitive ability while undergoing functional MRI. In one test, they found that carriers of the T allele had significantly increased activation in a region of subgenual cingulate cortex that has been implicated in schizophrenia, while C carriers showed decreased activation in the same region. This, the authors say, constitutes, “evidence that the identified risk allele is functional in a neural system relevant to the disorder.”
Both CCDC68 and TCF4 also show their faces in the second GWAS, a meta-analysis published in Human Molecular Genetics that, according to the authors, “buttresses the notion that larger sample sizes will allow the identification of additional common variants.” Kari Stefansson of deCODE Genetics, Reykjavik, Iceland, and colleagues from many other institutions drew from a combined dataset of an unprecedented 18,206 schizophrenia cases and 42,536 controls.
The group had previously conducted studies filtered for all SNPs that had attained a genomewide p-value greater than 1 x 10-5 in their well-known “SGENE-plus-ISC-MGS” GWAS and meta-analysis (see SRF related news story).
Here, first author Stacy Steinberg of deCODE and colleagues extended that work by examining loci reaching 1 x 10-4 in that dataset in an additional sample of 4,704 cases and 7,478 controls from Europe and the United States. In all, 39 SNPs from a variety of genomic regions were tested, including many in the major histocompatibility complex region (MHC) implicated in the 2009 work. In a subsequent follow-up consisting of 1,014 cases and 1,144 controls recruited in Germany, eight achieved genomewide significance (p <5 x 10-8), including two novel SNPs.
The newly identified SNPs were at 2p15.1, near VRK2 (vaccinia-related kinase), a gene thought to be involved in maintaining neuronal structure and preventing cell death, and at 18q21.2, smack between CCDC8 and TCF4. Though this SNP could influence either gene, or both, the growing evidence for a link between TCF4 and schizophrenia and other mental disorders (see Blake et al., 2010 for a discussion) suggests to the authors that the SNP acts through that gene.
Of the six replicated SNPs, four were found in two MHC subregions, one is located near NRGN, and one is in an intron of TCF4.
Divide and conquer
Another approach to solving the sample-size problem in genetics is to study subgroups of patients who share distinctive clinical phenotypes, a method chosen for a recent study of NRG3 (neuregulin-3) by an Australian group led by Assen Jablensky of the University of Western Australia in Perth. This growth factor gene, at 10q22-23, has been associated with schizophrenia in linkage studies of Ashkenazi, Scottish, and Han Chinese families (Fallin et al., 2003; Benzel et al., 2007; Faraone et al., 2006). An analysis of Ashkenazi case-control and familial datasets by a Johns Hopkins group (Chen et al., 2009) had found no association of the 10q22-23 region with schizophrenia per se, but a factor analysis of the data revealed an association between two SNPs (rs6584400 and rs10883866) in an intron of NRG3 with schizophrenia cases characterized by a “delusion factor.”
First author Bharti Morar and colleagues set out to replicate these findings in a group of 411 patients and 223 controls—the team aimed for rough ethnic homogeneity in their sample by recruiting only “Anglo-Irish” subjects—who were assigned to either a “pervasive cognitive deficit” or a “relatively spared cognition” group based on performance in a range of neuropsychological tests.
The researchers found a nominally significant association in the patient sample as a whole between one SNP studied by the Hopkins team, and a weak association for the other (odds ratios of 1.45 and 1.36, respectively). However, factor analysis revealed that these overall associations were entirely attributable to stronger associations (odds ratios of 1.67 and 1.49) in the subgroup with spared cognition.
Morar and colleagues take these findings as evidence that “the schizophrenia phenotype comprises heterogenous components influenced by multiple gene loci.”
Another recent paper bemoans the current state of research on CNVs in schizophrenia. Rolf Ophoff of the University of California, Los Angeles, and coauthors from a number of different institutions point out that informative meta-analyses such as those commonly performed on candidate gene data are difficult to conduct in the world of CNVs, where few complete datasets and not many raw data have been made publicly available. As a result, say the authors, the field’s findings so far have identified rare but recurrent deletions affecting multiple genes (e.g., at 1q21.1, 15q13.3, and 15q11.2) or genomic one-hit wonders, with little overlap between studies (see SRF related news story).
First author Jacobine E. Buizer-Voskamp of the University of Utrecht, The Netherlands, and colleagues thus hope to set an example by releasing the raw data from their own whole-genome study of 834 cases and 672 controls from The Netherlands, which targeted all CNVs 50 kb and larger. The group identified 2,437 CNVs in the subject pool overall, and confirmed results from other studies showing that deletions are more common in cases than controls, a pattern that becomes more pronounced as CNVs increase in size. The work corroborates previous linkages of the 1q42 region (the site of DISC1), 2p25, 15q13, and 22q11.2 with schizophrenia, findings given additional support in the authors’ comprehensive review of the literature on cytogenic and chromosomal anomalies in the disorder. Noting a clustering of reported cytogenic abnormalities at 5q35.1 in their literature review, the group also highlights a CNV they discovered there, in a region harboring candidate genes SLIT3, GABRP, and FGF18.
Another recent, but much larger, CNV study, from the Genetic Risk and Outcome in Psychosis (GROUP) consortium, focused on the 15q11.2-13.3 region, an imprinted area in which gene expression depends on parental origin. Involvement of this region in Prader-Willi syndrome and Angelman syndrome, caused respectively by deletions of paternal or maternal origin, is well documented, as is a more recently recognized syndrome characterized by autism that is associated with duplications of maternal origin (see, e.g., Cook et al., 1997). After turning up a microduplication at 15q11.2-13.1 in one patient with early-onset schizophrenia, the team searched more comprehensively in a European sample of 7,582 patients with schizophrenia or schizoaffective disorder and 41,370 controls.
As reported in the American Journal of Psychiatry, first author Andrés Ingason and corresponding author Thomas Werge of Copenhagen University Hospital, Denmark, and colleagues found 11 carriers of duplications in 15q11-13, five of whom had diagnoses of schizophrenia or schizoaffective disorder; all carried duplications of maternal origin. Two other patients carrying such duplications had received psychiatric diagnoses—of bipolar disorder and autism—and one control subject with a maternally derived duplication had been diagnosed with Alzheimer’s disease. By contrast, the only paternally derived duplications were observed in two control subjects, neither of whom had a psychiatric diagnosis. The authors highlight UBE3A as a gene of interest in this region because it is only expressed on the maternal chromosome, and it is related to synapse development and glutamate signaling.
A large target
In the exome-sequencing study from Karayiorgou and Gogos's group, first author Bin Xu of Columbia University and colleagues isolated sporadic schizophrenia cases by studying trios comprising subjects, 53 of whom were patients and 22 controls, and their unaffected parents, obtaining blood samples from all participants (for an account of the first published exome-sequencing effort in the field, see SRF related news story).
The researchers identified 40 de novo events affecting 40 genes in 27 (~51 percent) of the cases, including 35 point mutations, one dinucleotide substitution, and four indels; 10 cases carried more than one of these mutations. Of the 35 point mutations, 32 were non-synonymous, and were predicted to affect protein function, as were the four indels. Some others were determined to potentially disrupt splicing. In contrast, only seven controls carried these newly identified de novo mutations.
Of particular interest to the authors was a point mutation in DGCR2 on 22q11.1 in an otherwise structurally sound chromosome, which might contribute to the schizophrenia risk associated with 22q11.1 microdeletions.
The research group concludes that the “large mutational target” of 40 affected genes cited in the study supports a role for de novo events in schizophrenia and offers an explanation for the persistence of the disorder, despite the decrease in reproductive rate associated with it.
In the diverse research landscape of schizophrenia genetics (see SRF related news story), theories, methods, and data can all be contentious, but data appear to be emerging from all fronts, and perhaps all this controversy will move the field forward. Steinberg and colleagues propose that an ecumenical approach embracing GWAS, CNV research, and exome sequencing makes the most sense, in the hope that “eventually, a collection of variants—rare and common, structural and single-nucleotide—may account for a substantial portion of schizophrenia heritability, as has been shown for other common diseases such as type 2 diabetes.”—Pete Farley.
Buizer-Voskamp JE, Muntjewerff JW; Genetic Risk and Outcome in Psychosis (GROUP) Consortium, Strengman E, Sabatti C, Stefansson H, Vorstman JA, Ophoff RA. Genome-wide Analysis Shows Increased Frequency of Copy Number Variation Deletions in Dutch Schizophrenia Patients. Biol Psychiatry. 2011 Apr 12. Abstract
Ingason A, Kirov G, Giegling I, Hansen T, Isles AR, Jakobsen KD, Kristinsson KT, le Roux L, Gustafsson O, Craddock N, Möller HJ, McQuillin A, Muglia P, Cichon S, Rietschel M, Ophoff RA, Djurovic S, Andreassen OA, Pietiläinen OP, Peltonen L, Dempster E, Collier DA, St Clair D, Rasmussen HB, Glenthøj BY, Kiemeney LA, Franke B, Tosato S, Bonetto C, Saemundsen E, Hreidarsson SJ; GROUP Investigators, Nöthen MM, Gurling H, O'Donovan MC, Owen MJ, Sigurdsson E, Petursson H, Stefansson H, Rujescu D, Stefansson K, Werge T. Maternally derived microduplications at 15q11-q13: implication of imprinted genes in psychotic illness. Am J Psychiatry. 2011 Apr;168(4):408-17. Abstract
Morar B, Dragović M, Waters FA, Chandler D, Kalaydjieva L, Jablensky A. Neuregulin 3 (NRG3) as a susceptibility gene in a schizophrenia subtype with florid delusions and relatively spared cognition. Mol Psychiatry. 2011 Aug;16(8):860-6. Abstract
Rietschel M, Mattheisen M, Degenhardt F; GROUP Investigators; Genetic Risk and Outcome in Psychosis (GROUP Investigators), Kahn RS, Linszen DH, Os JV, Wiersma D, Bruggeman R, Cahn W, de Haan L, Krabbendam L, Myin-Germeys I, Mühleisen TW, Kirsch P, Esslinger C, Herms S, Demontis D, Steffens M, Strohmaier J, Haenisch B,Breuer R, Czerski PM, Giegling I, Strengman E, Schmael C, Mors O, Mortensen PB, Hougaard DM, Orntoft T, Kapelski P, Priebe L, Basmanav FB, Forstner AJ, Hoffmann P, Meier S, Nikitopoulos J, Moebus S, Alexander M, Mössner R, Wichmann HE, Schreiber S, Rivandeneira F, Hofman A, Uitterlinden AG, Wienker TF, Schumacher J, Hauser J, Maier W, Cantor RM, Erk S, Schulze TG; SGENE-plus Consortium; (Only those persons responsible for the samples of Replication 2 are listed), Stefansson H, Steinberg S, Gustafsson O, Sigurdsson E, Petursson H, Kong A, Stefansson K, Pietiläinen OP, Tuulio-Henriksson A, Paunio T, Lonnqvist J, Suvisaari J, Peltonen L, Ruggeri M, Tosato S, Walshe M, Murray R, Collier DA, Clair DS, Hansen T, Ingason A, Jakobsen KD, Duong L, Werge T, Melle I, Andreassen OA, Djurovic S, Bitter I, Réthelyi JM, Abramova L, Kaleda V, Golimbet V, Jönsson EG, Terenius L, Agartz I, Winkel RV, Kenis G, Hert MD, Veldink J, Wiuf C, Didriksen M, Craddock N, Owen MJ, O'Donovan MC, Børglum AD, Rujescu D, Walter H, Meyer-Lindenberg A, Nöthen MM, Ophoff RA, Cichon S. Association between genetic variation in a region on chromosome 11 and schizophrenia in large samples from Europe. Mol Psychiatry. 2011 Jul 12. Abstract
Steinberg S, de Jong S; Irish Schizophrenia Genomics Consortium, Andreassen OA, Werge T, Børglum AD, Mors O, Mortensen PB, Gustafsson O, Costas J, Pietiläinen OP, Demontis D, Papiol S, Huttenlocher J, Mattheisen M, Breuer R, Vassos E, Giegling I, Fraser G, Walker N, Tuulio-Henriksson A, Suvisaari J, Lönnqvist J, Paunio T, Agartz I, Melle I, Djurovic S, Strengman E; GROUP, Jürgens G, Glenthøj B, Terenius L, Hougaard DM, Orntoft T, Wiuf C, Didriksen M, Hollegaard MV, Nordentoft M, van Winkel R, Kenis G, Abramova L, Kaleda V, Arrojo M, Sanjuán J, Arango C, Sperling S, Rossner M, Ribolsi M, Magni V, Siracusano A, Christiansen C, Kiemeney LA, Veldink J, van den Berg L, Ingason A, Muglia P, Murray R, Nöthen MM, Sigurdsson E, Petursson H, Thorsteinsdottir U, Kong A, Rubino IA, De Hert M, Réthelyi JM, Bitter I, Jönsson EG, Golimbet V, Carracedo A, Ehrenreich H, Craddock N, Owen MJ, O'Donovan MC; Wellcome Trust Case Control Consortium, Ruggeri M, Tosato S, Peltonen L, Ophoff RA, Collier DA, St Clair D, Rietschel M, Cichon S, Stefansson H, Rujescu D, Stefansson K. Common Variants at VRK2 and TCF4 Conferring Risk of Schizophrenia. Hum Mol Genet. 2011 Jul 26. Abstract
Xu B, Roos JL, Dexheimer P, Boone B, Plummer B, Levy S, Gogos JA, Karayiorgou M. Exome sequencing supports a de novo mutational paradigm for schizophrenia. Nat Genet. 2011 Aug 7. Abstract