Molecular genetic strategies to identify Obsessive-compulsive disorder (OCD) and schizophrenia candidate genes in a South African sub-population group

Dissertation (PhD)--University of Stellenbosch, 2007.ENGLISH ABSTRACT: Obsessive-compulsive disorder is a severe, debilitating psychiatric disorder for which the underlying molecular aetiology still remains unclear. Evidence from family studies have suggested that OCD may be caused by a complex interplay of environmental and genetic factors. In order to identify the genetic factors that mediate OCD susceptibility, several genetic association studies have been undertaken, which have yielded inconsistent findings. Moreover, the majority of these studies have focused on a small number of candidate genes that encode components of the serotonin and dopamine neurotransmitter pathways. However, based on the complexity of clinical manifestations observed in OCD, it is likely that its pathogenesis is mediated by a broader complex of interrelated neurotransmitter systems and signal transduction pathways; consequently there is a need to identify and assess novel candidate genes. One method of identifying such novel OCD candidate genes is by utilising knowledge of diseases with phenomenological overlap with OCD, which lend themselves to better genetic dissection through linkage analysis and animal studies. Genetic loci for such disorders, identified though linkage analysis, could potentially harbour novel OCD candidate genes, while genes implicated through animal models may lead to the identification of additional susceptibility genes through delineation of pathways by, for instance, interactome analysis. One such disorder is schizophrenia, which manifests overlap in both symptoms and brain circuits with OCD. In schizophrenia, in addition to several case-control association studies having been performed, linkage data, studies of chromosomal aberrations and animal models have led to the identification of many chromosomal regions that may contain genes involved in its aetiology and thus may also contain OCD candidate genes. In the present investigation, this approach was employed using previously reported schizophrenia susceptibility loci to identify novel OCD candidate genes. All genes residing in each of these loci were catalogued and individually analysed using a battery of bioinformatic techniques in order to assess their potential candidature for OCD susceptibility. These analyses yielded 13 credible OCD candidate genes.Additional candidates were sought using information regarding a well-defined schizophrenia animal model, the heterozygous reeler mouse, that exhibits neurodevelopmental, neuroanatomical and behavioural abnormalities, similar to those displayed by patients with schizophrenia. The phenotype of these mice is caused by a mutation in Reln, which encodes reelin, a large extracellular matrix protein that plays a pivotal role in the ordered migration of neurons during the development of laminar brain structures. The fact that both reelin protein and mRNA levels have been shown to be reduced in post-mortem brain sections of schizophrenic patients, coupled with the observed behaviour and neurochemical similarities between the heterozygous reeler mouse and schizophrenic patients suggests that reelin may be involved in the pathogenesis of schizophrenia and hence also OCD. Furthermore, genes encoding proteins that interact with reelin may thus also be considered plausible candidate genes for both schizophrenia and OCD. For this reason, novel reelin-interacting proteins were sought using the N-terminal reeler-domain of reelin, a domain only found in proteins involved in neuronal migration, as “bait” in a yeast twohybrid screen of a foetal brain cDNA library. Putative reelin ligands were subsequently reevaluated using co-immunopreciptitation and mammalian two-hybrid analysis to corroborate the yeast two-hybrid findings. Results of these analyses showed that WDR47, a WD40-repeat domain protein, interacts with reelin via its reeler-domain; therefore, the gene encoding this ligand protein, as well as RELN itself, was also considered a credible OCD candidate gene. Each of the candidate genes identified using the afore-mentioned strategies were assessed for their potential role in the aetiology of OCD by case-control association studies of a cohort of Afrikaner OCD patients and control individuals. Statistically significant associations were detected for two genes, DLX6 and SYN3, with the disorder. These associations are exciting as they may point to novel mechanisms involved in OCD development. The identification of WDR47 as a novel reelin-interacting protein has significant implications for our understanding of reelin-dependant signalling. Using this protein as the starting point, further novel components of the reelin signalling pathway may be unravelled, an investigation which may lead to the identification of novel roles for reelin in neurodevelopment. Such novel components may, of course, also be considered OCD and schizophrenia candidate genes, which may, in turn, augment the existing knowledge of the pathophysiologies of OCD, schizophrenia and other neurodevelopmental disorders. Taken together, the current study yielded exciting results that warrants follow-up investigation in future. The identification of DLX6 and SYN3 as novel OCD susceptibility genes as well as the identification of WDR47 as a reelin-interacting protein may provide investigators with alternative avenues of research into potential pathological mechanisms involved both in OCD and schizophrenia, which may ultimately lead to alternative pharmacotherapy.AFRIKAANSE OPSOMMING: Obsessiewe kompulsiewe steuring (OKS) is `n ernstige, verswakkende psigiatriese steuring waarvan die onderliggende molekulêre etiologie steeds onbekend is. Bewyse verkry vanuit familiestudies het voorgestel dat OKS moontlik veroorsaak word deur `n komplekse interaksie van omgewings en genetiese faktore. Om die genetiese faktore te identifiseer wat OKS vatbaarheid veroorsaak, is `n hele aantal genetiese assosiasie studies onderneem, wat teenstrydige resultate gelewer het. Wat meer is, die grootste hoeveelheid van hierdie studies het gefokus op `n klein aantal kandidaatgene wat vir komponente van die serotonien en dopamine neurotransmittor weë enkodeer. Dit is egter, gebaseer op die kompleksiteit van die kliniese manifestasies wat waargeneem word in OKS, heel moontlik dat die patogenisiteit van die siekte bemiddel word deur `n breër kompleks van interverwante neurotransmittor sisteme en seintransduksie weë. Daar is dus `n behoefte na die identifikasie en ondersoek van nuwe kandidaatgene. Een metode om sulke nuwe OKS kandidaatgene te identifiseer, is deur die gebruik van bestaande kennis oor siektes wat fenomenologiese ooreenkomste het met OKS, siektes wat makliker geneties ontleed kan word deur koppelingsanalises en dierestudies. Genetiese lokusse vir sulke versteurings, geïdentifiseer deur koppelingsanalises, het die potensiaal om nuwe OKS kandidaatgene in te sluit, terwyl gene wat geïmpliseer word deur dierestudies mag lei tot die identifisering van bykomende vatbaarheidsgene deur die ondersoek van weë deur, byvoorbeeld, interaktoom analises. `n Voorbeeld van so `n versteuring is skisofrenie, wat in manifestasie oorvleuel in beide simptome en breinstroombane met OKS. In skisofrenie het, addisioneel tot verskeie geval-kontrole assosiasiestudies wat gedoen is, koppelingsdata, studies van chromosomale afwykings en dierestudies gelei tot die identifikasie van verskeie chromosomale gebiede wat gene mag bevat wat betrokke kan wees in die etiologie van die siekte, en dus ook OKS kandidaatgene mag bevat. In die huidige ondersoek is hierdie benadering gevolg en is gebruik gemaak van voorheen gerapporteerde skisofrenie vatbaarheidslokusse om nuwe OKS kandidaatgene te identifiseer. Alle gene wat in hierdie lokusse voorkom is gekatalogiseer en individueel geanaliseer deur gebruik te maak van `n battery van bioinformatika tegnieke om hul potensiaal as kandidate vir OKS vatbaarheid te bepaal. Hierdie analise het 13 geloofwaardige OKS kandidate opgelewer. Addisionele kandidate is gesoek deur inligting van `n goed gedefinieerde skisofrenie dieremodel te gebruik, naamlik die heterosigotiese “reeler” muismodel, wat neuro-ontwikkelings-, neuroanatomiese- en gedragsabnormaliteite vertoon, soortgelyk aan dié wat voorkom by pasiënte met skisofrenie. Die feit dat daar aangetoon is dat beide reelin protein en bRNS vlakke verlaag is in post-mortem brein seksies van skisofrenie pasiënte, gekoppel aan die gedrags- en neurochemiese ooreenkomste wat gesien word tussen heterosigotiese “reeler” muise en skisofrenie pasiënte, stel voor dat reelin betrokke is by die patogenese van skisofrenie en dus ook OKS. Vir hierdie rede is nuwe proteïene gesoek wat `n interaksie met reelin toon, deur gebruik te maak van die N-terminale reeler-domein van reelin, `n domein wat slegs gevind word in proteïene wat betrokke is by neuronale migrasie, as “aas” in `n gis-twee-hibried sifting van `n fetale brein cDNS biblioteek. Vermeende reelin ligande is vervolgens herevalueer deur gebruik te maak van koimmunopresipitasie en soogdier twee-hibried analises om die gis-twee-hibried bevindings te bevestig. Resultate van hierdie analises het getoon dat daar interaksie is tussen WDR47, `n WD40-herhalingsdomein protein, met reelin via sy reeler-domein. Die geen wat hierdie ligand protein enkodeer, sowel as RELN self, is dus beskou as ‘n geloofwaardige OKS kandidaatgeen. Elkeen van die kandidaatgene wat geïdentifiseer is deur gebruik te maak van bogenoemde strategieë is ondersoek vir `n potensiële rol in die etiologie van OKS deur gebruik te maak van geval-kontrole assosiasie studies met `n groep Afrikaner OKVS pasiënte en kontrole individue. Statisties-betekenisvolle assosiasies met die versteuring is vasgestel vir twee gene, DLX6 en SYN3. Hierdie assosiasies is opwindend aangesien hul nuwe meganismes betrokke by OKS ontwikkeling mag aantoon. Die identifikasie van WDR47 as ‘n nuwe protein wat interaksie met reelin vertoon, het betekenisvolle implikasies vir die verstaan van reelin-afhanklike seining. Deur hierdie proteïn as die beginpunt te gebruik kan vêrdere nuwe komponente van die reelin seinweg ontdek word, `n ondersoek wat mag lei tot die identifisering van nuwe funksies vir reelin in neuro-ontwikkeling. Sulke nuwe komponente mag, natuurlik, ook in aanmerking kom as OKS en skisofrenie kandidaatgene, wat op sy beurt weer die bestaande kennis van die patofisiologie van OKS, skisofrenie en ander neuro-ontwikkelings versteurings mag verbreed. In samevatting, hierdie studie het opwindende resultate gelewer wat opvolgondersoeke in die toekoms regverdig. Die identifikasie van DLX6 en Syn3 as nuwe OKS vatbaarheidsgene, sowel as die identifisering van WDR47 as ‘n protein wat interaksie vertoon met reelin, mag aan navorsers alternatiewe navorsingsweë voorsien om die moontlike patologiese meganismes wat betrokke is by beide OKS en skisofrenie te ondersoek, wat uiteindelik mag lei tot alternatiewe farmakoterapie