The identification of novel susceptibility genes involved in anxiety disorders

McGregor, Nathaniel Wade (2014-12)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: The etiology of anxiety disorders remains incompletely understood. Clear evidence for a genetic component has been proposed; however, there is also an increasing focus on environmental factors and the interaction between these and the genetic components that may mediate (anxiety) disorder pathogenesis. No single gene or genetic component has been explicitly identified as being involved in the development of anxiety disorders. This is most likely due to a number of reasons, which include, for example, the heterogeneity of anxiety disorders, the contribution of environmental factors and methodological limitations (e.g. small sample size) of research studies. Until now, genetic association studies usually focused on one particular psychiatric disorder at a time. However, with the difficulty in identifying susceptibility genes and/or loci in heterogeneous disorders like obsessive-compulsive disorder and other conditions in the anxiety spectrum, it is perhaps timely to consider multivariate genetics and epidemiological studies in a number of disorders sharing a core characteristic – such as anxiety. In addition to genetic underpinnings, a number of environmental variables have also been identified as risk factors for pathological anxiety, including adverse life events like childhood physical and sexual abuse. The hypothesis for this project is that a pre-existing genetic vulnerability (or genetic risk) interacts with the impact of adverse life events to result in the development of one or more anxiety disorder(s). Considering phenotypic overlap amongst the anxiety disorders, it is likely that diverse networks of genes and/ or interacting pathways are responsible for the phenotypic manifestations observed. Sprague Dawley rats exhibiting behaviours indicative of anxiety in the context of environmental stressors (maternal separation and restraint stress) were used as model for the identification of novel susceptibility genes for anxiety disorders in humans. The striatum has previously been implicated as a candidate in the brain architecture of anxiety pathogenicity, and is also a site exhibiting a high degree of synaptic plasticity. The synaptic plasticity pathway was investigated using the dorsal striatum of the rat brain and several genes were identified to be aberrantly expressed in “anxious” rats relative to controls (Mmp9, Bdnf, Ntf4, Egr2, Egr4, Grm2 and Arc). In humans, it was found that the severity of early adversity was significantly and positively associated with the presence of an anxiety disorder in adulthood. When the human homologues of the susceptibility candidate genes that were identified using the animal model were screened in a human cohort of patients with obsessive-compulsive disorder (OCD), panic disorder (PD) or social anxiety disorder (SAD) (relative to controls), five single nucleotide polymorphisms (SNPs) were found to be significantly associated with these conditions. Four of these SNPs were also found to significantly interact with the severity of childhood trauma. Haplotype analysis of variants within the identified susceptibility candidates revealed novel haplotype associations, four of which are located in the MMP9 gene. Notably, this the first study to link these particular mutations in the MMP9 gene with anxiety disorders and this finding is consistent with previous work suggesting that MMP9 is involved in conditions like cardiovascular disease and cancer which have been associated with increased prevalence of anxiety disorders. In conclusion, this project yielded important findings pertaining to the etiology of anxiety disorders. The use of a combined anxiety disorders cohort (OCD, PD and SAD) may suggest that the associations found here may hold true for anxiety disorders in general and not only for a particular clinically delineated condition. Childhood trauma was confirmed as an increased susceptibility risk for anxiety disorders. Also, this research contributed several novel susceptibility genes (MMP9, EGR2, EGR4, NTF4, and ARC), five significant SNP associations, four significant SNP-environment interactions and five haplotype associations (within MMP9 and BDNF) as candidates for anxiety pathogenicity. The identified polymorphisms and haplotypes were demonstrated to be associated with susceptibility to anxiety disorders in a gene-environment correlation and gene-environment interaction.

AFRIKAANSE OPSOMMING: Die oorsake van angssteurings word steeds nie volledig verstaan nie. Daar is duidelike bewyse vir 'n genetiese komponent, maar daar is ook toenemende fokus op omgewingsfaktore en die interaksie tussen hierdie omgewingsfaktore en genetiese komponente by angssteurings. Geen enkele geen of genetiese komponent is al geïdentifiseer as diè wat betrokke is by die ontwikkeling van angssteurings nie. Dit is waarskynlik weens 'n aantal redes, wat byvoorbeeld, die heterogeneïteit van angssteurings, die bydrae van omgewingsfaktore en metodologiese beperkings (bv. klein steekproef) van die navorsingstudies, insluit. Verder het genetiese assosiasiestudies tot nou toe gewoonlik net op een spesifieke psigiatriese versteuring op 'n slag gefokus. Maar, gegewe die uitdaging om vatbaarheidsgene en / of loci in heterogene steurings soos obsessief – kompulsiewe steuring (OKV) en ander toestande op die angsspektrum te identifiseer, is dit tyd om genetiese en kliniese studies in ‘n aantal steurings - met ‘n oorvleuende kern-element soos angs -, gesamentlik te oorweeg. Bykomend tot die genetiese boustene, is ‘n aantal omgewingsveranderlikes soos traumatiese lewenservarings tydens die kinderjare as risikofaktore vir patologiese angs geidentifiseer. Die hipotese vir hierdie projek is dat daar 'n interaksie tussen genetiese kwesbaarheid (of genetiese risiko) en traumatiese lewensevarings is en dat dit tot die ontwikkeling van 'n / veelvoudige angssteuring(s) kan lei. Inaggenome die fenotipiese oorvleueling tussen die angssteurings, is dit waarskynlik dat diverse netwerke van gene en / of interaktiewe geen-paaie vir die manifestasie van hierdie toestande verantwoordelik is. Sprague Dawley-rotte met gedragswyses aanduidend van angs, in die konteks van omgewingstressore (d.i. skeiding van die ma-rot en bedwang-stres [restraint stress]), is as model gebruik vir die identifisering van nuwe vatbaarheidsgene vir angssteurings in mense. Die striatum is voorheen as ‘n kandidaat in die brein-argitektuur van patologiese angs voorgehou, en is ook ‘n plek met ‘n hoë mate van sinaptiese plastisiteit. Die sinaptiese plastisiteit is ondersoek deur te fokus op die dorsale striatum van die rotbrein en daar is verskeie gene gevind wat anders is in “angstige” rotte in vergelyking met kontroles (Mmp9, Bdnf, Ntf4, Egr2, Egr4, Grm2 en Arc). In mense is daar gevind dat die ernstigheidsgraad van vroeë trauma beduidend en positief met die teenwoordigheid van ‘n angssteuring tydens volwassenheid verband hou. Toe die menslike ekwivalente van die vatbaarheidsgene wat met die dieremodel geïdentifiseer is in ‘n mens-kohort met obsessief-kompulsiewe steuring (OKS), panieksteuring (PS) en sosiale angssteuring (SAS) ondersoek is, is gevind dat daar 5 enkele nukleotied polimorfismes (ENPs) is wat met die toestande verband hou. Daar is ook gevind dat vier van hierdie ENPs beduidend verband hou met die ernstigheidsgraad van trauma tydens die kinderjare. Haplotipe analise van variante binne die geïdentifiseerde vatbaarheidsgene het op nuwe haplotipe assosiasies – waarvan 4 op die MMP9-geen geleë is – gedui. Hierdie is dus die eerste studie wat gevind het dat dié spesifieke mutasies van die MMP9-geen met angssteurings verband hou. Hierdie bevinding strook met vorige werk wat daarop dui dat die MMP9-geen by toestande soos kardiovaskulêre siekte en kanker wat ook met verhoogde voorkoms van angssteurings verband hou, betrokke is. Ter afsluiting kan ons sê dat hierdie projek belangrike bevindinge oor die oorsake van angssteurings gemaak het. Die gebruik van ‘n gekombineerde angssteurings-kohort (OKS. PS en SAS) kan moontlik suggereer dat die assosiasies wat ons hier gevind het, waar is vir alle angssteurings en nie net vir ‘n spesifieke afgebakende toestand nie. Traumatiese ervarings tydens die kinderjare is ook bevestig as ‘n risiko vir die ontwikkeling van angssteurings. Hierdie navorsing het ook verskeie nuwe vatbaarheidsgene (MMP9, EGR2, EGR4, NTF4, en ARC), 5 beduidende ENP assosiasies, 4 beduidende ENP-omgewings-interaksies en 5 haplotipe assosiasies (by MMP9 en BDNF) geïdentifiseer as moontlike kandidate wat ‘n rol speel by die ontstaan van patologiese angs. Daar is ook gevind dat die geïdentifiseerde polimorfismes en haplotipes met vatbaarheid vir angssteurings in ‘n geen-omgewing- korrelasie en geen-omgewing- interaksie verband hou. Stellenbosch University http://scholar.sun.ac.za

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