Cholinergic muscarinic M₄ receptor gene polymorphisms: A potential risk factor and pharmacogenomic marker for schizophrenia

Abstract

Although schizophrenia is a widespread disorder of unknown aetiology, we have previously shown that muscarinic M₄ receptor (CHRM4) expression is decreased in the hippocampus and caudate-putamen from subjects with the disorder, implicating the receptor in its pathophysiology.

These findings led us to determine whether variation in the CHRM4 gene sequence was associated with an altered risk of schizophrenia by sequencing the CHRM4 gene from the brains of 76 people with the disorder and 74 people with no history of psychiatric disorders. In addition, because the CHRM4 is a potential target for antipsychotic drug development, we investigated whether variations in CHRM4 sequence were associated with final recorded doses of, and life-time exposure to, antipsychotic drugs.

Gene sequencing identified two single nucleotide polymorphisms (SNPs; rs2067482 and rs72910092) in the CHRM4 gene. For rs2067482, our data suggested that both genotype (1341C/C; p = 0.05) and allele (C; p = 0.03) were associated with an increased risk of schizophrenia. In addition, there was a strong trend (p = 0.08) towards an association between CHRM4 sequence and increased lifetime exposure to antipsychotic drugs. Furthermore, there was a trend for people with the C allele to be prescribed benzodiazepines more frequently (p = 0.06) than those with the T allele.

These data, albeit on small cohorts, are consistent with genetic variance at rs2067482 contributing to an altered risk of developing schizophrenia which requires more forceful pharmacotherapy to achieve a clinical response.

Introduction

Schizophrenia is a complex psychiatric disorder (American Psychiatric Association, 2000). Although numerous studies have attempted to elucidate the cause of this disorder, the aetiology and pathophysiology remain ambiguous, making it difficult to design optimal treatments. Twin (Cardno et al., 2012) and adoption studies (see Ingraham and Kety, 2000) show that schizophrenia is a heritable disorder; this underpins the hypothesis that the genesis of schizophrenia should be able to be predicted by genetic factors (Agerbo et al., 2012). The genetic liability is further demonstrated by data showing that the risk of developing schizophrenia in the general population is less than 1% and rises incrementally if a single (7%) or both parents (27.3%) have the disorder (Gottesman et al., 2010). However, when considering the genetic load and schizophrenia it is important to note that approximately 90% of adults with schizophrenia do not have a parent with the disorder (Carter et al., 2002) and that only 3.8% of family groups affected by schizophrenia have more than one affected member (Lichtenstein et al., 2006). This data provides strong support for the concept that genetic risk is not the only factor that dictates whether an individual develops the disorder and that schizophrenia occurs in people with a genetic liability for the disorder who have encountered some environmental factor that has triggered the onset of illness (Tsuang, 2000). Thus, in order to better understand the epidemiology of the disorder, significant effort has been invested in identifying the genes that contribute to the genetic liability for the syndrome.

Current antipsychotic agents are effective in a proportion of patients but are not beneficial to many people with the disorder and cause unwanted side-effects (Tandon et al., 2008). Therefore, considerable energy is being expended in developing new drugs to treat schizophrenia. It is, therefore, significant that the muscarinic M₄ receptor (CHRM4) is viewed as a potential target to produce antipsychotic effects by a different mechanism of action to currently available drugs (Bymaster et al., 2002). A proof of principle drug trial supported this theory; xanomeline, a CHRM1/M4 preferring agonist (Shannon et al., 2000), reduced levels of psychotic symptoms in a group of people with schizophrenia who were treatment resistant (Shekhar et al., 2008). Importantly, pre-clinical studies (Gomeza et al., 1999, Salamone et al., 2001, Tzavara et al., 2003, Tzavara et al., 2004, Jeon et al., 2010) strongly support the hypothesis that the antipsychotic actions of xanomeline arise from its activation of CHRM4 (Woolley et al., 2009). Given these data, the discovery that muscarinic receptors have more than one potential drug binding site (Clark and Mitchelson, 1976) reinvigorated the development of drugs for these receptors. Importantly, these alternative (allosteric) binding sites, unlike the orthosteric sites, have not been preserved across evolution (Langmead et al., 2008). Therefore, they offer the potential to generate compounds that specifically activate CHRM4. CHRM4 allosteric activators are reported to be efficacious in models used to predict antipsychotic activity, such as conditioned avoidance response (Leach et al., 2010), prepulse inhibition (Chan et al., 2008) and amphetamine-induced hyperlocomotion (Brady et al., 2008). These preclinical data add further credibility to the concept that CHRM4 may be a viable drug target for the treatment of schizophrenia.

The involvement of CHRM4 in the pathophysiology of schizophrenia is supported by data showing that CHRM4 are decreased in the caudate-putamen (Dean et al., 2000) and hippocampus (Scarr et al., 2007) from people with the disorder. These data are the basis for our current study designed to determine if changes in the CHRM4 gene sequence are associated with an altered risk of schizophrenia. In addition, given the potential of CHRM4s as a novel target for antipsychotic drug development, and their role in modulating dopaminergic activity (Tzavara et al., 2004, Jeon et al., 2010), we determined whether changes in CHRM4 gene sequence was associated with the pharmacotherapy prescribed for the person.