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The catechol-O-methyltransferase (COMT) val158met polymorphism has received attention in schizophrenia due to its role in prefrontal dopamine catabolism. Given the rich dopaminergic innervations of the olfactory bulb and the influence of dopamine on the transmission of olfactory signals, we examined the influence of COMT genotype status on the olfactory processing impairment observed in schizophrenia. The University of Pennsylvania Smell Identification Test was administered unirhinally to individuals with schizophrenia (n = 42) and a demographically-matched sample of healthy controls (n = 30). Individuals were genotyped for the COMT val158met polymorphism. A statistically significant interaction of diagnosis and COMT genotype was observed, such that schizophrenia heterozygotes and Met homozygotes showed impaired odor identification accuracy relative to Val158 homozygotes. These findings could not be explained by factors such as antipsychotic medication status, clinical symptomatology, or demographic and illness characteristics. Notably, the schizophrenia Val158 homozygotes’ odor identification performance was comparable to that of the control group. These data indicate that odor identification impairments observed in schizophrenia are influenced by the COMT val158met polymorphism. This relationship is consistent with specific dopaminergic modulation of primary olfactory sensory afferents, rather than a broader effect on cognitive processes. Future studies examining the olfactory processing deficit in schizophrenia with respect to other olfactory measures and COMT haplotypes is warranted.
Disruptions in prefrontal circuitry and dopamine (DA) neurotransmission are often regarded as central to the neuropathophysiology and cognitive dysfunction observed in schizophrenia (Tan, Callicott, & Weinberger, 2007). Olfactory brain regions, medially located within the prefrontal cortex, are significantly affected by genetically-mediated neurodevelopmental processes (Anholt & Mackay, 2010; Rawson et al., 2010), and evidence from the extant human and animal literature has indicated an extensive role for DA in olfactory processing. Dopaminergic neurons are abundant within the olfactory bulb (OB), with greater density observed in the glomerular, mitral and granular cell layers of the OB than in the midbrain substantia nigra and ventral tegmentum combined (McLean & Shipley, 1988; Skagerberg, Lindvall, & Bjorklund, 1984). DA neurotransmission is thought to play a central role in olfactory neurogenesis and olfactory bulb development (Feron, Vincent, & Mackay-Sim, 1999). The synaptic organization, neural circuitry, and biochemistry of the olfactory system may, therefore, provide a unique model with which to investigate the effects of altered DA-mediated processes in schizophrenia.
Catechol-O-methyltransferase (COMT) located on chromosome 22q11.2 is one candidate gene for schizophrenia that has received widespread attention due to its role in prefrontal dopamine degradation. A relatively common deletion at chromosome 22q11.2, encompassing the entire COMT gene, is responsible for DiGeorge Syndrome (also known as velocardiofacial syndrome or VCFS), which manifests in adults with a high prevalence of a schizophrenia-like psychosis (Murphy, 2002). Consistent with this, genome-wide scans of rare copy number variants (CNVs) have revealed that microdeletions at chromosome 22q11.2 confer significant risk for schizophrenia (Karayiorgou et al., 1995; Levinson et al., 2011; Vacic et al., 2011). A common functional polymorphism of the COMT gene consists of a guanine to adenine transition at codon 158 in the membrane-bound isoform of the enzyme and at codon 108 in the short soluble form, which results in a valine (Val) to methionine (Met) amino acid mutation. The higher thermostability of the Val allele results in a four-fold increase in the rate of dopamine catabolism and significantly lower synaptic dopamine levels, compared to the thermolabile Met allele (Lachman et al., 1996). Mounting evidence from neuropsychological, structural and functional imaging studies have indicated that this biallelic single nucleotide polymorphism influences the cognitive and emotional disturbances seen in the illness. Though the nature of these impairments have been debated, there is general consensus that the Val allele is associated with greater working memory dysfunction, while the Met allele is associated with increased treatment resistance and limbic dysfunction (for a review, see: Bilder, Volavka, Lachman, & Grace, 2004).
Schizophrenia patients have marked olfactory deficits which are pervasive, observed across the lifespan and are independent of antipsychotic medication effects (Brewer et al., 2001; Moberg, Roalf, Gur, & Turetsky, 2004). Furthermore, olfactory impairment is often associated with increased negative symptomatology (Ishizuka et al., 2010) and is prevalent in individuals with deficit-syndrome schizophrenia (Malaspina et al., 2002). Thus, it is reasonable to consider whether the olfactory abnormalities observed in schizophrenia might also be related to abnormalities in the genetic mediation of dopamine catabolism. Prior studies have shown that individuals with 22q deletion syndrome are impaired on measures of odor identification, odor discrimination and olfactory sensitivity compared to healthy controls (Romanos et al., 2011; Sobin et al., 2006). Bassett and colleagues (2007) similarly examined birhinal odor identification performance in Met and Val hemizygotes with 22q deletion syndrome. Met hemizygotes had more pronounced odor identification impairment compared to Val hemizygotes. Based on these findings, it is likely that variations in the COMT genotype might similarly influence the olfactory processing deficits observed in patients with schizophrenia. However, this question has not been addressed previously.
Schizophrenia patients (n = 42) and healthy adults (n = 30) were recruited to the Schizophrenia Research Center at the University of Pennsylvania Medical Center. Following a full explanation of study procedures, written informed consent was obtained in compliance with guidelines established by the University of Pennsylvania Institutional Review Board and in accordance with The Code of Ethics of the World Medical Association (1964 Declaration of Helsinki). Patients were administered the Diagnostic Interview for Genetic Studies (DIGS; Nurnberger et al., 1994) or the Structured Clinical Interview for DSM-IV - Patient Edition (SCID; First, Spitzer, Gibbon, & Williams, 1996) and consensus DSM-IV-TR (American Psychiatric Association, 2000) diagnoses for schizophrenia were established. Healthy individuals were screened for Axis I Disorders using the SCID – Nonpatient Edition (First, Spitzer, Gibbon, & Williams, 1995). Controls were excluded for any history of Axis I psychiatric illness, Axis II diagnosis of schizotypal, schizoid or paranoid personality disorder, or a positive family history of schizophrenia or schizophrenia-spectrum disorder. Additional exclusion criteria for all groups included lack of English proficiency, a history of central nervous disease or head trauma with loss of consciousness, past substance dependence, current substance abuse within the preceding six months, or medical conditions affecting cerebral or olfactory functioning (e.g., sinusitis, nasal fracture/surgery, deviated septum, common cold).
In patients, clinical status was assessed using the Brief Psychiatric Rating Scale (BPRS; Rhoades & Overall, 1988) and the Scales for the Assessment of Negative Symptoms (SANS; Andreasen, 1984a) and Positive Symptoms (SAPS; Andreasen, 1984b). Medication status of schizophrenia patients was as follows: 1 was unmedicated, 9 were taking first generation antipsychotic medication, 23 were taking second generation antipsychotic medication (of which 1 individual was also on a mood-stabilizer), and 8 were not on an antipsychotic medication but were taking another psychotropic or anticholinergic agent at the time of testing. Medication data for 1 individual was unavailable. Sample characteristics are presented in Table 1.
Genotyping was performed at the Molecular Diagnosis and Genotyping Core of the Hospital of the University of Pennsylvania. Participants were genotyped for the COMT val158met polymorphism (rs4680) using TaqMan® based assays-on-demand (Assay ID: C__25746809_50) from Applied Biosystems Inc., Foster City, CA, USA.
Odor identification ability was assessed with the University of Pennsylvania Smell Identification Test (UPSIT; Doty, Shaman, & Dann, 1984). The UPSIT is a standardized, four-alternative, forced-choice test of olfactory identification comprised of four booklets containing 40 stimuli embedded in “scratch and sniff” microcapsules fixed and positioned on strips at the bottom of each page. The specific stimuli and the reliability and sensitivity of this test have been described in detail elsewhere (Doty, Frye, & Agrawal, 1989; Doty, et al., 1984). The UPSIT was administered to each participant unirhinally (each naris separately) with the contralateral naris occluded using a piece of Durapore™ tape (3M Corporation, Minneapolis, MN) fitted tightly over the edges of the naris. This procedure is employed in order to isolate the naris being examined and limits retronasal airflow (Bromley & Doty, 1995). A trained technician released the microencapsulated stimuli, placed them under the participant’s unoccluded nare, and recorded the answer following the subject’s response. Two booklets of 20 items were presented to the right naris and the remaining two booklets were presented to the left naris. Booklet order and naris presentation were systematically counterbalanced to avoid position effects.
Following UPSIT administration, participants were given the Picture Identification Test (PIT; Vollmecke, 1985). The PIT is identical to the UPSIT in item composition and response characteristics except that line drawings are presented instead of odors. This test was designed as a companion to the UPSIT to screen for individuals with gross cognitive deficits that might confound UPSIT scores.
An analysis of variance (ANOVA) was conducted to examine group differences in age, education, parental education, PIT scores, and smoking pack-days within and between schizophrenia and control groups. Pearson chi-square tests were conducted to examine differences in sex and racial distribution. In patients, additional ANOVAs were performance to examined differences in schizophrenia symptomatology and illness characteristics. Overall group differences in picture and odor identification performance were assessed using an ANOVA, with group (patient, control) and COMT genotype (Val/Val, Val/Met, Met/Met) as between-group factors. Naris (right, left) was included as the within-group factor for olfactory analysis.
Patient and controls did not differ significantly with respect to age (F1,70 = 2.85, p = .10), sex composition (χ2(1) = 0.20, p = .66), or racial distribution (χ2(2) = 3.64, p = .16). As expected, controls had a significantly higher level of educational attainment compared to patients (F1,70 = 15.82, p < .001), though groups did not differ with regard to parental educational (Wilks’ F2,62 = 0.06, p = .94). Patients and controls did not differ with respect to smoking burden (as quantified by pack-days) (F1,65 = 1.01, p = .32).
The distribution of genotypes was consistent with Hardy-Weinberg equilibrium expectations in both patients (χ2(1) = 1.22, p = .27) and controls (χ2(1) = 1.16, p = .28) and did not differ between groups (χ2(2) = 1.47, p = .48). Within the patient cohort, the Val/Val, Val/Met, and Met/Met groups did not differ with respect to age (F2,39 = 0.21, p = .81), race (χ2(2) = 3.52, p = .17), education (F2,39 = 0.52, p = .60), pack-days (F2,39 = 0.68, p = .51), antipsychotic medication dosage (F2,38 = 0.59, p = .56), age of onset (F2,38 = 0.20, p = .82), illness duration (F2,38 = 1.71, p = .19), general psychiatric symptoms (F2,37 = 0.67, p = .52), negative symptoms (F2,39 = 0.60, p = .55), or positive symptoms (F2,39 = 0.53, p = .59). Groups did differ with respect to sex composition (χ2(2) = 7.39, p = .02); a higher proportion of males were represented in the Val/Met group (81.3%) compared to the Met/Met (42.9%) and Val/Val group (36.8%). Within controls, the Val/Val, Val/Met, and Met/Met groups did not differ with respect to age (F2,27 = 0.63, p = .54), sex (χ2(2) = 2.33, p = .31), race (χ2(2) = 1.26, p = .53), education (F2,27 = 1.47, p = .25), or pack-days (F2,22 = 1.88, p = .18).
There was no statistically significant group difference (F1,60 = 0.21, p = .65) or interaction of COMT genotype by group (F2,60 = .16, p = .85; see Figure 1) on picture identification scores. Within the patient group, the main effect of COMT status was not statistically significant (F2,36 = .16, p = .85); inclusion of sex as a covariate did not alter these findings (F2,35 = .05, p = .95).
Consistent with previous findings, there was a highly significant main effect of diagnosis on olfactory identification performance (F1,66 = 23.76, p < .00001), with patients performing worse than controls. However, a statistically significant interaction of COMT genotype and group was also observed (F2,66 = 5.20, p < .01). Notably, schizophrenia Val158 homozygotes were indistinguishable from Val158 homozygote controls (F1,66 = 0.12, p = .73) at identifying odors. Val158Met heterozygote patients (F1,66 = 17.46, p < .0001) and Met158 homozygote patients (F1,66 = 14.00, p < .001) were significantly impaired at identifying odors relative to their control counterparts (see Figure 1). There were no significant main or interaction effects of naris (all p’s > .32). Inclusion of sex and smoking burden as covariates did not alter the significant effects observed. Exclusion of the 8 patients not actively taking antipsychotic medications also did not alter the observed effects.
Post-hoc paired comparisons of odor identification scores by COMT genotype within the patient group revealed that Val158 homozygote schizophrenia patients made significantly fewer errors across both nares compared to both Met158 homozygote (F1,39 = 7.07, p = .01) and Val158Met heterozygote patients (F1,39 = 14.81, p < .001). These results remained statistically significant after including sex and antipsychotic medication dosage as covariates in the analysis. Odor identification scores in the control group did not differ significantly by COMT genotype (all p’s > .26).
The results of our investigation replicate the well-documented observation of odor identification impairment in schizophrenia, and further suggest that this deficit is influenced by COMT genotype status. Notably, we found that schizophrenia patients with a Met allele were impaired at identifying odors relative to both Val158 homozygote patients and controls regardless of genotype. Val158 homozygote patients’ performance was comparable to healthy controls. This performance dissociation observed across patient subgroups could not be explained by factors such as antipsychotic medication status, clinical symptomatology, or demographic and illness characteristics. As the Met allele is associated with decreased dopamine catabolism, and therefore higher PFC dopamine levels, these findings raise the possibility that lower COMT activity contributes to increased olfactory impairment in schizophrenia.
COMT has been investigated extensively in schizophrenia due to its location on chromosome 22q, where a deletion encompassing the COMT gene results in the development of VCFS, a disorder associated with a high prevalence of schizophrenia-like psychotic symptoms (Murphy, 2002). VCFS is characterized by cardiovascular and craniofacial dysmorphology, as well as olfactory impairment (Romanos, et al., 2011). Individuals with schizophrenia have similarly shown minor midline physical anomalies (e.g., increased palate height, cleft palate; O’Callaghan, Larkin, Kinsella, & Waddington, 1991), as well as abnormalities on structural and functional olfactory indices (Moberg et al., 2006; Moberg, et al., 2004; Turetsky, Crutchley, Walker, Gur, & Moberg, 2009; Turetsky et al., 2007; Turetsky et al., 2000). Notably, the face, olfactory structures, and forebrain develop embryologically in close association with each other (Carstens, 2002; Diewert, Lozanoff, & Choy, 1993), and these early morphogenetic processes are affected in a dose-related fashion to diminished 22q11 genes (Meechan, Maynard, Tucker, & LaMantia, 2011; Meechan, Tucker, Maynard, & LaMantia, 2009). Thus, it is plausible that genetic disruption influences neurogenesis and, in turn, contributes to the observed abnormalities in the structure and function of olfactory regions observed in both schizophrenia and VCFS.
Alternatively, dopaminergic transmission in the prefrontal cortex may influence olfactory signal transduction. Olfactory receptor neurons (ORN) embedded in the nasal epithelium transduce and relay odor signals to the olfactory bulb (OB). Within the OB, the ORNs arborize within the glomeruli and terminate on mitral and tufted cell dendrites. It is here that the first point of olfactory information integration is initiated (for a review, see: Wachowiak & Shipley, 2006). In the glomerular layer, dopamine D2 receptors are highly expressed in juxtaglomerular neurons, which are involved in lateral inhibitory interactions within and between glomeruli. Though it is not entirely clear how dopamine influences transmission of olfactory signals, evidence from the extant animal literature indicates that dopamine suppresses the transmission of olfactory signals at the level of the OB via presynaptic action on ORN terminals (Berkowicz & Trombley, 2000; Ennis et al., 2001). Dopamine receptor agonists disrupt odor-induced metabolic activity in the OB glomeruli (Sallaz & Jourdan, 1992), reduce ORN to mitral cell synaptic transmission (Hsia, Vincent, & Lledo, 1999), and impair odor detection ability (Doty & Risser, 1989). These disruptive effects appear to be mediated specifically through D2 receptors, as they can be blocked by pre-treatment with selective D2 antagonists (Doty & Risser, 1989). In humans, the indirect dopamine receptor agonist, methylphenidate, has been shown to negatively affect olfactory performance. Children with attention-deficit/hyperactivity disorder (ADHD) exhibit enhanced abilities to detect and discriminate between odors, relative to healthy children, but this is attenuated following treatment with methylphenidate (Romanos et al., 2008; Schecklmann et al., 2011). Also, post-mortem studies of patients with Parkinson’s disease show a 100% increase in dopaminergic neurons in the olfactory bulb (Huisman, Uylings, & Hoogland, 2004). This dopaminergic increase was theorized to explain the olfactory impairment well-described in the illness.
In the current study, Val158 homozygote schizophrenia patients, who presumably have reduced levels of available dopamine, were significantly better at identifying odors compared to Met homozygotes. This is consistent with findings in VCFS patients (Bassett, et al., 2007) and with available research on the role of dopamine in olfactory processing. However, several studies examining correlates of the COMT val158met polymorphism in schizophrenia have found that the Val/Val genotype is generally associated with poorer performance on cognitive tasks reflecting prefrontal cortical activity (for a review, see: Kurnianingsih et al., 2011). Since accurate odor identification involves accessing the necessary verbal information from semantic stores, the task is thought to rely partly on verbal and executive functions (Hedner, Larsson, Arnold, Zucco, & Hummel, 2010) and we might therefore expect the Val158 homozygote patients to perform worse, rather than better. That this was not the case suggests that decreased prefrontal dopamine levels do not interfere with the verbal and executive components of odor identification performance in patients. This hypothesis is further supported by the finding that picture identification scores did not differ by group or COMT genotype status. Rather, it strongly suggests that the effect of the val158met polymorphism is a direct reflection of altered dopamine levels within the afferent olfactory neurocircuitry. It also implies that olfactory impairment in schizophrenia is not a byproduct of antipsychotic medications. Based on these findings, treatment with antipsychotic medications might actually enhance olfactory performance.
It is important to also note that the effect of the val158met polymorphism on olfactory performance was not observed in our healthy control sample. The reason for this is unclear. It may be that healthy subjects have sufficient olfactory reserve capacity, such that the lower dopamine levels associated with Val158 homozygote status does not result in impaired performance. Alternatively, the impact of the polymorphism may only manifest itself in the broader context of appropriate gene-gene or gene-environment interactions that exert a greater effect on olfactory function (Wishart et al., 2011). So, for example, altered dopaminergic modulation coupled with altered excitatory glutamate transmission could result in an observable deficit (e.g., Vorstman et al., 2009). Addressing the question of such interactions, however, requires much larger samples than those available for this study.
Several other limitations must be noted. The current study only employed one behavioral measure of olfactory processing. Odor identification tasks, like the one used here, are considered the most highly standardized and reliable of psychophysical olfactory processing measures in humans. Other olfactory measures, such as odor detection threshold and odor discrimination, typically load on one broad olfactory domain with odor identification (Doty, Smith, McKeown, & Raj, 1994). Nevertheless, studies like the aforementioned investigations by Romanos et al. (2008) and Schecklmann et al. (2011) have shown that methylphenidate differentially affects some aspects of olfactory performance, but not others. Thus, certain olfactory measures may be more sensitive to the influence of dopamine on odor processing despite sharing a common source of variance in the overall olfactory domain. Use of a broader olfactory battery would therefore be of interest in future studies. It would also be useful to examine the extent to which other structural and functional olfactory measures shown to be abnormal in schizophrenia (e.g., posterior nasal volume, olfactory sulcus depth, olfactory bulb volume, and olfactory event-related potentials) are influenced by COMT genotype status. Finally, as noted above, multiple gene interactions across multiple neural networks are tied to dopamine and olfactory processing in schizophrenia. While the current findings provide strong inferential support, replication and inclusion of additional functional COMT haplotypes and the examination of other candidate schizophrenia genes will be necessary to determine the extent to which the current findings are unique to the COMT val158met polymorphism.
Few studies have examined how olfactory deficits relate to current neurochemical or genetic theories of schizophrenia. The results of the current study suggest that odor identification impairment may be influenced, in part, by functional dopaminergic activity. Neuroimaging, genetic, neuropsychological, and animal studies converge in suggesting an important role of dopamine in olfactory processing. This finding is consistent with prior studies in individuals with VCFS and provides the impetus for future studies examining the role of genetic polymorphisms on olfactory performance in schizophrenia and other neuropsychiatric illnesses.
This study was funded in part by National Institutes of Health Grants MH63381 to Dr. Moberg, MH59852 to Dr. Turetsky, and an Independent Investigator Award from the Brain Behavior Research Foundation (awarding NARSAD grants) to Dr. Moberg. The funding source had no role in the study design, collection, analysis, or interpretation of this data or writing of this manuscript. The authors thank Stephen J. Kanes, M.D., Ph.D. for facilitating acquisition of the genotyping data and the Hoffman Trust for their support of this research through NARSAD.
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