• single nucleotide polymorphisms
• trace amines
• missense variants
• dopaminergic transmission
• human trace amine-associated receptor 1
• psychotic disorders
• bipolar disorder
• drug addiction

Background and objectives: Trace Amine-Associated Receptors 1 (TAAR1) is the prototype of a novel family of G protein-coupled receptor (GPCR) which turned out to be selectively activated by trace-amines (TAs) such as p-tyramine, β-phenylethylamine, tryptamine and p-octopamine. TAs are endogenous amines structurally related to classical biogenic amines but present within the mammalian brain and peripheral nervous tissues at low nanomolar concentrations (< 100 ng/g tissue).
TAAR1 is highly expressed in the limbic and monoaminergic brain areas, which are known to influence cognition, mood, motivated behaviors and movement, as well as in the prefrontal cortex, a region crucially implicated in executive functions and in the pathophysiology of schizophrenia. Recently, several evidences from preclinical studies have demostrated the role of TAAR1 in the modulation of dopaminergic, serotonergic, and glutamatergic transmission, suggesting its possible pathogenetic and therapeutic implication in several human conditions and neuropsychiatric disorders including obesity, schizophrenia, bipolar disorders, depression, Parkinson’s disease, attention-deficit/hyperactivity disorder, drug abuse and addiction.
As the human TAAR1 (hTAAR1) is located in a region on chromosome 6q23.2 that has been associated with schizophrenia and bipolar disorder in linkage and molecular genetic studies, we hypothesized a contribution of genotypic variants in hTAAR1 affecting protein function to the etiology of mental disorders.
The purpose of this study was the identification and characterization of hTAAR1 single-nucleotide missense variants in patients with mental disorders. We compared the minor allele frequency (MAF) of single nucleotide mutations between the population of patients and a population of healthy controls, and the detected variants were functionally characterized using in silico prediction tools. We hypothesized that single nucleotide missense variants may be more frequent in the patient population and associated to specific behavioral and psychopathological phenotypes.
Materials and methods: Screening for hTAAR1 single-nucleotide mutations was performed in a population of 96 patients affected by major psychiatric disorders and 97 healthy controls. All subjects were of Caucasian race. Patients were recruited at the Psychiatric Clinic of the Department of Clinical and Experimental Medicine, University of Pisa, and healthy controls among the voluntary blood donors at the Division of Transfusion Medicine and Transplant Biology, Azienda Ospedaliera Universitaria Pisana (June 2017-June 2018). The Structured Clinical Interview for DSM-5, Research Version (SCID-5-RV), was used to establish the psychiatric diagnosis in patients and to exclude current or past psychiatric illness in controls. An anamnestic questionnaire was administered to both patients and controls, and psychopathological assessments in patients were carried out by using the Positive and Negative Syndrome Scale (PANSS), the Young Mania Rating Scale (YMRS) and the Hamilton Rating Scale for Depression (HDRS). Specific recreational habits were also investigated through the administration of the Fagerström Test for Nicotine Dependence (FTND) and the Alcohol Use Disorder Identification Test (AUDIT).
Genomic DNA was isolated from saliva and blood samples of patients and healthy controls, respectively, by standard methods and quantified. We screened by Sanger sequencing three partially overlapping amplicons spanning the TAAR1 coding region and the 5’- and 3’- untranslated region (UTR). We calculated the MAF of the detected single nucleotide polymorphisms (SNPs) (MAF ≥ 1% ) or single nucleotide variants (SNVs) (MAF < 1%) in the population of patients and controls, and compared them with the MAF of the general population documented in public database [National Center for Biotechnology Information (NCBI), dbSNP, http://www.ncbi.nlm.nih.gov/SNP/]. Three in silico tools available in dbNSFP2.9 (SNP&GO, SNAP, and PhD-SNP) were used to assess the functional effect of the detected variants. The Residual Variation Intolerance Score (RVIS) was applied to assess the gene-level of intolerance to functional variation. The evolutionary conservation of the detected variants was also examined by the use of Blosum62 matrix.
Results: We detected 3 synonymous and 13 non-synonymous mutations in the hTAAR1 coding region, 2 mutations in the 3’ UTR, 2 mutations in the upstream region and 1 in the downstream region, among the study sample, 7 of which were novel. All the non-synonymous mutations were missense, and three of the missense SNVs, namely p.Arg23Cys (rs8192618), p.Tyr131Cys (rs41286174), and p.Cys263Arg (rs142169206), were predicted to be damaging by all the in silico tools used. These SNVs correspond to a MAF of 0.5% in the patient population, substantially higher than the MAF reported for the general population in public database (NCBI, dbSNP), and none of them were observed in healthy controls. We also found a significantly higher percentage of any non-synonymous SNPs/SNVs in the population of patients as compared to healthy controls. From RVIS analysis, TAAR1 resulted among 34.6% of the most intolerant of human genes.
The p.Arg23Cys variant, located at the transition between transmembrane helix 1 and the N-terminal tail at the extracellular side of the receptor, was detected in a male affected by schizoaffective disorder with comorbid obsessive-compulsive disorder. The p.Cys263Arg variant was located in a high conserved motif in the sixth transmembrane domain in a woman suffering from BD-II. Both these SNVs had been previously described to result in sub- or non-functional receptors from functional in vitro characterization. The p.Tyr131Cys variant, located in the second cytoplasmic loop, was associated with the apparently neutral p.Tyr123Ser variant (rs371440762) in a patient diagnosed as BD-I, manic episode with mixed and psychotic features and prominent cognitive abnormalities, that subsequently went on to develop a diagnosis of frontotemporal dementia, behavioral type. All the 3 missense variants are conserved among TAAR1 orthologue genes and paralogue genes in the TAAR family.
The separate comparison of clinical characteristics between subjects with and without individual mutations showed that carriers of the synonymous SNP rs8192621 (T > C, p.Arg312Arg, heterozygous genotype) had significantly higher rate of family history of more than one mental disorder as compared to wild-type subjects. Moreover, we found that the SNP rs9375907 (G > T, 3’ UTR) and the synonymous SNP rs8192620 (T > C, p.Val288Val), that were interconnected, were significantly more frequent in patients suffering from other bipolar disorders and other psychotic disorders. The homozygous variant genotype of these SNPs was significantly associated with substance use and alcohol use disorders.
Conclusions: In summary, we identified in the patient population three rare protein disturbing variants in hTAAR1, as well as three common SNPs located in highly conserved motifs of the protein or supposed to play a key role in ligand binding, proper mRNA translation and in maintaining protein structure, all of them associated to specific clinical phenotypes. These findings add evidence to the potential involvement of hTAAR1 variants in conferring susceptibility to bipolar or psychotic disorders, as well as addictive behaviors and neurodegenerative diseases.