Possible Association of the Ubiquitin-Specific Peptidase 46 Gene (USP46) with Affective Temperamental Traits in Healthy Korean Volunteers

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1 ORIGINAL ARTICLE htts://doi.org/ /i Print ISSN / On-line ISSN OPEN ACCESS Possible Association of the Ubiquitin-Secific Petidase 46 Gene (USP46) with Affective Temeramental Traits in Healthy Korean Volunteers Young Jun Boo 1,2, Chun Il Park 1,2, Hae Won Kim 2,3, Se Joo Kim 1,2, and Jee In Kang 1,2 1 Deartment of Psychiatry, Yonsei University College of Medicine, Seoul, Reublic of Korea 2 Institute of Behavioral Science in Medicine, Yonsei University College of Medicine, Seoul, Reublic of Korea 3 Deartment of Medical Education, Yonsei University College of Medicine, Seoul, Reublic of Korea Objective Ubiquitin-secific etidase 46 gene (USP46) olymorhisms is art of ubiquitin-roteasome system, which is resonsible for dynamic cellular rocesses such as the regulation of cell cycle. USP46 has been reorted to be associated with major deressive disorder. The objective of the resent study was to investigate the association of USP46 olymorhisms with affective temeramental traits in healthy subjects. Methods A total of 557 Korean healthy volunteers were recruited, and 545 subjects (328 male, 217 female) were included in the final analysis. The DNA of the subjects was isolated from saliva samles. Two single-nucleotide olymorhisms (SNPs) rs346005, rs in USP46 were genotyed. Affective temeraments were assessed using the Korean version of Temerament Evaluation of the Memhis, Pisa, Paris, and San Diego Autoquestionnaire (TEMPS-A). Results A significant association was found between rs genotyes and TEMPS-A only in male subjects. In articular, subjects with the CC genotye of rs showed a more deressive temerament than subjects with AA or CA genotyes in males. For rs , there were no associations between the rs genotyes and TEMPS-A scores. Conclusion Some affective temeraments may serve as a genetic redisosing factors for affective disorders, such as deressive disorder, via vulnerability genes related to the ubiquitin-roteasome system. Psychiatry Investig 2019;16(1):87-92 Key Words Affective temerament, Genetic association study, TEMPS-A, Ubiquitin-roteasome system, USP46. INTRODUCTION Affective temeraments refer to characteristic atterns in the quality and intensity of baseline mood levels and emotional and behavioral resonses and regulations to internal and external stimuli, which tend to aear early in life and be Received: Aril 30, 2018 Revised: August 14, 2018 Acceted: October 2, 2018 Corresondence: Se Joo Kim, MD, PhD Deartment of Psychiatry and Institute of Behavioral Science in Medicine, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Reublic of Korea Tel: , Fax: , kimsejoo@yuhs.ac Corresondence: Jee In Kang, MD, PhD Deartment of Psychiatry and Institute of Behavioral Science in Medicine, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Reublic of Korea Tel: , Fax: , jeeinkang@yuhs.ac cc This is an Oen Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (htts://creativecommons.org/licenses/bync/4.0) which ermits unrestricted non-commercial use, distribution, and reroduction in any medium, rovided the original work is roerly cited. temorally stable. 1-3 Akiskal et al. 1 roosed five affective temeramental traits: deressive, cyclothymic, hyerthymic, irritable, and anxious temeraments and develoed a valid assessment scale for affective temeraments, viz. the Temerament Evaluation of the Memhis, Pisa, Paris, and San Diego Autoquestionnaire (TEMPS-A). These affective temeraments are not only considered to be subclinical manifestation of affective disorders, 1,4,5 but are also known to lay a crucial role in the clinical rognosis of sychiatric illnesses. 6-9 A recent study of affective temerament and suicide showed that an anxious temerament is a strong risk factor for suicidal attemts, regardless of the resence of mental disorders, while deressive and irritable temeraments are risk factors for suicidal attemts in atients with mental disorders. 6 Therefore, a better understanding of affective temeraments is crucial to the understanding of the develoment of and treatments for mental disorders. Affective temeraments are known to be moderately heri- Coyright 2019 Korean Neurosychiatric Association 87

2 Affective Temerament and USP46 table, with heritability ranging from 21% to 52% for the five affective temeraments in 101 biolar families. 10 Additionally, several candidate gene association studies have been conducted to identify genes for affective temeraments. Previous genetic association aroaches of affective temeraments usually targeted monoaminergic systems, including serotoninrelated genes, and doaminergic genes. 13 A genome-wide association study has revealed several variants that are significantly associated with a secific affective temerament in biolar atients. 15 Since affective temeraments may be quantitative endohenotyes for affective disorders, reresenting genetic heterogeneity, the interest in genetic studies of affective temeraments has increased. Besides the monoaminergic genes, the ubiquitin-roteasome system (UPS), a major rotein degradation athway, which is resonsible for dynamic cellular rocesses, such as the regulation of cell cycle and the resonse to genotoxic and roteotoxic stress, 16 may be a otential candidate system contributing to affective temeraments as well as affective disorders. The UPS is a critical contributor to the construction of brain networks via extensive regulation of synatic lasticity. 17,18 In recent years, synatic lasticity has emerged as a comonent of the athohysiology of deression. 19,20 Interestingly, some studies have revealed direct associations between the UPS and deressive disorders In an animal study using quantitative trait locus maing, the ubiquitin-secific etidase 46 gene (USP46) was roosed to be resonsible for deressive-like behavior in mice in deression-inducing tests, such as tail susension and forced swimming tests. 23 Furthermore, USP46 knock-out mice also showed differences in deression-inducing test. 22 Moreover, a human study showed significant differences in USP46 single-nucleotide olymorhisms (SNPs) between atients with deressive disorder and normal controls. 21 Taken together, these findings may indicate that USP46 may be a candidate gene involved in the develoment of affective temeraments, articularly in deressive temeraments, as a subclinical manifestation of affective disorders. The objective of the resent study was to investigate the association of USP46 olymorhisms with affective temeramental traits in healthy subjects. Methods Subjects A total of 557 healthy Korean volunteers (334 males, 223 females) were recruited using advertisements. Most of the subjects were college students. All articiants were interviewed by sychiatrists, and subjects with any current or revious Axis I sychiatric disorders according to the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV), a family history of sychiatric disorders, or neurologic disorders were excluded. Twelve subjects (2.16% of the total subjects) were excluded based on the exclusion criteria. A total of 545 subjects (328 male, 217 female) were included in the final analysis. All subjects rovided informed consent before the study. The study rotocol was aroved by the Institutional Review Board of Severance Hosital (Severance IRB ). SNP selection To date, only one study has reorted ositive association between USP46 gene and sychiatric henotyes. Fukuo et al. 21 found significant association between halotyes consisting of three (rs rs rs346005) or four (rs rs rs rs346005) SNPs of seven tagging SNPs (r 2 0.8) caturing 30 SNPs (minor allele frequency; MAF 5) covering the USP46 gene region. 21 Therefore, we selected SNPs of rs , rs , rs , and rs in this study. However, we further excluded rs and rs , as MAFs were less than 0.1 (0.056 for rs , for rs ). Esecially, there were no minor homozygotes for rs in the HaMa database (release #27, oulation: Jaanese in Tokyo). Finally, we included two SNPs (rs and rs346005) in this study. SNP rs is located in intron, and SNP rs is located 2 kb ustream of USP46 start site (USP46; , rs346005; ). Genotying The DNA of the subjects was isolated from saliva collected in OG-500 Oragene self-collection kit (tube format OG-500 ), using standard techniques. Genotying of USP46 SNPs was erformed using a single-base rimer extension assay (ABI PRISM SNaPShot TM Multilex kit; ABI, Foster City, CA, USA) according to the manufacturer s instructions. Genotying was erformed by DNA Link, Inc. (Seoul, South Korea), in a manner blinded to any other study data. Assessments Affective temerament was assessed with the Korean version of the Memhis, Pisa, Paris, and San Diego Autoquestionnaire (TEMPS-A, a self-reorted temerament scale for evaluating temeramental dimensions, with five subscales, including deressive, cyclothymic, hyerthymic, irritable, and anxious temeraments. 1 This scale consists of 110 yes-or-no tye questions in total, of which 109 are alicable to male subjects. All articiants comleted the questionnaire. Statistical analyses Previous combined analysis showed the differences in TEMPS- A subscales scores between males and females. 24 Therefore, all the analyses in this study were conducted searately by 88 Psychiatry Investig 2019;16(1):87-92

3 YJ Boo et al. gender. Multile analyses of covariance (MANCOVA) were erformed using the total scores for each TEMPS-A affective temerament dimension as deendent variables, and USP46 SNP genotyes as fixed factors. Age was controlled as a covariate to control ossible effects. The two SNPs were analyzed searately for each gender. Further analyses were conducted for significant associations, with Fisher s least significant difference ost-hoc tests to evaluate differences among genotyes. Data analysis was erformed using SPSS 23.0 for Windows (IBM Cor., Armonk, NY, USA). The statistical significance was set at <0.05 for all tests. Table 1. Demograhic characteristics of the subjects Male (N=328) Female (N=217) Age (years) (2.64) (2.47) <0.001 Education (years) (1.46) (1.58) TEMPS-A Deressive 6.73 (3.03) 7.51 (2.90) Cyclothymic 6.83 (4.56) 8.03 (4.57) Hyerthymic (4.79) (4.54) Irritable 3.60 (3.27) 3.73 (3.45) Anxious 5.56 (5.19) 7.28 (5.42) <0.001 Mean (SD). SD: standard deviation, TEMPS-A: Temerament Evaluation of the Memhis, Pisa, Paris, and San Diego Autoquestionnaire Results Baseline characteristics Baseline demograhic data and characteristics, including TEMPS-A scores, are summarized in Table 1. The mean age of the articiants was years (standard deviation; SD=2.64 years) for males and years (SD=2.47 years) for females. The mean education level of the articiants were years (SD=1.46 years) for males and years (SD=1.58 years) for females. There were significant differences in age (<0.001) and deressive (=0.003), cyclothymic (=0.003), and anxious (<0.001) dimension scores between male subjects and female subjects. Association analyses between USP46 SNPs and five affective temeramental dimensions The genotye distribution of the two USP46 SNPs, rs and rs were in accordance with the Hardy-Weinberg equilibrium (χ 2 =0.068, =0.794 for rs346005, χ 2 =0.161, = for rs ). MANCOVA results are resented in Table 2 through Table 3. The MANCOVA showed a significant association between TEMPS-A and rs genotyes in male subjects (Wilks λ=0.929, F (10,640)=2.384, =0.009). Among temerament dimensions, there was a significant difference in deressive temerament among the genotyes of rs in male sub- Table 2. Multivariate analysis comaring scores for each affective temerament dimension for USP46 single-nucleotide olymorhism rs (mean±sem) after controlling for age Male (N=328) Female (N=217) AA (N=97) CA (N=161) CC (N=70) AA (N=65) CA (N=111) CC (N=41) MANCOVA Wilks λ=0.929, F (10,640)=2.384, =0.009 Wilks λ=0.979, F (10,418)=0.450, =0.921 Deressive 6.79± ± ±0.36 < ± ± ± Cyclothymic 6.47± ± ± ± ± ± Hyerthymic 11.46± ± ± ± ± ± Irritable 3.34± ± ± ± ± ± Anxious 5.97± ± ± ± ± ± SEM: standard error of mean, MANCOVA: multile analyses of covariance Table 3. Multivariate analysis comaring scores for each affective temerament dimension for USP46 gene single-nucleotide olymorhism rs (mean±sem) after controlling for age Male (N=328) Female (N=217) AA (N=198) GA (N=118) GG (N=12) AA (N=138) GA (N=68) GG (N=11) MANCOVA Wilks λ=0.966, F (10,640)=1.124, =0.341 Wilks λ=0.935, F (10,418)=1.421, =0.168 Deressive 6.62± ± ± ± ± ± Cyclothymic 6.62± ± ± ± ± ± Hyerthymic 11.64± ± ± ± ± ± Irritable 3.38± ± ± ± ± ± Anxious 5.63± ± ± ± ± ± SEM: standard error of mean, MANCOVA: multile analyses of covariance 89

4 Affective Temerament and USP Figure 1. Post-hoc analysis comaring the TEMPS-A deressive subscale score for USP46 single-nucleotide olymorhism rs Error bars indicate the standard error of mean. TEMPS- A: Temerament Evaluation of the Memhis, Pisa, Paris, and San Diego Autoquestionnaire. jects (F=7.79, <0.001, η 2 =0.046). The association was not observed in female subjects (F=0.75, =0.472, η 2 =0.007) (Table 2). In contrast, for rs , no associations were found between the genotyes and the five affective temeraments, in males or females (Table 3). Post-hoc comarisons showed that subjects who had the CC genotye (mean=7.89, SD=0.45) at rs showed a higher deressive temerament score than that of subjects with the AA genotye (mean=6.80, SD=0.30, <0.05) and the CA genotye (mean=6.18, SD=0.21, <0.0001) in male subjects. There is no significant difference in the deressive temerament score between subjects with the AA genotye and subjects with the CA genotye (=0.103) (Figure 1). Discussion <0.05 <0.001 USP46 rs AA CA CC We investigated the associations of the USP46 olymorhisms with the affective temeramental traits in healthy college students. The major finding was that the CC genotye of USP46 rs was related to a higher deressive temerament score in male subjects. These findings suggest that affective temeramental traits are influenced by USP46. Genetic associations between the ubiquitin roteasome system and affective temerament have not yet been reorted. Although there is little direct evidence, our finding of the involvement of USP46 in deressive temerament is suorted by indirect evidence. Theoretically, deubiquitinating enzymes, such as USP46, could lay a role in regulating the stability of cellular rotein and synatic trafficking. 25 Animal studies have shown that USP46-mutated mice exhibit a significantly shorter immobility time than wild tye mice in a tail susension test (TST), which indicates a change in deressive state. 23 Furthermore, a genetic association study of major deression showed significant involvement of a USP46 halotye in a Jaanese oulation with major deressive disorder. 21 In the study, rs was the only SNP that showed signification association with major deressive disorder. Considering that a deressive temerament is a subclinical form of major deressive disorder, it is ossible that USP46 may be involved in the athohysiology of deression. The biological mechanism underlying the association between USP46 and deressive temerament remains unclear. However, some ossible exlanations of the association can be roosed. First, USP46 may act as a mood regulator by modulating neurotransmitter systems, such as serotonin and GABA. The antideressant imiramine, a tricyclic antideressant that inhibits reutake of monoamines, such as serotonin and noreinehrine, reduced immobility in the TST of USP46 mutated mice, 23 which suorts the monoamine hyothesis. 26 In contrast, another study using USP46-knock-out mice reorted that deressive behaviors in these mice were changed by administration of nitrazeam, GABAA recetor agonist, 22 suorting the GABA hyothesis of major deressive disorder. 27 Second, the ubiquitin system is known to regulate the circadian system by controlling circadian clock rotein. 28 Associations between the circadian system and affective temerament have reviously been reorted. 29,30 In a revious study, a deressive temeramental trait aeared to be associated with evening-tye circadian references, along with cyclothymic, irritable, and anxious temeraments. 30 Therefore, it can be assumed that USP46 influences affective temeramental traits by modulating the circadian system. Third, USP46 may affect the affective temerament by modulating synatic lasticity. USP46 has been reorted to lay a role in regulating alha-amino-3-hydroxy-5-methyl-4-isoxazole-roinic acid recetor (AMPAR), one of the key recetors involved in longterm otentiation and deression. 25 USP46, which is known to be enriched at the neural synase, may modulate brain function by deubiquitinating AMPAR. Because evidence suorting these seculations is lacking, further studies elucidating the athways that may mediate the association between UPS and deressive temerament should be erformed. Unlike in male subjects, no main effects of USP46 olymorhisms were observed in female subjects. This finding could be exlained in several ways. In general, gene exression and slicing is known to differ between males and females. 31 From other ersectives, this gender difference might be related to an effect of sex hormones. Several studies have reorted that the sex hormone estrogen modulates the ubiquitin systems, 32,33 and lays an imortant role in synatic lasticity Psychiatry Investig 2019;16(1):87-92

5 YJ Boo et al. In this study, there was no difference in genotye distribution of rs between males and females. This suggested that USP46 rs is not the major cause of difference in deressive temerament between males and females. In addition, considering the fact that comlex traits, such as affective temeraments, are medicated by many genes (not one gene) and their interactions, the higher score of deressive temerament in females seems to be affected by other conditions, such as sexual hormone and/or X-chromosome. We could not find other studies showing the sexual dimorhisms of USP46 rs346005, since there is only a few existing studies on USP46. However, we found several other evidence, although not USP46, suggesting genetic sexual dimorhisms on temeramental traits. Stankova et al. 35 reorted that the sexual dimorhic effects of oxytocin recetor gene (OXTR) showed gender difference on harm avoidance; one of the asects of temeramental traits. In our revious study of doamine recetor D4 (DRD4) gene on affective temerament, the association between DRD4 olymorhism and affective temerament (cyclothymic and irritable) was found only in male subjects. 13 In addition, since this study involved a small number of female subjects (n=217), the small samle size may not have been sufficient to obtain significant results. There are several limitations in this study. First, most of our data were collected from college students, and it may not be generalizable. Although our samle can be considered a homogeneous grou, with similar age, educational levels, and ethnicity, the results of this study should be considered cautiously. The results need to be relicated in other oulations of different ages and ethnicities. Second, the resent study was erformed in a relatively small number of subjects. A further study in a larger samle size is needed to confirm the results. Third, we assessed only two SNPs. There are many more SNPs in USP46, and thus a more recise result could be obtained if additional SNPs were to be assessed. Fourth, affective temeramental scores could have been biased by social desirability, since TEMPS-A is a self-reorting scale. Fifth, we did not analyze gene-gene interaction and gene-environment interaction. Desite these limitations, this is the first study that reveals a ossible genetic association between the UPS and affective temeraments. In summary, the resent study showed an association between a SNP in USP46 and deressive temerament. Our findings suggest that that some affective temeraments may be genetically redisosed to develoing into affective disorders via vulnerable genes related to the UPS. In articular, USP46 may lay a role in the athohysiology of major deressive disorders by modulating a deressive temerament through dynamic roteolysis rocesses involving the UPS. Further research is required to elucidate influence of USP46 olymorhisms of affective temeraments in atients with mood disorders. Acknowledgments This study was suorted by a grant from the Disease Oriented Translational Research through the Korea Health Industry Develoment Institute (KHIDI), South Korea (grant number: HI14C0202). REFERENCES 1. Akiskal HS, Akiskal KK, Haykal RF, Manning JS, Connor PD. TEMPS- A: rogress towards validation of a self-rated clinical version of the Temerament Evaluation of the Memhis, Pisa, Paris, and San Diego Autoquestionnaire. J Affect Disord 2005;85: Kawamura Y, Akiyama T, Shimada T, Minato T, Umekage T, Noda Y, et al. Six-year stability of affective temeraments as measured by TEMPS- A. Psychoathology 2010;43: Nigg JT. Temerament and develomental sychoathology. J Child Psychol Psychiatry 2006;47: Kraeelin E. Manic-Deressive Insanity and Paranoia. Edinburgh: E. & S. Livingstone; Toda H, Inoue T, Tsunoda T, Nakai Y, Tanichi M, Tanaka T, et al. 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