Clozapine Pharmacogenetic Studies in Schizophrenia: Efficacy and Agranulocytosis

Clozapine is an efficacious atypical antipsychotic for treatment-refractory schizophrenia. Clinical response and appearance of adverse events vary among individual patients receiving clozapine, with genetic and non-genetic factors potentially contributing to individual variabilities. Pharmacogenetic studies investigate associations between genetic variants and drug efficacy and toxicity. To date, most pharmacogenetic studies of clozapine have been conducted through candidate gene approaches. A recent advance in technology made it possible to perform comprehensive genetic mapping underlying clinical phenotypes and outcomes, which allow novel findings beyond biological hypotheses based on current knowledge. In this paper, we will summarize the studies on clozapine pharmacogenetics that have extensively examined clinical response and agranulocytosis. While there is still limited evidence on clozapine efficacy, recent genome-wide studies provide further evidence of the involvement of the human leukocyte antigen (HLA) region in clozapine-induced agranulocytosis.

Pharmacogenetic studies investigate the associations between genetic variants and drug efficacy and toxicity (Jorgensen and Williamson, 2008). To date, a number of pharmacogenetic studies of clozapine have been reported. In this paper, we summarize the studies on clozapine pharmacogenetics that have extensively examined clinical response and agranulocytosis.

Clinical Response to Clozapine
Response rate for clozapine ranges from 32% in the short term to 39% in the long term among those with treatmentresistant schizophrenia (Siskind et al., 2017). Pharmacogenetic studies of clinical response to clozapine have focused on variations in the genes involved in the metabolism of clozapine [pharmacokinetics, e.g., the cytochrome P 450 (CYP) enzyme family] and the affinities of clozapine (pharmacodynamics, e.g., dopaminergic and serotonergic receptors). Positive candidate gene studies of the clinical response to clozapine are shown in Table 1.
With respect to the DRD1 gene (Potkin et al., 2003;Hwang et al., 2007;Lee et al., 2012), two studies found significant associations between genetic variants and the clozapine response (Potkin et al., 2003;Hwang et al., 2007). Potkin and colleagues demonstrated that rs4532 2/2 carriers were more likely to have a positive clozapine response following 5 weeks of treatment, while rs4532 1/2 carriers demonstrated a diminished clozapine response (N = 15) (Potkin et al., 2003). Hwang and colleagues demonstrated that rs265976 AC carriers were more likely to be non-responders to clozapine with a minimum of 6 months treatment in an African American sample (N = 49). They also investigated an association between three-marker haplotype (rs265981, rs4532, and rs686) and clozapine response, demonstrating that the T-G-A haplotype was associated with poor response in a Caucasian sample (N = 183), while the T-G-G haplotype was associated with better response in an African American sample (N = 49) (Hwang et al., 2007). With respect to the DRD2 gene (Arranz et al., 1998a;Malhotra et al., 1999;Hwang et al., 2005Hwang et al., , 2006Lee et al., 2012), three studies found significant associations between genetic variants in this gene and the clozapine response (Malhotra et al., 1999;Hwang et al., 2005Hwang et al., , 2006. Malhotra and colleagues investigated an association between 141C Ins/Del (rs1799732) and clozapine response in a 10 week treatment, demonstrating that Del-subjects had a fivefold greater reduction in psychotic symptoms as compared to Del+ subjects (N = 72) (Malhotra et al., 1999). Additionally, Hwang and colleagues investigated an association between 12 single nucleotide polymorphisms (SNPs) in the DRD2 gene and clozapine response for a minimum of 6 months treatment, identifying 3 SNPs (Taq1A, Taq1B, and rs1125394) only in an African-American sample (N = 49) and several haplotypes in both Caucasian (N = 183) and African-American samples (N = 49) as being predictive of a clozapine response (Hwang et al., 2005). This same group later investigated the effect of the same 12 SNPs in the DRD2 gene on clozapine response evaluated by overall, positive, and negative symptoms in smaller sample set (N = 35) and demonstrated that 2 SNPs (Taq1B and rs1125394) were associated with overall and positive symptom response to clozapine in an African-American sample (Hwang et al., 2006). However, a recent meta-analysis (total N = 596) suggests no association between 141C Ins/Del and a clozapine treatment response (Gressier et al., 2016). With respect to the DRD3 gene (Gaitonde et al., 1996;Shaikh et al., 1996;Malhotra et al., 1998;Scharfetter et al., 1999;Arranz et al., 2000b;Barlas et al., 2009;Hwang et al., 2010;Lee et al., 2012), three studies found significant associations between genetic variants and the clozapine response (Shaikh et al., 1996;Scharfetter et al., 1999;Hwang et al., 2010). Shaikh and colleagues investigated an association between Ser-9-Gly polymorphism (rs6280) in the DRD3 gene and clozapine response for at least 3 months of treatment (N = 79), demonstrating that the genotype Ser-9/Ser-9 was more frequent in the non-responders than in responders (Shaikh et al., 1996). Scharfetter and colleagues replicated this finding in patients treated with clozapine for 6 months (N = 32) (Scharfetter et al., 1999). However, a recent meta-analysis (total N = 852) suggests no association between Ser-9-Gly and a clozapine treatment response (Gressier et al., 2016). Hwang and colleagues demonstrated an association of better clozapine response for a minimum of 6 months with the A allele of  Frontiers in Pharmacology | www.frontiersin.org rs2134655 in a Caucasian sample (N = 183) and the T allele of rs1394016 in an African-American sample (N = 49) (Hwang et al., 2010). With respect to the DRD4 gene (Shaikh et al., 1993(Shaikh et al., , 1995Kerwin et al., 1994;Rao et al., 1994;Rietschel et al., 1996;Kohn et al., 1997;Kaiser et al., 2000;Zhao et al., 2005;Hwang et al., 2012;Lee et al., 2012;Rajagopal et al., 2018), two studies found significant associations between genetic variants and the clozapine response (Zhao et al., 2005;Hwang et al., 2012). Zhao and colleagues investigated an association between the 48 bp variant number tandem repeat (VNTR) polymorphism in the DRD4 gene and clozapine response for at least 2 months of treatment after clinical stabilization (N = 81), demonstrating that the frequencies of 5 allele and 5/5 genotype were higher among the non-responders than in responders (N = 81) (Zhao et al., 2005). Hwang and colleagues demonstrated an association between 120-bp 1-copy allele and intron I (G)n 142/140 bp genotype and poor clozapine responders for 6 months of treatment in an African American sample (N = 49), and an association between 48 bp repeat polymorphism and better clozapine response in a Caucasian sample (N = 183) (Hwang et al., 2012). With respect to the DRD5 gene, there were no significant associations between genetic variants and the clozapine response (Hwang et al., 2012). With respect to the COMT gene (Woodward et al., 2007;Bosia et al., 2015;Rajagopal et al., 2018), two studies found a significant association between the COMT Val108/158Met polymorphism (rs4680) and the clozapine response (Woodward et al., 2007;Bosia et al., 2015). Woodward and colleagues investigated an association between this polymorphism and clozapine response for 6 weeks and 6 months of treatment (N = 86), demonstrating that both the Met/Met and Val/Met groups showed greater improvement in attention and verbal fluency domains compared to the Val/Val group (Woodward et al., 2007). Bosia and colleagues demonstrated a greater improvement in the Val/Val group compared to both the Val/Met and Met/Met groups in the negative symptom response for 8 and 16 weeks of treatment with clozapine (N = 107) (Bosia et al., 2015). With respect to the SLC6A3 gene, Xu and colleagues demonstrated that the 71T allele of rs2975226 (T-71A) in the SLC6A3 gene occurred more frequently in the responders than in non-responders following at least 8 weeks of treatment with clozapine (Xu et al., 2010).
To date, genetic variants in the HTR2A, HTR2C, HTR6, and HTR7 genes as well as in 5-hydroxytryptamine receptor 1A (HTR1A), 5-hydroxytryptamine receptor 3A (HTR3A), 5hydroxytryptamine receptor 3B (HTR3B), 5-hydroxytryptamine receptor 5A (HTR5A), and solute carrier family 6 member 4 (SLC6A4) genes have been investigated in clozapine response studies, with positive associations observed in the HTR2A, HTR2C, HTR6, HTR1A, HTR3A, and SLC6A4 genes. With respect to the HTR2A gene (Arranz et al., 1995a(Arranz et al., , 1996(Arranz et al., , 1998b(Arranz et al., , 2000bMasellis et al., 1995Masellis et al., , 1998Nöthen et al., 1995;Malhotra et al., 1996a;Lin et al., 1999;Schumacher et al., 2000;Yu et al., 2001;Lee et al., 2012), six studies found significant associations between genetic variants and the clozapine response (Arranz et al., 1995a(Arranz et al., , 1996(Arranz et al., , 1998b(Arranz et al., , 2000bMasellis et al., 1998;Yu et al., 2001). Arransz and colleagues investigated an association between the T102C polymorphism (rs6313) in the HTR2A gene and clozapine response for at least 3 months of treatment following clinical stabilization (N = 149), demonstrating that homozygosity for the C102 allele was more frequent in the non-responders than in the responders (Arranz et al., 1995a). Yu and colleagues demonstrated an association between the 102C/C genotypes and higher N100 amplitude following clozapine treatment using event-related potentials to auditory stimuli (N = 98) . Consistent with these findings, a recent meta-analysis (total N = 868) suggests a significant association between the CC genotype and poor clozapine treatment response (Gressier et al., 2016). Arransz and colleagues also investigated an association between the his452tyr polymorphism (rs6314) in the HTR2A gene and clozapine response (N = 153), demonstrating that the Tyr452 allele occurred more frequently in the non-responders than in the responders (Arranz et al., 1996). Masellis and colleagues replicated this finding in subjects who received clozapine for a minimum of 6 month (N = 185) (Masellis et al., 1998). Consistent with these findings, a recent meta-analysis (total N = 671) suggests a significant association between C allele or C carriers or CC genotype and better clozapine treatment response compared to T allele or T carriers or TT genotype (Gressier et al., 2016). Arranz and colleagues also examined an association between the G-1438A polymorphism (rs6311) in the HTR2A gene and clozapine response (N = 274), demonstrating that homozygosity for the G-1438 allele was more frequent in the non-responders than the responders (Arranz et al., 1998b). However, a recent meta-analysis (total N = 547) suggests no association between the G-1438A polymorphism and clozapine treatment response (Gressier et al., 2016). With respect to the HTR2C gene (Sodhi et al., 1995;Malhotra et al., 1996b;Rietschel et al., 1997;Masellis et al., 1998;Arranz et al., 2000b;Schumacher et al., 2000), two studies found a significant association between genetic variants and the clozapine response (Sodhi et al., 1995;Arranz et al., 2000b). Sodhi and colleagues investigated an association between the Cys23Ser polymorphism (rs6318) in the HTR2C gene and clozapine response for at least 3 months treatment after clinical stabilization (N = 162), demonstrating that Ser allele carriers were more likely to show a response to clozapine (Sodhi et al., 1995). However, a recent meta-analysis suggests no association between the Cys23Ser polymorphism and clozapine treatment response in a Caucasian sample (Gressier et al., 2016). With respect to the HTR6 gene Masellis et al., 2001;Lee et al., 2012), one study found a significant association between a genetic variant and the clozapine response . Yu and colleagues examined the association between the C267T polymorphism in the HTR6 gene and clozapine response for at least 8 weeks of treatment (N = 99), demonstrating that the 267T/T genotype was more frequent in responders than in non-responders . With respect to the HTR7 gene, there was not a significant association between the pro279leu polymorphism and the clozapine response (Masellis et al., 2001). With respect to the HTR1A gene (Masellis et al., 2001;Bosia et al., 2015), one study found a significant association between a genetic variant and the clozapine response (Bosia et al., 2015). Bosia and colleagues examined the association between the −1019C/G (rs6295) polymorphism in the HTR1A gene and clozapine response for 8 and 16 weeks of treatment (N = 107), demonstrating a greater improvement in the G/G group compared to the C/C group (Bosia et al., 2015). With respect to the HTR3A gene (Arranz et al., 2000b;Gutiérrez et al., 2002;Souza et al., 2010;Lee et al., 2012;Rajkumar et al., 2012), two studies found significant associations between genetic variants and the clozapine response (Souza et al., 2010;Rajkumar et al., 2012). Souza and colleagues demonstrated the T allele of rs1062613 in the HTR3A gene was associated with a good clozapine treatment response when used for at least 6 months (N = 140) (Souza et al., 2010). Similarly, Rajkumar and colleagues investigated an association of rs1062613 and rs2276302 in the HTR3A gene with the clozapine response, demonstrating that the T allele of rs106263 and the G allele of rs2276302 were associated with a good clozapine treatment response when presented with a stable dose for at least 12 weeks (N = 101) (Rajkumar et al., 2012). Consistent with these findings, a recent meta-analysis suggested that there may be an association between the T allele of rs1062613 and an improved response to clozapine (Gressier et al., 2016). With respect to the HTR3B and HTR5A genes (Arranz et al., 2000b;Birkett et al., 2000;Gutiérrez et al., 2002;Souza et al., 2010), there were no significant associations between genetic variants and the clozapine response. With respect to the SLC6A4 gene (Arranz et al., 2000a,b;Schumacher et al., 2000;Tsai et al., 2000;Kohlrausch et al., 2010), two studies found significant associations between genetic variants and the clozapine treatment response (Arranz et al., 2000b;Kohlrausch et al., 2010). Arranz and colleagues demonstrated a significant association between the biallelic polymorphism in the promoter region of the SLC6A4 gene, 5-HTTLPR, and the clozapine response in a Caucasian sample (N = 200) (Arranz et al., 2000b), and they found a similar trend in a larger sample (N = 268) (p = 0.08) (Arranz et al., 2000a). Kohlrausch and colleagues demonstrated that the short allele of HTTLPR/rs25531 occurred more frequently in the non-responders than in responders (Kohlrausch et al., 2010)

Gene-Gene Interactions
Several trials have been performed to investigate a gene-gene interaction between multiple candidate genes and the clinical response to clozapine. Arranz and colleagues examined an association between 19 genetic polymorphisms in nine clozapinetargeted receptor subtypes and a neurotransmitter transporter and the response to clozapine, demonstrating that the strongest association was with a combination of six polymorphisms (HTR2A T102C, HTR2A his452tyr, HTR2C−330-GT/-244-CT, HTR2C Cys23Ser, SLC6A4 5-HTTLPR, and H2−1018-G/A) (N = 200) (Arranz et al., 2000b). However, Schumacher et al. could not replicate this finding (Schumacher et al., 2000). Hwang and colleagues demonstrated the most straightforward genegene-interaction effect of DRD1 rs686 and DRD3 Ser9Gly on the clozapine treatment response when taken for a minimum of 6 months in a Caucasian sample (N = 183) (Hwang et al., 2011). Bosia and colleagues demonstrated an additive effect of COMT Val158Met and HTR1A−1019 C/G on the clozapine treatment response when taken for 8 and 16 weeks (N = 107) (Bosia et al., 2015). Similarly, Rajagopal and colleagues demonstrated a significant gene-gene-interaction effect of DRD4 120-bp duplication and COMT Val158Met on the clozapine response when taken for at least 12 weeks (N = 93) (Rajagopal et al., 2018). Furthermore, Xu and colleagues conducted multivariate interaction analysis using 77 SNPs of 25 genes and demonstrated that the combination of rs6269 in the COMT gene and rs3813929 in the HTR2C gene may work as predictor to improve the clinical antipsychotic response (risperidone, clozapine, quetiapine, and chlorpromazine) in a large sample set (N = 995) (Xu et al., 2016).

Non-candidate Gene Approach
To date, the authors are unaware of any genome-wide association studies (GWAS) of clozapine efficacy that have been published. Recent pharmacogenetics GWAS suggest genetic overlap between antipsychotic response and susceptibility to schizophrenia (Ikeda et al., 2015;Ruderfer et al., 2016), and two genetic variants, identified by GWAS as schizophrenia risk loci (Cross-Disorder Group of the Psychiatric Genomics Consortium., 2013; Schizophrenia Working Group of the Psychiatric Genomics Consortium., 2014), have shown to be associated with a significant clinical response to clozapine (Brandl et al., 2016;Huang et al., 2016). Randl and colleagues demonstrated a significant association between rs2535629 in the inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) gene and an improvement of negative symptoms after 6 months of clozapine treatment (N = 105) (Brandl et al., 2016). Additionally, Huang and colleagues demonstrated a significant association between rs2514218, located upstream of the DRD2 gene, and clozapine response for 6 months of treatment (N = 208) .

Candidate Gene Approach Human leukocyte antigen (HLA) genes
In patients developing CIA (N = 6) compared with controls (N = 25), Lieberman and colleagues demonstrated that the occurrence of HLA-B38 as well as the haplotype of HLA-B38, DR4, and DQw3, was more frequent (Lieberman et al., 1990). The same laboratory subsequently analyzed them separately by Ashkenazi Jewish patients and non-Jewish patients, conforming their previous finding in the Ashkenazi Jewish samples and demonstrating a significant association of HLA-B7 and DR2 with CIA in non-Jewish patients . They also demonstrated that the variants in the heat-shock protein 70 (HSP-70) and tumor necrosis factor (TNF) genes were associated with CIA Turbay et al., 1997). Additionally, Amar and colleagues demonstrated that HLA-DQB1 * 020 significantly occurred more frequently in subjects who developed CIAG compared to those who did not develop those complications (5 CIAG cases vs. 13 controls) (Amar et al., 1998). Valevski and colleagues replicated the involvement of HLA-B38 in CIA in Jewish Israeli samples (11 CIA cases vs. 50 controls) (Valevski et al., 1998). Dettling and colleagues demonstrated a significant association of HLA-Cw * 7, DQB * 0502, DRB1 * 0101, and DRB3 * 0202 with CIA in non-Jewish Caucasian samples (31 CIA cases vs. 77 controls) (Dettling et al., 2001b). The same laboratory also demonstrated a significantly higher frequency of HLA-DRB5 * 02 and HLA-DQB * 0502 in patients who developed CIA (Dettling et al., 2001a). Later, the same laboratory performed an association study of CIA by combining the HLA class I and class II genetic variants, demonstrating that HLA-DRDB5 * 0201 and several haplotypes of the HLA genetic variants were associated with CIA (Dettling et al., 2007). Furthermore, Lahdelma and colleagues demonstrated that the HLA-B16 allele occurred more frequently in patients who developed CIAG (22 CIAG cases vs. 120 healthy controls) (Lahdelma et al., 2001), while Athanasiou and colleagues demonstrated that the HLA-DQB1 "REC 21G" occurred more frequently in patients who developed CIA compared with controls in two independent cohorts (Athanasiou et al., 2011).

Non-candidate Gene Approach
Tiwari and colleagues conducted exome sequence analysis and did not find any genetic variants associated with CIA in Finnish patients after Bonferroni correction (13 CIA cases and 11 cases with severe neutropenia vs. 26 controls) (Tiwari et al., 2014). Meanwhile, Goldstein and colleagues conducted GWAS, wholeexome sequencing, and HLA allele imputation and were able to show that HLA-B 158T and HLA-DQB1 126Q were associated with CIAG (Goldstein et al., 2014). Legge and colleagues utilized GWAS, imputed HLA alleles, exome array, and copy-number variation analyses in a European population and subsequently combined the data of Goldstein et al. (2014) and Legge et al. (2017). In their meta-analysis of GWAS, they demonstrated that rs149104283, an intronic transcript of SLCO1B3 and SLCO1B7, was associated with clozapine-induced neutropenia. The authors of this paper conducted GWAS of CIAG in Japanese samples (50 CIAG cases vs. 2,905 controls) and identified rs1800625 in the HLA region as the CIAG candidate locus (Saito et al., 2016). A classical HLA analysis was subsequently conducted, demonstrating a significant association of HLA-B * 59:01 with CIAG. However, we failed to replicate the risk SNPs on clozapineassociated neutropenia previously identified by Legge et al. (2017) in a Japanese population .

DISCUSSION
In the present article, we summarize clozapine pharmacogenetic studies, separated by clinical response and CIA. To date, pharmacogenetic studies of clozapine in schizophrenia have been mostly conducted through candidate gene approaches. Genetic variants in the CYP enzyme family, dopamine, and serotonin receptor genes have been extensively examined as pharmacokinetic and pharmacodynamics candidates related to   clozapine efficacy. Although there is limited evidence, the most recent meta-analyses indicate that only three SNPs (rs6313 and rs6314 in the HTR2A gene and rs1062613 in the HT3A gene) are associated with a significant clozapine response (Gressier et al., 2016). Genetic variants in the genes within the MHC region have been extensively examined with respect to CIA. The results of these candidate studies indicate the HLA variants are implicated in developing CIA. A comprehensive screening of genetic variants linked to a significant clinical response to clozapine and CIA is critical because the selection of candidate genes is restricted to current knowledge about underlying biological mechanisms . Recently, the field of clozapine pharmacogenetics has shifted from a candidate gene approach to a genome-wide approach. A genome-wide approach has successfully identified novel genes that are associated with CIA and provided further evidence of the involvement of the HLA variants in CIA (Goldstein et al., 2014;Saito et al., 2016). On the other hand, there are no known GWAS looking at the clinical response to clozapine, although there are several GWAS looking at the antipsychotic treatment response in patients with schizophrenia Drögemöller et al., 2016;Ruderfer et al., 2016;Li et al., 2017;Yu et al., 2018). Yu and colleagues conducted GWAS in a large cohort (n = 3,792) and detected five loci (CNTNAP5, MEGF10, PCDH7, SLC1A1, and TNIK) associated with general antipsychotic treatment response (Yu et al., 2018). Interestingly, these loci did not include DRD2, although D2 receptor blockade in the brain is a general pharmacodynamic property of antipsychotics (Mauri et al., 2014).
Pharmacogenetic testing has the potential to help improve patient outcome, lower healthcare costs, and increase patient medication adherence (Gardner et al., 2014). Pharmacogenetic testing in psychiatry is not yet a standard of practice, however, its utilization is steadily increasing (Eum et al., 2016;Fabbri et al., 2018). Several studies have, indeed, assessed the clinical utility for risk genetic variants of CIA. The sensitivity and specificity of the HLA-DQB1 6672G.>C polymorphism for CIA in patients treated with clozapine, identified though candidate approach, was 21.5 and 98.4%, respectively (Athanasiou et al., 2011). The sensitivity and specificity of the HLA-B * 59:01 for CIA, identified by GWAS, was 31.8 and 95.3%, respectively (Saito et al., 2016) and the sensitivity and specificity of HLA-B 158T and HLA-DQB1 126Q polymorphisms for CIA, identified by GWAS and whole-exome sequencing (Goldstein et al., 2014), was 41 and 85%, respectively (Girardin et al., 2018). Clinical application guidelines require HLA allele testing for CIA to have a sensitivity of ∼50% (Girardin et al., 2018), therefore none of these have reached an acceptable threshold for clinical application. Conversely, we examined the diagnostic performance of nonrisk allele (alleles except for HLA-B * 59:01) on non-CIA among CIG patients and demonstrated a moderate, positive predictive value for detecting non-CIA subjects in the CIG group without the risk allele, suggesting a potential candidate for re-challenging with clozapine treatment in a Japanese population (Saito et al., 2016). Based on this finding, a re-challenging with clozapine following neutropenia in a patient with a low risk of CIAG (HLA-B * 52:01/52:01) was successfully conducted (Yamaki et al., 2017).
The decision regarding clozapine re-challenge or withdrawal in case of CIAG should be based on careful consideration of risk factors, which can be facilitated by genetic testing in the future (Wicinski and Weclewicz, 2018). Further efforts to identify strong and reproducible genetic variants related to the clinical response to clozapine and CIA are needed to develop appropriate pharmacogenetic testing of clozapine.
The major issue of pharmacogenetics studies is inconsistent findings among studies. The discrepancies between these studies might be caused by statistical issues (i.e., sample size, multiple testing) and methodological issues (i.e., study design, phenotype definition, genetic polymorphism, population stratification) (Ross et al., 2012). Indeed, each sample size of the clozapine pharmacogenetic studies was relatively small. Large samples are needed to have enough statistical power to detect the effects of genetic variants on clinical outcomes by creating a clozapine consortium (Saito et al., 2016 or performing meta-analyses (Gressier et al., 2016;Legge et al., 2017). A sample size of more than 900 participants will be needed in a pharmacogenetic study if a common variant is anticipated with a large effect (Ross et al., 2012). Additionally, most of the clozapine pharmacogenetic studies did not adjust for the multiple statistical comparisons, resulting in type I error. To adjust for multiple testing, a false discovery rate (FDR) correction will be useful. Furthermore, selection bias and information bias are confounding factors in both prospective cohort and case-control studies (Ross et al., 2012). Clinical responses to clozapine have been determined by several different evaluation scales, including the Clinical Global Impressions Improvement (CGI-I) score, the global assessment scale (GAS), the Brief Psychiatric Rating Scale (BPRS), and Positive and Negative Symptom Scale (PANSS). Linkage disequilibrium (LD) varies among ethnic population, which may affect cross-subpopulation comparisons when causal SNPs are not directly genotyped but rather captures by proxy SNPs (Ross et al., 2012). Population stratification occurs when ethnic subpopulations within the entire study population differ in terms of genotype frequency and risk of disease (Thomas and Witte, 2002). In addition, clozapine doses and treatment length as well as types of antipsychotics preceding clozapine administration differ among studies. Although the clozapine treatment length in clinical response studies ranges from 5 weeks to more than 6 months, Lieberman and colleagues reported that 76% of patients responded to clozapine administered up to 52 weeks and slow clozapine responders experienced 70% of their total improvement after 12 weeks of treatment with clozapine (Lieberman et al., 1994). Standardized treatment protocols and evaluations of clinical outcomes will be needed. Furthermore, both non-genetic and genetic factors play an important role in the clinical outcome. For example, age at onset, gender, severity of the illness, negative symptoms, extrapyramidal side effects, clozapine concentration, history of catatonia, smoking, hypersomnolence, and cognitive deficits have all been associated with the clozapine treatment response (Perry et al., 1991;Lieberman et al., 1994;Miller et al., 1994;Umbricht et al., 2002;Semiz et al., 2007;Rajkumar et al., 2013a). To take these factors in analyses will be needed.
In conclusion, a number of clozapine pharmacogenetic studies have been performed based on candidate gene approaches. However, there is heterogeneity across studies and their results have been inconsistent. Reproducible genetic variants with large effect related to the clinical response to clozapine and CIA have not been detected so far. This field is beginning to shift from candidate gene approaches to more a comprehensive strategy, such as GWAS and whole genome sequencing, which will make it possible not only to identify novel genetic variants related to clinical outcomes, but also to analyze the effects of multiple genes on clinical outcomes. Extensive effort is required to apply pharmacogenetic information in clinical practice for a personalized medicine strategy of clozapine treatment.

AUTHOR CONTRIBUTIONS
SN and HU selected the articles and wrote the first draft of the manuscript. RH and TO supervised and contributed to the editing the manuscript. All authors have approved the final manuscript.

FUNDING
This work was supported in part by Japan Agency for Medical Research and development, AMED (JP15dk0310058, JP16dk0307065, SN and RH).