SCHRES-06021; No of Pages 7 Schizophrenia Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Schizophrenia Research journal homepage: www.elsevier.com/locate/schres
A genetic locus in 7p12.2 associated with treatment resistant schizophrenia Jiang Li, Herbert Y. Meltzer ⁎ Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine, United States
a r t i c l e
i n f o
Article history: Received 9 May 2014 Received in revised form 20 August 2014 Accepted 23 August 2014 Available online xxxx Keywords: Schizophrenia Genetic Treatment resistant Dopa decarboxylase Genome-wide association study Clozapine
a b s t r a c t Approximately 30% of patients with schizophrenia are treatment resistant (TRS), i.e. have persistent psychotic symptoms despite adequate trials of at least two antipsychotic drugs (APDs). Most TRS patients are candidates for clozapine treatment which is underutilized because of its side effects and difficulty in identifying TRS. We conducted a genome-wide association study (GWAS) of 79 TRS and 95 non-treatment resistant (NTRS) Caucasian schizophrenia patients to identify possible biomarkers for TRS, which might also provide insight into the pathobiology of TRS. The single nucleotide polymorphism, rs2237457, located in 7p12.2, a region reported to have imprinted inheritance, was found to have the lowest p value in an allelic association test (unadjusted p = 5.53 × 10−6). Haploview disclosed a 30 kb block flanking this SNP within GRB10, 70 kb upstream of L-dopa decarboxylase (DDC), an enzyme which is rate-limiting in the synthesis of trace amines and neurotransmitters implicated in schizophrenia and the action of APDs. This SNP or haplotype was identified as an exclusive cisacting eQTL for DDC in human dorsolateral prefrontal cortex by BrainCloud®. A replication sample genotyped for this SNP produced a weaker result, but in the same direction. After combining the two samples, rs2237457 remained significantly associated with TRS (unadjusted p = 5.66 × 10−7 in recessive mode; 9.42 × 10−5 in allelic association). If replicated in an independent sample, rs2237457 may provide a biomarker to identify a significant proportion of Caucasian TRS. The results implicate trace amines and their synthesis in the pathophysiology of TRS. © 2014 Published by Elsevier B.V.
1. Introduction It is estimated that 1% of adults meet current criteria for schizophrenia. Treatment with typical or atypical antipsychotic drugs (APDs) produces significant reductions in delusions and hallucinations in about 70% of people with schizophrenia. The other 30% of schizophrenia patients are referred to as treatment resistant (TRS) or refractory schizophrenia (Kane et al., 1988a; Conley and Kelly, 2001; Tiihonen et al., 2006). TRS is operationally defined as persistence of moderate to severe positive symptoms despite two or more trials of 4–6 week duration with typical or atypical APDs other than clozapine, the only drug approved for TRS (Kane et al., 1988a; Kane et al., 1988b; Meltzer, 1997; Suzuki et al., 2012). Although most TRS patients have poor functional outcome, due in part to persistent negative symptoms and cognitive impairment, these features, which are shared by many non-TRS (NTRS) patients, are not the basis for TRS as defined here (Meltzer, 1997). It is highly likely that the neurobiology of persistent positive symptoms is only partially related, if at all, to that of cognitive impairment and negative symptoms (Meltzer, 1997). ⁎ Corresponding author at: Department of Psychiatry and Behavioral Sciences, Ward 12-104, 303 Chicago Ave, Chicago, IL 60611, United States. Tel.: + 1 312 503 0309; fax: + 1 312 503 0348. E-mail address: [email protected] (H.Y. Meltzer).
Excluding inadequate APD trials, the differences in efficacy of APDs in TRS and NTRS patients may be based on pharmacokinetic or pharmacodynamic causes, or some combination of both. Pharmacokinetic differences in APD metabolism have rarely been shown to be causally related to poor response to APDs, but may be relevant for individual patients and drugs (Tugg et al., 1997; Meltzer, 2013). Indeed, TRS patients often receive higher doses of APDs and, despite that, have higher plasma levels. Since schizophrenia is a heterogeneous syndrome, differences in the causes of psychotic symptoms, e.g. excessive limbic dopaminergic activity, enhanced serotonin 2A receptor stimulation, or deficient GABAergic or glutamatergic activity, could be the basis for psychosis and, thus, difference in response to APDs, perhaps related to genetic and epigenetic differences. During the past decade, there have been a number of GWAS or candidate gene studies in search for genetic marker(s) related to schizophrenia and to TRS (Supplemental Table 1). The reported effect sizes are generally small and require replication. 2. Methods and materials 2.1. Subjects The discovery GWAS included 174 self-described Caucasian patients diagnosed with schizophrenia or schizoaffective disorder by DSM-IV
http://dx.doi.org/10.1016/j.schres.2014.08.018 0920-9964/© 2014 Published by Elsevier B.V.
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
2
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
Table 1 Demographic and clinical features for patients who are self-described Caucasians in GWAS dataset and additional dataset. Characteristics
GWAS sample
TR status Male (%) Age of first onset (years) Subdiagnosis
TR (N = 79) 53 (67.09%) 20.37 ± 0.75 69 (87.34%) — schizophrenia 10 (12.66%) — schizoaffective 32.96 ± 1.41 11.75 ± 0.61 26.68 ± 0.68
BPRS BPOS BMI
Additional sample
NTR (N = 95) 63 (66.32%) 22.55 ± 0.83 64 (67.37%) — schizophrenia 31 (32.63%) — schizoaffective 26.12 ± 1.11 8.42 ± 0.46 28.77 ± 0.79
TR (N = 70) 45 (64.29%) 19.71 ± 1.29 38 (54.29%) — schizophrenia 32 (45.71%) — schizoaffective 31.37 ± 1.02 11.04 ± 0.42 29.11 ± 0.90
p value
NTR (N = 125) 79 (63.20%) 20.94 ± 0.76 46 (36.80%) — schizophrenia 79 (63.20%) — schizoaffective 22.42 ± 1.17 6.62 ± 0.48 30.68 ± 1.00
(GWAS/additional) 0.914/0.880a 0.058/0.380b 0.002/0.018a b0.001/b0.001b b0.0001/b0.0001b 0.002/0.219c
Data was presented as Mean ± SE. BPRS represents Brief Psychiatric Rating Scale. BPOS is the sum of four positive symptoms (suspiciousness, hallucinatory behavior, unusual thought content, and conceptual disorganization) of 18 items in BPRS. a Chi-square test. b t-Test. c ANCOVA test adjusted by Gender.
criteria. Those subjects had participated in prospective clinical trial or longitudinal study of the effect of clozapine (Meltzer, 1997). An additional 195 subjects with these diagnoses who had been prospectively classified as TRS or NTRS were identified from subsequent clinical trials or cross-sectional study in our laboratory. 2.2. Genotyping and data analysis SNP genotyping was performed using 610K quad BeadChip® (Illumina) or Taqman® assay (Applied Biosystems). Quality control of genotyping data was described in Supplemental information. Association testing was conducted with PLINK 1.0.7 software (Purcell et al., 2009) and SPSS. QUANTO 1.2 was used for power test. BrainCloud (Colantuoni et al., 2011) was used to identify the potential cis-eQTL. ENCODE and UCSF Brain Methylation Database (Maunakea et al., 2010) were applied for functional prediction as cis-regulatory elements. 3. Results Clinical characteristics and demographic feature for the GWAS sample: TRS (n = 79) and NTRS (n = 95) patients, and for the additional subjects who were genotyped (TRS: n = 70 and NTRS: n = 125) are reported in Table 1. There was no significant difference between the
four groups in gender. Age at onset was nearly significantly earlier in the TRS patients, consistent with our previous report of a larger sample (Meltzer et al., 1998). SNPs which differentiated TRS and NTRS by allelic association test (Fig. 1A) and Cochran–Armitage trend test (Supplemental Fig. 2A) are demonstrated in a Manhattan plot (Fig. 1A). No SNP met genomewide significance for association with TRS. Six SNPs had unadjusted p b 10− 5 after relaxing the corrected p value for FDR-BH to 0.56 (Supplemental Table 2).
3.1. GRB10 rs2237457, a GRB10 SNP, one of the top hits associated with TRS, resides in a well-known imprinted genomic region, 7p12.2 (GRB10– DDC), in human (Blagitko et al., 2000; Yoshihashi et al., 2000) and mouse (Arnaud et al., 2003; Menheniott et al., 2008; Garfield et al., 2011). Due to unclear parental origin of risk allele, we re-analyze the GWAS dataset by excluding the 71 cases with heterozygous genotype for rs2237457, leaving 53 NTRS cases and 50 TRS cases. The association with TRS was significantly enhanced, from p = 5.53 × 10−6 with the heterozygotes to p = 4.99 × 10−12, by allelic association test. There were no other SNPs across the whole genome close to the same level of significance as rs2237457, or SNPs in LD with rs2237457, in a
Allelic test Chi-square on selfdescribed caucasian cases
B
A
3.0 2.0
5 3 1
-log10(P)
4.0
2
Observed -logP values
4
5.0
*
0
1.0
0
0.0
1
2
3
4
5
6
Expected -logP values
Chromosome
Fig. 1. Manhattan plot and Q–Q plot of genome-wide association with allelic Chi-square test. Panel A shows a Manhattan plot for all self-described Caucasian cases in the GWAS. “*” marks regions on chromosome 7 that reach highest genome-wide significance (p b 10−5). Values for each chromosome are shown in different colors for visual effect. Panel B shows the Q–Q plots for the corresponding test of association. This plot shows no deviation from the null distribution, except in the upper tail of the distribution, which corresponds to the SNPs with the strongest evidence for association. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
3
A
B SNP(s) Gene(s)
ENCORE
* UCSF Brain DNA Methylation
Fig. 2. Haplotype view of a genomic region surrounding rs2237457. A. Haplotype view of a region inside GRB10 but upstream of DDC. ±50 kb of rs2237457, was included. Color gradient from red to blue is correlated with D′ value from high to low in each box. rs2237457 is highlighted in the black box. B. UCSC Genome Browser Illustration of a custom track from haplotype block 2. Green bar represents rs2237457; Blue bar represents other associated SNPs from GWAS; Red bar represents imputed SNPs with r2 N 0.8. USCF Brain DNA Methylation tract and ENCODE Methylation tract were also added. This ~30 kb intronic region associated with TRS in GRB10 spans a variety of DNase I hypersensitivity clusters, suggesting a strong regulatory activity in this region, and enrichment of the modified histone mark, H3K4Me1, suggesting enhancers. According to UCSF brain methylation database, the TRS associated intronic haplotype we identified harbors an H3K4me3 site (promoter) as shown in “H3K4me3 Raw Signal” track in green (*). There is a strong overlap of H3K4me3 with an unmethylated intragenic CGI, as shown in “MRE CpG” track, suggesting that this is an alternate promoter site. This promoter site is uniquely neuronal or tissue-specific since it does not overlap with the histone methylation tracks (H3K4Me3) derived from ENCODE which has signal overlaid from nine non-neuronal cell lines from a variety of human tissues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
4
C
rs2237457
B
rs2329487
D
E
Fig. 3. BrainCloud suggests that this TRS associated SNP or haplotype in an intron region of GRB10 is cis-eQTL for gene expression of DDC. There were 105 and 130 SNPs within the 200 kb interval for DDC and GRB10, respectively. Scatterplots showed the distribution of gene expression data (normalized) of DDC (A, B) or GRB10 (C, D) for each genotype of rs2237457 (A, C) or rs2329487 (B, D) as a function of postnatal age. The LOESS fit curve was added to the corresponding scatterplot of that genotype as a function of age. E. Multiple custom annotation tracks were created at UCSC Genome Browser to demonstrate that this TRS associated SNP is cis-eQTL for gene expression of DDC. “Expression Probe” track visualizes the location of expression probes selected by BrainCloud. “TR haplotype LD” track represents all SNPs in LD (r square N 0.8), significantly associated with TR in GRB10–DDC genomic region. “GRB10 CisAssoc” track and “DDC CisAssoc” track represent all genotyped SNPs within ±100 kb up/down stream of GRB10 and DDC, respectively, by BrainCloud. Red signal represents SNPs with significant association with gene expression obtained from GLM based on the best fit model procedure. 6 out of 7 cis-eQTLs (red stick with p b 0.05) for DDC are aggregated in a small region of intron 4 of GRB10, suggesting an eQTL enrichment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
A
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
5
Table 2 Summary of GWAS and Joint analysis with additional samples for rs2237457. Testa
All samples
Homozygous only
GENO TREND ALLELIC DOM REC TREND ALLELIC
GWASb
Additionalb
Jointb
NTR
TR
CHISQ
p
NTR
TR
CHISQ
p
NTR
TR
CHISQ
p
3/42/50 48/142 48/142 45/50 3/92 6/100 6/100
24/29/26 77/81 77/81 53/26 24/55 48/52 48/52
25.03 18.54 20.65 6.818 24.38 23.85 47.69
3.67E−06 1.66E−05 5.53E−06 0.00902 7.90E−07 1.04E−06 4.99E−12
13/55/57 81/169 81/169 68/57 13/112 26/114 26/114
15/22/33 52/88 52/88 37/33 15/55 30/66 30/66
5.62 0.801 0.8983 0.04298 4.438 2.529 5.058
0.060 0.371 0.343 0.846 0.035 0.112 0.025
16/97/107 129/311 129/311 113/107 16/204 32/214 32/214
39/51/59 129/169 129/169 90/59 39/110 78/118 78/118
25.06 13.64 15.25 2.933 25.02 20.94 41.88
3.61E−06 0.00022 9.42E−05 0.0868 5.66E−07 4.75E−06 9.73E−11
a Test includes Cochran–Armitage trend test (TREND), genotypic (2 df) test (GENO), dominant gene action (1 df) test (DOM), and recessive gene action (1 df) test (REC) in addition to the basic allelic test (ALLELIC). b Represents no significant deviation from Hardy–Weinberg Equilibrium (p N 10−2) in this dataset for rs2237457. Data in NTR/TR represents the number of cases with the corresponding genotypes as TT/TC/CC, T/C, and TT/CC.
30 kb region of GRB10 (Fig. 2A and B). This method identified 48% patients with TRS, while 52% diagnosed as TRS were not identified by this allele, suggesting that at least half of TRS cases are due to other genetic variants or non-genetic influences. As will be discussed, the TRS cases identified by rs2237457 may constitute a subtype of TRS. Logistic regression was performed for the association of TRS with rs2237457, after controlling for age of onset, gender, subdiagnosis (schizophrenic or schizoaffective), and PCA1/2 of population stratification (p = 7.64 × 10−5 for all cases, p = 6.13 × 10−5 for homozygous cases). None of the covariates had a significant impact on this association (p N 0.05). 3.2. Dopa decarboxylase Previous studies showed that this SNP was associated with diabetes and adiposity traits (Fox et al., 2007; Rampersaud et al., 2007). However, no significant SNP × environment (obesity and diabetic) interaction was observed (Supplemental information). Of particular importance, the 30 kb haplotype is located 70 kb upstream of the closest gene, dopa decarboxylase (DDC). DDC, also known as aromatic L-amino acid decarboxylase (AADC), catalyzes the decarboxylation of L -3,4-dihydroxyphenylalanine (DOPA) to dopamine (DA), L -5hydroxytryptophan to serotonin (5-HT), and L-tryptophan to tryptamine. There is extensive information relating DDC to schizophrenia (Davis et al., 1991; O'Reilly et al., 1991; Reith et al., 1994; Borglum et al., 2001; Grunder et al., 2003; Ikemoto et al., 2003). Expression data of DDC or GRB10 for the three genotypes of rs2237457 or rs2329487 are plotted as a function of age in Fig. 3. Normal people with TT genotype for rs2237457 or rs2329487 have significantly lower levels of expression of DDC compared to CC (p = 0.002 or p = 0.001, respectively) or CT genotypes (p = 0.016 or p = 0.225, respectively). No significantly different expression of GRB10 was observed between the genotypes for rs2237457 or rs2329487 (p N 0.05). Additional samples (70 TRS and 125 NTRs) were genotyped for rs2237457. There was a trend for rs2237457 to be more common in TRS in this sample, but less significant than the GWAS sample (Table 2). After the two samples were combined (TRS = 149; NTRS = 220), rs2237457 remained significantly associated with TRS (p = 5.66 × 10− 7 in recessive mode). The %TT in the entire TRS sample (26.2%) was significantly higher than that in the NTR sample (7.3%). Each additional T allele increased the odds of being TRS by 1.74. In other words, 73.8% TRS have either TC or CC genotype, suggesting that the predictability of TRS through rs2237457 is limited (PPV = 70.91% & NPV = 64.97% under recessive mode). The Power test showed that if each additional T allele (allele frequency = 0.36) increased the odds of having TRS by 1.74, population risk (Kp) = 0.30, and 149 TRS cases and 220 NTR controls were genotyped, the power to detect an association with significance p b 0.01 was 79%.
4. Discussion 4.1. Subtyping TRS schizophrenia, first-episode and late-onset Our results indicate genetic heterogeneity for TRS patients; those with rs2237457, homozygotes and those heterozygotes with possibly imprinted T alleles, may have an etiologically distinct form of TRS. The TRS subgroup (TT) and perhaps some heterozygotes may be one or more additional subtypes of TRS. The hypothesis that some TRS patients represent a biologically, and, possibly, genetically distinct subgroup of schizophrenia, is supported by the evidence that positive symptoms in patients with schizophrenia, and response of positive symptoms to APD treatment, are likely to be heritable (Hitzemann et al., 1991; Northup and Nimgaonkar, 2004). This is most likely to be the case for first-episode TRS patients who never respond to APDs at adequate dosages, as opposed to the subset of TRS patients who initially experience improvement in positive symptoms during treatment with APDs, but subsequently become refractory, so-called late-onset TRS (Meltzer et al., 1997). These two types of TRS patients have been identified by others as well (Kolakowska et al., 1985). In this study, we did not stratify the TRS cases based on this dimension due to lack of data in a sufficient number of subjects. We further evaluated symptom improvement of those TRS patients in a longitudinal study of treatment with atypical APDs for up to 6 months (Supplemental information). No significant association between symptom improvement, either total or subcategories of psychopathology, and rs2237457 genotype was observed, suggesting that response to APDs by TRS patients is based upon factors other than those which lead to TRS. 4.2. DDC and trace amines DDC is not considered to be the rate-limiting enzyme for catecholamine or indoleamine synthesis, but is rate-limiting for the synthesis of trace amines such as 2-phenylethylamine (2-PE), p-tyramine, and tryptamine, which are thought to be present at least two orders of magnitude below the level of neurotransmitters such as DA, NE and 5-HT. Trace amines, through the action of trace amine-associated receptor 1 (TAAR1), are believed to function as neuromodulators to maintain neuronal activity of monoamine neurotransmitters, possibly also glutamate, within defined physiological limits (Berry, 2007; Miller, 2011; Revel et al., 2011; Revel et al., 2013). A TAAR1 agonist has been shown to have antipsychotic activity in animal models and is currently in clinical testing (Revel et al., 2013). DDC expression and activity are altered by APD administration, including clozapine, in rodents, suggesting that it could be relevant to response to APD treatment (Neff et al., 2006). DDC has been reported to be regulated by immediate and delayed mechanisms involving enzyme activation and gene expression induction (Neff et al., 2000). Several PET studies on striatal DDC activity in schizophrenic patients reported increased DDC activity (Reith et al.,
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
6
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
1994; Hietala et al., 1995; Hietala et al., 1999; Lindstrom et al., 1999; Meyer-Lindenberg et al., 2002; McGowan et al., 2004). As a ratelimiting enzyme for trace amine, DDC activity could play a critical role in determining response to APDs. A deficit in DDC activity or its response to treatment with APDs leading to inadequate stimulation of TAAR1 could contribute to inadequate response to APDs in a subgroup of TRS. If so, TAAR1 agonists are expected to be particularly useful in the treatment of TRS patients, alone, or as an adjunct to atypical APDs. The limitations in this study include a relatively small number of subjects. Replication of these results is essential. However, possible biological significance of DDC to the action of clozapine and the emerging role of trace amines and TAAR1 agonists as a treatment for schizophrenia adds to the importance of reporting our findings and stimulating additional research on this important topic. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.schres.2014.08.018. Role of funding source Supported, in part, by grants from the Weissman Foundation and Mike Burke (SureGene, LLC). These groups had no further role in the design of the study, collection and analysis of the data, interpretation of the data, writing of the report, and the decision to submit the paper. Contributors Dr Meltzer had full access to all of the data in the study and takes responsibility for the integrity of the clinical and genetic data. Study concept and design: Meltzer; Acquisition of data: Meltzer; Analysis and interpretation of data: Li & Meltzer; Drafting of the manuscript: Li; Critical revision of the manuscript for important intellectual content: Meltzer; Statistical analysis: Li. Conflict of interest Herbert Y. Meltzer is a stockholder of ACADIA and SureGene. He has received grant support in the last 3 years from BioLine Rx, Cephalon, Dainippon Sumitomo, Eli Lilly, EnVivo, Janssen, Otsuka, Pfizer, and Sunovion. He is, or has been, a consultant to ACADIA, Alkemes, Astellas, Boehringer Mannheim, Bristol Myers Squibb, Cypress, Janssen, Lundbeck, Ovation, Merck, Novartis, Pfizer, Teva, and Valeant (BioVail). Jiang Li does not have any conflicts of interest. Acknowledgments We thank Anna Need and David Goldstein (Duke University) for SNP array genotyping.
References Arnaud, P., Monk, D., Hitchins, M., Gordon, E., Dean, W., Beechey, C.V., Peters, J., Craigen, W., Preece, M., Stanier, P., Moore, G.E., Kelsey, G., 2003. Conserved methylation imprints in the human and mouse GRB10 genes with divergent allelic expression suggests differential reading of the same mark. Hum. Mol. Genet. 12 (9), 1005–1019. Berry, M.D., 2007. The potential of trace amines and their receptors for treating neurological and psychiatric diseases. Rev. Recent Clin. Trials 2 (1), 3–19. Blagitko, N., Mergenthaler, S., Schulz, U., Wollmann, H.A., Craigen, W., Eggermann, T., Ropers, H.H., Kalscheuer, V.M., 2000. Human GRB10 is imprinted and expressed from the paternal and maternal allele in a highly tissue- and isoform-specific fashion. Hum. Mol. Genet. 9 (11), 1587–1595. Borglum, A.D., Hampson, M., Kjeldsen, T.E., Muir, W., Murray, V., Ewald, H., Mors, O., Blackwood, D., Kruse, T.A., 2001. Dopa decarboxylase genotypes may influence age at onset of schizophrenia. Mol. Psychiatry 6 (6), 712–717. Colantuoni, C., Lipska, B.K., Ye, T., Hyde, T.M., Tao, R., Leek, J.T., Colantuoni, E.A., Elkahloun, A.G., Herman, M.M., Weinberger, D.R., Kleinman, J.E., 2011. Temporal dynamics and genetic control of transcription in the human prefrontal cortex. Nature 478 (7370), 519–523. Conley, R.R., Kelly, D.L., 2001. Management of treatment resistance in schizophrenia. Biol. Psychiatry 50 (11), 898–911. Davis, B.A., Shrikhande, S., Paralikar, V.P., Hirsch, S.R., Durden, D.A., Boulton, A.A., 1991. Phenylacetic acid in CSF and serum in Indian schizophrenic patients. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 15 (1), 41–47. Fox, C.S., Heard-Costa, N., Cupples, L.A., Dupuis, J., Vasan, R.S., Atwood, L.D., 2007. Genomewide association to body mass index and waist circumference: the Framingham Heart Study 100K project. BMC Med. Genet. 8 (Suppl. 1), S18. Garfield, A.S., Cowley, M., Smith, F.M., Moorwood, K., Stewart-Cox, J.E., Gilroy, K., Baker, S., Xia, J., Dalley, J.W., Hurst, L.D., Wilkinson, L.S., Isles, A.R., Ward, A., 2011. Distinct physiological and behavioural functions for parental alleles of imprinted Grb10. Nature 469 (7331), 534–538. Grunder, G., Vernaleken, I., Muller, M.J., Davids, E., Heydari, N., Buchholz, H.G., Bartenstein, P., Munk, O.L., Stoeter, P., Wong, D.F., Gjedde, A., Cumming, P., 2003. Subchronic haloperidol downregulates dopamine synthesis capacity in the brain of schizophrenic patients in vivo. Neuropsychopharmacology 28 (4), 787–794.
Hietala, J., Syvalahti, E., Vuorio, K., Rakkolainen, V., Bergman, J., Haaparanta, M., Solin, O., Kuoppamaki, M., Kirvela, O., Ruotsalainen, U., et al., 1995. Presynaptic dopamine function in striatum of neuroleptic-naive schizophrenic patients. Lancet 346 (8983), 1130–1131. Hietala, J., Syvalahti, E., Vilkman, H., Vuorio, K., Rakkolainen, V., Bergman, J., Haaparanta, M., Solin, O., Kuoppamaki, M., Eronen, E., Ruotsalainen, U., Salokangas, R.K., 1999. Depressive symptoms and presynaptic dopamine function in neuroleptic-naive schizophrenia. Schizophr. Res. 35 (1), 41–50. Hitzemann, R., Dains, K., Bier-Langing, C.M., Zahniser, N.R., 1991. On the selection of mice for haloperidol response and non-response. Psychopharmacology 103 (2), 244–250. Ikemoto, K., Nishimura, A., Oda, T., Nagatsu, I., Nishi, K., 2003. Number of striatal Dneurons is reduced in autopsy brains of schizophrenics. Leg. Med. (Tokyo) 5 (Suppl. 1), S221–S224. Kane, J., Honigfeld, G., Singer, J., Meltzer, H., 1988a. Clozapine for the treatment-resistant schizophrenic. A double-blind comparison with chlorpromazine. Arch. Gen. Psychiatry 45 (9), 789–796. Kane, J.M., Honigfeld, G., Singer, J., Meltzer, H., 1988b. Clozapine in treatment-resistant schizophrenics. Psychopharmacol. Bull. 24 (1), 62–67. Kolakowska, T., Williams, A.O., Ardern, M., Reveley, M.A., Jambor, K., Gelder, M.G., Mandelbrote, B.M., 1985. Schizophrenia with good and poor outcome. I: early clinical features, response to neuroleptics and signs of organic dysfunction. Br. J. Psychiatry J. Ment. Sci. 146, 229–239. Lindstrom, L.H., Gefvert, O., Hagberg, G., Lundberg, T., Bergstrom, M., Hartvig, P., Langstrom, B., 1999. Increased dopamine synthesis rate in medial prefrontal cortex and striatum in schizophrenia indicated by L-(beta-11C) DOPA and PET. Biol. Psychiatry 46 (5), 681–688. Maunakea, A.K., Nagarajan, R.P., Bilenky, M., Ballinger, T.J., D'Souza, C., Fouse, S.D., Johnson, B.E., Hong, C., Nielsen, C., Zhao, Y., Turecki, G., Delaney, A., Varhol, R., Thiessen, N., Shchors, K., Heine, V.M., Rowitch, D.H., Xing, X., Fiore, C., Schillebeeckx, M., Jones, S.J., Haussler, D., Marra, M.A., Hirst, M., Wang, T., Costello, J.F., 2010. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466 (7303), 253–257. McGowan, S., Lawrence, A.D., Sales, T., Quested, D., Grasby, P., 2004. Presynaptic dopaminergic dysfunction in schizophrenia: a positron emission tomographic [18F] fluorodopa study. Arch. Gen. Psychiatry 61 (2), 134–142. Meltzer, H.Y., 1997. Treatment-resistant schizophrenia—the role of clozapine. Curr. Med. Res. Opin. 14 (1), 1–20. Meltzer, H.Y., 2013. Update on typical and atypical antipsychotic drugs. Annu. Rev. Med. 64, 393–406. Meltzer, H.Y., Rabinowitz, J., Lee, M.A., Cola, P.A., Ranjan, R., Findling, R.L., Thompson, P.A., 1997. Age at onset and gender of schizophrenic patients in relation to neuroleptic resistance. Am. J. Psychiatry 154 (4), 475–482. Meltzer, H.Y., Lee, M., Cola, P., 1998. The evolution of treatment resistance: biologic implications. J. Clin. Psychopharmacol. 18 (2 Suppl 1), 5S–11S. Menheniott, T.R., Woodfine, K., Schulz, R., Wood, A.J., Monk, D., Giraud, A.S., Baldwin, H.S., Moore, G.E., Oakey, R.J., 2008. Genomic imprinting of Dopa decarboxylase in heart and reciprocal allelic expression with neighboring Grb10. Mol. Cell. Biol. 28 (1), 386–396. Meyer-Lindenberg, A., Miletich, R.S., Kohn, P.D., Esposito, G., Carson, R.E., Quarantelli, M., Weinberger, D.R., Berman, K.F., 2002. Reduced prefrontal activity predicts exaggerated striatal dopaminergic function in schizophrenia. Nat. Neurosci. 5 (3), 267–271. Miller, G.M., 2011. The emerging role of trace amine-associated receptor 1 in the functional regulation of monoamine transporters and dopaminergic activity. J. Neurochem. 116 (2), 164–176. Neff, N.H., Wemlinger, T.A., Hadjiconstantinou, M., 2000. SCH 23390 enhances exogenous L-DOPA decarboxylation in nigrostriatal neurons. J. Neural Transm. 107 (4), 429–443. Neff, N.H., Wemlinger, T.A., Duchemin, A.M., Hadjiconstantinou, M., 2006. Clozapine modulates aromatic L-amino acid decarboxylase activity in mouse striatum. J. Pharmacol. Exp. Ther. 317 (2), 480–487. Northup, A., Nimgaonkar, V.L., 2004. Genetics of schizophrenia: implications for treatment. Expert. Rev. Neurother. 4 (4), 725–731. O'Reilly, R., Davis, B.A., Durden, D.A., Thorpe, L., Machnee, H., Boulton, A.A., 1991. Plasma phenylethylamine in schizophrenic patients. Biol. Psychiatry 30 (2), 145–150. Purcell, S.M., Wray, N.R., Stone, J.L., Visscher, P.M., O'Donovan, M.C., Sullivan, P.F., Sklar, P., 2009. Common polygenic variation contributes to risk of schizophrenia and bipolar disorder. Nature 460 (7256), 748–752. Rampersaud, E., Damcott, C.M., Fu, M., Shen, H., McArdle, P., Shi, X., Shelton, J., Yin, J., Chang, Y.P., Ott, S.H., Zhang, L., Zhao, Y., Mitchell, B.D., O'Connell, J., Shuldiner, A.R., 2007. Identification of novel candidate genes for type 2 diabetes from a genomewide association scan in the Old Order Amish: evidence for replication from diabetes-related quantitative traits and from independent populations. Diabetes 56 (12), 3053–3062. Reith, J., Benkelfat, C., Sherwin, A., Yasuhara, Y., Kuwabara, H., Andermann, F., Bachneff, S., Cumming, P., Diksic, M., Dyve, S.E., Etienne, P., Evans, A.C., Lal, S., Shevell, M., Savard, G., Wong, D.F., Chouinard, G., Gjedde, A., 1994. Elevated dopa decarboxylase activity in living brain of patients with psychosis. Proc. Natl. Acad. Sci. U. S. A. 91 (24), 11651–11654. Revel, F.G., Moreau, J.L., Gainetdinov, R.R., Bradaia, A., Sotnikova, T.D., Mory, R., Durkin, S., Zbinden, K.G., Norcross, R., Meyer, C.A., Metzler, V., Chaboz, S., Ozmen, L., Trube, G., Pouzet, B., Bettler, B., Caron, M.G., Wettstein, J.G., Hoener, M.C., 2011. TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity. Proc. Natl. Acad. Sci. U. S. A. 108 (20), 8485–8490. Revel, F.G., Moreau, J.L., Pouzet, B., Mory, R., Bradaia, A., Buchy, D., Metzler, V., Chaboz, S., Groebke Zbinden, K., Galley, G., Norcross, R.D., Tuerck, D., Bruns, A., Morairty, S.R., Kilduff, T.S., Wallace, T.L., Risterucci, C., Wettstein, J.G., Hoener, M.C., 2013. A new perspective for schizophrenia: TAAR1 agonists reveal antipsychotic- and antidepressant-
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx like activity, improve cognition and control body weight. Mol. Psychiatry 18 (5), 543–556. Suzuki, T., Remington, G., Mulsant, B.H., Uchida, H., Rajji, T.K., Graff-Guerrero, A., Mimura, M., Mamo, D.C., 2012. Defining treatment-resistant schizophrenia and response to antipsychotics: a review and recommendation. Psychiatry Res. 197 (1–2), 1–6. Tiihonen, J., Wahlbeck, K., Lonnqvist, J., Klaukka, T., Ioannidis, J.P., Volavka, J., Haukka, J., 2006. Effectiveness of antipsychotic treatments in a nationwide cohort of patients in community care after first hospitalisation due to schizophrenia and schizoaffective disorder: observational follow-up study. BMJ 333 (7561), 224.
7
Tugg, L.A., Desai, D., Prendergast, P., Remington, G., Reed, K., Zipursky, R.B., 1997. Relationship between negative symptoms in chronic schizophrenia and neuroleptic dose, plasma levels and side effects. Schizophr. Res. 25 (1), 71–78. Yoshihashi, H., Maeyama, K., Kosaki, R., Ogata, T., Tsukahara, M., Goto, Y., Hata, J., Matsuo, N., Smith, R.J., Kosaki, K., 2000. Imprinting of human GRB10 and its mutations in two patients with Russell–Silver syndrome. Am. J. Hum. Genet. 67 (2), 476–482.
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
Contents lists available at ScienceDirect
Schizophrenia Research journal homepage: www.elsevier.com/locate/schres
A genetic locus in 7p12.2 associated with treatment resistant schizophrenia Jiang Li, Herbert Y. Meltzer ⁎ Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine, United States
a r t i c l e
i n f o
Article history: Received 9 May 2014 Received in revised form 20 August 2014 Accepted 23 August 2014 Available online xxxx Keywords: Schizophrenia Genetic Treatment resistant Dopa decarboxylase Genome-wide association study Clozapine
a b s t r a c t Approximately 30% of patients with schizophrenia are treatment resistant (TRS), i.e. have persistent psychotic symptoms despite adequate trials of at least two antipsychotic drugs (APDs). Most TRS patients are candidates for clozapine treatment which is underutilized because of its side effects and difficulty in identifying TRS. We conducted a genome-wide association study (GWAS) of 79 TRS and 95 non-treatment resistant (NTRS) Caucasian schizophrenia patients to identify possible biomarkers for TRS, which might also provide insight into the pathobiology of TRS. The single nucleotide polymorphism, rs2237457, located in 7p12.2, a region reported to have imprinted inheritance, was found to have the lowest p value in an allelic association test (unadjusted p = 5.53 × 10−6). Haploview disclosed a 30 kb block flanking this SNP within GRB10, 70 kb upstream of L-dopa decarboxylase (DDC), an enzyme which is rate-limiting in the synthesis of trace amines and neurotransmitters implicated in schizophrenia and the action of APDs. This SNP or haplotype was identified as an exclusive cisacting eQTL for DDC in human dorsolateral prefrontal cortex by BrainCloud®. A replication sample genotyped for this SNP produced a weaker result, but in the same direction. After combining the two samples, rs2237457 remained significantly associated with TRS (unadjusted p = 5.66 × 10−7 in recessive mode; 9.42 × 10−5 in allelic association). If replicated in an independent sample, rs2237457 may provide a biomarker to identify a significant proportion of Caucasian TRS. The results implicate trace amines and their synthesis in the pathophysiology of TRS. © 2014 Published by Elsevier B.V.
1. Introduction It is estimated that 1% of adults meet current criteria for schizophrenia. Treatment with typical or atypical antipsychotic drugs (APDs) produces significant reductions in delusions and hallucinations in about 70% of people with schizophrenia. The other 30% of schizophrenia patients are referred to as treatment resistant (TRS) or refractory schizophrenia (Kane et al., 1988a; Conley and Kelly, 2001; Tiihonen et al., 2006). TRS is operationally defined as persistence of moderate to severe positive symptoms despite two or more trials of 4–6 week duration with typical or atypical APDs other than clozapine, the only drug approved for TRS (Kane et al., 1988a; Kane et al., 1988b; Meltzer, 1997; Suzuki et al., 2012). Although most TRS patients have poor functional outcome, due in part to persistent negative symptoms and cognitive impairment, these features, which are shared by many non-TRS (NTRS) patients, are not the basis for TRS as defined here (Meltzer, 1997). It is highly likely that the neurobiology of persistent positive symptoms is only partially related, if at all, to that of cognitive impairment and negative symptoms (Meltzer, 1997). ⁎ Corresponding author at: Department of Psychiatry and Behavioral Sciences, Ward 12-104, 303 Chicago Ave, Chicago, IL 60611, United States. Tel.: + 1 312 503 0309; fax: + 1 312 503 0348. E-mail address: [email protected] (H.Y. Meltzer).
Excluding inadequate APD trials, the differences in efficacy of APDs in TRS and NTRS patients may be based on pharmacokinetic or pharmacodynamic causes, or some combination of both. Pharmacokinetic differences in APD metabolism have rarely been shown to be causally related to poor response to APDs, but may be relevant for individual patients and drugs (Tugg et al., 1997; Meltzer, 2013). Indeed, TRS patients often receive higher doses of APDs and, despite that, have higher plasma levels. Since schizophrenia is a heterogeneous syndrome, differences in the causes of psychotic symptoms, e.g. excessive limbic dopaminergic activity, enhanced serotonin 2A receptor stimulation, or deficient GABAergic or glutamatergic activity, could be the basis for psychosis and, thus, difference in response to APDs, perhaps related to genetic and epigenetic differences. During the past decade, there have been a number of GWAS or candidate gene studies in search for genetic marker(s) related to schizophrenia and to TRS (Supplemental Table 1). The reported effect sizes are generally small and require replication. 2. Methods and materials 2.1. Subjects The discovery GWAS included 174 self-described Caucasian patients diagnosed with schizophrenia or schizoaffective disorder by DSM-IV
http://dx.doi.org/10.1016/j.schres.2014.08.018 0920-9964/© 2014 Published by Elsevier B.V.
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
2
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
Table 1 Demographic and clinical features for patients who are self-described Caucasians in GWAS dataset and additional dataset. Characteristics
GWAS sample
TR status Male (%) Age of first onset (years) Subdiagnosis
TR (N = 79) 53 (67.09%) 20.37 ± 0.75 69 (87.34%) — schizophrenia 10 (12.66%) — schizoaffective 32.96 ± 1.41 11.75 ± 0.61 26.68 ± 0.68
BPRS BPOS BMI
Additional sample
NTR (N = 95) 63 (66.32%) 22.55 ± 0.83 64 (67.37%) — schizophrenia 31 (32.63%) — schizoaffective 26.12 ± 1.11 8.42 ± 0.46 28.77 ± 0.79
TR (N = 70) 45 (64.29%) 19.71 ± 1.29 38 (54.29%) — schizophrenia 32 (45.71%) — schizoaffective 31.37 ± 1.02 11.04 ± 0.42 29.11 ± 0.90
p value
NTR (N = 125) 79 (63.20%) 20.94 ± 0.76 46 (36.80%) — schizophrenia 79 (63.20%) — schizoaffective 22.42 ± 1.17 6.62 ± 0.48 30.68 ± 1.00
(GWAS/additional) 0.914/0.880a 0.058/0.380b 0.002/0.018a b0.001/b0.001b b0.0001/b0.0001b 0.002/0.219c
Data was presented as Mean ± SE. BPRS represents Brief Psychiatric Rating Scale. BPOS is the sum of four positive symptoms (suspiciousness, hallucinatory behavior, unusual thought content, and conceptual disorganization) of 18 items in BPRS. a Chi-square test. b t-Test. c ANCOVA test adjusted by Gender.
criteria. Those subjects had participated in prospective clinical trial or longitudinal study of the effect of clozapine (Meltzer, 1997). An additional 195 subjects with these diagnoses who had been prospectively classified as TRS or NTRS were identified from subsequent clinical trials or cross-sectional study in our laboratory. 2.2. Genotyping and data analysis SNP genotyping was performed using 610K quad BeadChip® (Illumina) or Taqman® assay (Applied Biosystems). Quality control of genotyping data was described in Supplemental information. Association testing was conducted with PLINK 1.0.7 software (Purcell et al., 2009) and SPSS. QUANTO 1.2 was used for power test. BrainCloud (Colantuoni et al., 2011) was used to identify the potential cis-eQTL. ENCODE and UCSF Brain Methylation Database (Maunakea et al., 2010) were applied for functional prediction as cis-regulatory elements. 3. Results Clinical characteristics and demographic feature for the GWAS sample: TRS (n = 79) and NTRS (n = 95) patients, and for the additional subjects who were genotyped (TRS: n = 70 and NTRS: n = 125) are reported in Table 1. There was no significant difference between the
four groups in gender. Age at onset was nearly significantly earlier in the TRS patients, consistent with our previous report of a larger sample (Meltzer et al., 1998). SNPs which differentiated TRS and NTRS by allelic association test (Fig. 1A) and Cochran–Armitage trend test (Supplemental Fig. 2A) are demonstrated in a Manhattan plot (Fig. 1A). No SNP met genomewide significance for association with TRS. Six SNPs had unadjusted p b 10− 5 after relaxing the corrected p value for FDR-BH to 0.56 (Supplemental Table 2).
3.1. GRB10 rs2237457, a GRB10 SNP, one of the top hits associated with TRS, resides in a well-known imprinted genomic region, 7p12.2 (GRB10– DDC), in human (Blagitko et al., 2000; Yoshihashi et al., 2000) and mouse (Arnaud et al., 2003; Menheniott et al., 2008; Garfield et al., 2011). Due to unclear parental origin of risk allele, we re-analyze the GWAS dataset by excluding the 71 cases with heterozygous genotype for rs2237457, leaving 53 NTRS cases and 50 TRS cases. The association with TRS was significantly enhanced, from p = 5.53 × 10−6 with the heterozygotes to p = 4.99 × 10−12, by allelic association test. There were no other SNPs across the whole genome close to the same level of significance as rs2237457, or SNPs in LD with rs2237457, in a
Allelic test Chi-square on selfdescribed caucasian cases
B
A
3.0 2.0
5 3 1
-log10(P)
4.0
2
Observed -logP values
4
5.0
*
0
1.0
0
0.0
1
2
3
4
5
6
Expected -logP values
Chromosome
Fig. 1. Manhattan plot and Q–Q plot of genome-wide association with allelic Chi-square test. Panel A shows a Manhattan plot for all self-described Caucasian cases in the GWAS. “*” marks regions on chromosome 7 that reach highest genome-wide significance (p b 10−5). Values for each chromosome are shown in different colors for visual effect. Panel B shows the Q–Q plots for the corresponding test of association. This plot shows no deviation from the null distribution, except in the upper tail of the distribution, which corresponds to the SNPs with the strongest evidence for association. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
3
A
B SNP(s) Gene(s)
ENCORE
* UCSF Brain DNA Methylation
Fig. 2. Haplotype view of a genomic region surrounding rs2237457. A. Haplotype view of a region inside GRB10 but upstream of DDC. ±50 kb of rs2237457, was included. Color gradient from red to blue is correlated with D′ value from high to low in each box. rs2237457 is highlighted in the black box. B. UCSC Genome Browser Illustration of a custom track from haplotype block 2. Green bar represents rs2237457; Blue bar represents other associated SNPs from GWAS; Red bar represents imputed SNPs with r2 N 0.8. USCF Brain DNA Methylation tract and ENCODE Methylation tract were also added. This ~30 kb intronic region associated with TRS in GRB10 spans a variety of DNase I hypersensitivity clusters, suggesting a strong regulatory activity in this region, and enrichment of the modified histone mark, H3K4Me1, suggesting enhancers. According to UCSF brain methylation database, the TRS associated intronic haplotype we identified harbors an H3K4me3 site (promoter) as shown in “H3K4me3 Raw Signal” track in green (*). There is a strong overlap of H3K4me3 with an unmethylated intragenic CGI, as shown in “MRE CpG” track, suggesting that this is an alternate promoter site. This promoter site is uniquely neuronal or tissue-specific since it does not overlap with the histone methylation tracks (H3K4Me3) derived from ENCODE which has signal overlaid from nine non-neuronal cell lines from a variety of human tissues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
4
C
rs2237457
B
rs2329487
D
E
Fig. 3. BrainCloud suggests that this TRS associated SNP or haplotype in an intron region of GRB10 is cis-eQTL for gene expression of DDC. There were 105 and 130 SNPs within the 200 kb interval for DDC and GRB10, respectively. Scatterplots showed the distribution of gene expression data (normalized) of DDC (A, B) or GRB10 (C, D) for each genotype of rs2237457 (A, C) or rs2329487 (B, D) as a function of postnatal age. The LOESS fit curve was added to the corresponding scatterplot of that genotype as a function of age. E. Multiple custom annotation tracks were created at UCSC Genome Browser to demonstrate that this TRS associated SNP is cis-eQTL for gene expression of DDC. “Expression Probe” track visualizes the location of expression probes selected by BrainCloud. “TR haplotype LD” track represents all SNPs in LD (r square N 0.8), significantly associated with TR in GRB10–DDC genomic region. “GRB10 CisAssoc” track and “DDC CisAssoc” track represent all genotyped SNPs within ±100 kb up/down stream of GRB10 and DDC, respectively, by BrainCloud. Red signal represents SNPs with significant association with gene expression obtained from GLM based on the best fit model procedure. 6 out of 7 cis-eQTLs (red stick with p b 0.05) for DDC are aggregated in a small region of intron 4 of GRB10, suggesting an eQTL enrichment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
A
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
5
Table 2 Summary of GWAS and Joint analysis with additional samples for rs2237457. Testa
All samples
Homozygous only
GENO TREND ALLELIC DOM REC TREND ALLELIC
GWASb
Additionalb
Jointb
NTR
TR
CHISQ
p
NTR
TR
CHISQ
p
NTR
TR
CHISQ
p
3/42/50 48/142 48/142 45/50 3/92 6/100 6/100
24/29/26 77/81 77/81 53/26 24/55 48/52 48/52
25.03 18.54 20.65 6.818 24.38 23.85 47.69
3.67E−06 1.66E−05 5.53E−06 0.00902 7.90E−07 1.04E−06 4.99E−12
13/55/57 81/169 81/169 68/57 13/112 26/114 26/114
15/22/33 52/88 52/88 37/33 15/55 30/66 30/66
5.62 0.801 0.8983 0.04298 4.438 2.529 5.058
0.060 0.371 0.343 0.846 0.035 0.112 0.025
16/97/107 129/311 129/311 113/107 16/204 32/214 32/214
39/51/59 129/169 129/169 90/59 39/110 78/118 78/118
25.06 13.64 15.25 2.933 25.02 20.94 41.88
3.61E−06 0.00022 9.42E−05 0.0868 5.66E−07 4.75E−06 9.73E−11
a Test includes Cochran–Armitage trend test (TREND), genotypic (2 df) test (GENO), dominant gene action (1 df) test (DOM), and recessive gene action (1 df) test (REC) in addition to the basic allelic test (ALLELIC). b Represents no significant deviation from Hardy–Weinberg Equilibrium (p N 10−2) in this dataset for rs2237457. Data in NTR/TR represents the number of cases with the corresponding genotypes as TT/TC/CC, T/C, and TT/CC.
30 kb region of GRB10 (Fig. 2A and B). This method identified 48% patients with TRS, while 52% diagnosed as TRS were not identified by this allele, suggesting that at least half of TRS cases are due to other genetic variants or non-genetic influences. As will be discussed, the TRS cases identified by rs2237457 may constitute a subtype of TRS. Logistic regression was performed for the association of TRS with rs2237457, after controlling for age of onset, gender, subdiagnosis (schizophrenic or schizoaffective), and PCA1/2 of population stratification (p = 7.64 × 10−5 for all cases, p = 6.13 × 10−5 for homozygous cases). None of the covariates had a significant impact on this association (p N 0.05). 3.2. Dopa decarboxylase Previous studies showed that this SNP was associated with diabetes and adiposity traits (Fox et al., 2007; Rampersaud et al., 2007). However, no significant SNP × environment (obesity and diabetic) interaction was observed (Supplemental information). Of particular importance, the 30 kb haplotype is located 70 kb upstream of the closest gene, dopa decarboxylase (DDC). DDC, also known as aromatic L-amino acid decarboxylase (AADC), catalyzes the decarboxylation of L -3,4-dihydroxyphenylalanine (DOPA) to dopamine (DA), L -5hydroxytryptophan to serotonin (5-HT), and L-tryptophan to tryptamine. There is extensive information relating DDC to schizophrenia (Davis et al., 1991; O'Reilly et al., 1991; Reith et al., 1994; Borglum et al., 2001; Grunder et al., 2003; Ikemoto et al., 2003). Expression data of DDC or GRB10 for the three genotypes of rs2237457 or rs2329487 are plotted as a function of age in Fig. 3. Normal people with TT genotype for rs2237457 or rs2329487 have significantly lower levels of expression of DDC compared to CC (p = 0.002 or p = 0.001, respectively) or CT genotypes (p = 0.016 or p = 0.225, respectively). No significantly different expression of GRB10 was observed between the genotypes for rs2237457 or rs2329487 (p N 0.05). Additional samples (70 TRS and 125 NTRs) were genotyped for rs2237457. There was a trend for rs2237457 to be more common in TRS in this sample, but less significant than the GWAS sample (Table 2). After the two samples were combined (TRS = 149; NTRS = 220), rs2237457 remained significantly associated with TRS (p = 5.66 × 10− 7 in recessive mode). The %TT in the entire TRS sample (26.2%) was significantly higher than that in the NTR sample (7.3%). Each additional T allele increased the odds of being TRS by 1.74. In other words, 73.8% TRS have either TC or CC genotype, suggesting that the predictability of TRS through rs2237457 is limited (PPV = 70.91% & NPV = 64.97% under recessive mode). The Power test showed that if each additional T allele (allele frequency = 0.36) increased the odds of having TRS by 1.74, population risk (Kp) = 0.30, and 149 TRS cases and 220 NTR controls were genotyped, the power to detect an association with significance p b 0.01 was 79%.
4. Discussion 4.1. Subtyping TRS schizophrenia, first-episode and late-onset Our results indicate genetic heterogeneity for TRS patients; those with rs2237457, homozygotes and those heterozygotes with possibly imprinted T alleles, may have an etiologically distinct form of TRS. The TRS subgroup (TT) and perhaps some heterozygotes may be one or more additional subtypes of TRS. The hypothesis that some TRS patients represent a biologically, and, possibly, genetically distinct subgroup of schizophrenia, is supported by the evidence that positive symptoms in patients with schizophrenia, and response of positive symptoms to APD treatment, are likely to be heritable (Hitzemann et al., 1991; Northup and Nimgaonkar, 2004). This is most likely to be the case for first-episode TRS patients who never respond to APDs at adequate dosages, as opposed to the subset of TRS patients who initially experience improvement in positive symptoms during treatment with APDs, but subsequently become refractory, so-called late-onset TRS (Meltzer et al., 1997). These two types of TRS patients have been identified by others as well (Kolakowska et al., 1985). In this study, we did not stratify the TRS cases based on this dimension due to lack of data in a sufficient number of subjects. We further evaluated symptom improvement of those TRS patients in a longitudinal study of treatment with atypical APDs for up to 6 months (Supplemental information). No significant association between symptom improvement, either total or subcategories of psychopathology, and rs2237457 genotype was observed, suggesting that response to APDs by TRS patients is based upon factors other than those which lead to TRS. 4.2. DDC and trace amines DDC is not considered to be the rate-limiting enzyme for catecholamine or indoleamine synthesis, but is rate-limiting for the synthesis of trace amines such as 2-phenylethylamine (2-PE), p-tyramine, and tryptamine, which are thought to be present at least two orders of magnitude below the level of neurotransmitters such as DA, NE and 5-HT. Trace amines, through the action of trace amine-associated receptor 1 (TAAR1), are believed to function as neuromodulators to maintain neuronal activity of monoamine neurotransmitters, possibly also glutamate, within defined physiological limits (Berry, 2007; Miller, 2011; Revel et al., 2011; Revel et al., 2013). A TAAR1 agonist has been shown to have antipsychotic activity in animal models and is currently in clinical testing (Revel et al., 2013). DDC expression and activity are altered by APD administration, including clozapine, in rodents, suggesting that it could be relevant to response to APD treatment (Neff et al., 2006). DDC has been reported to be regulated by immediate and delayed mechanisms involving enzyme activation and gene expression induction (Neff et al., 2000). Several PET studies on striatal DDC activity in schizophrenic patients reported increased DDC activity (Reith et al.,
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
6
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx
1994; Hietala et al., 1995; Hietala et al., 1999; Lindstrom et al., 1999; Meyer-Lindenberg et al., 2002; McGowan et al., 2004). As a ratelimiting enzyme for trace amine, DDC activity could play a critical role in determining response to APDs. A deficit in DDC activity or its response to treatment with APDs leading to inadequate stimulation of TAAR1 could contribute to inadequate response to APDs in a subgroup of TRS. If so, TAAR1 agonists are expected to be particularly useful in the treatment of TRS patients, alone, or as an adjunct to atypical APDs. The limitations in this study include a relatively small number of subjects. Replication of these results is essential. However, possible biological significance of DDC to the action of clozapine and the emerging role of trace amines and TAAR1 agonists as a treatment for schizophrenia adds to the importance of reporting our findings and stimulating additional research on this important topic. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.schres.2014.08.018. Role of funding source Supported, in part, by grants from the Weissman Foundation and Mike Burke (SureGene, LLC). These groups had no further role in the design of the study, collection and analysis of the data, interpretation of the data, writing of the report, and the decision to submit the paper. Contributors Dr Meltzer had full access to all of the data in the study and takes responsibility for the integrity of the clinical and genetic data. Study concept and design: Meltzer; Acquisition of data: Meltzer; Analysis and interpretation of data: Li & Meltzer; Drafting of the manuscript: Li; Critical revision of the manuscript for important intellectual content: Meltzer; Statistical analysis: Li. Conflict of interest Herbert Y. Meltzer is a stockholder of ACADIA and SureGene. He has received grant support in the last 3 years from BioLine Rx, Cephalon, Dainippon Sumitomo, Eli Lilly, EnVivo, Janssen, Otsuka, Pfizer, and Sunovion. He is, or has been, a consultant to ACADIA, Alkemes, Astellas, Boehringer Mannheim, Bristol Myers Squibb, Cypress, Janssen, Lundbeck, Ovation, Merck, Novartis, Pfizer, Teva, and Valeant (BioVail). Jiang Li does not have any conflicts of interest. Acknowledgments We thank Anna Need and David Goldstein (Duke University) for SNP array genotyping.
References Arnaud, P., Monk, D., Hitchins, M., Gordon, E., Dean, W., Beechey, C.V., Peters, J., Craigen, W., Preece, M., Stanier, P., Moore, G.E., Kelsey, G., 2003. Conserved methylation imprints in the human and mouse GRB10 genes with divergent allelic expression suggests differential reading of the same mark. Hum. Mol. Genet. 12 (9), 1005–1019. Berry, M.D., 2007. The potential of trace amines and their receptors for treating neurological and psychiatric diseases. Rev. Recent Clin. Trials 2 (1), 3–19. Blagitko, N., Mergenthaler, S., Schulz, U., Wollmann, H.A., Craigen, W., Eggermann, T., Ropers, H.H., Kalscheuer, V.M., 2000. Human GRB10 is imprinted and expressed from the paternal and maternal allele in a highly tissue- and isoform-specific fashion. Hum. Mol. Genet. 9 (11), 1587–1595. Borglum, A.D., Hampson, M., Kjeldsen, T.E., Muir, W., Murray, V., Ewald, H., Mors, O., Blackwood, D., Kruse, T.A., 2001. Dopa decarboxylase genotypes may influence age at onset of schizophrenia. Mol. Psychiatry 6 (6), 712–717. Colantuoni, C., Lipska, B.K., Ye, T., Hyde, T.M., Tao, R., Leek, J.T., Colantuoni, E.A., Elkahloun, A.G., Herman, M.M., Weinberger, D.R., Kleinman, J.E., 2011. Temporal dynamics and genetic control of transcription in the human prefrontal cortex. Nature 478 (7370), 519–523. Conley, R.R., Kelly, D.L., 2001. Management of treatment resistance in schizophrenia. Biol. Psychiatry 50 (11), 898–911. Davis, B.A., Shrikhande, S., Paralikar, V.P., Hirsch, S.R., Durden, D.A., Boulton, A.A., 1991. Phenylacetic acid in CSF and serum in Indian schizophrenic patients. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 15 (1), 41–47. Fox, C.S., Heard-Costa, N., Cupples, L.A., Dupuis, J., Vasan, R.S., Atwood, L.D., 2007. Genomewide association to body mass index and waist circumference: the Framingham Heart Study 100K project. BMC Med. Genet. 8 (Suppl. 1), S18. Garfield, A.S., Cowley, M., Smith, F.M., Moorwood, K., Stewart-Cox, J.E., Gilroy, K., Baker, S., Xia, J., Dalley, J.W., Hurst, L.D., Wilkinson, L.S., Isles, A.R., Ward, A., 2011. Distinct physiological and behavioural functions for parental alleles of imprinted Grb10. Nature 469 (7331), 534–538. Grunder, G., Vernaleken, I., Muller, M.J., Davids, E., Heydari, N., Buchholz, H.G., Bartenstein, P., Munk, O.L., Stoeter, P., Wong, D.F., Gjedde, A., Cumming, P., 2003. Subchronic haloperidol downregulates dopamine synthesis capacity in the brain of schizophrenic patients in vivo. Neuropsychopharmacology 28 (4), 787–794.
Hietala, J., Syvalahti, E., Vuorio, K., Rakkolainen, V., Bergman, J., Haaparanta, M., Solin, O., Kuoppamaki, M., Kirvela, O., Ruotsalainen, U., et al., 1995. Presynaptic dopamine function in striatum of neuroleptic-naive schizophrenic patients. Lancet 346 (8983), 1130–1131. Hietala, J., Syvalahti, E., Vilkman, H., Vuorio, K., Rakkolainen, V., Bergman, J., Haaparanta, M., Solin, O., Kuoppamaki, M., Eronen, E., Ruotsalainen, U., Salokangas, R.K., 1999. Depressive symptoms and presynaptic dopamine function in neuroleptic-naive schizophrenia. Schizophr. Res. 35 (1), 41–50. Hitzemann, R., Dains, K., Bier-Langing, C.M., Zahniser, N.R., 1991. On the selection of mice for haloperidol response and non-response. Psychopharmacology 103 (2), 244–250. Ikemoto, K., Nishimura, A., Oda, T., Nagatsu, I., Nishi, K., 2003. Number of striatal Dneurons is reduced in autopsy brains of schizophrenics. Leg. Med. (Tokyo) 5 (Suppl. 1), S221–S224. Kane, J., Honigfeld, G., Singer, J., Meltzer, H., 1988a. Clozapine for the treatment-resistant schizophrenic. A double-blind comparison with chlorpromazine. Arch. Gen. Psychiatry 45 (9), 789–796. Kane, J.M., Honigfeld, G., Singer, J., Meltzer, H., 1988b. Clozapine in treatment-resistant schizophrenics. Psychopharmacol. Bull. 24 (1), 62–67. Kolakowska, T., Williams, A.O., Ardern, M., Reveley, M.A., Jambor, K., Gelder, M.G., Mandelbrote, B.M., 1985. Schizophrenia with good and poor outcome. I: early clinical features, response to neuroleptics and signs of organic dysfunction. Br. J. Psychiatry J. Ment. Sci. 146, 229–239. Lindstrom, L.H., Gefvert, O., Hagberg, G., Lundberg, T., Bergstrom, M., Hartvig, P., Langstrom, B., 1999. Increased dopamine synthesis rate in medial prefrontal cortex and striatum in schizophrenia indicated by L-(beta-11C) DOPA and PET. Biol. Psychiatry 46 (5), 681–688. Maunakea, A.K., Nagarajan, R.P., Bilenky, M., Ballinger, T.J., D'Souza, C., Fouse, S.D., Johnson, B.E., Hong, C., Nielsen, C., Zhao, Y., Turecki, G., Delaney, A., Varhol, R., Thiessen, N., Shchors, K., Heine, V.M., Rowitch, D.H., Xing, X., Fiore, C., Schillebeeckx, M., Jones, S.J., Haussler, D., Marra, M.A., Hirst, M., Wang, T., Costello, J.F., 2010. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466 (7303), 253–257. McGowan, S., Lawrence, A.D., Sales, T., Quested, D., Grasby, P., 2004. Presynaptic dopaminergic dysfunction in schizophrenia: a positron emission tomographic [18F] fluorodopa study. Arch. Gen. Psychiatry 61 (2), 134–142. Meltzer, H.Y., 1997. Treatment-resistant schizophrenia—the role of clozapine. Curr. Med. Res. Opin. 14 (1), 1–20. Meltzer, H.Y., 2013. Update on typical and atypical antipsychotic drugs. Annu. Rev. Med. 64, 393–406. Meltzer, H.Y., Rabinowitz, J., Lee, M.A., Cola, P.A., Ranjan, R., Findling, R.L., Thompson, P.A., 1997. Age at onset and gender of schizophrenic patients in relation to neuroleptic resistance. Am. J. Psychiatry 154 (4), 475–482. Meltzer, H.Y., Lee, M., Cola, P., 1998. The evolution of treatment resistance: biologic implications. J. Clin. Psychopharmacol. 18 (2 Suppl 1), 5S–11S. Menheniott, T.R., Woodfine, K., Schulz, R., Wood, A.J., Monk, D., Giraud, A.S., Baldwin, H.S., Moore, G.E., Oakey, R.J., 2008. Genomic imprinting of Dopa decarboxylase in heart and reciprocal allelic expression with neighboring Grb10. Mol. Cell. Biol. 28 (1), 386–396. Meyer-Lindenberg, A., Miletich, R.S., Kohn, P.D., Esposito, G., Carson, R.E., Quarantelli, M., Weinberger, D.R., Berman, K.F., 2002. Reduced prefrontal activity predicts exaggerated striatal dopaminergic function in schizophrenia. Nat. Neurosci. 5 (3), 267–271. Miller, G.M., 2011. The emerging role of trace amine-associated receptor 1 in the functional regulation of monoamine transporters and dopaminergic activity. J. Neurochem. 116 (2), 164–176. Neff, N.H., Wemlinger, T.A., Hadjiconstantinou, M., 2000. SCH 23390 enhances exogenous L-DOPA decarboxylation in nigrostriatal neurons. J. Neural Transm. 107 (4), 429–443. Neff, N.H., Wemlinger, T.A., Duchemin, A.M., Hadjiconstantinou, M., 2006. Clozapine modulates aromatic L-amino acid decarboxylase activity in mouse striatum. J. Pharmacol. Exp. Ther. 317 (2), 480–487. Northup, A., Nimgaonkar, V.L., 2004. Genetics of schizophrenia: implications for treatment. Expert. Rev. Neurother. 4 (4), 725–731. O'Reilly, R., Davis, B.A., Durden, D.A., Thorpe, L., Machnee, H., Boulton, A.A., 1991. Plasma phenylethylamine in schizophrenic patients. Biol. Psychiatry 30 (2), 145–150. Purcell, S.M., Wray, N.R., Stone, J.L., Visscher, P.M., O'Donovan, M.C., Sullivan, P.F., Sklar, P., 2009. Common polygenic variation contributes to risk of schizophrenia and bipolar disorder. Nature 460 (7256), 748–752. Rampersaud, E., Damcott, C.M., Fu, M., Shen, H., McArdle, P., Shi, X., Shelton, J., Yin, J., Chang, Y.P., Ott, S.H., Zhang, L., Zhao, Y., Mitchell, B.D., O'Connell, J., Shuldiner, A.R., 2007. Identification of novel candidate genes for type 2 diabetes from a genomewide association scan in the Old Order Amish: evidence for replication from diabetes-related quantitative traits and from independent populations. Diabetes 56 (12), 3053–3062. Reith, J., Benkelfat, C., Sherwin, A., Yasuhara, Y., Kuwabara, H., Andermann, F., Bachneff, S., Cumming, P., Diksic, M., Dyve, S.E., Etienne, P., Evans, A.C., Lal, S., Shevell, M., Savard, G., Wong, D.F., Chouinard, G., Gjedde, A., 1994. Elevated dopa decarboxylase activity in living brain of patients with psychosis. Proc. Natl. Acad. Sci. U. S. A. 91 (24), 11651–11654. Revel, F.G., Moreau, J.L., Gainetdinov, R.R., Bradaia, A., Sotnikova, T.D., Mory, R., Durkin, S., Zbinden, K.G., Norcross, R., Meyer, C.A., Metzler, V., Chaboz, S., Ozmen, L., Trube, G., Pouzet, B., Bettler, B., Caron, M.G., Wettstein, J.G., Hoener, M.C., 2011. TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity. Proc. Natl. Acad. Sci. U. S. A. 108 (20), 8485–8490. Revel, F.G., Moreau, J.L., Pouzet, B., Mory, R., Bradaia, A., Buchy, D., Metzler, V., Chaboz, S., Groebke Zbinden, K., Galley, G., Norcross, R.D., Tuerck, D., Bruns, A., Morairty, S.R., Kilduff, T.S., Wallace, T.L., Risterucci, C., Wettstein, J.G., Hoener, M.C., 2013. A new perspective for schizophrenia: TAAR1 agonists reveal antipsychotic- and antidepressant-
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
J. Li, H.Y. Meltzer / Schizophrenia Research xxx (2014) xxx–xxx like activity, improve cognition and control body weight. Mol. Psychiatry 18 (5), 543–556. Suzuki, T., Remington, G., Mulsant, B.H., Uchida, H., Rajji, T.K., Graff-Guerrero, A., Mimura, M., Mamo, D.C., 2012. Defining treatment-resistant schizophrenia and response to antipsychotics: a review and recommendation. Psychiatry Res. 197 (1–2), 1–6. Tiihonen, J., Wahlbeck, K., Lonnqvist, J., Klaukka, T., Ioannidis, J.P., Volavka, J., Haukka, J., 2006. Effectiveness of antipsychotic treatments in a nationwide cohort of patients in community care after first hospitalisation due to schizophrenia and schizoaffective disorder: observational follow-up study. BMJ 333 (7561), 224.
7
Tugg, L.A., Desai, D., Prendergast, P., Remington, G., Reed, K., Zipursky, R.B., 1997. Relationship between negative symptoms in chronic schizophrenia and neuroleptic dose, plasma levels and side effects. Schizophr. Res. 25 (1), 71–78. Yoshihashi, H., Maeyama, K., Kosaki, R., Ogata, T., Tsukahara, M., Goto, Y., Hata, J., Matsuo, N., Smith, R.J., Kosaki, K., 2000. Imprinting of human GRB10 and its mutations in two patients with Russell–Silver syndrome. Am. J. Hum. Genet. 67 (2), 476–482.
Please cite this article as: Li, J., Meltzer, H.Y., A genetic locus in 7p12.2 associated with treatment resistant schizophrenia, Schizophr. Res. (2014), http://dx.doi.org/10.1016/j.schres.2014.08.018
