Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115
Contents lists available at ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
Genetic modulation of working memory deficits by ankyrin 3 gene in schizophrenia Chen Zhang a,b, Jun Cai a, Jiangtao Zhang c, Zezhi Li d, Zhongwei Guo c, Xu Zhang e, Weihong Lu a, Yi Zhang a, Aihua Yuan f, Shunying Yu f,⁎, Yiru Fang g,⁎ a
Schizophrenia Program, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming, Yunnan, China Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang, China d Department of Neurology, Shanghai Changhai Hospital, Secondary Military Medical University, Shanghai, China e Department of Psychiatry, Tongji Hospital, Tongji University School of Medicine, Shanghai, China f Department of Genetics, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China g Division of Mood Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China b c
a r t i c l e
i n f o
Article history: Received 11 November 2013 Received in revised form 9 December 2013 Accepted 13 December 2013 Available online 19 December 2013 Keywords: ANK3 First-episode schizophrenia SNP Working memory
a b s t r a c t Neuropsychological endophenotype approach is an emerging strategy in schizophrenia research to understand and identify the functional importance of genetically transmitted, brain-based deficits present in this disorder. Accumulating evidence indicated that working memory deficit is a core neuropsychological dysfunction in schizophrenia and a primary endophenotype indexing the liability to develop schizophrenia. Genetic variation in ankyrin 3 gene (ANK3) is likely to have widespread cognitive effects. Our previous study has identified a significant association of ANK3 SNPs and schizophrenia. In this study, we aimed to examine whether the schizophrenia-risk SNPs within ANK3 may affect working memory deficits in schizophrenia patients. Herein, we assess the working memory performance in 163 patients with first-episode, antipsychotic-naïve schizophrenia and 42 sex, agematched healthy subjects using N-back task. Two SNPs rs10761482 and rs10994336 were genotyped among the patients and 209 controls. Our results showed that schizophrenia patients showed significantly poorer performance than healthy controls on N-back task (ps b 0.01). After adjusting for the scores of intelligence quotient, memory quotient and the demographic factors, there was a significant genotype effect of the rs10994336 on the accuracy rate and reaction time of 2-back item (p = 0.048 and 0.024, respectively). Post-hoc analyses showed that patients with rs10994336T/T genotype had significantly lower accuracy rate and more reaction time at 2back task than those with T/C and C/C genotypes. The association of SNP rs10994336 with schizophrenia was replicated in our sample (genotypic p = 0.024 and allelic p = 0.006). However, we did not find any significant association of rs10761482 with schizophrenia and parameters in N-back task. Our results indicated that genetic variation within ANK3 may exert gene-specific modulating effects on working memory deficits in schizophrenia. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Schizophrenia is a chronic, severe and disabling brain disorder manifested by a disruption in cognition and along with positive and negative symptoms, affecting ~1% of the adult population worldwide (Fatemi and Folsom, 2009). Although the current understanding of the etiology of schizophrenia is fragmentary, epidemiological studies have demonstrated that schizophrenia is a familiar disorder with a complex mode of inheritance and heritability reaching upwards of 80% (Goldman et al.,
Abbreviations: ANK3, ankyrin 3 gene; SCID-P, Structured Clinical Interview of the DSM-IV, Patient Edition; SNP, single-nucleotide polymorphism; WMS-R, Wechsler Memory Scale-Revised; WAIS-R, Wechsler Adult Intelligence Scale-Revised; ANCOVA, analysis of covariance; IQ, intelligence quotient; MQ, memory quotient. ⁎ Corresponding authors. E-mail addresses: [email protected] (S. Yu), [email protected] (Y. Fang). 0278-5846/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2013.12.010
2008; Zhang et al., 2009). Schizophrenia has likewise long been suspected as being both a clinically and genetically heterogeneous disorder (Tsuang et al., 2001). Accordingly, Turetsky et al. (2007) observed that in recent years research on schizophrenia has turned at a remarkable pace to using an endophenotype strategy to reveal susceptibility genes ensconced in human genome, likely because identifying schizophrenia susceptibility genes may be made easier by investigating the genetic determinants of these observable endophenotypes (Greenwood et al., 2007). Cognitive dysfunctions have been widely pursued as targeted endophenotype in investigating schizophrenia, in part because deficits in working memory is presumed to be a core neuropsychological dysfunction at the heart of schizophrenia as well as a primary endophenotype indexing the liability to develop schizophrenia (Schmidt-Hansen and Honey, 2009; Silver et al., 2003). To that end, Hansell et al. (2006) performed a linkage analyses in a working memory task and found the ankyrin 3 gene (ANK3) under a linkage peak,
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115
111
et al., 2012, 2013). All subjects from both groups were of East Chinese Han origin.
suggesting that this gene may influence the levels of neural activity at play in working memory. A major neuron-enriched gene, ANK3 encodes the ankyrin G protein, which is involved in the anchoring of voltagegated sodium ion channels to the nodes of Ranvier and the axon initial segment, two sub-compartments of neurons responsible for action potential generation (Iqbal et al., 2013; Kosaka et al., 2008). Accordingly, ANK3 is considered as a neurodevelopmental gene (Talkowski et al., 2012) and neurodevelopmental factors have been implicated in the pathophysiology of several mental disorders (Sanches et al., 2008). To date, the accumulated evidence strongly supports the notion of ANK3 as a risk gene for a number of mental disorders, including bipolar disorder, schizophrenia and autism (Bi et al., 2012; Green et al., 2013; Leussis et al., 2013; Rueckert et al., 2013; Tesli et al., 2011). Recently, Iqbal et al. (2013) provided compelling further evidence that ANK3 plays an important role in intellectual functioning in a Drosophila ANK3 knockdown model. Together, these findings imply that a suggestive association of ANK3 with mental disorders, one which may underlie ANK3's function on cognitive function. One of our recent work identified a significant association between two single-nucleotide polymorphisms (SNPs) within ANK3— rs10761482 and rs10994336—and schizophrenia in a Chinese Han population, an association supported by follow-up meta-analysis (Yuan et al., 2012). Given the endophenotypic role of working memory in schizophrenia, we therefore hypothesized that the schizophrenia-risk SNPs within ANK3 may affect working memory performance among Chinese Han patients with schizophrenia. Evidence from clinical observations and preclinical studies indicated that the differential medication effect of antipsychotics on the treatment of working memory impairment among schizophrenia patients or schizophrenia-like animal model (Ettinger et al., 2011; Gunduz-Bruce et al., 2013; Nagai et al., 2007; Reilly et al., 2007; Schlagenhauf et al., 2010). Subsequently, taking this evidence into account, we first used a set of antipsychotic-naïve patients with first-episode schizophrenia to examine whether the schizophreniaassociated ANK3 SNPs influence performance of working memory. As a secondary aim, a replication study was employed to evaluate the association of the rs10761482 and rs10994336 with schizophrenia in an independent cohort of East Chinese Han subjects.
Blood samples were obtained from the participants and then Genomic DNA was isolated from whole blood using a Tiangen DNA isolation kit (Tiangen Biotech, Beijing, China). Genotyping of rs10761482 and rs10994336 was carried out using the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). PCR was performed following standard protocols, with 5 μl reaction volumes for each well in a 384-well plate and 5 ng of DNA. SDS 2.0 (Applied Biosystems) was used for genotypic identification. For quality control, all genotypes were determined without knowledge of case or control status during the genotyping process. Genotyping assays were then repeated for 10% of the samples, and the results were 100% concordant.
2. Methods
2.4. Statistical analysis
2.1. Subjects
Demographic characteristics and test results were compared between the schizophrenia and control groups, with averages presented as means. Group comparisons of demographic data were performed using analysis of covariance (ANCOVA), the t-test, or chi-square, where appropriate. Hardy–Weinberg equilibrium testing, allele and genotype frequency analysis were all conducted using SHEsis (http:// analysis.bio-x.cn) (Shi and He, 2005). kANCOVA was used to examine the association between genotype and performance on the N-back tasks among schizophrenia patients. These analyses were constructed with the rs10761482 or rs10994336 genotypes as the independent variable, and the working memory scores shown from the accuracy rate and reaction time as dependent variables, with age, education, intelligence quotient (IQ) and memory quotient (MQ) as covariates. Post-hoc comparisons between the genotypic subgroups were made using the Fisher's least significant difference (LSD) procedure. The power of the sample was calculated with Quanto 1.2.3 (available at http://hydra.usc.edu/GxE) with a log-additive inheritance mode. With the false positive rate controlled as 0.05, the statistical power to detect the odds ratio (OR) value at 1.5 for risk allele was expected to be 68% for rs10761482 and 76% for rs10994336. To correct for multiple testing, Bonferroni correction was used, and the corrected p values were set at an uncorrected p value multiplied by k (independent significance tests). All p values were two-tailed, and p values less than 0.05 were considered statistically significant following Bonferroni correction.
All procedures for this study were reviewed and approved by the Institutional Review Boards of the Shanghai Mental Health Center and other participating institutions. This study and its procedures were strictly performed in accordance with the Declaration of Helsinki as revised. Written informed consent was provided by each participant prior to any procedures related to this study being performed. In total, 163 antipsychotic-naïve patients with first-episode schizophrenia were recruited from Shanghai Mental Health Center and Tongde Hospital of Zhejiang Province. Detailed information on the recruitment procedures has been described in one of our previous studies (Lu et al., 2012). Briefly, patients were enrolled based on the following criteria: (1) first-episode and diagnosed with schizophrenia according to the DSM-IV (American Psychiatric Association, 2000); diagnoses were made using modified sections of the structural clinical interview for DSM-IV disorders by at least two trained psychiatrist and all relevant diagnostic information for each patient was reviewed; (2) never given antipsychotic medications; (3) able to complete the clinical assessments; and (4) suffering from no physical disease or other psychiatric disorder besides schizophrenia was present. A further 209 control subjects were enrolled from hospital staff and students of the School of Medicine in Shanghai, all of which were interviewed by a specialized psychiatrist using the SCID-P. Subjects with any psychiatric disorder and chronic physical disease were excluded from our analysis (Wang
2.2. Cognitive assessments Cognitive tests were performed to all of the patients and 42 control subjects who were also part of the control group in genetic study. The Wechsler Memory Scale-Revised (WMS-R) and full versions of the Wechsler Adult Intelligence Scale-Revised (WAIS-R) were administrated as described previously (Lu et al., 2012). Working memory was evaluated with the N-back task, which is similar to the version introduced by Zhang et al. (2007). In brief, a visually paced motor task served as a non-memory-guided control condition (0-back) that presented the same stimuli, but simply required participants to identify the stimulus currently being seen. In the working memory condition, the task required the recollection of a stimulus from two stimuli previously seen (2-back) while continuing to encode additional incoming stimuli. Performance was recorded as an accuracy rate, that is the percentage of correct responses (%) and reaction time (ms).
2.3. Genotyping
112
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115
Table 1 Demographic and clinical characteristics of healthy controls and schizophrenia patients. Genetic study sample
Control (n = 209)
Schizophrenia (n = 163)
Sex (male/female) Age (years)
103/106 28.3 (7.1)
91/72 25.8 (5.6)
Test statistics
p
1.57 3.74
0.25 b0.01
Cognitive assessments sample
Control (n = 42)
Schizophrenia (n = 163)
Sex (male/female) Age (years) Education (years)
20/22 26.9 (4.6) 10.5 (0.8)
91/72 25.8 (5.6) 9.9 (2.0)
WAIS-R Intelligence quotient (IQ)a
110.5 (8.3)
93.6 (7.5)
162.2
b0.01
WMS Memory quotient (MQ)a
108.0 (6.9)
81.9 (7.4)
419.9
b0.01
N-back Accuracy rate (%) 0-backa 2-backa
97.9 (1.1) 88.4 (5.4)
95.0 (1.8) 71.1 (9.6)
106.0 78.8
b0.01 b0.01
Reaction times (ms) 0-backa 2-backa
694.3 (43.7) 947.9 (152.4)
928.4 (86.6) 1245.6 (131.7)
−283.8 −157.2
b0.01 b0.01
1.05 1.21 3.03
0.39 0.23 0.003
Data presented as mean (SD). a F values adjusted for education.
3. Results Demographic characteristics for the control and schizophrenia groups are summarized in Table 1. Among the cognitive assessments samples, there was a significant difference in years of education between the two groups (p = 0.003). Consequently, we used these two parameters as covariates to compare the scores of cognitive assessments between the groups. Table 1 shows that the patients had significantly poorer performance than controls on all of the cognitive assessments, including IQ, MQ and N-back task (ps b 0.01). Working memory storage capacity is well-established to be robustly associated with general cognitive ability (Ackerman et al., 2005; Johnson et al., 2013), prompting us to restrict patients by IQ and MQ, and then to examine whether either the SNPs rs10761482 or rs10994336 had an important role in working memory performance. After adjusting for the scores of IQ, MQ, and demographic factors, there was a significant genotype effect of the rs10994336 on the accuracy rate and reaction time of 2-back item (p = 0.012 and 0.006; p = 0.048 and 0.024 after Bonferroni correction, respectively) (Table 3). However, no significant association was observed between the scores of N-back task and the SNP rs10761482 among the patients (Table 2). Post-hoc analyses showed that patients with rs10994336T/T genotype had significantly lower accuracy rate and greater reaction times at the 2-back task than those with either the T/C or C/C genotype (Fig. 1). None of the genotype distributions in the patients and control population deviated from Hardy–Weinberg equilibrium. Table 4 shows the genotype and allele frequencies for rs10761482 or rs10994336 among the 163 patients and 209 controls. The SNP rs10994336 showed Table 2 Working memory performance comparisons of rs10761482 genotypic groups in schizophrenia. N-back
C/C (N = 104)
Accuracy rate (%) 0-back 94.9 (1.7) 2-back 71.1 (9.6) Reaction times (ms) 0-back 924.22 (88.0) 2-back 1241.9 (137.4)
C/T (N = 52)
T/T (N = 7)
Fa
pb
95.2 (1.9) 70.7 (9.2)
94.8 (1.8) 73.2 (14.0)
0.42 0.41
0.66 0.67
941.5 (82.1) 1239.7 (114.7)
892.9 (96.3) 1345.7 (140.9)
1.32 2.95
0.27 0.06
Data presented as mean (SD). a F values adjusted for age, education, scores of full-scale IQ and memory quotient. b p values not corrected for multiple testing.
significant association with schizophrenia among those sampled in the present study (p = 0.012; p = 0.024 after Bonferroni correction). Similarly, the frequency of T allele of rs10994336 was significantly higher in patients than among the controls (OR = 1.60, 95%CI: 1.17–2.20, p = 0.003; p = 0.006 after Bonferroni correction). There was no significant difference in either genotype or allele frequencies of rs10761482 found between the controls and patients. 4. Discussion The experiments we conducted as part of this present study yielded three major findings. First, that our results further confirmed the previously reported connections in working memory impairment among those with schizophrenia. Second, that ANK3 rs10994336 is significantly correlated with the deficits of working memory among patients with schizophrenia. Third, there was a significant association of the SNP rs10994336 with schizophrenia found among an independent cohort, which replicated our previous study (Yuan et al., 2012). Our previous work revealed a wide range of cognitive functions that were substantially impaired in antipsychotic-naïve patients with firstepisode schizophrenia (Lu et al., 2012), which supports the view that cognitive impairment may be the core of this disorder (Elvevag and Goldberg, 2000). Furthermore, Silver et al. (2003) proposed that impaired working memory is the core underlying multiple cognitive deficits among schizophrenia patients. This idea is not implausible, given that neuropsychological studies have consistently reported that schizophrenia patients and their healthy first-degree relatives exhibit stable Table 3 Working memory performance comparisons of rs10994336 genotypic groups in schizophrenia. N-back
T/T (N = 23)
Accuracy rate (%) 0-back 95.5 (2.0) 2-back 65.4 (8.1) Reaction times (ms) 0-back 911.6 (102.3) 2-back 1322.6 (94.2)
Fa
pb
pc
95.0 (1.7) 72.8 (9.7)
1.52 4.54
0.22 0.012
0.048
934.0 (88.3) 1219.5 (130.5)
0.46 5.23
0.63 0.006
0.024
T/C (N = 69)
C/C (N = 71)
94.7 (1.7) 71.2 (9.4)
928.2 (79.7) 1246.9 (134.8)
Data presented as mean (SD). a F values adjusted for age, education, scores of full-scale IQ and memory quotient. b p values not corrected for multiple testing. c p values adjusted for Bonferroni correction.
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115
113
Fig. 1. 2-back performance results across genotypes of rs10994336 polymorphism. (A) Performance is represented as the accuracy rate at 2-back task across the genotypes of rs10994336 polymorphism. (B) Performance is represented as the reaction time at 2-back across the genotypes of rs10994336 polymorphism. Error bars represent the standard error of the mean. Significant post hoc differences are denoted via asterisks (*p b 0.05, **p b 0.01).
and reliable working memory deficits across diverse paradigms, methods, and techniques (Glahn et al., 2003; Lee and Park, 2005; Myles-Worsley and Park, 2002). Accordingly, working memory impairment may be an endophenotypic marker for schizophrenia, thus making it a potential ally in discovering the genetic basis of this disorder (Lee et al., 2008). To the best of our knowledge, this study is the first report to investigate the relationship between working memory deficits and ANK3 in schizophrenia. The N-back task used herein is a useful tool for the experimental investigation of working memory processes. However, a previous study showed that this task is insufficiently reliable and often predicts inter-individual differences at high levels of load (Jaeggi et al., 2010), so we chose to use a 0 and 2-back task model (Deserno et al., 2012) in this study. Our results showed that SNP rs10994336 is associated with either accuracy rate or reaction time at 2-back task. This implies that ANK3 may have a specific effect on the working memory deficits in schizophrenia (Fig. 2). In the human brain, ANK3 is most highly expressed in the frontal cortex, cingulated cortex, hippocampus, thalamus and cerebellum (Leussis et al., 2012; Rueckert et al., 2013), all of which are within the neural circuits implicated in working memory (Kuperberg and Heckers, 2000; LaBar et al., 1999). Neuroimaging studies likewise showed that carriers of the T allele of rs10994336 had reduced fractional anisotropy, less stringent directional alignment or loss of axonal fibers and impaired connections between subcortical and frontal brain regions, suggesting that the T allele of rs10994336 may likely change the neural circuit (Linke et al., 2012). This view is supported by neuropsychological evidence that the T allele impacts a series of cognitive processes, such as sustained attention, alterations in set-shifting and decision-making (Hatzimanolis et al., 2012; Linke et al., 2012; Ruberto et al., 2011). In this study, we observed that individuals with T/T genotype of rs10994336 scored worse on the 2-back condition tests than those with T/C and C/C genotypes, suggesting that the T allele may worsen working memory deficits among schizophrenia patients. Taken on the whole, the aforementioned evidence may reflect a potential functional effect of rs10994336.
Recently, Roussos et al. (2012) found an association between lower ANK3 mRNA expression level and poorer working memory in healthy individuals, and concluded that the molecular mechanism of ANK3 genetic susceptibility for schizophrenia may underlie the reduced gene and protein expression of ANK3/AnkG, with disease-relevant anatomic specificity. However, there is no extant information regarding the effect of rs10994336 on the ANK3 mRNA expression in brain. According to the Ensembl database (http://asia.ensembl.org/index.html), this SNP is located in intronic regions of ANK3 (Tesli et al., 2011). If rs10994336 directly affects the expression of ANK3, it follows that it may be via cisregulatory or trans-regulatory mechanisms (Quinn et al., 2010). If not, however, is the real risk allele is in a high degree of linkage disequilibrium with rs10994336? Though an interesting question, there is no available answer to date. As such, using reporter gene assays, all known polymorphisms in strong linkage disequilibrium with rs10994336 can be investigated for their functional relevance upon ANK3 expression. Independent replication is an important step towards endorsing susceptibility genes because it not only confirms the validity of genetic association studies, but also verifies the contribution of a given locus to disease pathogenesis (Cunninghame Graham, 2009). We previously reported two schizophrenia-risk SNPs—rs10761482 and rs10994336— within ANK3 (Yuan et al., 2012). In this study, we determined whether the association could be replicated in another independent set of schizophrenia patients. Our results confirmed the speculative association of rs10994336, but not of rs10761482. One reason for our failure to replicate the previous findings could be due to insufficient sample size, since in this study we only had had a power of 68% to replicate association between rs10761482 and schizophrenia. However, despite an endophenotypic approach used in this study, we still failed to find any significant association between rs10761482 and working memory deficits among those with schizophrenia. Moreover, the lack of association between rs10761482 and schizophrenia could also be observed from another study (Gella et al., 2011), indicating that more studies with large-scale sample sizes are necessary to determine whether its functional effect and genetic variation may have notable impact on schizophrenia susceptibility.
Table 4 Genotype and allele distributions for SNPs in ANK3 between control and schizophrenia groups. SNP
Group
rs10761482 Control Case rs10994336 Control Case a b c
pa
Genotype, N (%) C/C 129 (61.7) 104 (63.8) T/T 13 (6.2) 23 (14.1)
p values not corrected for multiple testing. p values adjusted for Bonferroni correction. Cases vs controls.
C/T 65 (31.1) 52 (31.9) T/C 80 (38.3) 69 (42.3)
T/T 15 (7.2) 7 (4.3) C/C 116 (55.5) 71 (43.6)
pb
0.50
0.012
0.024
pa
Allele, N (%) C 323 (77.3) 260 (79.8) T 106 (25.4) 115 (35.3)
T 95 (22.7) 66 (20.2) C 312 (74.6) 211 (64.7)
pb
0.41
0.003
OR (95%CI)c 1.16 (0.81–1.65)
0.006
1.60 (1.17–2.20)
114
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115
References
Fig. 2. Schematic representation of the putative effect of ANK3 on working memory deficits in schizophrenia.
One strength of this study worth noting was the use of a clinically well-defined and characterized sample of antipsychotic-naïve subjects with first-episode schizophrenia without any other comorbid psychiatric or physical conditions. Additionally, the random selection of the control subjects from the general population and the subsequent psychiatric screening for mental disorders likewise bolsters some of our results. When interpreting the results of this study, we would be remiss in not noting some limitations. While all enrolled patients were from East China, hidden population stratification could confound our findings, and cross-sectional association analyses have the potential for population stratification. Given these possibilities, a replication study with genomic controls or a family-based population study would be helpful in addressing this limitation and giving a more definitive view of the results reported in this present study (Hui et al., 2013). In conclusion, we found that the rs10994336 polymorphism may play an important role in the development of working memory deficits in schizophrenia. Moreover, our results provided evidence that support a genetic contribution of ANK3 to schizophrenia, though we failed to replicate findings of an association between rs10761482 polymorphism and schizophrenia among the studied population of Han Chinese. While intriguing, these findings are still only suggestive, and require replication in other independent and more ethnically diverse samples to offer any concrete conclusions.
Acknowledgment We are deeply grateful to all participants. We thank the two reviewers for their insightful comments. This work was supported by the National Natural Science Foundation of China (81000581), the China Postdoctoral Science Foundation (2013M530410), the Shanghai Science & Technology Development Foundation (12140904200) and the National Key Clinical Disciplines at Shanghai Mental Health Center (OMA-MH, 2011–873). Conflict of interest There are no conflicts of interest to report.
Ackerman PL, Beier ME, Boyle MO. Working memory and intelligence: the same or different constructs? Psychol Bull 2005;131:30–60. American Psychiatric Association. Handbook of psychiatric measures. Washington: American Psychiatric Association; 2000. Bi C, Wu J, Jiang T, Liu Q, Cai W, Yu P, et al. Mutations of ANK3 identified by exome sequencing are associated with autism susceptibility. Hum Mutat 2012;33:1635–8. Cunninghame Graham DS. Genome-wide association studies in systemic lupus erythematosus: a perspective. Arthritis Res Ther 2009;11:119. Deserno L, Sterzer P, Wustenberg T, Heinz A, Schlagenhauf F. Reduced prefrontal-parietal effective connectivity and working memory deficits in schizophrenia. J Neurosci 2012;32:12–20. Elvevag B, Goldberg TE. Cognitive impairment in schizophrenia is the core of the disorder. Crit Rev Neurobiol 2000;14:1–21. Ettinger U, Williams SC, Fannon D, Premkumar P, Kuipers E, Moller HJ, et al. Functional magnetic resonance imaging of a parametric working memory task in schizophrenia: relationship with performance and effects of antipsychotic treatment. Psychopharmacology (Berl) 2011;216:17–27. Fatemi SH, Folsom TD. The neurodevelopmental hypothesis of schizophrenia, revisited. Schizophr Bull 2009;35:528–48. Gella A, Segura M, Durany N, Pfuhlmann B, Stober G, Gawlik M. Is Ankyrin a genetic risk factor for psychiatric phenotypes? BMC Psychiatry 2011;11:103. Glahn DC, Therman S, Manninen M, Huttunen M, Kaprio J, Lonnqvist J, et al. Spatial working memory as an endophenotype for schizophrenia. Biol Psychiatry 2003;53:624–6. Goldman AL, Pezawas L, Mattay VS, Fischl B, Verchinski BA, Zoltick B, et al. Heritability of brain morphology related to schizophrenia: a large-scale automated magnetic resonance imaging segmentation study. Biol Psychiatry 2008;63:475–83. Green EK, Hamshere M, Forty L, Gordon-Smith K, Fraser C, Russell E, et al. Replication of bipolar disorder susceptibility alleles and identification of two novel genome-wide significant associations in a new bipolar disorder case–control sample. Mol Psychiatry 2013;18:1302–7. Greenwood TA, Braff DL, Light GA, Cadenhead KS, Calkins ME, Dobie DJ, et al. Initial heritability analyses of endophenotypic measures for schizophrenia: the consortium on the genetics of schizophrenia. Arch Gen Psychiatry 2007;64:1242–50. Gunduz-Bruce H, Oliver S, Gueorguieva R, Forselius-Bielen K, D'Souza DC, Zimolo Z, et al. Efficacy of pimozide augmentation for clozapine partial responders with schizophrenia. Schizophr Res 2013;143:344–7. Hansell NK, Medland SE, Ferreira MA, Geffen GM, Zhu G, Montgomery GW, et al. Linkage analyses of event-related potential slow wave phenotypes recorded in a working memory task. Behav Genet 2006;36:29–44. Hatzimanolis A, Smyrnis N, Avramopoulos D, Stefanis CN, Evdokimidis I, Stefanis NC. Bipolar disorder ANK3 risk variant effect on sustained attention is replicated in a large healthy population. Psychiatr Genet 2012;22:210–3. Hui L, Zhang X, Yu YQ, Han M, Huang XF, Chen DC, et al. Association between DBH 19 bp insertion/deletion polymorphism and cognition in first-episode schizophrenic patients. Schizophr Res 2013;147:236–40. Iqbal Z, Vandeweyer G, van der Voet M, Waryah AM, Zahoor MY, Besseling JA, et al. Homozygous and heterozygous disruptions of ANK3: at the crossroads of neurodevelopmental and psychiatric disorders. Hum Mol Genet 2013;22:1960–70. Jaeggi SM, Buschkuehl M, Perrig WJ, Meier B. The concurrent validity of the N-back task as a working memory measure. Memory 2010;18:394–412. Johnson MK, McMahon RP, Robinson BM, Harvey AN, Hahn B, Leonard CJ, et al. The relationship between working memory capacity and broad measures of cognitive ability in healthy adults and people with schizophrenia. Neuropsychology 2013;27:220–9. Kosaka T, Komada M, Kosaka K. Sodium channel cluster, betaIV-spectrin and ankyrinG positive “hot spots” on dendritic segments of parvalbumin-containing neurons and some other neurons in the mouse and rat main olfactory bulbs. Neurosci Res 2008;62:176–86. Kuperberg G, Heckers S. Schizophrenia and cognitive function. Curr Opin Neurobiol 2000;10:205–10. LaBar KS, Gitelman DR, Parrish TB, Mesulam M. Neuroanatomic overlap of working memory and spatial attention networks: a functional MRI comparison within subjects. Neuroimage 1999;10:695–704. Lee J, Park S. Working memory impairments in schizophrenia: a meta-analysis. J Abnorm Psychol 2005;114:599–611. Lee J, Folley BS, Gore J, Park S. Origins of spatial working memory deficits in schizophrenia: an event-related fMRI and near-infrared spectroscopy study. PloS One 2008;3: e1760. Leussis MP, Madison JM, Petryshen TL. Ankyrin 3: genetic association with bipolar disorder and relevance to disease pathophysiology. Biol Mood Anxiety Disord 2012;2:18. Leussis MP, Berry-Scott EM, Saito M, Jhuang H, de Haan G, Alkan O, et al. The ANK3 bipolar disorder gene regulates psychiatric-related behaviors that are modulated by lithium and stress. Biol Psychiatry 2013;73:683–90. Linke J, Witt SH, King AV, Nieratschker V, Poupon C, Gass A, et al. Genome-wide supported risk variant for bipolar disorder alters anatomical connectivity in the human brain. Neuroimage 2012;59:3288–96. Lu WH, Zhang C, Yi ZH, Li ZZ, Wu ZG, Fang YR. Association between BDNF Val66Met polymorphism and cognitive performance in antipsychotic-naive patients with schizophrenia. J Mol Neurosci 2012;47:505–10. Myles-Worsley M, Park S. Spatial working memory deficits in schizophrenia patients and their first degree relatives from Palau, micronesia. Am J Med Genet 2002;114:609–15. Nagai T, Takuma K, Dohniwa M, Ibi D, Mizoguchi H, Kamei H, et al. Repeated methamphetamine treatment impairs spatial working memory in rats: reversal by clozapine but not haloperidol. Psychopharmacology (Berl) 2007;194:21–32.
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115 Quinn EM, Hill M, Anney R, Gill M, Corvin AP, Morris DW. Evidence for cis-acting regulation of ANK3 and CACNA1C gene expression. Bipolar Disord 2010;12:440–5. Reilly JL, Harris MS, Khine TT, Keshavan MS, Sweeney JA. Antipsychotic drugs exacerbate impairment on a working memory task in first-episode schizophrenia. Biol Psychiatry 2007;62:818–21. Roussos P, Katsel P, Davis KL, Bitsios P, Giakoumaki SG, Jogia J, et al. Molecular and genetic evidence for abnormalities in the nodes of Ranvier in schizophrenia. Arch Gen Psychiatry 2012;69:7–15. Ruberto G, Vassos E, Lewis CM, Tatarelli R, Girardi P, Collier D, et al. The cognitive impact of the ANK3 risk variant for bipolar disorder: initial evidence of selectivity to signal detection during sustained attention. PloS One 2011;6:e16671. Rueckert EH, Barker D, Ruderfer D, Bergen SE, O'Dushlaine C, Luce CJ, et al. Cis-acting regulation of brain-specific ANK3 gene expression by a genetic variant associated with bipolar disorder. Mol Psychiatry 2013;18:922–9. Sanches M, Keshavan MS, Brambilla P, Soares JC. Neurodevelopmental basis of bipolar disorder: a critical appraisal. Prog Neuropsychopharmacol Biol Psychiatry 2008;32:1617–27. Schlagenhauf F, Dinges M, Beck A, Wustenberg T, Friedel E, Dembler T, et al. Switching schizophrenia patients from typical neuroleptics to aripiprazole: effects on working memory dependent functional activation. Schizophr Res 2010;118:189–200. Schmidt-Hansen M, Honey RC. Working memory and multidimensional schizotypy: dissociable influences of the different dimensions. Cogn Neuropsychol 2009;26:655–70. Shi YY, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res 2005;15:97–8. Silver H, Feldman P, Bilker W, Gur RC. Working memory deficit as a core neuropsychological dysfunction in schizophrenia. Am J Psychiatry 2003;160:1809–16.
115
Talkowski ME, Rosenfeld JA, Blumenthal I, Pillalamarri V, Chiang C, Heilbut A, et al. Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries. Cell 2012;149:525–37. Tesli M, Koefoed P, Athanasiu L, Mattingsdal M, Gustafsson O, Agartz I, et al. Association analysis of ANK3 gene variants in nordic bipolar disorder and schizophrenia case– control samples. Am J Med Genet B Neuropsychiatr Genet 2011;156B:969–74. Tsuang MT, Stone WS, Faraone SV. Genes, environment and schizophrenia. Br J Psychiatry 2001;40:s18–24. Turetsky BI, Calkins ME, Light GA, Olincy A, Radant AD, Swerdlow NR. Neurophysiological endophenotypes of schizophrenia: the viability of selected candidate measures. Schizophr Bull 2007;33:69–94. Wang Z, Li Z, Chen J, Huang J, Yuan C, Hong W, et al. Association of BDNF gene polymorphism with bipolar disorders in Han Chinese population. Genes Brain Behav 2012;11: 524–8. Wang Z, Fan J, Gao K, Li Z, Yi Z, Wang L, et al. Neurotrophic tyrosine kinase receptor type 2 (NTRK2) gene associated with treatment response to mood stabilizers in patients with bipolar I disorder. J Mol Neurosci 2013;50:305–10. Yuan AH, Yi ZH, Wang Q, Sun JH, Li ZQ, Du YS, et al. ANK3 as a risk gene for schizophrenia: new data in Han Chinese and meta analysis. Am J Med Genet B Neuropsychiatr Genet 2012;159B:997–1005. Zhang Y, Bertolino A, Fazio L, Blasi G, Rampino A, Romano R, et al. Polymorphisms in human dopamine D2 receptor gene affect gene expression, splicing, and neuronal activity during working memory. Proc Natl Acad Sci U S A 2007;104: 20552–7. Zhang C, Fang Y, Xie B, Cheng W, Du Y, Wang D, et al. DNA methyltransferase 3B gene increases risk of early onset schizophrenia. Neurosci Lett 2009;462:308–11.
Contents lists available at ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp
Genetic modulation of working memory deficits by ankyrin 3 gene in schizophrenia Chen Zhang a,b, Jun Cai a, Jiangtao Zhang c, Zezhi Li d, Zhongwei Guo c, Xu Zhang e, Weihong Lu a, Yi Zhang a, Aihua Yuan f, Shunying Yu f,⁎, Yiru Fang g,⁎ a
Schizophrenia Program, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming, Yunnan, China Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang, China d Department of Neurology, Shanghai Changhai Hospital, Secondary Military Medical University, Shanghai, China e Department of Psychiatry, Tongji Hospital, Tongji University School of Medicine, Shanghai, China f Department of Genetics, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China g Division of Mood Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China b c
a r t i c l e
i n f o
Article history: Received 11 November 2013 Received in revised form 9 December 2013 Accepted 13 December 2013 Available online 19 December 2013 Keywords: ANK3 First-episode schizophrenia SNP Working memory
a b s t r a c t Neuropsychological endophenotype approach is an emerging strategy in schizophrenia research to understand and identify the functional importance of genetically transmitted, brain-based deficits present in this disorder. Accumulating evidence indicated that working memory deficit is a core neuropsychological dysfunction in schizophrenia and a primary endophenotype indexing the liability to develop schizophrenia. Genetic variation in ankyrin 3 gene (ANK3) is likely to have widespread cognitive effects. Our previous study has identified a significant association of ANK3 SNPs and schizophrenia. In this study, we aimed to examine whether the schizophrenia-risk SNPs within ANK3 may affect working memory deficits in schizophrenia patients. Herein, we assess the working memory performance in 163 patients with first-episode, antipsychotic-naïve schizophrenia and 42 sex, agematched healthy subjects using N-back task. Two SNPs rs10761482 and rs10994336 were genotyped among the patients and 209 controls. Our results showed that schizophrenia patients showed significantly poorer performance than healthy controls on N-back task (ps b 0.01). After adjusting for the scores of intelligence quotient, memory quotient and the demographic factors, there was a significant genotype effect of the rs10994336 on the accuracy rate and reaction time of 2-back item (p = 0.048 and 0.024, respectively). Post-hoc analyses showed that patients with rs10994336T/T genotype had significantly lower accuracy rate and more reaction time at 2back task than those with T/C and C/C genotypes. The association of SNP rs10994336 with schizophrenia was replicated in our sample (genotypic p = 0.024 and allelic p = 0.006). However, we did not find any significant association of rs10761482 with schizophrenia and parameters in N-back task. Our results indicated that genetic variation within ANK3 may exert gene-specific modulating effects on working memory deficits in schizophrenia. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Schizophrenia is a chronic, severe and disabling brain disorder manifested by a disruption in cognition and along with positive and negative symptoms, affecting ~1% of the adult population worldwide (Fatemi and Folsom, 2009). Although the current understanding of the etiology of schizophrenia is fragmentary, epidemiological studies have demonstrated that schizophrenia is a familiar disorder with a complex mode of inheritance and heritability reaching upwards of 80% (Goldman et al.,
Abbreviations: ANK3, ankyrin 3 gene; SCID-P, Structured Clinical Interview of the DSM-IV, Patient Edition; SNP, single-nucleotide polymorphism; WMS-R, Wechsler Memory Scale-Revised; WAIS-R, Wechsler Adult Intelligence Scale-Revised; ANCOVA, analysis of covariance; IQ, intelligence quotient; MQ, memory quotient. ⁎ Corresponding authors. E-mail addresses: [email protected] (S. Yu), [email protected] (Y. Fang). 0278-5846/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2013.12.010
2008; Zhang et al., 2009). Schizophrenia has likewise long been suspected as being both a clinically and genetically heterogeneous disorder (Tsuang et al., 2001). Accordingly, Turetsky et al. (2007) observed that in recent years research on schizophrenia has turned at a remarkable pace to using an endophenotype strategy to reveal susceptibility genes ensconced in human genome, likely because identifying schizophrenia susceptibility genes may be made easier by investigating the genetic determinants of these observable endophenotypes (Greenwood et al., 2007). Cognitive dysfunctions have been widely pursued as targeted endophenotype in investigating schizophrenia, in part because deficits in working memory is presumed to be a core neuropsychological dysfunction at the heart of schizophrenia as well as a primary endophenotype indexing the liability to develop schizophrenia (Schmidt-Hansen and Honey, 2009; Silver et al., 2003). To that end, Hansell et al. (2006) performed a linkage analyses in a working memory task and found the ankyrin 3 gene (ANK3) under a linkage peak,
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115
111
et al., 2012, 2013). All subjects from both groups were of East Chinese Han origin.
suggesting that this gene may influence the levels of neural activity at play in working memory. A major neuron-enriched gene, ANK3 encodes the ankyrin G protein, which is involved in the anchoring of voltagegated sodium ion channels to the nodes of Ranvier and the axon initial segment, two sub-compartments of neurons responsible for action potential generation (Iqbal et al., 2013; Kosaka et al., 2008). Accordingly, ANK3 is considered as a neurodevelopmental gene (Talkowski et al., 2012) and neurodevelopmental factors have been implicated in the pathophysiology of several mental disorders (Sanches et al., 2008). To date, the accumulated evidence strongly supports the notion of ANK3 as a risk gene for a number of mental disorders, including bipolar disorder, schizophrenia and autism (Bi et al., 2012; Green et al., 2013; Leussis et al., 2013; Rueckert et al., 2013; Tesli et al., 2011). Recently, Iqbal et al. (2013) provided compelling further evidence that ANK3 plays an important role in intellectual functioning in a Drosophila ANK3 knockdown model. Together, these findings imply that a suggestive association of ANK3 with mental disorders, one which may underlie ANK3's function on cognitive function. One of our recent work identified a significant association between two single-nucleotide polymorphisms (SNPs) within ANK3— rs10761482 and rs10994336—and schizophrenia in a Chinese Han population, an association supported by follow-up meta-analysis (Yuan et al., 2012). Given the endophenotypic role of working memory in schizophrenia, we therefore hypothesized that the schizophrenia-risk SNPs within ANK3 may affect working memory performance among Chinese Han patients with schizophrenia. Evidence from clinical observations and preclinical studies indicated that the differential medication effect of antipsychotics on the treatment of working memory impairment among schizophrenia patients or schizophrenia-like animal model (Ettinger et al., 2011; Gunduz-Bruce et al., 2013; Nagai et al., 2007; Reilly et al., 2007; Schlagenhauf et al., 2010). Subsequently, taking this evidence into account, we first used a set of antipsychotic-naïve patients with first-episode schizophrenia to examine whether the schizophreniaassociated ANK3 SNPs influence performance of working memory. As a secondary aim, a replication study was employed to evaluate the association of the rs10761482 and rs10994336 with schizophrenia in an independent cohort of East Chinese Han subjects.
Blood samples were obtained from the participants and then Genomic DNA was isolated from whole blood using a Tiangen DNA isolation kit (Tiangen Biotech, Beijing, China). Genotyping of rs10761482 and rs10994336 was carried out using the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). PCR was performed following standard protocols, with 5 μl reaction volumes for each well in a 384-well plate and 5 ng of DNA. SDS 2.0 (Applied Biosystems) was used for genotypic identification. For quality control, all genotypes were determined without knowledge of case or control status during the genotyping process. Genotyping assays were then repeated for 10% of the samples, and the results were 100% concordant.
2. Methods
2.4. Statistical analysis
2.1. Subjects
Demographic characteristics and test results were compared between the schizophrenia and control groups, with averages presented as means. Group comparisons of demographic data were performed using analysis of covariance (ANCOVA), the t-test, or chi-square, where appropriate. Hardy–Weinberg equilibrium testing, allele and genotype frequency analysis were all conducted using SHEsis (http:// analysis.bio-x.cn) (Shi and He, 2005). kANCOVA was used to examine the association between genotype and performance on the N-back tasks among schizophrenia patients. These analyses were constructed with the rs10761482 or rs10994336 genotypes as the independent variable, and the working memory scores shown from the accuracy rate and reaction time as dependent variables, with age, education, intelligence quotient (IQ) and memory quotient (MQ) as covariates. Post-hoc comparisons between the genotypic subgroups were made using the Fisher's least significant difference (LSD) procedure. The power of the sample was calculated with Quanto 1.2.3 (available at http://hydra.usc.edu/GxE) with a log-additive inheritance mode. With the false positive rate controlled as 0.05, the statistical power to detect the odds ratio (OR) value at 1.5 for risk allele was expected to be 68% for rs10761482 and 76% for rs10994336. To correct for multiple testing, Bonferroni correction was used, and the corrected p values were set at an uncorrected p value multiplied by k (independent significance tests). All p values were two-tailed, and p values less than 0.05 were considered statistically significant following Bonferroni correction.
All procedures for this study were reviewed and approved by the Institutional Review Boards of the Shanghai Mental Health Center and other participating institutions. This study and its procedures were strictly performed in accordance with the Declaration of Helsinki as revised. Written informed consent was provided by each participant prior to any procedures related to this study being performed. In total, 163 antipsychotic-naïve patients with first-episode schizophrenia were recruited from Shanghai Mental Health Center and Tongde Hospital of Zhejiang Province. Detailed information on the recruitment procedures has been described in one of our previous studies (Lu et al., 2012). Briefly, patients were enrolled based on the following criteria: (1) first-episode and diagnosed with schizophrenia according to the DSM-IV (American Psychiatric Association, 2000); diagnoses were made using modified sections of the structural clinical interview for DSM-IV disorders by at least two trained psychiatrist and all relevant diagnostic information for each patient was reviewed; (2) never given antipsychotic medications; (3) able to complete the clinical assessments; and (4) suffering from no physical disease or other psychiatric disorder besides schizophrenia was present. A further 209 control subjects were enrolled from hospital staff and students of the School of Medicine in Shanghai, all of which were interviewed by a specialized psychiatrist using the SCID-P. Subjects with any psychiatric disorder and chronic physical disease were excluded from our analysis (Wang
2.2. Cognitive assessments Cognitive tests were performed to all of the patients and 42 control subjects who were also part of the control group in genetic study. The Wechsler Memory Scale-Revised (WMS-R) and full versions of the Wechsler Adult Intelligence Scale-Revised (WAIS-R) were administrated as described previously (Lu et al., 2012). Working memory was evaluated with the N-back task, which is similar to the version introduced by Zhang et al. (2007). In brief, a visually paced motor task served as a non-memory-guided control condition (0-back) that presented the same stimuli, but simply required participants to identify the stimulus currently being seen. In the working memory condition, the task required the recollection of a stimulus from two stimuli previously seen (2-back) while continuing to encode additional incoming stimuli. Performance was recorded as an accuracy rate, that is the percentage of correct responses (%) and reaction time (ms).
2.3. Genotyping
112
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115
Table 1 Demographic and clinical characteristics of healthy controls and schizophrenia patients. Genetic study sample
Control (n = 209)
Schizophrenia (n = 163)
Sex (male/female) Age (years)
103/106 28.3 (7.1)
91/72 25.8 (5.6)
Test statistics
p
1.57 3.74
0.25 b0.01
Cognitive assessments sample
Control (n = 42)
Schizophrenia (n = 163)
Sex (male/female) Age (years) Education (years)
20/22 26.9 (4.6) 10.5 (0.8)
91/72 25.8 (5.6) 9.9 (2.0)
WAIS-R Intelligence quotient (IQ)a
110.5 (8.3)
93.6 (7.5)
162.2
b0.01
WMS Memory quotient (MQ)a
108.0 (6.9)
81.9 (7.4)
419.9
b0.01
N-back Accuracy rate (%) 0-backa 2-backa
97.9 (1.1) 88.4 (5.4)
95.0 (1.8) 71.1 (9.6)
106.0 78.8
b0.01 b0.01
Reaction times (ms) 0-backa 2-backa
694.3 (43.7) 947.9 (152.4)
928.4 (86.6) 1245.6 (131.7)
−283.8 −157.2
b0.01 b0.01
1.05 1.21 3.03
0.39 0.23 0.003
Data presented as mean (SD). a F values adjusted for education.
3. Results Demographic characteristics for the control and schizophrenia groups are summarized in Table 1. Among the cognitive assessments samples, there was a significant difference in years of education between the two groups (p = 0.003). Consequently, we used these two parameters as covariates to compare the scores of cognitive assessments between the groups. Table 1 shows that the patients had significantly poorer performance than controls on all of the cognitive assessments, including IQ, MQ and N-back task (ps b 0.01). Working memory storage capacity is well-established to be robustly associated with general cognitive ability (Ackerman et al., 2005; Johnson et al., 2013), prompting us to restrict patients by IQ and MQ, and then to examine whether either the SNPs rs10761482 or rs10994336 had an important role in working memory performance. After adjusting for the scores of IQ, MQ, and demographic factors, there was a significant genotype effect of the rs10994336 on the accuracy rate and reaction time of 2-back item (p = 0.012 and 0.006; p = 0.048 and 0.024 after Bonferroni correction, respectively) (Table 3). However, no significant association was observed between the scores of N-back task and the SNP rs10761482 among the patients (Table 2). Post-hoc analyses showed that patients with rs10994336T/T genotype had significantly lower accuracy rate and greater reaction times at the 2-back task than those with either the T/C or C/C genotype (Fig. 1). None of the genotype distributions in the patients and control population deviated from Hardy–Weinberg equilibrium. Table 4 shows the genotype and allele frequencies for rs10761482 or rs10994336 among the 163 patients and 209 controls. The SNP rs10994336 showed Table 2 Working memory performance comparisons of rs10761482 genotypic groups in schizophrenia. N-back
C/C (N = 104)
Accuracy rate (%) 0-back 94.9 (1.7) 2-back 71.1 (9.6) Reaction times (ms) 0-back 924.22 (88.0) 2-back 1241.9 (137.4)
C/T (N = 52)
T/T (N = 7)
Fa
pb
95.2 (1.9) 70.7 (9.2)
94.8 (1.8) 73.2 (14.0)
0.42 0.41
0.66 0.67
941.5 (82.1) 1239.7 (114.7)
892.9 (96.3) 1345.7 (140.9)
1.32 2.95
0.27 0.06
Data presented as mean (SD). a F values adjusted for age, education, scores of full-scale IQ and memory quotient. b p values not corrected for multiple testing.
significant association with schizophrenia among those sampled in the present study (p = 0.012; p = 0.024 after Bonferroni correction). Similarly, the frequency of T allele of rs10994336 was significantly higher in patients than among the controls (OR = 1.60, 95%CI: 1.17–2.20, p = 0.003; p = 0.006 after Bonferroni correction). There was no significant difference in either genotype or allele frequencies of rs10761482 found between the controls and patients. 4. Discussion The experiments we conducted as part of this present study yielded three major findings. First, that our results further confirmed the previously reported connections in working memory impairment among those with schizophrenia. Second, that ANK3 rs10994336 is significantly correlated with the deficits of working memory among patients with schizophrenia. Third, there was a significant association of the SNP rs10994336 with schizophrenia found among an independent cohort, which replicated our previous study (Yuan et al., 2012). Our previous work revealed a wide range of cognitive functions that were substantially impaired in antipsychotic-naïve patients with firstepisode schizophrenia (Lu et al., 2012), which supports the view that cognitive impairment may be the core of this disorder (Elvevag and Goldberg, 2000). Furthermore, Silver et al. (2003) proposed that impaired working memory is the core underlying multiple cognitive deficits among schizophrenia patients. This idea is not implausible, given that neuropsychological studies have consistently reported that schizophrenia patients and their healthy first-degree relatives exhibit stable Table 3 Working memory performance comparisons of rs10994336 genotypic groups in schizophrenia. N-back
T/T (N = 23)
Accuracy rate (%) 0-back 95.5 (2.0) 2-back 65.4 (8.1) Reaction times (ms) 0-back 911.6 (102.3) 2-back 1322.6 (94.2)
Fa
pb
pc
95.0 (1.7) 72.8 (9.7)
1.52 4.54
0.22 0.012
0.048
934.0 (88.3) 1219.5 (130.5)
0.46 5.23
0.63 0.006
0.024
T/C (N = 69)
C/C (N = 71)
94.7 (1.7) 71.2 (9.4)
928.2 (79.7) 1246.9 (134.8)
Data presented as mean (SD). a F values adjusted for age, education, scores of full-scale IQ and memory quotient. b p values not corrected for multiple testing. c p values adjusted for Bonferroni correction.
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115
113
Fig. 1. 2-back performance results across genotypes of rs10994336 polymorphism. (A) Performance is represented as the accuracy rate at 2-back task across the genotypes of rs10994336 polymorphism. (B) Performance is represented as the reaction time at 2-back across the genotypes of rs10994336 polymorphism. Error bars represent the standard error of the mean. Significant post hoc differences are denoted via asterisks (*p b 0.05, **p b 0.01).
and reliable working memory deficits across diverse paradigms, methods, and techniques (Glahn et al., 2003; Lee and Park, 2005; Myles-Worsley and Park, 2002). Accordingly, working memory impairment may be an endophenotypic marker for schizophrenia, thus making it a potential ally in discovering the genetic basis of this disorder (Lee et al., 2008). To the best of our knowledge, this study is the first report to investigate the relationship between working memory deficits and ANK3 in schizophrenia. The N-back task used herein is a useful tool for the experimental investigation of working memory processes. However, a previous study showed that this task is insufficiently reliable and often predicts inter-individual differences at high levels of load (Jaeggi et al., 2010), so we chose to use a 0 and 2-back task model (Deserno et al., 2012) in this study. Our results showed that SNP rs10994336 is associated with either accuracy rate or reaction time at 2-back task. This implies that ANK3 may have a specific effect on the working memory deficits in schizophrenia (Fig. 2). In the human brain, ANK3 is most highly expressed in the frontal cortex, cingulated cortex, hippocampus, thalamus and cerebellum (Leussis et al., 2012; Rueckert et al., 2013), all of which are within the neural circuits implicated in working memory (Kuperberg and Heckers, 2000; LaBar et al., 1999). Neuroimaging studies likewise showed that carriers of the T allele of rs10994336 had reduced fractional anisotropy, less stringent directional alignment or loss of axonal fibers and impaired connections between subcortical and frontal brain regions, suggesting that the T allele of rs10994336 may likely change the neural circuit (Linke et al., 2012). This view is supported by neuropsychological evidence that the T allele impacts a series of cognitive processes, such as sustained attention, alterations in set-shifting and decision-making (Hatzimanolis et al., 2012; Linke et al., 2012; Ruberto et al., 2011). In this study, we observed that individuals with T/T genotype of rs10994336 scored worse on the 2-back condition tests than those with T/C and C/C genotypes, suggesting that the T allele may worsen working memory deficits among schizophrenia patients. Taken on the whole, the aforementioned evidence may reflect a potential functional effect of rs10994336.
Recently, Roussos et al. (2012) found an association between lower ANK3 mRNA expression level and poorer working memory in healthy individuals, and concluded that the molecular mechanism of ANK3 genetic susceptibility for schizophrenia may underlie the reduced gene and protein expression of ANK3/AnkG, with disease-relevant anatomic specificity. However, there is no extant information regarding the effect of rs10994336 on the ANK3 mRNA expression in brain. According to the Ensembl database (http://asia.ensembl.org/index.html), this SNP is located in intronic regions of ANK3 (Tesli et al., 2011). If rs10994336 directly affects the expression of ANK3, it follows that it may be via cisregulatory or trans-regulatory mechanisms (Quinn et al., 2010). If not, however, is the real risk allele is in a high degree of linkage disequilibrium with rs10994336? Though an interesting question, there is no available answer to date. As such, using reporter gene assays, all known polymorphisms in strong linkage disequilibrium with rs10994336 can be investigated for their functional relevance upon ANK3 expression. Independent replication is an important step towards endorsing susceptibility genes because it not only confirms the validity of genetic association studies, but also verifies the contribution of a given locus to disease pathogenesis (Cunninghame Graham, 2009). We previously reported two schizophrenia-risk SNPs—rs10761482 and rs10994336— within ANK3 (Yuan et al., 2012). In this study, we determined whether the association could be replicated in another independent set of schizophrenia patients. Our results confirmed the speculative association of rs10994336, but not of rs10761482. One reason for our failure to replicate the previous findings could be due to insufficient sample size, since in this study we only had had a power of 68% to replicate association between rs10761482 and schizophrenia. However, despite an endophenotypic approach used in this study, we still failed to find any significant association between rs10761482 and working memory deficits among those with schizophrenia. Moreover, the lack of association between rs10761482 and schizophrenia could also be observed from another study (Gella et al., 2011), indicating that more studies with large-scale sample sizes are necessary to determine whether its functional effect and genetic variation may have notable impact on schizophrenia susceptibility.
Table 4 Genotype and allele distributions for SNPs in ANK3 between control and schizophrenia groups. SNP
Group
rs10761482 Control Case rs10994336 Control Case a b c
pa
Genotype, N (%) C/C 129 (61.7) 104 (63.8) T/T 13 (6.2) 23 (14.1)
p values not corrected for multiple testing. p values adjusted for Bonferroni correction. Cases vs controls.
C/T 65 (31.1) 52 (31.9) T/C 80 (38.3) 69 (42.3)
T/T 15 (7.2) 7 (4.3) C/C 116 (55.5) 71 (43.6)
pb
0.50
0.012
0.024
pa
Allele, N (%) C 323 (77.3) 260 (79.8) T 106 (25.4) 115 (35.3)
T 95 (22.7) 66 (20.2) C 312 (74.6) 211 (64.7)
pb
0.41
0.003
OR (95%CI)c 1.16 (0.81–1.65)
0.006
1.60 (1.17–2.20)
114
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115
References
Fig. 2. Schematic representation of the putative effect of ANK3 on working memory deficits in schizophrenia.
One strength of this study worth noting was the use of a clinically well-defined and characterized sample of antipsychotic-naïve subjects with first-episode schizophrenia without any other comorbid psychiatric or physical conditions. Additionally, the random selection of the control subjects from the general population and the subsequent psychiatric screening for mental disorders likewise bolsters some of our results. When interpreting the results of this study, we would be remiss in not noting some limitations. While all enrolled patients were from East China, hidden population stratification could confound our findings, and cross-sectional association analyses have the potential for population stratification. Given these possibilities, a replication study with genomic controls or a family-based population study would be helpful in addressing this limitation and giving a more definitive view of the results reported in this present study (Hui et al., 2013). In conclusion, we found that the rs10994336 polymorphism may play an important role in the development of working memory deficits in schizophrenia. Moreover, our results provided evidence that support a genetic contribution of ANK3 to schizophrenia, though we failed to replicate findings of an association between rs10761482 polymorphism and schizophrenia among the studied population of Han Chinese. While intriguing, these findings are still only suggestive, and require replication in other independent and more ethnically diverse samples to offer any concrete conclusions.
Acknowledgment We are deeply grateful to all participants. We thank the two reviewers for their insightful comments. This work was supported by the National Natural Science Foundation of China (81000581), the China Postdoctoral Science Foundation (2013M530410), the Shanghai Science & Technology Development Foundation (12140904200) and the National Key Clinical Disciplines at Shanghai Mental Health Center (OMA-MH, 2011–873). Conflict of interest There are no conflicts of interest to report.
Ackerman PL, Beier ME, Boyle MO. Working memory and intelligence: the same or different constructs? Psychol Bull 2005;131:30–60. American Psychiatric Association. Handbook of psychiatric measures. Washington: American Psychiatric Association; 2000. Bi C, Wu J, Jiang T, Liu Q, Cai W, Yu P, et al. Mutations of ANK3 identified by exome sequencing are associated with autism susceptibility. Hum Mutat 2012;33:1635–8. Cunninghame Graham DS. Genome-wide association studies in systemic lupus erythematosus: a perspective. Arthritis Res Ther 2009;11:119. Deserno L, Sterzer P, Wustenberg T, Heinz A, Schlagenhauf F. Reduced prefrontal-parietal effective connectivity and working memory deficits in schizophrenia. J Neurosci 2012;32:12–20. Elvevag B, Goldberg TE. Cognitive impairment in schizophrenia is the core of the disorder. Crit Rev Neurobiol 2000;14:1–21. Ettinger U, Williams SC, Fannon D, Premkumar P, Kuipers E, Moller HJ, et al. Functional magnetic resonance imaging of a parametric working memory task in schizophrenia: relationship with performance and effects of antipsychotic treatment. Psychopharmacology (Berl) 2011;216:17–27. Fatemi SH, Folsom TD. The neurodevelopmental hypothesis of schizophrenia, revisited. Schizophr Bull 2009;35:528–48. Gella A, Segura M, Durany N, Pfuhlmann B, Stober G, Gawlik M. Is Ankyrin a genetic risk factor for psychiatric phenotypes? BMC Psychiatry 2011;11:103. Glahn DC, Therman S, Manninen M, Huttunen M, Kaprio J, Lonnqvist J, et al. Spatial working memory as an endophenotype for schizophrenia. Biol Psychiatry 2003;53:624–6. Goldman AL, Pezawas L, Mattay VS, Fischl B, Verchinski BA, Zoltick B, et al. Heritability of brain morphology related to schizophrenia: a large-scale automated magnetic resonance imaging segmentation study. Biol Psychiatry 2008;63:475–83. Green EK, Hamshere M, Forty L, Gordon-Smith K, Fraser C, Russell E, et al. Replication of bipolar disorder susceptibility alleles and identification of two novel genome-wide significant associations in a new bipolar disorder case–control sample. Mol Psychiatry 2013;18:1302–7. Greenwood TA, Braff DL, Light GA, Cadenhead KS, Calkins ME, Dobie DJ, et al. Initial heritability analyses of endophenotypic measures for schizophrenia: the consortium on the genetics of schizophrenia. Arch Gen Psychiatry 2007;64:1242–50. Gunduz-Bruce H, Oliver S, Gueorguieva R, Forselius-Bielen K, D'Souza DC, Zimolo Z, et al. Efficacy of pimozide augmentation for clozapine partial responders with schizophrenia. Schizophr Res 2013;143:344–7. Hansell NK, Medland SE, Ferreira MA, Geffen GM, Zhu G, Montgomery GW, et al. Linkage analyses of event-related potential slow wave phenotypes recorded in a working memory task. Behav Genet 2006;36:29–44. Hatzimanolis A, Smyrnis N, Avramopoulos D, Stefanis CN, Evdokimidis I, Stefanis NC. Bipolar disorder ANK3 risk variant effect on sustained attention is replicated in a large healthy population. Psychiatr Genet 2012;22:210–3. Hui L, Zhang X, Yu YQ, Han M, Huang XF, Chen DC, et al. Association between DBH 19 bp insertion/deletion polymorphism and cognition in first-episode schizophrenic patients. Schizophr Res 2013;147:236–40. Iqbal Z, Vandeweyer G, van der Voet M, Waryah AM, Zahoor MY, Besseling JA, et al. Homozygous and heterozygous disruptions of ANK3: at the crossroads of neurodevelopmental and psychiatric disorders. Hum Mol Genet 2013;22:1960–70. Jaeggi SM, Buschkuehl M, Perrig WJ, Meier B. The concurrent validity of the N-back task as a working memory measure. Memory 2010;18:394–412. Johnson MK, McMahon RP, Robinson BM, Harvey AN, Hahn B, Leonard CJ, et al. The relationship between working memory capacity and broad measures of cognitive ability in healthy adults and people with schizophrenia. Neuropsychology 2013;27:220–9. Kosaka T, Komada M, Kosaka K. Sodium channel cluster, betaIV-spectrin and ankyrinG positive “hot spots” on dendritic segments of parvalbumin-containing neurons and some other neurons in the mouse and rat main olfactory bulbs. Neurosci Res 2008;62:176–86. Kuperberg G, Heckers S. Schizophrenia and cognitive function. Curr Opin Neurobiol 2000;10:205–10. LaBar KS, Gitelman DR, Parrish TB, Mesulam M. Neuroanatomic overlap of working memory and spatial attention networks: a functional MRI comparison within subjects. Neuroimage 1999;10:695–704. Lee J, Park S. Working memory impairments in schizophrenia: a meta-analysis. J Abnorm Psychol 2005;114:599–611. Lee J, Folley BS, Gore J, Park S. Origins of spatial working memory deficits in schizophrenia: an event-related fMRI and near-infrared spectroscopy study. PloS One 2008;3: e1760. Leussis MP, Madison JM, Petryshen TL. Ankyrin 3: genetic association with bipolar disorder and relevance to disease pathophysiology. Biol Mood Anxiety Disord 2012;2:18. Leussis MP, Berry-Scott EM, Saito M, Jhuang H, de Haan G, Alkan O, et al. The ANK3 bipolar disorder gene regulates psychiatric-related behaviors that are modulated by lithium and stress. Biol Psychiatry 2013;73:683–90. Linke J, Witt SH, King AV, Nieratschker V, Poupon C, Gass A, et al. Genome-wide supported risk variant for bipolar disorder alters anatomical connectivity in the human brain. Neuroimage 2012;59:3288–96. Lu WH, Zhang C, Yi ZH, Li ZZ, Wu ZG, Fang YR. Association between BDNF Val66Met polymorphism and cognitive performance in antipsychotic-naive patients with schizophrenia. J Mol Neurosci 2012;47:505–10. Myles-Worsley M, Park S. Spatial working memory deficits in schizophrenia patients and their first degree relatives from Palau, micronesia. Am J Med Genet 2002;114:609–15. Nagai T, Takuma K, Dohniwa M, Ibi D, Mizoguchi H, Kamei H, et al. Repeated methamphetamine treatment impairs spatial working memory in rats: reversal by clozapine but not haloperidol. Psychopharmacology (Berl) 2007;194:21–32.
C. Zhang et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 110–115 Quinn EM, Hill M, Anney R, Gill M, Corvin AP, Morris DW. Evidence for cis-acting regulation of ANK3 and CACNA1C gene expression. Bipolar Disord 2010;12:440–5. Reilly JL, Harris MS, Khine TT, Keshavan MS, Sweeney JA. Antipsychotic drugs exacerbate impairment on a working memory task in first-episode schizophrenia. Biol Psychiatry 2007;62:818–21. Roussos P, Katsel P, Davis KL, Bitsios P, Giakoumaki SG, Jogia J, et al. Molecular and genetic evidence for abnormalities in the nodes of Ranvier in schizophrenia. Arch Gen Psychiatry 2012;69:7–15. Ruberto G, Vassos E, Lewis CM, Tatarelli R, Girardi P, Collier D, et al. The cognitive impact of the ANK3 risk variant for bipolar disorder: initial evidence of selectivity to signal detection during sustained attention. PloS One 2011;6:e16671. Rueckert EH, Barker D, Ruderfer D, Bergen SE, O'Dushlaine C, Luce CJ, et al. Cis-acting regulation of brain-specific ANK3 gene expression by a genetic variant associated with bipolar disorder. Mol Psychiatry 2013;18:922–9. Sanches M, Keshavan MS, Brambilla P, Soares JC. Neurodevelopmental basis of bipolar disorder: a critical appraisal. Prog Neuropsychopharmacol Biol Psychiatry 2008;32:1617–27. Schlagenhauf F, Dinges M, Beck A, Wustenberg T, Friedel E, Dembler T, et al. Switching schizophrenia patients from typical neuroleptics to aripiprazole: effects on working memory dependent functional activation. Schizophr Res 2010;118:189–200. Schmidt-Hansen M, Honey RC. Working memory and multidimensional schizotypy: dissociable influences of the different dimensions. Cogn Neuropsychol 2009;26:655–70. Shi YY, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res 2005;15:97–8. Silver H, Feldman P, Bilker W, Gur RC. Working memory deficit as a core neuropsychological dysfunction in schizophrenia. Am J Psychiatry 2003;160:1809–16.
115
Talkowski ME, Rosenfeld JA, Blumenthal I, Pillalamarri V, Chiang C, Heilbut A, et al. Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries. Cell 2012;149:525–37. Tesli M, Koefoed P, Athanasiu L, Mattingsdal M, Gustafsson O, Agartz I, et al. Association analysis of ANK3 gene variants in nordic bipolar disorder and schizophrenia case– control samples. Am J Med Genet B Neuropsychiatr Genet 2011;156B:969–74. Tsuang MT, Stone WS, Faraone SV. Genes, environment and schizophrenia. Br J Psychiatry 2001;40:s18–24. Turetsky BI, Calkins ME, Light GA, Olincy A, Radant AD, Swerdlow NR. Neurophysiological endophenotypes of schizophrenia: the viability of selected candidate measures. Schizophr Bull 2007;33:69–94. Wang Z, Li Z, Chen J, Huang J, Yuan C, Hong W, et al. Association of BDNF gene polymorphism with bipolar disorders in Han Chinese population. Genes Brain Behav 2012;11: 524–8. Wang Z, Fan J, Gao K, Li Z, Yi Z, Wang L, et al. Neurotrophic tyrosine kinase receptor type 2 (NTRK2) gene associated with treatment response to mood stabilizers in patients with bipolar I disorder. J Mol Neurosci 2013;50:305–10. Yuan AH, Yi ZH, Wang Q, Sun JH, Li ZQ, Du YS, et al. ANK3 as a risk gene for schizophrenia: new data in Han Chinese and meta analysis. Am J Med Genet B Neuropsychiatr Genet 2012;159B:997–1005. Zhang Y, Bertolino A, Fazio L, Blasi G, Rampino A, Romano R, et al. Polymorphisms in human dopamine D2 receptor gene affect gene expression, splicing, and neuronal activity during working memory. Proc Natl Acad Sci U S A 2007;104: 20552–7. Zhang C, Fang Y, Xie B, Cheng W, Du Y, Wang D, et al. DNA methyltransferase 3B gene increases risk of early onset schizophrenia. Neurosci Lett 2009;462:308–11.
