Polymorphism in the LASP1 gene promoter region alters cognitive functions of patients with schizophrenia

Schizophrenia’s pathogenesis remains elusive. Cognitive dysfunction is the endophenotype and outcome predictor of schizophrenia. The LIM and SH3 domain protein (LASP1) protein, a component of CNS synapses and dendritic spines, has been related to the N-methyl-D-aspartate receptor (NMDAR) dysfunction hypothesis and schizophrenia. A single-nucleotide polymorphism (rs979607) in the LASP1 gene promoter region has been also implicated in schizophrenia susceptibility. The aim of this study was to investigate the role of the LASP1 rs979607 polymorphism in the cognitive functions of patients with schizophrenia. Two hundred and ninety-one Han Taiwanese patients with schizophrenia were recruited. Ten cognitive tests and two clinical rating scales were assessed. The scores of cognitive tests were standardized to T-scores. The genotyping of the LASP1 rs979607 polymorphism was performed using TaqMan assay. Among the 291 patients, 85 were C/C homozygotes of rs979607, 141 C/T heterozygotes, and 65 T/T homozygotes, which fitted the Hardy-Weinberg equilibrium. After adjusting age, gender, and education with general linear model, the C/C homozygotes performed better than C/T heterozygotes in overall composite score (p = 0.023), Category Fluency test (representing processing speed and semantic memory) (p = 0.045), and Wechsler Memory Scale (WMS)-III backward Spatial Span test (p = 0.025), albeit without correction for multiple comparisons for the latter two individual tests. To the best of our knowledge, this is the first study suggesting that the genetic variation of LASP1 may be associated with global cognitive function, category verbal fluency, and spatial working memory of patients with schizophrenia. The finding also lends support to the NMDAR dysfunction hypothesis of schizophrenia. More studies with longitudinal designs are warranted.


Methods
The study was approved by institutional review board of China Medical University Hospital, Taiwan and conducted in accordance with the current revision of the Declaration of Helsinki. All participants were recruited from the chronic wards of China Medical University Hospital, Taiwan. The patients were included if they were (1) aged between 18 and 65; (2) diagnosed as schizophrenia by research psychiatrists using the Structured Clinical Interview for DSM-IV (1994); (3) with laboratory assessments (including blood routine, biochemical tests) within normal range; (4) with sufficient education to communicate effectively and complete the assessments of the study; and (5) under stable dosages of antipsychotics treatment for at least two months before test enrollment. Patients were excluded when they (1) presented with other comorbid psychiatric disorders, substance use disorder, mental retardation; (2) had other existing physical or neurological illnesses; and (3) failed to cooperate with the study. A total of 291 Han Taiwanese schizophrenic patients were recruited after they agreed to participate in the study and provided written informed consent after complete description of the study.
Patients' clinical manifestations were measured by Positive and Negative Syndrome Scale (PANSS) 37 , Scale for Assessment of Negative Symptoms (SANS) 38,39 . All the participants received clinical ratings by trained and experienced research psychiatrist.
Interrater reliability was analyzed with the ANOVA test. Only raters achieving intra-class correlation coefficients of 0.90 or higher during pre-study training were allowed to rate the study patients.
Patients' cognitive functions were measured by a battery, which included 7 domains: (1) speed of processing, which is composed of 3 subtests: Category Fluency 40-42 , Trail Making A 43 , and Wechsler Adult Intelligence Scale (WAIS)-III Digit Symbol-Coding 44 ; (2) Continuous Performance Test for sustained attention 45,46 ; (3) working memory, which is composed of verbal subtest (backward digit span) 47 49 , and (7) Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT) 50 used for social cognition scaling, as recommended by the committee of US National Institute of Mental Health Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) as the solitary measure of social cognition in schizophrenia 51 . The Chinese version of MSCEIT tasks was translated from English to Mandarin Chinese with satisfactory reliability, validity 52 , and applicability 53 . The cognitive battery had been successfully applied in previous clinical trials on schizophrenia 54,55 .
To analyze the rs979607 polymorphism of LASP1, the Master Pure DNA Purification Kit for Blood Version II (EPICENTRE, Madison, Wisconsin, USA) was used to isolate DNA from patients' blood. We used ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific Inc.) to determine the concentration of DNA by absorbance at 260 nM. DNA was then diluted to a concentration of 50 ng/ul and underwent PCR amplification reaction. For all SNP genotyping, the Taqman SNP genotyping assay (ABI: Applied Biosystems Inc., Foster City, CA, USA) was used. DNA samples of known genotypes were used in every reaction as positive controls.
The PCR reaction was conducted in the 10 μl reaction volume, containing 0.4 μl DNA sample, 5 μl PCR master mix, and 0.25 μl primer pairs and probes. A pre-incubation at 95 °C for 10 min was used to activate the Hot-Start DNA polymerase and denature DNA and was followed by 40 amplification cycles of 92 °C denaturation for 15 Sec; and 60 °C for 60 Sec. The probe fluorescence signal detection was performed using the ABI Prism 7500 Real-Time PCR System. Statistical analysis. Statistical Package for the Social Sciences (SPSS) version 17 (IBM Inc.) was used to analyze the data. The scores of the cognitive tests were all standardized to T-score. Among the genetic groups, the demographic and clinical symptoms groups differences were tested by Chi-Square test, Analysis of Variance (ANOVA) or Kruskal-Wallis test, according to the normality of the data. One-way ANOVA was performed to examine the effects of different SNP genotypes on cognitive functions. In addition, general linear models (GLM) was applied to further investigate the genetic effect of LASP1 on cognitive functions while controlling for patients' gender, age, and duration of education. Finally, to test the Hardy-Weinberg equilibrium of the genotype counts, a Chi-Square goodness-of-fit test was employed.

Results
Two hundred and ninety-one patients with stable, chronic schizophrenia were enrolled and successfully genotyped for the LAPS1 rs979607 SNP. The mean age of the patients was 38.2 ± 9.4 years old. The mean education level was 10.9 ± 2.4 years. The mean age of illness onset was 23.1 ± 6.7 years old, and mean illness duration lasted 188.4 ± 252.5 months. The mean dosage of antipsychotics used, shown as the equivalent to chlorpromazine, was 531.0 ± 497.3 mg per day. No significant difference between genotypes of LASP1 rs979607 and demographic data was observed ( Table 1).
Of the 291 patients, all were assayed using the Taqman SNP genotyping method: 85 showed the C/C genotype of the LASP1 rs979607 SNP, 141 had the C/T genotype, and 65 had the T/T genotype. This genotype distribution was in equilibrium with the Hardy-Weinberg law (p = 0.653). Regarding the allele distribution, the minor allele frequency of rs979607 in our study (T allele: 46.6%) was similar to that of Han Chinese populations (43.0%) from HapMap database (www.hapmap.org). However, our study yielded a different minor allele (T) from that (C) of the Korean patients (Table 3). Three positive controls (C/C, C/T and T/T) were added while analyzing the LASP1 rs979607 genotype, and the results of the positive control genotyping were in line with the expected types. Therefore, the genotyping error rate could be regarded as 0.
We further investigated the genetic effect of LASP1 on cognitive functions (Table 4). Cognitive functions among three genotypic groups of LASP1 rs979607 failed to reach significant difference. However, schizophrenic patients with C/C homozygotes showed an insignificant trend of better performance in overall composite score (p = 0.070), Category Fluency (p = 0.089), and WMS-III Spatial Span (representing non-verbal working memory) (p = 0.079).    Table 4. Cognitive function in patients with different LASP1 genotypes. 1 All data standardized to T scores and presented by mean (SD). 2 All data were analyzed by ANOVA. 3 verall composite score: an overall composite T scores including all seven domains was calculated by standardizing the sum of T scores. 4 Speed of processing: an overall composite T score including all 3 domains (Category fluency, Trail marking A, WAIS-III Digit symbol-coding) was calculated by standardizing the sum of T scores. 5 Working memory: an overall composite T score including 2 domains (Backward digit span, WMS-III, Spatial span) was calculated by standardizing the sum of T scores. 6 WAIS-III, Wechsler adult intelligence scale, 3 rd version. 7 WMS-III, Wechsler memory scale, 3 rd version. 8 WISC-III, Wechsler intelligence scale for children, 3 rd version. 9 MSCEIT, Mayer-Salovey-Caruso emotional intelligence test.

LASP1 rs979607 C/C vs C/T C/C vs T/T C/T vs T/T
Overall composite score Mean difference (SD  Table 5. Analysis of the effects of LASP1 genetic polymorphism on cognitive functions in schizophrenic patients by general linear model 1 All data were standardized to T scores. 2 95% CI = 95% confidence interval. P-value was based on the cognitive function between genetic groups multiple regression model analysis of by general linear model. Gender, age, and years of education were adjusted in the model. www.nature.com/scientificreports www.nature.com/scientificreports/ Next, we applied general linear model (GLM) to adjust age, gender, and education. Schizophrenic patients with LASP1 rs979607 C/C homozygotes performed significantly better than C/T heterozygotes in overall composite score (p = 0.023) ( Table 5). At this first step, there was no need for multiple comparisons due to only one analysis.
After obtaining a positive finding, we then examined the significance of various cognitive domains. Schizophrenic patients with LASP1 rs979607 C/C homozygotes performed significantly better than C/T heterozygotes in category fluency (p = 0.045), and WMS-III-Spatial Span (p = 0.025) ( Table 5). In addition, the C/C homozygotes also showed an insignificant trend to perform better than C/T heterozygotes in overall composite score, category fluency, and WMS-III-Spatial Span (Table 5). At this secondary step, correction of multiple comparisons could be imposed. In this case, none of the cognitive domains reached statistical significance.

Discussion
The results of this study showed that schizophrenic patients with the C/C genotype of LASP1 rs979607 performed better than those with the C/T genotype in general cognitive function, category verbal fluency, and non-verbal (spatial) working memory ( Table 5). Since LASP1 protein has been related to the NMDA hypofunction theory of schizophrenia 34 and NMDAR-related neurotransmission has been associated with cognitive function in schizophrenia patients 56 , it is reasonable that LASP1 also plays a role in the modulation of cognitive function.
Schizophrenia patients with homozygote alleles (particularly the C/C group) performed better than heterozygotes (C/T group) in some cognitive tests. The hypothesis named molecular heterosis implies that heterozygote subjects result in a greater or lesser impact on specific traits compared with homozygotes for a specific genetic polymorphism. Examples include smoking and cognitive functions in schizophrenic patients [57][58][59] . Our findings appeared in accordance with molecular heterosis.
Schizophrenia patients with C/C genotype performed better than C/T heterozygotes on category fluency in this study. Deficits in category fluency have been observed in schizophrenia patients 60,61 and also in dementia patients 62,63 . Category fluency is reflective of speed of processing 64,65 and semantic memory 66 . NMDARs specifically in CA3 pyramidal cells regulate speed of processing 67 , and NMDAR modulations (both agonism and antagonism) alter semantic memory 68,69 . In accordance, the current study suggests that LASP1 genetic polymorphism may be related with different category fluency. More studies are needed to elucidate the mechanism of LASP1's influence on speed of processing and semantic memory.
Spatial working memory varied with LASP1 rs979607 polymorphisms of schizophrenia patients in this study. Deficit in spatial working memory is considered one of the core neurocognitive impairments and an endophenotype of schizophrenia [70][71][72] . NMDARs, specifically NR2B-containing NMDARs, are vital for spatial working memory 73,74 . Of note, functional loss of NMDARs in the dentate gyrus impairs spatial working memory but spares spatial reference memory 75 . Therefore, it is expectable that LASP1 may be involved in spatial working memory.
The limitations of this study included the following: First, schizophrenia is a heterogeneous disease, in which many factors contribute to its development and presentation. A SNP might only have limited influence on the cognitive function of schizophrenia. More studies are warranted to explore the potential roles of other markers. Second, the sample size was modest. Third, only Han Taiwanese patients were enrolled. Whether the findings could be extrapolated to other ethnicity populations remain unclear. Fourth, no healthy individuals were included in this study for comparison. Fifth, the study was cross-sectional, without longitudinal follow-up. In addition, whether the finding could be observed even during the prodromal phase of the illness also deserves research.
To the best of our knowledge, this is the first study describing genetic polymorphisms of LASP1 may have impact on cognitive functions in patients with schizophrenia. The finding also lends support to the NMDA dysfunction theory of schizophrenia. Further studies with larger sample sizes of both schizophrenia patients and controls, various ethnicities, and longitudinal designs are needed to clarify the role of LASP1 in schizophrenia and cognitive functions.