Background
Cognitive impairments are a core feature of schizophrenia and a major determinant of functional outcome [
1]. Cognitive domains that are particularly impaired in patients with chronic schizophrenia are verbal memory, working memory, motor speed, verbal fluency, attention, and executive function [
2,
3]. These cognitive domains, specifically working memory and executive function, are heritable traits that have a potential association with genetic variants [
4,
5]. Therefore, these traits are useful as an intermediate phenotype to investigate the functional association of susceptibility genes for schizophrenia [
6,
7].
22q12 is one of the regions that show high logarithm of the odds ratio scores in schizophrenia [
8‐
10]. The Hermansky–Pudlak Syndrome Type 4 (
HPS4) gene (OMIM: 606682) maps to human chromosome 22q12.1, and it consists of 14 exons that span approximately 32 kb of genomic DNA encoding an intracellular trafficking-related cytoplasmic protein named HPS4 [
11,
12]. The human HPS4 has 708 amino acids and a predicted molecular mass of 77 kDa and makes up BLOC-3 jointly with HPS1 as a 1:1 heterodimer [
13‐
15]. Hermansky–Pudlak Syndrome (HPS; MIM: 203300) is a group of rare autosomal recessive diseases characterized by clinical symptoms, such as oculocutaneous albinism and bleeding diathesis, and is caused by defective biogenesis of lysosome-related organelles, such as melanosomes and platelet-dense granules [
16]. In our previous case–control association study, haplotypes that were composed of five tagging single-nucleotide polymorphisms (SNPs) of
HPS4 (rs4822724, rs61276843, rs9608491, rs713998, and rs2014410) were found to be significantly associated with schizophrenia, a finding that was revealed by a sliding window approach for 2–5 locus haplotypes, including rs9608491 as an essential SNP [
17]. In addition, we have reported that two Japanese siblings who suffered from the comorbidity of major mental disorders, schizophrenia and major depression, and HPS harbored a nonsense mutation in
HPS4[
17].
HPS is caused by a mutation in one of the genes named
HPS1 to
HPS9, which encode subunit proteins that consist of multiprotein complexes, the adaptor protein complex-3 (AP-3), and the biogenesis of lysosome-related organelles complex (BLOC)-1, 2, and 3 [
18,
19]. BLOC-1, BLOC-2, and AP-3 interact to play a pivotal role in sorting and trafficking membrane proteins on early endosomes [
20‐
24]. Dysbindin, which is encoded by the dystrobrevin binding protein 1 (
DTNBP1) gene (also known as
HPS7), is one of the subunit proteins of BLOC-1 [
25]. Several lines of evidence from linkage, association, and postmortem brain studies have indicated an association between
DTNBP1 and schizophrenia [
26]. Genetic variants and haplotypes of
DTNBP1 have frequently been reported to be associated with a number of cognitive functions, including verbal, visual, and general memory [
27‐
30], attention [
31,
32], and executive function [
30,
32], in patients with schizophrenia and/or healthy controls. These genetic studies on
DTNBP1[
26‐
32] led to several research studies on identification of binding partners for dysbindin [
21‐
24]. By contrast, BLOC-3, which comprises HPS1 and HPS4, modulates the intracellular movement and distribution of late endosomes and lysosome-related organelles in cells [
14,
15,
33]. Although the precise function of BLOC-3 remains to be clarified, the interaction between BLOC-3 and Rab GTPases has recently been found [
34,
35].
In order to investigate the association between putative candidate genes and cognitive impairments in schizophrenia, a portable instrument with a high reliability and validity is required to assess the cognition that is specifically impaired in patients with schizophrenia. Most of the neurocognitive assessment batteries that have previously been used in schizophrenia studies involve detailed assessments of the entire profile of neuropsychological function, including normal functioning, but require lengthy and complex procedures. The Brief Assessment of Cognition in Schizophrenia (BACS) was developed as a neurocognitive battery that enables brief administration (about 40 min) and portability and has high reliability for specifically assessing the domains of cognitive function that are consistently impaired in schizophrenia, including verbal memory, working memory, motor speed, verbal fluency, attention, and executive function [
3,
36]. The Japanese-language version of BACS (BACS-J) has been developed for the assessment of cognition in Japanese patients with schizophrenia, and its reliability and validity have been confirmed [
37].
To the best of our knowledge, HPS4 has not been reported in relation to cognitive functions in patients with schizophrenia or healthy controls. The aim of the present study was to investigate whether HPS4 is associated with cognitive functions in patients with schizophrenia and healthy controls and with the clinical profiles of patients with schizophrenia. We initially tested the association of five tagging SNPs in HPS4 with the clinical profiles and cognitive domains as measured by BACS-J in Japanese patients with schizophrenia, and the association of HPS4 SNPs with cognitive domains was also tested in healthy controls. In addition, we examined the association of HPS4 haplotypes and cognitive domains by using a sliding window approach in patients with schizophrenia and healthy controls.
Discussion
In the present study, which was based on our previous reports indicating an association of
HPS4 with schizophrenia [
17], we evaluated the associations of
HPS4 with the clinical profiles of patients with schizophrenia and the cognitive functions of patients with schizophrenia and healthy controls. The major findings of our study were as follows: (1) the
HPS4 rs713998 polymorphism was significantly associated with executive function in Japanese patients with schizophrenia, and (2) the polymorphisms (rs9608491 and rs713998) and haplotypic variants (rs9608491-713998 and rs61276843-9608491-713998) in
HPS4 were significantly associated with working memory in Japanese healthy controls. These significances survived after corrections for multiple testing. Associations of the clinical profiles of patients with schizophrenia (PANSS scores, ages at onset, and premorbid IQs as measured by JART) with each SNP in
HPS4 were not observed.
Culminating lines of evidence have suggested that patients with schizophrenia show deficits of executive function, which is mainly mediated by the frontal lobe and which is involved in the ability of goal formation, planning, executing goal-directed plans, and effective performance [
4,
54]. The TOL task, which was employed as a task in order to assess executive function in the BACS [
3], is known to be involved in multifaceted aspects of executive function, including working memory, planning, problem-solving, and inhibition [
55,
56]. Task complexity in performing the TOL correlated with activation of the dorsolateral prefrontal cortex, the lateral premotor cortex, the rostral anterior cingulate cortex, and the right dorsal caudate nucleus in a positron emission tomographic study of healthy subjects [
57]. Moreover, decreased prefrontal activation during the TOL has been reported in subjects with first-episode schizophrenia [
58]. We showed that the minor allele A that is carried at rs713998 was significantly associated with higher performance in executive function that was measured by the TOL task in patients with schizophrenia under the dominant genetic model, but there was no association between any of the
HPS4 SNPs and executive function in healthy controls. Because the sample size, age, and percentage of males were almost comparable between the patients with schizophrenia and the healthy controls, these results suggested that the rs713998 polymorphism confers distinct properties in frontal cortex-mediated executive function, specifically in patients with schizophrenia.
HS4 is unique in binding to the counterpart protein HPS1 and in forming a 1:1 heterodimer called BLOC-3 that is involved in late endosomal trafficking [
14,
15,
33]. Both the N-terminal (amino acid: 1–274) and C-terminal domains (534–708) of HPS4 have been found to be required for assembly with a full-length HPS1 to form a soluble and stable BLOC-3 complex [
34]. Rs713998 is a nonsynonymous SNP that is located in exon 9 (p.G229E) and that has been reported to be completely linked with three other nonsynonymous SNPs (rs5752330: exon 11, p.M552V; rs1894706: exon 12, p.Y606H; rs1894704: exon 13, p.H625G) of
HPS4[
17]. Given that the four SNPs (rs713998, rs5752330, rs1894706, and rs1894704) that completely link with each other occur in domains that are required for HPS1–HPS4 interaction (N-terminal domain: rs713998; C-terminal domain: rs5752330, rs1894706, and rs1894704), it is plausible that the amino-acid substitutions that are caused by these SNPs affect the HPS1–HPS4 interactions and BLOC-3 complex stability. The HPS1-HPS4 interaction is also indispensable for the recently identified function of BLOC-3 as a guanine nucleotide exchange factor (GEF) for the small GTPases Rab32/38 [
35]. Although the longin domain that was bioinformatically predicted to occur in the N-terminal portion of HPS4 may be implicated in Rab GEF activity [
35], no exonic tagging SNP corresponding to this domain was included in the present study.
Working memory impairments have been consistently reported in patients with schizophrenia, even those with first-episode schizophrenia with highly preserved IQ [
59]. A digit-sequencing task, which has been employed as a task for assessing working memory in BACS [
3], is akin to a number-letter sequencing task. The latter was found to activate the dorsolateral prefrontal cortex, the orbital frontal lobe, the premotor cortex, and the posterior parietal cortex, mainly in the right hemisphere, in a positron emission tomographic study of healthy subjects [
60]. An SNP-based association analysis that was conducted under the recessive model and a haplotype-based association analysis have consistently shown that the C-allele at rs9608491 and the G-allele at 713998 conferred high performance in working memory in healthy controls. Rs9608491 would contribute to a greater proportion of the effect of this two-marker haplotype in the performance of working memory in this population due to its higher regression coefficient and smaller
P value (β = 0.701,
P value = 0.001) than rs713998 under the recessive model. However, the finding of the involvement of rs713998 in working memory is apparently paradoxical because rs713998 at the A-allele state exerts a beneficial effect on executive function in patients with schizophrenia. Thus, working memory and executive function that is measured by the TOL may reflect different aspects of cognition, even though they are intimately related to each other, and rs713998 may exert its effects on these cognitive subdomains across controls and patients. Moreover, the finding that two alleles carried at rs713998 showed differential effects on cognitive aspects in cases versus controls could indicate a kind of ceiling effect that cannot be accounted for by a simple dose–response relationship of the alleles. For example, it has been suggested that the catechol-O-methyltransferase Val158Met polymorphism has an opposite effect on verbal fluency in healthy controls compared with patients with schizophrenia [
61]. In patients with schizophrenia, there has been no association of working memory with any polymorphism or haplotype that has been examined in
HPS4, although medication and clinical symptoms may have the potential to obscure any effects of genetic variance. We previously found that protective haplotypes always contained the C-allele carried at rs9608491 in
HPS4 in a case–control study [
17]. As discussed in our previous study [
17], an argument for the involvement of variants of
HPS4, particularly rs9608491, in the susceptibility to schizophrenia awaits further research. Collectively, the C-allele at rs9608491 may have a certain role in protecting against the susceptibility to schizophrenia and in enhancing working memory performance in controls only.
Very little information has thus far been found regarding the neurobiological and behavioral aspects of
HPS4, although it is ubiquitously expressed in most tissues, including brain [
11]. To the best of our knowledge, the Allen Brain Atlas [
62] has only shown that HPS4 is expressed in the hippocampal formation, the olfactory areas, the cortical subplate, and the cerebellum of the male adult mouse using in situ hybridization. Notwithstanding the uncertainty about the role of this gene in these brain regions, the data obtained here regarding the association between rs9608491 and working memory suggested that
HPS4 plays a certain role in working memory that is driven by the appropriate function of the prefrontal cortex. Further analysis is required to reveal a putative role of
HPS4 in the neurobiology related to the cognitive deficits in schizophrenia.
Several limitations of the present study must be mentioned. First, although the total sample size was moderate for an association study of cognitive functions, a relatively small number of individuals were included as carriers of minor homozygotes for each SNP, except for rs4822724. This could have resulted in a selection bias and decreased the power under the recessive model when individuals carrying the minor homozygotes were assigned to a distinct group. The associations of working memory with rs9608491 and rs713998 in the healthy controls was revealed by the recessive model, which yielded a minor homozygote group with a small number of individuals (rs9608491 C/C, n = 13; rs713998 A/A, n = 11). Replication of these findings in an independent large cohort or in different ethnic cohorts is needed. Second, several lines of evidence have indicated that cognitive performance in patients with schizophrenia is influenced by psychopharmacological medications [
63,
64]. In our positive finding of an association between rs713998 and executive function in patients with schizophrenia, we confirmed there was no significant difference (
P > 0.05) in the dosages of antipsychotics, anticholinergics, or the prescription status of anxiolytics/hypnotics under the dominant model. However, in a real-world clinical setting, the extent to which various kinds of antipsychotics or their combination, particularly in the context of the polypharmacy of antipsychotic medication, exerts an effect, if any, on the cognitive performance of patents with schizophrenia is difficult to analyze. Third, rs9608491 is an intronic SNP located in between the exons encoding the N-terminal domain. Its function is uncertain because it is unlikely to affect the stability of HPS4 mRNA [
17]. This is reminiscent of the relevant SNPs of the
DTNBP1 gene, which are mostly intronic and have been significantly implicated in the executive function of healthy controls [
30], in cases belonging to early-onset families with functional psychosis disorders [
65], and in the spatial working memory in patients with schizophrenia [
66]. This general aspect regarding the question as to why intronic SNPs are involved in cognitive function awaits further genetic study.
Competing interests
Kazufumi Akiyama is a paid consultant to Taisho Toyama Pharmaceutical Co., Ltd. This consultancy had no further role in the study design, the collection, analysis and interpretation of data, the writing of the report, or the decision to submit the paper for publication. None of the remaining authors declare any conflicts of interest.
Authors’ contributions
GK was involved in the design of the study, performed the statistical analysis, and drafted the manuscript. AS was involved in the design of the study, sample collection, and genotyping. YO, TW, KF, KS, TI, HM and KO were involved in participation in the design of the study and coordination for sample collection. KA was involved in the design of the study, laboratory coordination, sample collection, and editing the manuscript. All authors read and approved final manuscript.