Analysis of the association between MICA gene polymorphisms and schizophrenia

Abstract Background The major histocompatibility complex (MHC) has been implicated in schizophrenia. This study aimed to explore the correlation between the major histocompatibility complex class I polypeptide‐related sequence A (MICA) polymorphisms and schizophrenia. Methods A total of 220 Han schizophrenia patients, 47 Han healthy controls, 155 Li schizophrenia patients, and 48 Li controls were selected from Hainan Province, China. The diagnosis was made according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, criteria. Sequencing‐based‐typing (PCR‐SBT) technology was used for MICA allele typing, and the correlation analyses of MICA gene polymorphism and schizophrenia were performed. Results In the Han group, the three allele frequencies of MICA*002:01, MICA*A4, and MICA*A9 in the schizophrenia group were significantly higher than those in the healthy control group, and the differences were statistically significant (pc < 0.05; pc values were 0.024, 0.030, and 0.031, respectively). Yet, there was no difference in the MICA gene between the schizophrenia group and the healthy controls group in the Li population. Conclusion We found MICA*002:01, MICA*A4, and MICA*A9 may be susceptibility alleles for schizophrenia in the Han population, while the MICA allele polymorphism in the Li population is not associated with schizophrenia in Chinese.


| INTRODUC TI ON
Schizophrenia is a chronic and disabling mental disorder characterized by hallucinations, delusions, disorganized speech or behavior, and impaired cognitive ability. Most patients present with highly heterogeneous typical symptoms in late adolescence or early adulthood. 1 A 2016 study showed that schizophrenia's global agestandardized time-point prevalence was about 0.28%, with high early mortality. 2 Moreover, patients with schizophrenia have a high risk of suicide, and their life expectancy is approximately 20 years shorter than in the general population. 3 Intermittent and long-term mental problems, chronic symptoms, and even disability often lead to extremely high unemployment among people with schizophrenia, with profound implications for individuals and societies. 4 The etiology and pathogenesis of schizophrenia are still unclear.
Genetics, environment, neurodevelopment, inflammation, and immunity are possible pathogenic factors. It is generally accepted that the interaction of genetics and the environment has an important influence on schizophrenia. 5,6 Genome-wide association studies identified a number of risk loci with polymorphisms, and numerous studies have demonstrated the important role of genetics in susceptibility to schizophrenia. 7 Moreover, epidemiological and animal model evidence suggests that maternal infection is a risk factor for schizophrenia and that maternal immune activation (MIA) alone is sufficient to lead to lifelong neurological and behavioral changes in offspring. 8 Subsequently, another study pointed out that the cause of MIA in schizophrenia is associated with the production of cytokines and complement proteins in the immune system that affects neural development. 9 It has been suggested that complement levels are potential peripheral biomarkers in schizophrenia. 10,11 Inflammation has an important role in pathogenesis and maintenance, and cytokine disturbance is associated with disease staging. 12 Schizophrenia has also been associated with chronic low-grade inflammation, while an abnormal immune system has been identified as a risk factor for schizophrenia. 13 Clinical trials have demonstrated that immunomodulation improves psychiatric symptoms in patients with schizophrenia. 14 Recent multiple genome-wide association studies identified a strong genetic association between MHC locus and schizophrenia. 15 MHC, also known as human leukocyte antigen (HLA), is located on the short arm of chromosome 6 (6p21.3-22.1) and has a high degree of polymorphism and extensive linkage disequilibrium (LD). 16 The MHC region is divided into class I, class II, class III, and extended class I and II genes. Several MHC-related single nucleotide polymorphisms in schizophrenia have been previously reported. 17,18 HLA-G, a non-classical MHC class I gene, is associated with the risk of developing schizophrenia and the severity of its clinical symptoms. 19,20 The major histocompatibility complex class I chainassociated gene A (MICA) is located at the centromeric terminal of the HLA class I-associated region, adjacent to HLA-B, and belongs to the non-classical HLA class I family. MICA does not present any antigen but acts as a ligand for natural killer (NK) cells, γδ T cells, and αβ CD8+ T cells, which express a common NK cell receptor natural killer group 2D (NKG2D). 21 MICA protein is absent from most cells but can be induced by infections and oncogenic transformation. 22 MICA is highly polymorphic. Previous studies have linked MICA to rheumatoid arthritis, ankylosing spondylitis, Behçet's disease, celiac disease, and type 1 diabetes. [23][24][25][26][27] In addition, some studies have shown a bidirectional association between these autoimmune diseases and an increased risk of schizophrenia. 28,29 This study aimed to analyze the association between MICA gene polymorphisms and schizophrenia in Han and Li populations in Hainan Province, located at the southernmost tip of China, isolated from the mainland, where the Li and Han nationalities account for more than 98% of the province's population. To the best of our knowledge, this is the first study that explored the role of MICA gene polymorphism in the pathogenesis of schizophrenia.

| DNA extraction
Genomic DNA was extracted from subjects' peripheral blood (EDTA anticoagulated) using standard salting-out methods. All samples were stored in a −20°C freezer.

| Pre-processing of PCR amplification products for gene sequencing
After the PCR reaction, 3.6 μl of the mixture of alkaline phosphatase and exonuclease I were added to the PCR product solution before sequencing. After incubation at 37°C for 30 min, the product was placed at −80°C for 20 min to inactivate all enzyme activities.

| PCR reaction before sequencing
The strategies for primers and gene sequencing 31 are shown in Figure 1. Four different sequencing reactions were performed for each DNA product. The sequencing reaction system included: 3 μl of purified PCR product, 2 μl of BigDye 5× buffer, 2 μl of sequencing primer 10 pmol/μl, 1.5 μl of BigDye, and 6.5 μl of ddH2O. The total volume of the above reaction system was 15 μl. PCR conditions for the sequencing reaction were 95°C for 1 min, 96°C for 10 s, 50°C for 5 s, 60°C for 2 min, 25 cycles. Finally, the product was stored at 4°C.

| Pre-processing for sequencing
After the PCR reaction before sequencing was completed, 1.

| Hardy-Weinberg Equilibrium (HWE) test for MICA loci
There was no significant difference between the observed and theoretical frequencies of the selected subjects' genotypes (p > 0.05) between Han and Li schizophrenia populations and healthy controls. Also, all data were in line with the Hardy-Weinberg equilibrium law. Province. Even after Bonferroni correction, the allele frequencies of MICA*002:01, MICA*A4, and MICA*A9 significantly differed between Han schizophrenia patients and Han healthy controls, indicating that these alleles may be related to the susceptibility of schizophrenia in the Han population. Thus, these data imply that carrying the MICA*002:01, MICA*A4, and MICA*A9 alleles may increase schizophrenia risk. Although the correlation between MICA*007 allele frequency and schizophrenia in the Han population was not significant, it also seems to be related to schizophrenia, because it was absent in healthy controls. In the Li population, the MICA*010 allele frequency of schizophrenia patients was relatively higher than that of healthy controls, but the difference was not statistically significant after Bonferroni correction. Also, no significant differences were found for the remaining MICA alleles. We speculate that the MICA gene may increase the risk of schizophrenia by affecting immune regulation or fetal neurodevelopment. In addition, in this study, no MICA alleles significantly associated with schizophrenia in the Li population were found, which may imply that the effect of MICA on schizophrenia is associated with ethnic differences.  18,37,38 MHC has a more important role in susceptibility to schizophrenia than other psychiatric disorders. 37 Numerous studies also provided support for the involvement of the immune system in the pathogenesis of schizophrenia; HLA class I (classical and non-classical), class II, and class III (complement system) have been reported in schizophrenia. 19,[39][40][41][42][43] Inflammation has also been identified as a risk factor for schizophrenia, possibly interacting with genetic variants in schizophrenia-associated HLA loci to alter the risk of developing schizophrenia. 44 MICA is a member of the non-classical HLA class I family with the greatest degree of polymorphism. 45  The strength of this article is that it simultaneously studied two ethnicities and that the effect of racial differences on the findings was taken into account. Also, there is no research report on the relationship between MICA and schizophrenia. In our future studies, we plan to investigate the effect of overexpression of MICA on cell proliferation and apoptosis through corresponding cell function experiments and explore the pathophysiological relationship between MICA and schizophrenia. Meanwhile, we need to expand the sample size and use multi-ethnic studies to fully describe the impact of MICA on schizophrenia to verify and evaluate whether and how MICA is associated with schizophrenia. Moreover, further research will reveal the link between genotype and animal phenotype by establishing gene mutation animal models. Also, further studies are needed to establish whether the MICA gene mutation is widespread in the schizophrenia population and to determine its exact pathogenesis. Elucidating the etiology and specific pathogenesis of schizophrenia will help prevent and treat schizophrenia, which in turn will reduce the burden of disease treatment.

| CON CLUS IONS
We found MICA*002:01, MICA*A4, and MICA*A9 may be susceptibility alleles for schizophrenia in the Han population, while the MICA allele polymorphism in the Li population is not associated with schizophrenia in Chinese.

ACK N OWLED G M ENTS
This study was supported by the Finance Science and Technology in study design, data collection and analysis, decision to publish, or preparation of the article.

CO N FLI C T O F I NTE R E S T
All authors declare no competing interests.

FU N D I N G I N FO R M ATI O N
The study was supported by the Finance Science and Technology Project of Hainan Province, Grant/Award Number: ZDYF2022SHFZ2952, ZDYF(XGFY)2020002 and ZDYF2018132.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.