Association between polymorphism rs2421943 of the insulin‐degrading enzyme and schizophrenia: Preliminary report

Abstract Background Insulin‐degrading enzyme (IDE) is an important gene in studies of the pathophysiology of type 2 diabetes mellitus (T2DM). Recent studies have suggested a possible link between type 2 diabetes mellitus (T2DM) and the pathophysiology of schizophrenia (SZ). At the same time, significant changes in insulin‐degrading enzyme (IDE) gene expression have been found in the brains of people with schizophrenia. These findings highlight the need to further investigate the role of IDE in schizophrenia pathogenesis. Methods We enrolled 733 participants from the Czech Republic, including 383 patients with schizophrenia and 350 healthy controls. Our study focused on the single nucleotide polymorphism (SNP) rs2421943 in the IDE gene, which has previously been associated with the pathogenesis of Alzheimer's disease. The SNP was analyzed using the PCR‐RFLP method. Results The G allele of the rs2421943 polymorphism was found to significantly increase the risk of developing SZ (p < 0.01) when a gender‐based analysis showed that both AG and GG genotypes were associated with a more than 1.55 times increased risk of SZ in females (p < 0.03) but not in males. Besides, we identified a potential binding site at the G allele locus for has‐miR‐7110‐5p, providing a potential mechanism for the observed association. Conclusion Our results confirm the role of the IDE gene in schizophrenia pathogenesis and suggest that future research should investigate the relationship between miRNA and estrogen influence on IDE expression in schizophrenia pathogenesis.

the possible involvement of the IGF-I pathway in SZ, along with disturbed insulin signaling pathways, and suggest an association of T2DM with SZ in drug-naïve patients, providing a reason to believe that brain IDE may be involved in SZ. 18,24 The expression of IDE has been found to be significantly reduced in the dorsolateral prefrontal cortex of patients with schizophrenia compared with controls, suggesting a possible role in the disturbed insulin signaling cascades and altered neuropeptide metabolism in the disease. 18 Previous studies have shown an association between the IDE gene polymorphisms rs1887922 and rs2149632 and T2DM. 25,26 However, to date, no study has investigated whether IDE gene SNPs are associated with an increased risk of SZ. Therefore, this study aimed to investigate the association of polymorphism (SNP) rs2421943 of the IDE gene, previously associated with an increased risk of AD and mild cognitive impairment (MCI), 22 with SZ using a case-control association approach.

| Ethics statement
The study was conducted in accordance with the Helsinki Declaration of 1975 (revised 2000) and approved by the Ethics Committee of the Faculty of Medicine, University of Ostrava (08/2012). Informed consent was obtained from all participants or their legal representatives.

| Participants
In our case-control study, we enrolled a total of 733 participants, including 383 patients with schizophrenia (SZ) and 350 healthy controls from the Czech Republic who gave informed consent.  Table 1.

| Genetic analysis
Genomic DNA was isolated from EDTA containing whole blood

| Statistical and in silico analysis
Statistical analysis was performed using R software. 28 Fisher's exact test was used to assess the association between discrete variables, including disease onset, genotype, cannabis use, and drug use. The risk ratio (RR) and odds ratio (OR) were used to determine the differences between genotypes or alleles in the calculated risk and odds. The confidence interval (CI) for each OR was calculated using the logit method as previously described. 29 The R package statmod 30 was used to calculate the power of Fisher's exact test by simulation.
We also performed an analysis of the potential involvement of the intronic rs2421943 polymorphism in the enhancer regulatory element of neighboring genes using the GeneHancer database and in miRNA regulation using miRBase and mirDIP. Table 1 presents the descriptive statistics for both patients and controls. We found that in the control group, the A allele had to be associated with SZ risk using Fisher's exact test. The G allele was found to significantly increase the risk of developing SZ (p < 0.01) compared with the A allele in subjects. The GG genotype was also found to be associated with a significant increase in SZ risk (p = 0.0141) with a risk of over 1.29 times that of the A allele. Notably, the statistically significant risk was observed only in homozygotes (GG -p < 0.0141), while heterozygotes (AG) did not exhibit a significant risk of SZ ( Table 2).

Moreover, a gender-based analysis showed that both AG and GG
genotypes of the rs2421943 polymorphism were associated with a 1.55 times increased risk of SZ in females, indicating a dominant effect of the G allele (p < 0.03). The power of the tests, calculated based on the observed risks and sample sizes, was higher than 0.56 in all comparisons that found a statistically significant difference in schizophrenia risk between genotypes. No statistically significant association was observed between SZ and any genotype in males ( Note: Mean ± standard deviation is given for age distribution. To investigate the possible role of intronic rs2421943 polymorphism, we performed an analysis of its potential involvement in the enhancer regulatory element of nearby genes using the GeneHancer database, 31 as well as an analysis of miRNA regulation with miRBase for mature miRNAs specific to humans 32 and mirDIP. 33 Our findings indicated no presence of enhancer regulatory elements at the polymorphism site. However, we did find that the G allele was the target of the has-miR-7110-5p miRNA, as identified by both miRbase and miDIP. To further support this observation, we employed the RNA22 v2 program 34 to analyze parameters of the hsa-miR-7110-5p interaction, using a sensitivity of 45% and specificity of 78%, with a seed size of 7 and a maximum of 1 unpaired base in the seed. The analysis indicated a high probability of an interaction site for the hsa-miR-7110-5p molecule in the case of the G allele ( Table 5).

| DISCUSS ION
In our study, we investigated the association between the rs2421943 polymorphism of the IDE gene and the risk of SZ. Although the G allele and GG genotype appear to be risk factors for the predis- and there is a suggested association between T2DM and SZ in drugnaïve patients. 24 Many metabolic genes that are downregulated in SZ are regulated by insulin, 42 and IDE is a downstream target of the insulin receptor signaling pathway. In addition, several linkage analyses have suggested an association between SZ and chromosomal loci between 10q23 and 10q25, where the IDE gene is located. [43][44][45] Previous research has reported a significant reduction in the number of IDE-expressing neurons and IDE protein levels in post-mortem brains of SZ patients compared with controls. 18 Our in silico analysis revealed that the IDE rs2421943 polymorphism is located in a region highly likely to be targeted by the miRNA hsa-miR-7110-5p, with the G allele at position 13 increasing the affinity of hsa-miR-7110-5p for this binding site ( Table 5). This finding provides a potential explanation for the observed differences in allele and genotype frequencies and their association with SZ risk. The increased affinity of the G allele for hsa-miR-7110-5p may lead to altered gene regulation, potentially contributing to the development of SZ. The G allele of the IDE rs2421943 polymorphism may potentially hinder the transition of hnRNA to mRNA, leading to a slower translation of the IDE protein in the brain, which has previously been reported in postmortem brains of SZ patients. 18 This reduction in IDE enzymatic activity would lead to increased levels of insulin interacting with brain regions involved in cognitive function. 46 This study has some limitations that need to be addressed.