Functional variants in the promoter region of Chitinase 3-like 1 (CHI3L1) and susceptibility to schizophrenia

Abstract

The chitinase 3-like 1 gene (CHI3L1) is abnormally expressed in the hippocampus of subjects with schizophrenia and may be involved in the cellular response to various environmental events that are reported to increase the risk of schizophrenia. Here, we provide evidence that the functional variants at the CHI3L1 locus influence the genetic risk of schizophrenia. First, using case-control and transmission/disequilibrium-test (TDT) methodologies, we detected a significant association between schizophrenia and haplotypes within the promoter region of CHI3L1 in two independent cohorts of Chinese individuals. Second, the at-risk CCC haplotype (P=.00058 and .0018 in case-control and TDT studies, respectively) revealed lower transcriptional activity (P=2.2 x 10(-7)) and was associated with lower expression (P=3.1 x 10(-5)) compared with neutral and protective haplotypes. Third, we found that an allele of SNP4 (rs4950928), the tagging SNP of CCC, impaired the MYC/MAX-regulated transcriptional activation of CHI3L1 by altering the transcriptional-factor consensus sequences, and this may be responsible for the decreased expression of the CCC haplotype. In contrast, the protective TTG haplotype was associated with a high level of CHI3L1 expression. Our findings identify CHI3L1 as a potential schizophrenia-susceptibility gene and suggest that the genes involved in the biological response to adverse environmental conditions are likely to play roles in the predisposition to schizophrenia.

Figure  1.

Genomic structure of CHI3L1 and the location of SNPs included in the present study.

Figure  2.

Luciferase reporter assay of SNP2, SNP3, and SNP4 common haplotypes in U251 cells. The transcriptional activity of the pCBG99 control was set at 100%. Each CHI3L1 haplotype was tested eight times. Among the haplotypes, the risk haplotype (CCC) revealed the lowest activity, whereas the protective haplotype (TTG) showed the highest activity (P=2.2×10^-7, by one-way ANOVA).

Figure  3.

In vitro binding of U251 nuclear proteins to CHI3L1 promoter sequences containing SNP3 and SNP4. In lanes 1–4 and 9–10, probes of major alleles for each SNP were loaded. In lanes 5–8, probes of minor alleles for each SNP were loaded. Competitors are the unlabeled oligonucleotides with same allele as probes, and cross-competitors are the oligonucleotides with the other allele of probes. a, No obvious difference shown between the two alleles of SNP3. b, G-allele probes of SNP4 generating two binding complexes (lane 2), whereas C-allele probes generated one (lane 6). Loading 100× cross-competitors (C allele) does not eliminate the G-allele–specific band (lane 4). c, Supershift assay for the G-allele probes of SNP4 with use of antibodies of AP-2 and MYC. The presence of either of the antibodies resulted in a visible supershift band.

Figure  4.

Effect of inhibition of the MYC/MAX signaling pathways on CHI3L1 expression. In both cell lines, MYC-inhibitor trial severely reduces expression level of CHI3L1 (76% decreased in U251 and 82% decreased in THP-1). Double asterisk (**) indicates P<.01.

Figure  5.

Expression analyses of CHI3L1 in vivo. a, Expression level of CHI3L1 in the PBC from patients with schizophrenia and unaffected controls. The average level of CHI3L1 in unaffected controls was defined as 100. No significant difference in expression level between the two groups was detected (t test P=.081 ). b and c, Expression of CHI3L1 was associated with the genotype of both SNP4 (b) and SNP3 (c) in PBC (Kruskal-Wallis H test P<.01). d, Haplotypes in the promoter region of CHI3L1 associated with expression level of the gene in PBC. e, Relative allele-specific expression of the SNP5 data expressed as allele ratios of A/G. Patients who were heterozygotes for SNP4 revealed highly significant allele differences (t test P=9.1×10^-6, corrected for 5 tests) f, Risperidone-treated mice did not change the mRNA level of CHI3L1 in blood and brain (t test P>.05). The expression level of CHI3L1 in untreated mice was defined as 100.

Figure  6.

Hypothetical scenario for CHI3L1 in the pathophysiology of schizophrenia. Impairment of AKT signaling and overactivation of immune responses were observed in the subjects with schizophrenia and were considered to contribute to the risk of schizophrenia. The CHI3L1 gene may partly compensate the alteration of the processes and, therefore, may play a role in preventing the progression of psychosis.