Rare genetic variants in CX3CR1 and their contribution to the increased risk of schizophrenia and autism spectrum disorders

Abstract

CX3CR1, a G protein-coupled receptor solely expressed by microglia in the brain, has been repeatedly reported to be associated with neurodevelopmental disorders including schizophrenia (SCZ) and autism spectrum disorders (ASD) in transcriptomic and animal studies but not in genetic studies. To address the impacts of variants in CX3CR1 on neurodevelopmental disorders, we conducted coding exon-targeted resequencing of CX3CR1 in 370 Japanese SCZ and 192 ASD patients using next-generation sequencing technology, followed by a genetic association study in a sample comprising 7054 unrelated individuals (2653 SCZ, 574 ASD and 3827 controls). We then performed in silico three-dimensional (3D) structural modeling and in vivo disruption of Akt phosphorylation to determine the impact of the detected variant on CX3CR1-dependent signal transduction. We detected a statistically significant association between the variant Ala55Thr in CX3CR1 with SCZ and ASD phenotypes (odds ratio=8.3, P=0.020). A 3D structural model indicated that Ala55Thr could destabilize the conformation of the CX3CR1 helix 8 and affect its interaction with a heterotrimeric G protein. In vitro functional analysis showed that the CX3CR1-Ala55Thr mutation inhibited cell signaling induced by fractalkine, the ligand for CX3CR1. The combined data suggested that the variant Ala55Thr in CX3CR1 might result in the disruption of CX3CR1 signaling. Our results strengthen the association between microglia-specific genes and neurodevelopmental disorders.

Figure 1

Genomic locations of the variants in CX3CR1 detected in this study. Note: Localization of a protein domain is based on the human protein reference database (http://www.hprd.org/index.html) (HPRD). Genomic position is based on NCBI builds GRCh38 (Ensembl Transcript ID ENST000000399220). Amino acid position is based on the NCBI reference sequences NP_001328.1 and DDBJ DRA004490. UTR, untranslated regions.

Figure 2

3D model of the structure of the CX3CR1-Ala55Thr mutant. (a) 3D model of the structure of CX3CR1, fractalkine, a heterotrimeric G protein (Gα, Gβ, Gγ) and the cellular membrane. Three CX3CR1-mutated sites (Ala55, Gly112, Met138) are indicated in dotted black circles. (b) Enlarged view of the 55th residue of wild-type CX3CR1. (c) Enlarged view of the 55th residue of the CX3CR1-Ala55Thr mutant.

Figure 3

Effect of CX3CR1-Ala55Thr on Akt phosphorylation-mediated signaling. (a) Subcellular localization of CX3CR1 wild-type (WT) or its Ala55Thr mutant. mCherry-fused CX3CR1 WT or mutant-expressing vectors were transfected into HEK293 cells and cultured for 36 h before immunofluorescent analysis. DIC, differential interference contrast. Scale bar, 25 μm. (b) Ala55Thr mutation in CX3CR1 inhibits Akt phosphorylation upon fractalkine (FKN) treatment. HEK293 cells transfected with an mCherry-conjugated CX3CR1 WT or Ala55Thr-expressing vector were cultured for 36 h, and then the cells were stimulated with the CX3CR1 ligand fractalkine for 15 min. Cell lysates were analyzed by western blotting using the indicated antibodies. The data are expressed as means±s.e.m. and statistical significance was tested with analysis of variance followed by Tukey–Kramer’s multiple comparison test. **P<0.01, *P<0.05. n=5.