Evidence that ITGB3 promoter variants increase serotonin blood levels by regulating platelet serotonin transporter trafficking

Abstract

Elevated serotonin (5-HT) blood levels, the first biomarker identified in autism research, has been consistently found in 20-30% of patients with Autism Spectrum Disorder (ASD). Hyperserotonemia is mainly due to greater 5-HT uptake into platelets, mediated by the 5-HT transporter (SERT) located at the platelet plasma membrane. The protein complex involved in platelet SERT trafficking and externalization includes integrin β3, the beta subunit of the platelet membrane adhesive GP IIb/IIIa. Integrin β3 is encoded by the ITGB3 gene, previously identified as a quantitative trait locus (QTL) for 5-HT blood levels in ASD at single nucleotide polymorphism (SNP) rs2317385. The present study aims to identify the functional ITGB3 gene variants contributing to hyperserotonemia. ITGB3 gene sequencing in 20 individuals selected on the basis of rs2317385 genotypes defined four haplotypes encompassing six SNPs located in the ITGB3 gene promoter region, all in linkage disequilibrium with rs2317385. Luciferase assays in two hematopoietic cell lines, K-562 and HEL 92.1.7, demonstrate that ITGB3 gene promoter activity is enhanced by the presence of the C allele at rs55827077 specifically during differentiation into megakaryocytes (P < 0.01), with modulatory effects by flanking SNPs. This same allele is strongly associated with (a) higher 5-HT blood levels in 176 autistic individuals (P < 0.001), (b) greater platelet integrin β3 protein expression (P < 0.05) and (c) enhanced SERT trafficking from the cytosol toward the platelet plasma membrane (P = 4.05 × 10-11). Our results support rs55827077 as the functional ITGB3 gene promoter variant contributing to elevated 5-HT blood levels in ASD and define a mechanistic chain of events linking ITGB3 to hyperserotonemia.