AbstractGene-environment interactions, by means of abnormal macromolecular intestinal adsorption, is one of the possible causes of autism spectrum disorders (ASD) predominantly in patients with gastrointestinal disorders. Pre-haptoglobin-2 (zonulin), encoded by the Haptoglobin (HP) allele-2 gene, enhances the intestinal permeability by modulation of intercellular tight junctions. The two alleles of HP, HP1 and HP2, differ for 2 extra exons in HP2 that result in exon duplication undetectable by classic genome-wide association studies. To evaluate the role of HP2 in ASD pathogenesis and to set up a method to discriminate HP alleles, Italian subjects with ASD (n = 398) and healthy controls (n = 379) were genotyped by PCR analysis; subsequently, the PCR results were integrated with microarray genotypes (Illumina Human Omni 1S-8), obtained using a subset from the same subjects, and then we developed a computational method to predict HP alleles. On the contrary to our expectations, there was no association between HP2 and ASD (P > 0.05), and there was no significant allele association in subjects with ASD with or without gastrointestinal disorders (P > 0.05). With the aid of bioinformatics analysis, from a window frame of ~2 Mb containing 314 SNPs, we obtain imputation accuracy (r²) between 0.4 and 0.9 (median 0.7) and correct predictions were between 70% and 100% (median 90%). The conclusions endorse that enhanced intestinal permeability in subjects with ASD should not be imputed to HP2 but to other members of the zonulin family and/or to environmental factors.