Figure 2.

Identification of mutations in PEX7 in a family with ASD. (A) AU-3500, a family with three children affected with ASD. Shaded symbols indicate affected individuals. WES was performed on samples from individuals indicated with a star. Genotyping by Sanger sequencing in additional family members was performed where indicated (+: reference allele, −: alternate allele). (B) Mapping to a locus on chromosome 6. Genome-wide linkage plot (top) and maximum obtainable LOD score in the family across the interval (bottom). A homozygous missense mutation in the AU-3500 family disrupts a conserved tryptophan (p.W75C) (C) in the first WD40 repeat of PEX7, the receptor responsible for import of PTS2-containing proteins into the peroxisome (D), and an established cause of rhizomelic chondrodysplasia punctata. (E) PEX7 W75C and S25F, a previously characterized mutation from a patient with mild RCDP and ASD, result in partial loss-of-function in a peroxisomal import assay. In fibroblasts from patients with RCDP, PTS2-tagged mCherry accumulates in the cytoplasm due to lack of PEX7 transport activity (top). Transfection of wildtype PEX7 cDNA causes PTS2-mCherry to redistribute to small puncta, indicative of restoration of peroxisomal import (bottom). Transfection of mutant PEX7 W75C or S25F does not restore complete import, with a minority of cells showing partial import (middle). Quantification of defects in peroxisomal import mediated by ASD-associated PEX7 alleles, scored by visual assay. Error bars represent standard error (right). (F) Immunoblotting of whole cell lysates illustrates deficient maturation of PTS2-targeted proteins in a RCDP cell line transfected with ASD-associated PEX7 alleles. AGPS, PhyH, and thiolase are imported into the peroxisome and undergo cleavage into a smaller, mature peptide. p: preprotein, m: mature protein, Beta-tubulin: loading control. Quantification of processing defects by densitometry of transfected cell lysates (right).