Fig. 4
Functional analysis of the FRRS1L regulatory variants. (A) Changes in transcription factor binding motifs by the fixed trinucleotide with positive selection signatures and its flanking SRV. Calculation of the affinity changes is provided in Supplementary Fig. 5. (B) Expression level of FRRS1L measured by qRT-PCR in SH-SY5Y cells before (red) and after (blue) CRISPR/Cas9-mediated genome editing. Relative expression levels were computed by dividing with the wildtype measure. Three technical replicates were obtained for each of the three independent experiments. Shown data is a representative of the three experiments. P values were derived from two-tailed Student’s t-tests. Error bars, SEM. (C) Confirmation of CRISPR-induced edits with Sanger sequencing chromatographs for the autism/macaque variant (upper) and the ancestral sequences at the human-specific fixation site (lower). (B-C) WT, wildtype sequence; sg-Empty, no sgRNA; Mut, intended mutation. (D) eQTL mapping results for the rs2176692 genotypes and normalized FRRS1L expression levels. (E) Decay of the EHH statistic22, as a function of distance for haplotypes that carry the derived and ancestral allele of rs2176692. qRT-PCR, quantitative real-time reverse-transcription; SEM, standard error of mean; EHH, extended haplotype homozygosity.
