Our top SNP rs2273500 is a splice acceptor variant that changes the sequence at the 3′ end of the intron between exon 4 and an ancillary exon, designated as exon 4.1 in Supplementary Figure 1; the resulting alternate transcript is truncated and predicted to be targeted for nonsense-mediated decay.43 To investigate the rs2273500 effect on splicing, we obtained RNA-seq and genotype data from the GTEx project.44 Liver (N=32) showed the highest CHRNA4 expression among the GTEx tissues, and transcripts containing exon 4.1 were detected. Using split read counts (reads crossing an exon:exon boundary) to test the efficiency of splicing events that utilize the exon 4 splice donor, we found that rs2273500-C resulted in significant reduction of splicing to exon 4.1 in favor of increased splicing to exon 4.2 and to a cryptic splice acceptor in exon 4.1 (P=5.4 × 10−58, Supplementary Table 10). These results provide strong evidence that rs2773500 functions as a splicing QTL.
