a transcriptional target of MEF2. Because the KCl depolarization assay identified fewer than 5% of the transcriptome as altered in expression, the identification in this assay of two of three genes associated with the two largest deletions is quite unlikely by chance alone (binomial test, P < 0.006 for two or more genes in the “altered expression” category). Enrichment of genes induced by membrane depolarization remains significant even when genes closest to all of the five homozygous deletions are considered (two of six transcripts found on the array, binomial test, P < 0.027). Moreover, a separate screen using RNAi knock-down of NPAS4, a transcription factor activated in response to depolarization, showed that NHE9 was one of 292 out of 22,407 cDNAs (1.3%) whose transcription was significantly altered (in this case, increased) (Fig. 3D) (22), although NHE9 expression was not detectably altered by membrane depolarization alone. These transcriptional effects suggest that the homozygous deletions in autism patients may preferentially involve activity-regulated genes, either mutating their coding sequence (e.g., DIA1) or potentially affecting conserved DNA sequences (NHE9, PCDH10) that may be critical for proper transcriptional regulation.
