, 2012; O’Roak et al., 2012; Sanders et al., 2012). Among the 1,000 families assessed by the four studies, the rate of de novo loss-of-function (LoF) variation was consistently found to be significantly higher in cases compared to controls, allowing for the development of rigorous statistical approaches to identifying specific risk genes. Indeed, six ASD genes were identified, CHD8, DYRK1A, GRIN2B, KATNAL2, POGZ, and Y-27632 supplier SCN2A, because they carried recurrent,

highly damaging de novo events. While SCN2A has been previously implicated in epilepsy, none of these genes were known to carry ASD risk. Another key finding, one that will prove useful for gene discovery, was that roughly half of all de novo LoF mutations seen in ASD probands fall in ASD genes, with about 12% of ASD subjects

showing a de novo LoF mutation. These WES studies found a background rate of missense de novo variation that is more than 10-fold higher than that for LoF alleles. These missense changes undoubtedly include risk alleles; however, only a 5%–10% excess of such mutations was found in ASD cohorts, a difference that did not reach significance collectively across studies. Accordingly, it is not yet possible to confidently assign risk to this broad category of mutation, nor to establish an agreed upon threshold for the significance of observing “recurrent” de novo missense mutations in a given gene. Given the relevance of LoF alleles, this difficulty surely reflects the signal-to-noise problems engendered by neutral background variation and the difficulties that attend differentiating the subset of truly functional missense ISRIB datasheet variations. The interpretation of case-control exome sequencing has also not been as straightforward as family studies evaluating de novo LoF events. For example, WES of a sample of 1,000 cases and 1,000 controls and inspection of the six novel ASD genes just described showed, in hindsight, only a slight excess of LoF mutations in KATNAL2 and CHD8 in cases, a difference that did not approach statistical significance

( Neale et al., 2012). Indeed, across the entire genome, no genes were found to harbor a sufficiently large excess of rare alleles in cases versus controls to support a significant association after accounting mafosfamide for multiple comparisons (Liu et al., personal communication). These results are consistent with the multiple lines of evidence supporting a large number of ASD risk genes scattered throughout the genome. Methods to extract signal from case-control studies, alone and in combination with de novo data, are rapidly evolving. Still, it seems reasonable to conclude that large studies, involving tens of thousands of subjects, will be necessary to identify risk loci using standard analyses of mutation burden in case-control samples. The path forward is either WES or WGS in large cohorts.