This is evident both in overall differential in de novo mutations, but also from the effects of purifying selection on sets of genes (Table 7). Missense mutation should contribute to autism to some degree, as gene function can be severely altered by single-amino-acid substitutions. However, we see no statistical evidence in our work

for the hypothesis that de novo missense mutations contribute to autism. The number of de novo Everolimus missense events we observe is not greater in probands than in siblings. Moreover, the ratio of numbers of missense mutations in probands to siblings is not significantly different than the observed ratio of numbers of synonymous mutations. Even when we filter for genes expressed in brain, count missense mutations that cause nonconservative amino acid changes, or count missense mutations at positions conserved among vertebrates (Table S1, columns BA–BJ), we see no statistical evidence for contribution from this type of mutation. This is also true when we look for overlap of de novo missense mutations with FMRP-associated genes (Table 5). The

lack of signal is not attributable to the type of population we study, as we observe de novo copy number imbalance of the expected magnitude in this very same population Gemcitabine ic50 (Levy et al., 2011 and Sanders et al., 2011). But given the size of the population and background mutation rate, we are unable to find signal in the present study. A simple power calculation indicates that we cannot rule out confidently even a 20% contribution to autism from de novo missense mutation. Despite these caveats, it is worth considering that de novo

mutation causing merely amino acid substitution may only rarely create a dominant allele of strong effect. We make a strikingly different observation for mutations that are likely to disrupt gene function. In contrast to de novo missense mutation, we do get signal from de novo mutations likely to severely disrupt coding: mutations at splice sites, nonsense mutations, and small indels, particularly indels that cause frame shifts. We observe 59 likely gene disruptions MycoClean Mycoplasma Removal Kit (LGD) in affected and 28 in siblings, a ratio of two to one. We note that girls on the autistic spectrum have a higher rate (9/29) than boys (50/314), a bias we have previously noted for de novo CNV events. The total contribution from LGD mutations can be estimated as 31 events in 343 families (59 events in probands minus 28 events in siblings), or roughly 10% of affected children. We observed de novo point mutations in children at the rate expected from other studies (Awadalla et al., 2010 and Conrad et al., 2011), about 120 point mutations per genome per generation. We observe that the frequency of de novo mutation is dependent on parental age, and know this with a high degree of statistical certainty.