We next utilized a maximum likelihood estimation (MLE) procedure to estimate the number of genes contributing risk to TD, based on vulnerability to de novo damaging variants, as has been done recently in congenital heart disease (Homsy et al., 2015). We observed 192 confirmed damaging de novo variants in 484 TD probands. Therefore, for every possible number of risk genes, from 1 to 2,500, we simulated 192 variants. 50,000 permutations were conducted: in each permutation, we randomly selected risk genes and then, based on the fraction of damaging variants estimated to carry risk, randomly assigned a percentage of variants to the risk genes and the rest of the variants to the non-risk genes. We weighted the probability of variation by gene size and GC content (He et al., 2013). We then determined the combined number of risk and non-risk genes harboring multiple de novo variants and recorded when the number of genes with two variants and the number of genes with three or more variants in the simulated data matched the number observed in our study (4 and 1, respectively).
