We also conducted gene-level association tests grouping nonsynonymous, stop gain, stop loss and splice variants within each gene, using rareMETALS version 6.0(44). Variant annotation was conducted using SEQMINER with RefSeq 1.9(45). Two complementary gene-level association tests were performed: the sequence kernel association test (SKAT; 46, 47) with a MAF cutoff of 1% and a simple burden test(48) that summed the number of rare alleles within a given gene, again with a maximum MAF=1%. We chose variants with MAF≤1% as we were interested in the contribution of variants with a frequency lower than that which has been reliably imputed and tested in past GWAS meta-analyses. We considered a gene association to be significant if the p-value surpassed a Bonferroni correction for the number of genes tested for a given phenotype and test, assuming approximately 20,000 genes in the genome (.05/20,000 = 2.5×10 −6).
