2.7. Ingenuity Pathway Analysis (IPA)

IPA (Qiagen; http://www.qiagen.com/ingenuity; analysis date 08/04/2019) was used to perform a core analysis to identify relationships, mechanisms, functions, networks, and pathways relevant to the genes affected by variants that passed the mean allele frequency cut-off, fulfilled family-based segregation criteria, had CADD scores >10 and were not intergenic or intronic variants. Data were analyzed for all five families together. Top canonical pathways were identified from the IPA pathway library and ranked according to their significance to our input data. This significance was determined by p-values calculated using the right tailed Fisher’s exact test. These values indicated the probability of association of genes from the input dataset with the canonical pathway by random chance alone. Ratios were also calculated for each pathway by dividing the number of genes from the input dataset that map to the pathway by the total number of genes in that pathway. The ratios did not influence the ranking of the canonical pathways.

IPA was also used to generate gene networks in which upstream regulators were connected to the input dataset genes while taking advantage of paths that involved more than one link (i.e., through intermediate regulators). These connections represent experimentally observed cause-effect relationships that relate to expression, transcription, activation, molecular modification and transport as well as binding events.