Using Ingenuity Pathway Analysis to Study Gene Relationships Under Linkage Peaks of Interest in ASD

Background: Over the past 10 years there has been compelling evidence supporting a genetic basis for autism using a combination of behavioral family studies and genetic linkage and association studies. Of particular interest for several research groups has been the attempt to define and then replicate significant linkage signals using language-based phenotypes in ASD probands with the outcome objective of finding genes that are associated with a language phenotype.

Objectives: While several linkage and association studies have used onset of first words and phrases as potential phenotypes, we suggest that there is a subset of individuals with and without ASD who have an expressive language impairment that is beyond the scope of tracking the onset of language. We have already identified strong linkage peaks based on two such behavioral phenotypes and are now defining the peaks and investigating genes of interest under each peak.

Methods: All individuals from the Autism Genetics Resource Exchange (AGRE) who received an ADI-R and had 550K genetic data available were included in our analyses regardless of their final ASD status.  Variables used to develop two speech/language phenotypes were derived from specific items on the Autism Diagnostic Interview (1995, 2003).Linkage analyses were performed using the Posterior Probability of Linkage (PPL). A broad definition was used to define each linkage peak, including the entire contiguous region with PPL greater than the baseline of 2%. Genes under these regions were identified using the UCSC Genome Browser (NCBI Build 36.1).  Ingenuity Pathway Analysis software (v. 7.0) was used to identify known relationships among the genes identified in the linkage regions.  Relationships between these genes and other genes reported as Autism Susceptibility Genes (ASG) were also identified. 

Results: Regions of linkage were identified for both phenotypes.  NRXN3 was identified within the linkage region on Chromosome 14.  IPA identified a direct binding relationship of NXRN3 to NLGN3, which was identified by IPA as an ASG. NXPH3 was identified within the linkage region on Chromosome 17 and IPA identified a direct binding relationship to NRXN1, an IPA identified ASG. Relationships between PTEN, another identified ASG, and 5 genes located within the linkage regions on Chromosomes 4, 5, 14, and 17 were identified with IPA. 

Conclusions: Linkage analyses using the PPL algorithm has revealed several suggested regions of linkage for two unique language phenotypes associated with ASD and other disorders with a language component.  We have demonstrated that IPA software can be used as a useful method of prioritizing susceptibility loci by identifying relationships between specified molecules and thus allow us to narrow and refine further analyses.