Aberrations in the excitatory/inhibitory balance within the brain have been associated with both intellectual disability (ID) and schizophrenia (SZ). The bHLH-PAS transcription factors NPAS3 and NPAS4 have been implicated in controlling the excitatory/inhibitory balance, and targeted disruption of either gene in mice results in a phenotype resembling ID and SZ. However, there are few human variants in NPAS3 and none in NPAS4 that have been associated with schizophrenia or neurodevelopmental disorders. From a clinical exome sequencing database we identified three NPAS3 variants and four NPAS4 variants that could potentially disrupt protein function in individuals with either developmental delay or ID. The transcriptional activity of the variants when partnered with either ARNT or ARNT2 was assessed by reporter gene activity and it was found that variants which truncated the NPAS3/4 protein resulted in a complete loss of transcriptional activity. The ability of loss-of-function variants to heterodimerise with neuronally enriched partner protein ARNT2 was then determined by co-immunoprecipitation experiments. It was determined that the mechanism for the observed loss of function was the inability of the truncated NPAS3/4 protein to heterodimerise with ARNT2. This further establishes NPAS3 and NPAS4 as candidate neurodevelopmental disorder genes.

Single-Minded 2 (SIM2) is a neuron enriched basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) transcription factor essential for mammalian survival. SIM2 is located within the Down Syndrome Critical Region (DSCR) of chromosome 21, and manipulation in mouse models suggests Sim2 may play a role in brain development and function. During screening of a clinical exome sequencing database, nine SIM2 non-synonymous mutations were found which were subsequently investigated for impaired function using cell-based reporter gene assays. A number of these human variants attenuated abilities to activate transcription and were further characterized to determine the mechanisms underpinning their deficiencies. These included impaired partner protein dimerization, reduced DNA binding and reduced expression and nuclear localization. This study highlighted several SIM2 variants found in patients with disabilities and validated a candidate set as potentially contributing to pathology.