A novel framework for functional annotation of variants of uncertain significance in ID/ASD risk gene CC2D1A

Document Type

Journal Article

Publication Date



Human molecular genetics




CC2D1A; CREB; VUS; autism; intellectual disability


Intellectual disability (ID) and autism spectrum disorder (ASD) are genetically heterogeneous with hundreds of identified risk genes, most affecting only a few patients. Novel missense variants in these genes are being discovered as clinical exome sequencing is now routinely integrated into diagnosis, yet most of them are annotated as variants of uncertain significance (VUS). VUSs are a major roadblock in using patient genetics to inform clinical action. We developed a framework to characterize VUSs in Coiled-coil and C2 domain containing 1A (CC2D1A), a gene causing autosomal recessive ID with comorbid ASD in 40% of cases. We analyzed seven VUSs (p.Pro319Leu, p.Ser327Leu, p.Gly441Val, p.Val449Met, p.Thr580Ile, p.Arg886His and p.Glu910Lys) from four cases of individuals with ID and ASD. Variants were cloned and overexpressed in HEK293 individually and in their respective heterozygous combination. CC2D1A is a signaling scaffold that positively regulates PKA-CREB signaling by repressing phosphodiesterase 4D (PDE4D) to prevent cAMP degradation. After testing multiple parameters including direct interaction between PDE4D and CC2D1A, cAMP levels and CREB activation, we found that the most sensitive readout was CREB transcriptional activity using a luciferase assay. Compared to WT CC2D1A, five VUSs (p.Pro319Leu, p.Gly441Val, p.Val449Met, p.Thr580Ile, and p.Arg886His) led to significantly blunted response to forskolin induced CREB activation. This luciferase assay approach can be scaled up to annotate ~150 CC2D1A VUSs that are currently listed in ClinVar. Since CREB activation is a common denominator for multiple ASD/ID genes, our paradigm can also be adapted for their VUSs.


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