Functional assessment of the "two-hit" model for neurodevelopmental defects in Drosophila and X. laevis

We previously identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. We hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while "second hits" in the genetic background modulate the phenotypic trajectory. To test this model, we examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of "second hit" genes in Drosophila melanogaster and Xenopus laevis. We observed that knockdown of 16p12.1 homologs affect multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. In contrast to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs additively interacted and were less connected to each other in a human brain-specific interaction network, suggesting that interactions with second-hit genes confer higher impact towards neurodevelopmental phenotypes. Assessment of 358 pairwise interactions in Drosophila between 16p12.1 homologs and 76 homologs of patient-specific "second-hit" genes (such as ARID1B and CACNA1A), genes within neurodevelopmental pathways (such as PTEN and UBE3A), and transcriptomic targets (such as DSCAM and TRRAP) identified both additive (47%) and epistatic (53%) effects. In 11 out of 15 families, homologs of patient-specific "second-hits" showed distinct patterns of interactions, enhancing or suppressing the phenotypic effects of one or many 16p12.1 homologs. In fact, homologs of SETD5 synergistically interacted with homologs of MOSMO in both Drosophila and X. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. Our results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with "second-hit" genes determine the ultimate phenotypic manifestation. Author SummaryCopy-number variants, or deletions and duplications in the genome, are associated with multiple neurodevelopmental disorders. The developmental delay-associated 16p12.1 deletion is mostly inherited, and severely affected children carry an excess of "second-hits" variants compared to mildly affected carrier parents, suggesting that additional variants modulate the clinical manifestation. We studied this "two-hit" model using Drosophila and Xenopus laevis, and systematically tested how homologs of "second-hit" genes modulate neurodevelopmental defects observed for 16p12.1 homologs. We observed that 16p12.1 homologs independently led to multiple neurodevelopmental features and additively interacted with each other, suggesting that interactions with "second-hit" homologs could have a higher impact towards neurodevelopmental defects than interactions between 16p12.1 homologs. We tested 358 pairwise interactions of 16p12.1 homologs with "second-hit" homologs and genes within conserved neurodevelopmental pathways, and identified interacting homologs in 11 out of 15 families studied, which modified neurodevelopmental defects of individual or multiple 16p12.1 homologs through complex interactions. Interestingly, we observed that SETD5 homologs interacted with homologs of MOSMO, which modified cellular and brain defects, and conferred neuronal phenotypes not observed with knockdown of individual homologs. We propose that the 16p12.1 deletion sensitizes the genome to multiple neurodevelopmental defects, and complex interactions with "second-hit" genes determine the final manifestation.