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

Abstract

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. Compared to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs were less likely to interact with each other in Drosophila models or a human brain-specific interaction network, suggesting that interactions with “second-hit” genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 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 genetic interactions in 63% of the tested pairs. In 11 out of 15 families, patient-specific “second-hits” enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs in 32/96 pairwise combinations tested. 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.

Mosmo Is Required for Zebrafish Craniofacial Formation

“…Knock-down of mosmo in Xenopus instead shows a craniofacial phenotype in which both craniofacial and cartilage development appears affected, in association with alteration of neural crest cell proliferation and migration (Lasser et al, 2020). Interestingly, the coexistence of neurodevelopmental and craniofacial defects were observed in experiments performed in both Drosophila and Xenopus aimed at testing the importance of "a two-hit model" as trigger of neurodevelopmental disorders (Pizzo et al, 2021). Notably the study demonstrated a synergistic interaction between mutated mosmo and setd5 (Pizzo et al, 2021), a gene encoding a histone methyltransferase, which has been associated with intellectual disability (Grozeva et al, 2014).…”

“…In the same study, Mosmo was demonstrated to encode a membrane tetraspan protein, which promoted the endocytosis of the Hh transducer Smo, thereby lowering its levels at the cell plasma membrane (Pusapati et al, 2018). To what extent Mosmo participates in Hh signaling regulation in vivo, however, it is just beginning to be elucidated (Lasser et al, 2020;Kong et al, 2021;Pizzo et al, 2021).…”

Combinatorial patterns of gene expression changes contribute to variable expressivity of the developmental delay-associated 16p12.1 deletion

“…For example, we found that genes differentially expressed by the deletion were preferentially expressed in multiple brain tissues during development and were enriched for core signaling and developmental pathways. In fact, knockdown of individual homologs of 16p12.1 genes in Drosophila melanogaster models showed neuronal phenotypes and transcriptome disruptions, suggesting that the individual effects of multiple genes in the deletion sensitize the genome for neuropsychiatric phenotypes [ 109 ]. Interestingly, we found several examples of biological functions and mechanisms that were simultaneously dysregulated by both the deletion and “second-hit” variants in the carrier children.…”

Phenotypic Trade-Offs: Deciphering the Impact of Neurodiversity on Drug Development in Fragile X Syndrome

“…The genetic background makeup may contribute to the phenotypes in a manner not directly related to the target gene mutation. This has been labeled as the two-hit hypothesis ( 160 ). This implies that a better understanding of the genetic background and its targeting by the drug may shed light on complex trade-offs.…”

“…In FMR1 KO mice, systematic comparative analysis between strains revealed important differences between strains from the masking of some phenotypes to the exacerbation of others (156)(157)(158). However, this has been seen as well in other neurological disorders such as Huntington's disease (HD) (159). The genetic background makeup may contribute to the phenotypes in a manner not directly related to the target gene mutation.…”