RESUMEN
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.
Asunto(s)
Encéfalo/metabolismo , Deleción Cromosómica , Cromosomas Humanos Par 16/genética , Trastornos del Neurodesarrollo/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Encéfalo/patología , Canales de Calcio/genética , Moléculas de Adhesión Celular/genética , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epistasis Genética/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Metiltransferasas/genética , Trastornos del Neurodesarrollo/patología , Proteínas Nucleares/genética , Fosfohidrolasa PTEN/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development.
Asunto(s)
Encéfalo/embriología , Cromosomas Humanos Par 3/genética , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Discapacidad Intelectual/genética , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Proteínas de Xenopus/genética , Animales , Apoptosis/genética , Encéfalo/patología , Ciclo Celular/genética , Deleción Cromosómica , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Humanos , Discapacidad Intelectual/patología , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
As opposed to syndromic CNVs caused by single genes, extensive phenotypic heterogeneity in variably-expressive CNVs complicates disease gene discovery and functional evaluation. Here, we propose a complex interaction model for pathogenicity of the autism-associated 16p11.2 deletion, where CNV genes interact with each other in conserved pathways to modulate expression of the phenotype. Using multiple quantitative methods in Drosophila RNAi lines, we identify a range of neurodevelopmental phenotypes for knockdown of individual 16p11.2 homologs in different tissues. We test 565 pairwise knockdowns in the developing eye, and identify 24 interactions between pairs of 16p11.2 homologs and 46 interactions between 16p11.2 homologs and neurodevelopmental genes that suppress or enhance cell proliferation phenotypes compared to one-hit knockdowns. These interactions within cell proliferation pathways are also enriched in a human brain-specific network, providing translational relevance in humans. Our study indicates a role for pervasive genetic interactions within CNVs towards cellular and developmental phenotypes.
