Variant-specific pathophysiological mechanisms ofAFF3differently influence transcriptome profiles
Authors: Bassani Sissy , Chrast Jacqueline , Ambrosini Giovanna , Voisin Norine , Schütz Frédéric , Brusco Alfredo , Sirchia Fabio , Turban Lydia , Schubert Susanna , Jamra Rami Abou , Schlump Jan-Ulrich , DeMille Desiree , Bayrak-Toydemir Pinar , Nelson Gary Rex , Wong Kristen Nicole , Duncan Laura , Mosera Mackenzie , Gilissen Christian , Vissers Lisenka E.L.M. , Pfundt Rolph , Kersseboom Rogier , Yttervik Hilde , Hansen Geir Åsmund Myge , Smeland Marie Falkenberg , Butler Kameryn M. , Lyons Michael J. , Carvalho Claudia M.B. , Zhang Chaofan , Lupski James R. , Potocki Lorraine , Flores-Gallegos Leticia , Morales-Toquero Rodrigo , Petit Florence , Yalcin Binnaz , Tuttle Annabelle , Elloumi Houda Zghal , Mccormick Lane , Kukolich Mary , Klaas Oliver , Horvath Judit , Scala Marcello , Iacomino Michele , Operto Francesca , Zara Federico , Writzl Karin , Maver Ales , Haanpää Maria K. , Pohjola Pia , Arikka Harri , Iseli Christian , Guex Nicolas , Reymond Alexandre 
Published: January 2024

AbstractBackgroundWe previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney,caused byde novovariants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative (DN) mode-of-action, wherein an increased level of AFF3 resulted in pathological effects.MethodsEvolutionary constraints suggest that other mode-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be deleterious variants inAFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants.ResultsWe identified an individual with a KINSSHIP-like phenotype carrying ade novopartial duplication ofAFF3further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous LoF or biallelic missense variants inAFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing humanAFF3mRNA, confirming their association with the ablation ofaff3. Conversely, some of the humanAFF3mRNAs carrying missense variants identified in affected individuals did not complement. Overexpression of mutatedAFF3mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect ofAFF3variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring +/+, DN/DN, LoF/+, LoF/LoF or DN/LoFAFF3genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the DN/DN or the LoF/LoF lines. While the same pathways are affected, only about one-third of the differentially expressed genes are common to these homozygote datasets, indicating thatAFF3LoF and DN variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation.ConclusionsOur results and the high pleiotropy shown by variation at this locus suggest that minute changes inAFF3function are deleterious.