Mutation-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles

We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with de novo variants in the AFF3 degron , a sequence involved in its binding to ubiquitin ligase. 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. In line with this hypothesis, we describe an individual presenting a KINSSHIP-like phenotype carrying a partial duplication of AFF3. Further screening of intellectual disability cohorts revealed nine individuals with heterozygous and three with homozygous loss-of-function (LoF) variants, as well as two probands with compound LoF/missense and six with biallelic missense mutations in AFF3, who displayed a milder syndrome. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA confirming their association with the ablation of aff3. Conversely, the missense isoforms did not complement demonstrating the deleteriousness of the variants identified in affected individuals. To assess the different effect of these variants, we profiled the transcriptome of fibroblasts of affected individuals and isogenic cells harboring DN/DN, LoF/+, LoF/LoF or DN/LoF AFF3 genotypes. While the same pathways are affected, only one-third of the differentially expressed genes are common to both homozygote datasets, indicating that AFF3 LoF and DN mutations largely modulate transcriptomes differently. In particular, the apical junction and DNA repair pathways displayed opposite modulation. Our results and the high pleiotropy shown by this locus in GWASes suggest that even slight changes in the AFF3 function might be deleterious. To assess the effect of AFF3 biallelic missense variants we established primary fibroblasts lines of four healthy controls and two affected individuals carrying biallelic missense variants in AFF3, i.e. patient B1 who is homozygous for the p.(Lys528Arg) variants and patient B7 who is compound heterozygote for the p.(Val1036Ile) and p.(Arg1186Gln) variants. We profiled the transcriptome of primary fibroblasts of the two affected individuals carrying biallelic missense variants in AFF3 and compared them to those of fibroblasts of three of the healthy controls (the fourth one was removed due to QC issues). The transcriptome profile of each line was analyzed in biological triplicate.