Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles.

Background: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney,caused by variants 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.

Methods: Evolutionary 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 in . We used both animal and cellular models to assess the deleteriousness of the identified variants.

Results: We identified an individual with a KINSSHIP-like phenotype carrying a partial duplication of further 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 in . 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 human mRNA, confirming their association with the ablation of . Conversely, some of the human mRNAs carrying missense variants identified in affected individuals did not complement. Overexpression of mutated mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring +/+, DN/DN, LoF/+, LoF/LoF or DN/LoF genotypes. 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 that LoF and DN variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation.

Conclusions: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in function are deleterious.