背景:我们之前描述了KINSSHIP综合征,与智力障碍(ID)相关的常染色体显性疾病,间质发育不良和马蹄肾,由AFF3的degron中的从头变体引起的。斑马鱼中的小鼠敲入和过表达提供了显性阴性作用模式的证据,其中AFF3水平升高导致病理效应。
方法:进化约束表明,其他继承模式可能会发挥作用。我们通过筛选具有AFF3中预测的破坏性变体的个体的ID队列来挑战这一假设。我们使用动物和细胞模型来评估所鉴定的变体的有害性。
结果:我们确定了一个具有KINSSHIP样表型的个体,该个体携带AFF3从头部分复制,进一步加强了AFF3水平升高是病理性的假设。我们还检测到17名个体表现出轻度综合征,在AFF3中具有杂合功能丧失(LoF)或双等位基因错义变异。与半支配地位一致,我们发现了三名纯合LoF患者和一个复合杂合子的LoF和一个错义变体,他们比他们的杂合父母表现出更严重的表型。匹配斑马鱼击倒表现出神经系统缺陷,可以通过表达人AFF3mRNA来挽救,确认它们与aff3消融的关联。相反,在受影响个体中鉴定的一些携带错义变异的人AFF3mRNA不能挽救这些表型.与野生型过表达相比,斑马鱼胚胎中突变的AFF3mRNA的过表达导致异常幼虫的显着增加,进一步证明了有害性。为了进一步评估AFF3变异的影响,我们分析了来自受影响个体的成纤维细胞的转录组和带有+/+的工程等基因细胞,KINSSHIP/KINSSHIP,LoF/+,LoF/LoF或KINSSHIP/LoFAFF3基因型。超过三分之一的AFF3结合基因座的表达在KINSSHIP/KINSSHIP或LoF/LoF系中被修饰。虽然同样的途径受到影响,只有大约三分之一的差异表达基因是纯合子数据集共有的,表明AFF3LoF和KINSSHIP变体在很大程度上不同地调节转录组,例如,DNA修复途径显示出相反的调节。
结论:我们的结果和该位点变异显示的高多效性表明AFF3功能的微小变化是有害的。
BACKGROUND: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney, caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative mode of action, wherein an increased level of AFF3 resulted in pathological effects.
METHODS: Evolutionary constraints suggest that other modes-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be damaging variants in AFF3. 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 de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous Loss-of-Function (LoF) or biallelic missense variants in AFF3. 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 AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not rescue these phenotypes. Overexpression of mutated AFF3 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 AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring + / + , KINSSHIP/KINSSHIP, LoF/ + , LoF/LoF or KINSSHIP/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the KINSSHIP/KINSSHIP or the LoF/LoF lines. While the same pathways are affected, only about one third of the differentially expressed genes are common to the homozygote datasets, indicating that AFF3 LoF and KINSSHIP 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 AFF3 function are deleterious.