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Abstract：
KBG syndrome is a rare autosomal dominant condition characterized by multisystem developmental disorder, primarily caused by loss-of-function variants in ankyrin repeat domain-containing protein 11 (ANKRD11). Approximately 80 % of ANKRD11 variants associated with KBG syndrome, are frameshift and nonsense variants. Current insight into the pathogenesis of KBG syndrome resulting from ANKRD11 truncating variants remains limited. Here, we presented two members from a non-consanguineous Chinese pedigree both exhibiting characteristics fitting the KBG syndrome-associated phenotypic spectrum. Whole-exome sequencing identified a novel heterozygous frameshift variant in ANKRD11 (NM_013275.6, c.2280_2281delGT, p.Y761Qfs*20) in the proband. Sanger sequencing confirmed that the variant was inherited from her mother and co-segregated with KBG syndrome phenotype. In vitro functional assays revealed that the frameshift variant escaped nonsense-mediated mRNA decay, and resulting in a truncated protein with significantly increased expression levels compared to full-length ANKRD11. Immunofluorescence results demonstrated that truncated protein was predominantly expressed in the nucleus of HEK293 cells, while wild-type ANKRD11 was equally distributed in both the nucleus and cytoplasm. Moreover, the truncated protein significantly reduced CDKN1A/P21-promoter luciferase activity in comparison to wild-type ANKRD11 protein, as well as a remarkably decrease in the endogenous CDKN1A/P21 mRNA level in HEK293 cells. These findings suggest a loss of transcriptional activation function and potentially a dominant-negative mechanism. Overall, our study expands the mutational spectrum of ANKRD11 gene and provides new insights into the pathogenic mechanism of KBG syndrome caused by ANKRD11 truncating variants.
摘要：
KBG综合征是一种罕见的常染色体显性遗传病，以多系统发育障碍为特征，主要由含有锚蛋白重复结构域的蛋白11（ANKRD11）的功能丧失变体引起。大约80%的ANKRD11变异与KBG综合征相关，是移码和无意义的变体。目前对由ANKRD11截断变体引起的KBG综合征的发病机理的认识仍然有限。这里,我们介绍了来自中国非血缘谱系的两名成员,他们均表现出符合KBG综合征相关表型谱的特征.全外显子组测序在ANKRD11中鉴定出一种新的杂合移码变体（NM_013275.6，c.2280_2281delGT，p.Y761Qfs*20）在先证中。Sanger测序证实该变体是从她的母亲遗传的并且与KBG综合征表型共分离。体外功能测定表明，移码变体逃脱了无义介导的mRNA衰减，并且产生与全长ANKRD11相比具有显著增加的表达水平的截短蛋白。免疫荧光结果显示,截短蛋白主要在HEK293细胞核中表达,而野生型ANKRD11在细胞核和细胞质中分布均匀。此外，与野生型ANKRD11蛋白相比，截短的蛋白显着降低了CDKN1A/P21启动子荧光素酶的活性，以及HEK293细胞中内源性CDKN1A/P21mRNA水平的显着降低。这些发现表明转录激活功能的丧失和潜在的显性负机制。总的来说,我们的研究扩展了ANKRD11基因的突变谱,并为ANKRD11截短变异导致KBG综合征的致病机制提供了新的见解.
