National Institutes of Health Undiagnosed Diseases Program, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
Center for Genomic Integrity, Institute for Basic Science, Ulsan, Republic of Korea; Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea.
Am J Hum Genet. 2024 Sep 5;111(9):1970-1993. doi: 10.1016/j.ajhg.2024.07.008. Epub 2024 Aug 5.
The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.
DNA 复制的精确调控对于细胞分裂和基因组完整性至关重要。复制因子 C(RFC)复合物是这一过程的核心,包含五个亚基,它将增殖细胞核抗原加载到 DNA 上,以促进复制和修复蛋白的招募,并增强 DNA 聚合酶的进程性。虽然 RFC1 在小脑共济失调、神经病和前庭反射消失综合征(CANVAS)中的作用是已知的,但 RFC2-5 亚基在人类孟德尔疾病中的贡献在很大程度上尚未得到探索。我们的研究将编码核心 RFC 复合物亚基的 RFC4 中的双等位基因变异与一种未诊断的疾病联系起来,这种疾病的特征是协调障碍和肌肉无力、听力障碍和体重减轻。我们在 9 名受影响的个体中发现了罕见的、保守的、预测致病性的 RFC4 变异,这些变异可能破坏 RFC 复合物形成所必需的 C 末端结构域。对先前确定的与增殖细胞核抗原结合的 RFC 的冷冻电镜结构的分析表明,这些变异破坏了 RFC4 内的相互作用,或使 RFC 复合物不稳定。使用 RFC4 缺陷的 HeLa 细胞和原代成纤维细胞进行的细胞研究表明,RFC4 蛋白减少,其他 RFC 复合物亚基的稳定性受损,并且 RFC 复合物形成受到干扰。此外,对 RFC4 变异体的功能研究证实了 RFC 复合物形成减少,细胞周期研究表明 DNA 复制和细胞周期进程受到干扰。我们综合运用计算机模拟、结构、细胞和功能分析的方法,提供了令人信服的证据,证明双等位基因失活 RFC4 变异体导致了这种多系统疾病的发病机制。这些见解拓宽了我们对 RFC 复合物及其在人类健康和疾病中的作用的理解。