International Registry for Werner Syndrome

项目摘要 Werner综合征国际登记招募Werner综合征(WS)和一系列病例 来自世界各地的其他节段性进展性综合征，目的是阐明 加速衰老的机制。详细的临床信息、基因分析结果和生物学 标本可广泛提供给各种合格的老年学家。 在这一应用中，我们建议将我们先前的研究扩展到包括全基因组范围的系统 寻找导致78例早孕症的基因变异，目前我们还无法找到 从基因上描述。我们研究议程的另一个重要扩展是启动翻译 可能导致潜在治疗药物的研究。 我们将采用下一代测序、阵列计算全息和桑格测序的组合， 然后进行验证性Western分析和定量聚合酶链式反应。这些方法已经成功地 发现了WRN(一种DNA解旋酶)、LMNA(核结构的一种成分)的新致病变种， POLD1(DNA聚合酶增量)、SPRTN(DNA聚合酶招募者)、ERCC4(核苷酸切除修复)、 CTC1(端粒复制)、MDM2(P53的抑制物)和SAMHD1(dNTP池的调节)。这些基因座 强调基因组不稳定的主要作用，基因组不稳定现在被广泛接受为老龄化的标志之一。我们也 在识别疾病突变方面取得了进展，这些突变表明了加速衰老的其他机制， 如BSCL2(脂滴形成)和Smad4(SASP的一种成分的细胞内信号转导，转化生长因子β)。 如上所述，我们将继续进行翻译研究，并有可能开发 为我们的早衰症患者提供的改善疗法。基于我们之前的研究，我们的合作者横德博士 日本Werner联盟的Koutaro正在评估一种NAD中间体和一种mTOR的效果 WS患者的抑制剂，二甲双胍。我们将开始对抑制者的影响进行独立研究 慢性炎症，即JAK抑制剂，来自WS患者和对照组的培养。这 这项工作的动机是我们的发现，Smad4突变的成纤维细胞表现出DNA积累的增加 WRN突变的成纤维细胞表现出Smad4的表达增加和水平显著升高 与对照细胞相比，表明持续的DNA损伤和炎症的协同作用 可能是导致衰老加速的常见关键机制之一。和谐的实验将会 通过高通量筛选细胞培养来探索这些新的治疗靶点的效果 有其他孕激素综合征的患者。一项涉及siRNA筛选的初步小规模实验已经完成 发现siRNAs和改变核内dNTP浓度的药物能够调节细胞 POLD1突变体的疾病表型。将采用更大规模的siRNA筛选来识别其他 孕激素基因座新的相关靶向途径以及未知的功能相互作用。

Project Summary The International Registry of Werner Syndrome recruits cases of Werner syndrome (WS) and a range of other segmental progeroid syndromes from around the world with the goal of elucidating underlying mechanisms of accelerated aging. Detailed clinical information, the results of genetic analyses, and biological specimens are made available to a wide range of qualified geroscientists. In this application, we propose to extend our previous studies to include systematic genome-wide searches for the genetic variants responsible for the 78 progeroid cases that we have so far been unable to genetically characterize. An additional important extension of our research agenda is to initiate translational research that can lead to potential therapeutic agents. We will employ a combination of next generation sequencings, array CGH, and Sanger sequencing, followed by confirmatory Western analysis and quantitative PCR. These approaches have successfully identified novel pathogenic variants of WRN (a DNA helicase), LMNA (a component of nuclear structure), POLD1 (DNA polymerase delta), SPRTN (recruiter of DNA polymerase), ERCC4 (nucleotide excision repair), CTC1 (telomere replication), MDM2 (an inhibitor of P53) and SAMHD1 (regulation of dNTP pools). These loci highlight major roles for genome instability, now widely accepted as one of the hallmarks of aging. We also made progress in the identification of disease mutations that suggest other mechanisms of accelerated aging, such as BSCL2 (lipid droplet formation) and SMAD4 (intracellular signaling of a component of SASP, TGFβ). As indicated above, we will pursue translational research with the potential for the development of ameliorative therapies for our progeroid patients. Based on our previous studies, our collaborator, Dr. Yokote Koutaro at the Japanese Werner Consortium, is evaluating the efficacy of an NAD intermediate and an mTOR inhibitor, metformin, in WS patients. We shall begin independent studies of the effects of suppressors of chronic inflammation, namely Janus kinase (JAK) inhibitors, in cultures from WS patients and controls. This effort was motivated by our findings that SMAD4 mutant fibroblasts exhibited increased accumulation of DNA damage and that WRN mutant fibroblasts showed elevated SMAD4 expressions and dramatically higher levels of SASP compared to control cells, suggesting that a synergy of persistent DNA damage and inflammation may be one of the common key mechanisms leading to the accelerated aging. Concordant experiments will explore the effects of these novel therapeutic targets with high throughput screening of cell cultures from patients with other progeroid syndromes. An initial small scale experiment involving siRNA screening has revealed that siRNAs and drugs that alter the intranuclear dNTP concentration are able to modulate the cellular disease phenotypes of POLD1 mutants. Larger scale siRNA screening will be employed to identify additional novel relevant target pathways as well as previously unknown functional interactions of progeroid genetic loci.