A20 Gene Polymorphisms in LDLT

描述（申请人提供）：我们的长期工作表明，A20 (tnfaip3) 增强肝脏对损伤的综合抗炎、抗凋亡和抗氧化反应，并促进肝细胞增殖和肝再生。在实验动物模型中，我们发现肝脏中 A20 的过度表达在根治性致死性肝切除术和严重肝脏缺血再灌注损伤后具有保护作用，并允许边缘肝移植的成功植入。功能丧失实验进一步证实了 A20 在支持肝再生方面的重要生理作用。事实上，我们发现部分 A20 敲低，如杂合子小鼠 (A20) 中所见，会显着损害肝脏再生，并增加 2/3 部分肝切除术后的致死率。最近，在人类全基因组关联研究中，tnfaip3基因多态性已被确定为多种炎症和自身免疫性疾病的易感位点。在本提案中，我们希望确定 A20 基因多态性以及 A20 及其靶基因在移植物中的表达水平是否可以作为活体肝移植 (LDLT) 中肝再生和功能的可靠预后标志物。 LDLT 已成为缓解原位肝移植 (OLT) 器官短缺问题的解决方案。然而，其在成人中的广泛使用受到可安全收获的移植物大小的限制。迫切需要能够限制捐献者风险同时提高移植成功率的生物标志物，以安全扩展该领域，这将有助于缓解可用于 OLT 的器官的严重短缺。我们计划： 1. 探测所有活体肝脏捐献者的 tnfaip3 基因多态性。 2. 评估肝移植前、再灌注后（受者）或手术结束前（供者）的前瞻性患者队列中肝移植活检中 A20 及其靶基因的表达水平。作为该目标的一部分，我们还将使用捐赠者的外周血单核细胞测定 A20 mRNA 和蛋白质水平。 3. 测量移植物再灌注（受者）或肝切除（供者）前后受者和供者血清中引发细胞因子（TNF 和 IL-6）的循环水平。 4. 将 tnfaip3 基因多态性、A20 表达水平及其靶基因的表达水平与启动细胞因子的循环水平和再生状态相关联，通过动态对比增强多探测器 CT (DCE-MDCT) 确定，随后进行定性和定量 3D 分析和 MeVis 重建、肝功能（肝酶和凝血测试）以及早期临床结果（出院时间、原发性功能障碍、重新移植的需要）。这项工作中收集的数据将促进使用 A20 基因多态性和表达水平作为 LDLT 中供体和受体配对的可靠选择标记，以降低供体发病率并改善受体结果。 公共卫生相关性：原位肝移植（OLT）是急性或慢性肝衰竭患者的一种挽救生命的措施。然而，需求远远超过了器官的供应量，导致许多患者在等待名单上死亡。活体肝移植 (LDLT) 已成为缓解 OLT 器官短缺问题的解决方案。然而，在成人中，LDLT 受到可安全采集的移植物大小的限制。由 tnfaip3/A20 基因多态性确定的保肝蛋白 A20 的表达水平和功能是有前途的工具，可用作 LDLT 中供体和受体配对的选择生物标志物，这将降低供体发病率并改善受体结果。验证 A20 作为 LDLT 可靠的预后生物标志物可以作为供体充足性的客观衡量标准，并有助于扩大 LDLT、​​劈裂肝和 OLT 的总体领域。

DESCRIPTION (provided by applicant): Our longstanding work demonstrates that A20 (tnfaip3) enhances the liver's combined anti-inflammatory, anti- apoptotic, and anti-oxidant response to injury, and promotes hepatocyte proliferation and liver regeneration. In experimental animal models, we have shown that overexpression of A20 in the liver is protective following radical lethal hepatectomy and severe liver ischemia reperfusion injury, and allows for successful engraftment of marginal liver transplants. Loss-of-function experiments further confirmed the essential physiologic role of A20 in supporting liver regeneration. Indeed, we showed that partial A20 knockdown, as seen in heterozygote mice (A20 ) significantly impairs liver regeneration and increases lethality following 2/3 partial hepatectomy. Recently, in human genome-wide association studies, tnfaip3 gene polymorphisms have been identified as susceptibility loci for several inflammatory and autoimmune diseases. In this proposal, we wish to determine whether A20 gene polymorphisms and the expression level of A20 and of its target genes in the graft could serve as reliable prognostic markers for liver regeneration and function in living donor liver transplantation (LDLT). LDLT has emerged as a solution to ease the shortage of organs available for orthotopic liver transplantation (OLT). However its widespread use in adults is limited by the size of the graft that can be safely harvested. Biomarkers that would limit the risk to the donor while improving graft success are direly needed to allow for safe expansion of the field, which would help in easing the severe shortage of organs that are available for OLT. We plan to: 1. Probe for tnfaip3 gene polymorphisms in all living liver donors. 2. Evaluate the expression levels of A20 and of its target genes in liver transplant biopsies before liver transplantation, and in the prospective cohort of patients, after reperfusion (recipient), or before the end of surgery (donor). As part of this aim, we will also determine A20 mRNA and protein levels using peripheral blood mononuclear cells from the donor. 3. Measure circulating levels of priming cytokines (TNF and IL-6) in recipient and donor sera before and after graft reperfusion (recipient) or liver resection (donor). 4. Correlate tnfaip3 gene polymorphisms, A20 expression levels, and expression levels of its target genes with circulating levels of priming cytokines, and regeneration status, as determined by Dynamic Contrast Enhanced Multi-Detector CT (DCE-MDCT) with subsequent qualitative and quantitative 3D analysis and MeVis reconstruction, liver function (liver enzymes, and coagulation tests), and early clinical outcomes (time to discharge, primary dysfunction, need for re-transplantation). Data gathered in this work will promote the use of A20 gene polymorphisms and expression levels as reliable selection markers for pairing donors and recipients in LDLT to reduce donor morbidity and improve recipient outcomes. PUBLIC HEALTH RELEVANCE: Orthotopic liver transplantation (OLT) is a life-saving measure for patients suffering from acute or chronic liver failure. However, demand greatly exceeds organ availability, causing a number of patients to die while on the waiting list. Living donor liver transplantation (LDLT) has emerged as a solution to ease the shortage in organs that are available for OLT. However, in adults, LDLT is limited by the size of the graft that can be safely harvested. Expression levels and function of the hepatoprotective protein A20, as determined by tnfaip3/A20 gene polymorphisms, are promising tools that can be used as selection biomarkers for pairing donors and recipients in LDLT, which would reduce donor morbidity and improve recipient outcomes. Validation of A20 as a reliable prognostic biomarker in LDLT could serve as an objective measure of donor adequacy and could help to expand the field of LDLT, split livers and OLT in general.