ANKYRIN MUTATIONS IN HEREDITARY SPHEROCYTOSIS

遗传性球形红细胞增多症中的锚蛋白突变

• 批准号: 3083138
• 负责人: HANI HASSOUN
• 金额: $ 7.07万
• 依托单位: STEWARD ST. ELIZABETH'S MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3083138
• 关键词: affinity chromatography; ankyrins; binding proteins; computer assisted sequence analysis; denaturing gradient gel electrophoresis; family genetics; gene mutation; hereditary spherocytosis; human subject; molecular cloning; nucleic acid hybridization; polymerase chain reaction; protein structure; restriction mapping; spectrin; transcription factor

Having completed a clinical fellowship in Hematology/Oncology, I have spent the past two years working with Dr. Jiri Palek's group under the guidance of Drs. Jiri Palek, Ken Sahr and Shih-Chun David Liu. I have spent equal time learning the basics of molecular biology and protein biochemistry of the red cell membrane. This training has lead me, with the guidance of Dr. Jiri Palek to propose the project described in this grant application which relates to the primary molecular defect in hereditary spherocytosis. Hereditary spherocytosis (HS) is caused by abnormalities of the erythrocyte membrane skeleton. In the majority of patients with HS the erythrocyte membrane has a deficiency in spectrin, the major protein of the membrane skeleton. The primary defect responsible for this deficiency appears heterogeneous and has been defined in a small number of patients only. We have identified in our laboratory several distinct subsets of patients with HS, including a subset with deficiency in spectrin but a normal ankyrin content. Because of the growing data linking HS to ankyrin, as well as the important role of ankyrin as the principal attachment site of the spectrin skeleton we have asked whether a subset of HS patients could have a defect of ankyrin leading to a poor binding to spectrin and a subsequent deficiency in spectrin. In preliminary experiments, we have examined the spectrin/ankyrin binding interaction in three unrelated patients in this subset of HS patients. We have found, in two unrelated probands, a decreased binding of normal spectrin to patients' inside-out-vesicles (IOVs) as compared to normal IOVs. The specific aim of this work is to investigate the possibility that, in this subset of patients with HS, a defect in the spectrin binding domain of ankyrin could be responsible for an abnormal binding and a subsequent deficiency in spectrin. First, we will further characterize this binding abnormality in these 2 patients, focusing on the spectrin binding domain of ankyrin: (i) At the protein level, by examining the binding of normal spectrin to purified ankyrin or its proteolytic 72 kD fragment (which contains the spectrin binding domain) or its subfragments. (ii) At the gene level, we will try to uncover mutations of the portion of the gene coding for the spectrin binding domain using the denaturing gradient gel electrophoresis technique. If a mutation is uncovered, we will examine the co- inheritance of this mutation with the HS phenotype by analyzing DNA from family members, using the DGGE, allele specific oligonucleotide hybridization or restriction enzyme analysis. Second, we will study the frequency of ankyrin defects characterized by a weak binding to spectrin in other HS patients in the same subset. At the protein level, we will examine the spectrin/ankyrin interaction using binding assays as above. At the gene level, we will try to uncover new mutations of the spectrin binding domain of ankyrin using the DGGE. I strongly believe that the group of Dr. Jiri Palek and his colleagues will provide an optimal environment both for my future career development and for a successful execution of this project as it combines strong expertise in protein biochemistry of the red cell membrane, a strong molecular biology training as well as a broad clinical expertise in hemolytic anemias and a wide patient referral population.

在完成了血液学/肿瘤学的临床研究后，我 在过去的两年里， Jiri Palek博士、Ken Sahr博士和Shih-Chun大卫刘博士的指导。 我有 花同样的时间学习分子生物学和蛋白质的基础知识 红细胞膜的生物化学。 这次培训使我， 在Jiri Palek博士的指导下，提出了本报告所述的项目。 与主要分子缺陷有关的补助申请 遗传性球形红细胞增多症 遗传性球形红细胞增多症（HS）是由异常的 红细胞膜骨架 大多数HS患者， 红细胞膜缺乏血影蛋白，血影蛋白是红细胞的主要蛋白质。 膜骨架造成这种缺陷的主要缺陷是 似乎是异质性的，并已在少数患者中定义 只. 我们已经在实验室中确定了几个不同的子集， HS患者，包括血影蛋白缺乏但 正常锚蛋白含量。 由于越来越多的数据将HS与 锚蛋白，以及锚蛋白的重要作用，作为主要的 我们已经问过是否一个子集 的HS患者可能存在锚蛋白缺陷，导致结合不良 血影蛋白和血影蛋白的后续缺陷。 初步 实验中，我们研究了血影蛋白/锚蛋白结合相互作用 在该HS患者亚组中的三名无关患者中。 我们有 在两个不相关的先证者中发现，正常血影蛋白的结合减少， 与正常的IOV相比，患者的由内而外的囊泡（IOV）。 的 这项工作的具体目标是调查的可能性，在这一点上， HS患者的一个子集，在血影蛋白结合结构域的缺陷， 锚蛋白可能负责异常结合和随后的 血影蛋白缺乏 首先，我们将进一步描述这2个细胞中的这种结合异常。 患者，集中于锚蛋白的血影蛋白结合结构域：（i）在 蛋白水平，通过检查正常血影蛋白与纯化的 锚蛋白或其蛋白水解的72 kD片段（其含有血影蛋白 结合结构域）或其亚片段。 (ii)在基因层面，我们将尝试 为了揭示编码血影蛋白的基因部分的突变 变性梯度凝胶电泳 法 如果发现了突变，我们会检查共- 通过分析DNA，可以确定这种突变与HS表型的遗传关系。 家族成员，使用DGGE，等位基因特异性寡核苷酸 杂交或限制酶分析。 其次，我们将研究锚蛋白缺陷的频率，其特征在于： 在同一亚组的其他HS患者中，与血影蛋白的结合较弱。 在 蛋白质水平，我们将研究血影蛋白/锚蛋白的相互作用， 结合试验如上所述。 在基因水平上，我们将尝试发现新的 使用DGGE分析锚蛋白血影蛋白结合结构域的突变。 我坚信Jiri Palek博士和他的同事们 我将为我未来的职业发展提供一个最佳的环境 为了成功执行这个项目，因为它结合了强大的 专业研究红细胞膜蛋白生物化学， 分子生物学培训以及广泛的临床专业知识， 溶血性贫血和广泛的患者转诊人群。