ANKYRIN MUTATIONS IN HEREDITARY SPHEROCYTOSIS

在完成了血液学/肿瘤学的临床奖学金后，我 在过去的两年里，Jiri Palek博士的团队在 由Jiri Palek博士、Ken Sahr博士和Shih-Chun David Liu博士指导。我有过 花同样多的时间学习分子生物学和蛋白质基础知识 红细胞膜的生物化学。这次培训给了我很大的帮助 Jiri Palek博士的指导，以提出本文件中描述的项目 与初级分子缺陷有关的授予申请 遗传性球形红细胞增多症。 遗传性球形细胞增多症(HS)是由 红细胞膜骨架。在大多数HS患者中， 红细胞膜缺乏血影蛋白，这是红细胞膜的主要蛋白质。 膜骨架。造成这一缺陷的主要缺陷 看起来是异质性的，在少数患者中被定义为 只有这样。我们在实验室中已经确定了几个不同的子集 HS患者，包括一个血影蛋白缺乏但有 锚定蛋白含量正常。因为不断增长的数据将HS与 锚蛋白，以及锚蛋白作为主体的重要作用 幽灵蛋白骨架的附着部位我们曾询问过是否有一个子集 的HS患者可能存在强直蛋白缺陷，导致结合不良 与幽灵蛋白和随后的幽灵蛋白缺陷有关。在预赛中 实验中，我们考察了血影蛋白/锚定蛋白的结合作用 在这个HS患者亚群中的三个无关患者中。我们有 在两个无关的先证者中，发现正常血影蛋白的结合减少 与正常的IOV相比，患者的由内向外的囊泡(IOV)。这个 这项工作的具体目的是调查在这种情况下， HS患者中的一个亚组，其血影蛋白结合域存在缺陷 锚蛋白可能导致异常结合和随后的 血影蛋白缺乏。 首先，我们将进一步刻画这两个中的绑定异常 患者，重点研究Ankyrin的幽灵蛋白结合域：(I)在 蛋白质水平，通过检测正常的血影蛋白与纯化的结合 Ankyrin或其蛋白水解性72kD片段(包含血影蛋白 结合结构域)或其亚片段。(Ii)在基因层面，我们会尝试 以发现编码幽灵蛋白的基因部分的突变 变性梯度凝胶电泳法结合结构域的研究 技术。如果发现突变，我们将检查联合- 用DNA分析Hs表型基因突变的遗传 使用DGGE的家族成员，等位基因特异的寡核苷酸 杂交或限制性内切酶分析。 第二，我们将研究锚蛋白缺陷的频率表征为 与同一亚群中其他HS患者的血影蛋白结合较弱。在… 在蛋白质水平上，我们将使用 结合分析如上所述。在基因层面上，我们将努力发现新的 应用DGGE技术检测锚蛋白的血影蛋白结合区突变。 我坚信Jiri Palek博士和他的同事们 将为我未来的职业发展提供一个最佳的环境 为了这个项目的成功执行，因为它结合了强大的 在红细胞膜蛋白质生物化学方面的专业知识，一个强大的 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。

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.