【作者】 王改锋；

【导师】 王兆钺；

【作者基本信息】 苏州大学 ， 内科学， 2011， 硕士

【摘要】 巨大血小板病(giant platelet disorders, GPD)是一组异质性疾病,表现为血小板数量减少,体积增大,可伴有不同程度的出血。近年来,随着人类基因库的公布以及分子生物学技术的发展,巨大血小板病新的发病机制不断地被发现,除血小板本身的原因外,脂质代谢异常的患者也可表现为血小板数量减少和体积增大。最近我们遇到了一种罕见的病例,7例来自4个不同家系的患者表现为巨大血小板减少和口形红细胞增多,同时血浆中植物固醇含量明显升高、ABCG5或ABCG8存在纯合或双重杂合突变。对这种伴有典型血液学改变的植物固醇血症患者进行临床特性的研究,有助于血液科医生正确的认识、诊断和治疗此病。目的:研究4个伴有巨大血小板减少和溶血性贫血的植物固醇血症家系的临床表型和基因诊断。方法:光学显微镜观察红细胞及血小板的形态;流式细胞仪(FCM)检测血小板膜糖蛋白(GP)Ⅰb/Ⅸ和Ⅱb/Ⅲa;比浊法分析血小板聚集;高效液相色谱法(HPLC)检测血浆中豆固醇、二氢胆固醇和谷固醇;PCR结合测序分析先证者和家系成员ABCG5和ABCG8基因外显子和侧翼序列,寻找基因突变;同时用PCR-RFLP法分析基因多态性。结果:7例患者均有巨大血小板减少和溶血性贫血的表现,其中6例伴有皮肤黄褐瘤,1例仅有血液系统的改变。外周血涂片可见口形红细胞增多,血小板体积增大,甚至达红细胞大小;血浆中植物固醇尤其是谷固醇显著升高,是正常人30倍左右;基因分析发现5个突变,ABCG5 C20896T (R446X)、G19839A (R419H)和A20883G;ABCG8 del43683-43724、del43866C-43867G/ins43866T,其中后3个是新的突变位点。结论1.鉴定了7例植物固醇血症患者,患者均有巨大血小板减少和口形红细胞增多,基因分析发现ABCG5 A20883G;ABCG8 del43683-43724、del43866C-43867G/ ins43866T三个国际上尚未报道的突变。2.植物固醇血症患者可仅有血液系统的改变,植物固醇血症可作为巨大血小板减少伴或不伴溶血性贫血患者一个新的鉴别诊断。3.对血小板减少长期常规治疗效果不佳的患者,应注意血细胞的形态变化,对巨大血小板减少伴有口形红细胞增多的患者应考虑植物固醇血症的可能。更多还原

【Abstract】 Giant platelet disorders (GPD) are a heterogeneous group of disease with thrombocytopenia, large platelets and variable hemorrhagic phenomena. With the publication of gene bank and the development of protocols in molecular biology, the pathogenesis of GPD are constantly discovered;Apart from the intrinsic reasons of platelet, lipid dysbolism can be associated with thrombocytopenia. Recently, we found 7 patients from 4 unrelated families who presented with macrothrombocytopenia and stomatocytosis, increased plasma levels of phytosterols, and had homozygous or compound heterozygous mutations in ABCG5 or ABCG8 genes. Investigation of clinical characteristics identification of gene mutations would be essential for correct diagnosis and proper treatment of phytosterolemia with macrothrombocytopenia and stomatocytosis.Objective: To study the clinical features and ABCG5/ABCG8 gene mutations of four pedigrees of phytosterolemia involved in macrothrombocytopenia and hemolytic anemia.Methods: Erythrocyte and platelet morphology was examined under light microscope; Platelet glycoproteins (GP) (Ⅰb/ⅨandⅡb/Ⅲa ) were analyzed using flow cytometry; Platelet aggregation tests were performed with aggregometer; Plasma phytosterols were measured by high pressure/performance liquid chromatography (HPLC) method; All of ABCG5 and ABCG8 exons and intron-exon boundaries were amplified by PCR and directly sequenced to identify mutations. In order to exclude the probability of polymorphism, the corresponding gene mutation sites of four family members and healthy individuals were also analyzed by PCR-RFLP. Results :All the patients presented with macrothrombocytopenia and hemolytic anemia, six of them also had xanthomatosis, and one patient exclusively showed hematologic abnormalities. Large platelets (some as large as erythrocytes) and abnormal erythrocyte shapes such as stomatocytes were found on the blood smears; Plasma concentrations of phytosterols, especially sitosterol, were markedly elevated (30 fold) in the affected patients. Five mutations were identified in the four pedigrees, C20896T (R446X), G19839A ( R419H ) and A20883G in ABCG5, del43683-43724 and del1938C-1939G/ins1938T in ABCG8, the three latters are novel mutations.Conclusions1. 7 phytosterolemia patients were identified, and all of them associated with macrothrombocytopenia and stomatocytosis; Three novel mutations were found firstly: A20883G in ABCG5 and del43683-43724、del43866C-43867G/ins43866T in ABCG8.2. The patients with phytosterolemia might exclusively present with hematologic abnormalities, patients with macrothrombocytopenia and/or hemolytic anemia should be included in differentiation from phytosterolemia.3. When the patients with thrombocytopenia had no response to conventional managements, the shapes of red cells and platelets should be examined and phytosterolemia should be considered if prominent macrothrombocytopenia and stomatocytosis coexist.更多还原