【作者】 张淑芳；

【导师】 余平；

【作者基本信息】 中南大学 ， 医学免疫学， 2011， 博士

【摘要】 第一章非肌球蛋白重链9基因相关疾病家系收集、家系诊断和临床特点分析目的：收集一非肌球蛋白重链9基因相关疾病(MYH9-RD)家系患者的详细遗传资料。通过分析该MYH9-RD家系的临床特点、遗传方式和实验室特点来对该家系进行诊断,以探讨该家系MYH9-RD的致病机理。方法：登记家系所有成员并进行详细的临床体征检查和实验室检测。每一个家系成员进行临床体征包括白内障、神经性耳聋、异常出血、肾损害等检查及其它并发症调查和实验室评估等；分别采用自动血细胞计数仪法和手工法计数血小板数量,同时分析巨大血小板和包涵体的特点和百分率；应用染色体核型分析技术进行细胞学诊断；应用光学显微镜和电子显微镜观察中性粒细胞包涵体和血小板的形态特点和超微结构特点。结果：通过实验室评估和临床表现显示：该家系为常染色体显性遗传性MYH9-RD,外周血染色体核型分析正常。调查的该MYH9-RD家系4代32人中有15人患病。所有患者均具有典型的“血小板减少、巨大血小板和粒细胞包涵体”三联症,都有轻至中度的出血倾向,并且与血小板的数量或大小无关。而且大多数患者的临床表型高度复杂,其中一些患者还伴有严重的白血病、青光眼、心功能不全、转氨酶升高、血脂升高、哮喘、鼻炎及白内障等多种疾病,不同于目前国内外报道的家系。MYH9-RD家系患者机数血小板数明显低于镜数血小板数(P<0.01)。电镜下可见清晰的巨大血小板和包涵体的超微结构,包涵体电镜下的结构为椭圆形或梭形,位于中性粒细胞的边缘。此外,正常对照者血小板数量、形态和结构均正常,而且中性粒细胞中未见包涵体。结论：通过临床表现和实验室特点分析,该家系MYH9-RD诊断成立。该家系具有规模大、患者人数多、临床表型复杂多样的特点,是一个不同于目前国内外报道的大家系。该家系将为MYH9相关疾病的深入研究奠定基础。此外,为了正确反映巨大血小板患者的血小板数量,我们应使用人工计数法而不是细胞自动计数仪法来计数血小板的数量。笔二章MYH9-RD家系MYH9基因突变检测与分析目的：探讨和分析—MYH9-RD家系非肌球蛋白重链9(MYH9)基因的突变情况。方法：签定知情同意书后,采集患者和家系成员的外周血。应用聚合酶链反应和直接测序的方法对15例家系患者MYH9基因的全部40个外显子和侧翼区进行突变检测和分析,选择家系正常个体为对照。测序结果通过DNAMAN软件与Genebank中的正常MYH9基因序列进行比对。结果：家系患者MYH9基因的所有外显子与侧翼区均未检测到致病性突变,只有25号和18号外显子各有一同义突变。结论：本家系可能存在新的基因突变类型或此类疾病的另一个新的致病基因或其它原因,其临床表型复杂多样可能与此有关。对该家系的深入研究将对进一步阐明该遗传性疾病形成的分子机制具有重要的意义。第三章MYH9-RD家系非肌性肌球蛋白重链ⅡA在中性粒细胞中的定位与表达目的：观察该MYH9-RD家系患者非肌性肌球蛋白重链ⅡA (nonmuscle myosin heavy chain-ⅡA, NMMHC-ⅡA)在中性粒细胞中的定位和表达,阐明MYH9-RD中性粒细胞包涵体的性质和形成特点,以探讨MYH9-RD的致病机制。方法：分离MYH9-RD患者和正常对照者外周血中性粒细胞。应用免疫荧光染色技术,观察MYH9-RD患者与正常对照者NMMHC-ⅡA在胞浆中的分布情况；应用Westernblot技术分析MHY9-RD中患者和正常对照者之间NMMHC-ⅡA蛋白的表达水平。结果：MYH9-RD患者中性粒细胞胞浆中存在“椭圆形及梭形”包涵体的形态和轮廓,与瑞特姬姆萨染色显示的包涵体形状、大小基本一致,但更为清晰；而正常对照中性粒细胞胞浆中的荧光呈弥散分布,无丛集现象,未见包涵体；Westernblot结果显示患者NMMHC-ⅡA含量较正常对照降低。结论：免疫荧光技术和Westernblot技术均显示患者和正常人之间MYH9基因编码蛋白NMMHC-ⅡA在中性粒细胞中的表达和分布存在异常,提示NMMHC-ⅡA的显性负性效应导致了中性粒细胞包涵体的产生,表明本家系MYH9-RD患者中性粒细胞包涵体的主要构成成份和性质是非肌性肌球蛋白ⅡA, NMMHC-ⅡA是本家系致病机理的一个重要方面。虽然本家MYH9基因40个外显子和侧翼区未检测到致病性突变,但本家系的致病基因有可能还是由MYH9基因异常导致的。同时,免疫荧光染色技术较瑞特姬姆萨染色法更敏感,特异性更强,对诊断MYH9-RD具有重要的意义。第四章MYH9-RD家系血小板中NMMHC-ⅡA和膜糖蛋白的检测与分析目的：通过分析该MYH9-RD家系患者血小板膜糖蛋白表达差异和NMMHC-ⅡA在血小板中的分布特点,来进一步探讨该家系血小板与MYH9-RD之间的致病机理。方法：采取家系患者和正常对照者的外周血,并分离血小板。应用流式细胞术对MYH9-RD家系患者与正常个体之间的血小板膜糖蛋白GPⅡb/IIIa(CD41/61)、GP I a(CD49b)、GP I b/IX/V (CD42a) GPⅠb(CD42b)和GPⅣ(CD36)进行检测,运用免疫荧光方法观察NMMHC-ⅡA在正常人和患者之间的分布。结果：家系患者血小板膜糖蛋白GPⅡb/Ⅲa(CD41/61)、GP I a(CD49b)、GPⅠb/Ⅸ/Ⅴ(CD42a) GPⅠb(CD42b)和(GPIV(CD36)检测结果正常,血小板膜糖蛋白表达量在家系患者和正常对照者之间,无统计学意义(P>0.05)；但家系患者个体之间血小板的活化水平不同。免疫荧光结果显示患者血小板中NMMHC-ⅡA在血小板中央区染色分布均匀,但是在血小板的外围染色较密集；而正常人结果显示NMMHC-ⅡA在血小板中是呈均匀分布的。结论：家系患者中血小板中NMMHC-ⅡA蛋白分布存在异常,提示家系患者巨大血小板的形成可能与NMMHC-ⅡA异常分布有关；虽然家系患者和正常个体之间的血小板膜糖蛋白表达没有差异,但是家系患者之间血小板的活化水平差异却较大,提示MYH9-RD本家系患者临床表型复杂多样可能与血小板活化水平相关。更多还原

【Abstract】 PART I Family collection, diagnosis and clinical features analysis of non-myosin heavy chain 9 related diseaseObjective To collect the detailed genetic resource in this family with non-muscle myosin heavy chain 9 related disease(MYH9-RD) and make a diagnosis for this family by analyzing the clinical manifestations,inheritance, laboratory features to explore the pathgenic mechanism of MYH9-RD in this family.Methods All of the family members were enrolled in the current study and a detailed screening including clinical and laboratory features were made for this family members. Family members were screened for the presence or absence of clinical manifestations including nephritis, sensorineural hearing loss, cataract, abnormal bleeding or renal disease etc, and other complications and laboratory evaluation. Platelet counts were examined by automatic blood cell counter and morphology, respectively. Meanwhile characteristic and percentage of giant platelet and neutrophil inclusion bodies were analyzed by morphology.Technique of chromosome karyotyping was applied to carry diagnostic cytogenetics, technique of light microscope and electron microscope were applied to analyze the cell morphology and ultrastructure of platelet and inclusion bodies in granulocytes.Results Based on the laboratory evulation of peripheral blood and clinical manifestations,15 patients with a autosomal dominant disorder markedly showed thrombocytopenia, giant platelets and inclusion bodies in granulocytes among 32 menbers of four generations investigated in this family. All the patients not only have thrombocytopenia, giant platelets and neutrophils inclusion, but also have easy bruising and mild to moderate bleeding tendency which is not associated with platelet count or diameter. Moreover, most patients presented complicated clinical manifestations, and some even suffered from serious phenotypes, such as leucocythemia, glaucoma, cataract, proteinuria, abnormal hepatic function, hyperlipemia and disordered action of heart etc, which is distinguished from those families reported previously. Chromosome karyotyping of patients are normal. Platelet count from cell counter was lower than that from microscope (P<0.01). Ultrastructurally, giant platelets and neutrophil inclusions were clearly observed and neutrophil inclusions were oval or spindle-like, localized in the periphery of the neutrophils. Meanwhile, the unaffected members from the family were chosen as control among which normal platelet count, normal platelet volume and without neutrophils inclusions.Conclusion The family patients were diagnosed as MYH9-RD based on clinical and laboratory features. Also not only this family is large, but also the phenotypes of the patients from the family had complicated clinical manifestations which were more serious than those families ever reporeted. Therefore, this large family is established and will lay the foundation for the further studying of MYH9-RD. Moreover,microscopic cell counter rather than automatic blood cell counter should be used to examine the correct platelet counts for the giant platelets patients. PARTⅡMutation screening and analysis of non-myosin heavy chain 9 gene of the MYH9-RD familyObjective Mutation screening and analysis of non-myosin heavy chain 9 (MYH9) gene in this family patients with MYH9-RD were given to explore the pathogenic mechanism of MYH9-RD in this family.Methods After informed consent, we obtained blood samples from fifteen patients and their normal family members in the family with MYH9-RD. Under standard PCR conditions, genomic DNA was extract-ed from peripheral blood leukocytes. All the 40 exons and franking regions of MYH9 gene from 15 patiens and the normal family member were amplified by polymerase chain reaction (PCR), and PCR products were analyzed by using direct sequcing method.The normal family members were chosen as control subjects, and results of sequence was analyzed comparing with the normal MYH9 gene in the Genebank by DNAMAN software.Results Of all the patients, there is no pathogenic mutation in the exons and franking regions of MYH9 gene except a synonymous mutation in the 25 and 18 exon in patients. Meanwhile, there is no mutation in the 40 exons and franking regions of MYH9 gene in normal family members.Conclusion There may be a novel mutation or another novel pathogenic gene or other reasons with MYH9-RD which is related to its complex clinicl phenotype of this family. It is of significance to reveal the molecular pathogenesis mechanism of MYH9-RD by further studying this large family. PartⅢLocalization and expression of non-myosin heavy chain-ⅡA in granulocytes of the MYH9-RD familyObjective To infer the formation characterics of neutrophils inclusions and explore the. pathologic mechanism of this MYH9-RD family by detecting and analyzing the localization or distribution and the expression of NMMHCⅡA in neutrophils of MYH9-RD patients.Methods Neutrophils were isolated from periperal blood of MYH9-RD patients and normal family members. Normal family members were chosen as control subjects. Immunofluorescence technique was used to observe localization or distribution of NMMHCⅡA in MYH9-RD patients and normal family members. NMMHCⅡA levels in neutrophils from patients and normal family members were analyzed by westernblot.Results Oval or spindle-like neutrophils inclusions with green fluorescence in MYH9-RD patients were markly showed by immunofluorescence technique, which matched very well in localization, size and shape, compared with the inclusions, displayed by light microscope (Wright-Giemsa’s stain). Meanwhile, no inclusions in the normal family members were observed except a diffusive distribution of fluorescent spot in neutrophils cytoplasm. The result of western blot revealed that the expression of NMMHC-ⅡA in neutrophils of MYH9-RD patients was down regulated than normal family members.Conclusion The distribution and expression of NMMHC-ⅡA in neutrophils between patients and the normal family members are all differently revealed by immunofluorescence technology and westernblot technology. It is concluded that the molecular pathogenesis mechanism of this MYH9-RD family may be also resulted from abnormal MYH9 gene because the major component of neutrophils inclusions from this MYH9-RD family patients are NMMHC-ⅡA, and dominant negative effect of NMMHC-ⅡA is involved in the formation of inclusion bodies even though there is no pathogenic mutation in the 40 exons and franking regions of MYH9 gene in this family.It is of significance to reveal the molecular pathogenic mechanism of this MYH9-RD family by the study of NMMHC-ⅡA. Meanwhile, immunofluorescence technology is more sensitive and specific than Wright-Giemsa’s staining in detecting inclusions of MYH9-RD patients. PartⅣDetection and analy sis of NMMHC-ⅡA and membrane glycoprotein in platelets of the MYH9-RD familyObjective To further explore the pathogenic mechanism by detect ing and analyzing the distribution of NMMHC-ⅡA and expression of membrane glycoprotein in the platelets of MYH9-RD patients.Methods we obtained periperal blood from MYH9-RD patients and normal family members. Meanwhile, platelets were isolated. The platelet membrane glycoprotein including GPⅡb/Ⅲa(CD41/61),GP I a(CD49b), GP I b/IX/V (CD42a) GP I b(CD42b) and GPIV(CD36) was examined by flowcytometry and the distribution of NMMHCⅡA in the platelet was observed by immunofluorescence technique. Normal family members were chosen as control subjects.Results Platelet membrane glycoprotein GPⅡb/Ⅲa(CD41/61), GPIa(CD49b), GP I b/Ⅸ/Ⅴ(CD42a) GP I b(CD42b)和GP IV (CD36)were in normal range in the family patients. There is no difference in the expression of the platelet membrane glycoprotein between patients and normal family members by flowcytometry(P> 0.05),while the activated level of platelet membrane protein among the family patients was different. Immunofluorescence technique showed well-distributed staining of NMMHC-ⅡA in the central zone and intensive stainning in the periphery in platelets of the patients, while only showed well-distributed staining in the normal family members.Conclusion The distribution of NMMHC-ⅡA in platelets between patients and normal family members was differently displayed by immunofluorescence technology. It revealed that the formation of giant platelets may be related to the abnormal distribution of NMMHC-ⅡA. The difference for the level of platelet membrane glycoprotein between family patients and normal family members is normal while the difference among family patients are quite differently.It revealed that the difference for the activated level of platelet may be involved in the clinical manifestations of family patients.更多还原