背景：血小板无力症（GT）是一种罕见的遗传性出血性疾病，患者的血小板数量和形态多正常，但表现出对胶原、花生四烯酸和ADP等生理性激动剂的聚集反应低下或缺如，对瑞斯托霉素的反应正常。患者自幼出现不同程度的皮肤黏膜等部位出血，严重者可危及生命。不同患者之间的出血情况差别很大，甚至随着年龄增长，同一患者的出血症状也会有所改变。该病的遗传方式为常染色体隐性遗传，纯合突变患者多见于近亲结婚的家庭。ITGA2B和ITGB3基因均位于17号染色体长臂，分别编码αIIb和β3亚单位。血小板无力症患者存在ITGA2B或ITGB3基因突变，会导致αIIbβ3质或量的缺陷，从而导致疾病的发生。疾病的突变数据库在不断更新中，目前人类基因突变数据库（HGMD）（http://www.hgmd.org）总共列出了236个ITGA2B基因突变和170个ITGB3基因突变。

目的：统计和分析血小板无力症患者的临床和实验室特征。比较不同性别、年龄和不同类型GT患者之间的出血严重程度，探索影响临床特征异质性的因素。对血小板无力症家系中的患者采用高通量测序的方法进行ITGA2B和ITGB3的测序，并进行家系验证。分析GT患者的出血表型和基因型之间的关联。对突变利用生物信息学方法进行功能预测和遗传学分析，描述中国GT患者的分子学特征。

方法：使用病历查询和随访的方式记录104名血小板无力症患者的基本特征、实验室检查、出血部位和严重程度等。出血严重程度使用WHO出血分数评价体系。根据性别、年龄和不同GT类型进行亚组分析，比较各组间患者的出血分数。对ITGA2B和ITGB3基因的所有外显子、剪接位点和相邻的内含子区域，进行高通量测序，测序完成后对结果进行分析，所有的突变位点使用sanger测序在家系内验证。对新发现的位点利用生物信息学工具进行致病性预测。使用单倍型分析的方法分析两种重现性突变是否参与“建立者效应”。

结果：患者自幼表现出中至重度的出血，诊断时的中位年龄为5岁。最常见的出血部位依次为皮肤出血、鼻衄、牙龈出血。女性患者出血症状明显比男性患者严重。多数患者为I型血小板无力症，其次为II型和III型，三组患者之间的出血分数无明显差异。对54名GT患者基因检测并进行家系验证，发现了56个基因突变（其中ITGA2B 35个，ITGB3 21个），包括个29错义突变，11个无义突变，7个剪切位点突变，9个移码突变。不同突变类型和基因型的患者，出血严重程度无明显差异。有18个突变为新报道的突变，通过生物信息学分析，9个位点被认定为“致病性变异”，9个为“可能致病性变异”。ITGA2B基因上的c.2333A>C和c.1750C>T分别在16和10个无关家系中检测到。通过单倍型分析，存在c.2333A>C突变的多个家系患者拥有部分相同的单倍型，表明这些家系可能来自于同一祖先，而c.2333A>C可能起“建立者效应”。

结论：本研究展示了目前我国最大的血小板无力症患者的临床队列。患者的临床表现主要为出血，出血类型多样。出血严重程度因人而异，与性别有关，与基因型和疾病分型无关。在54名患者中，共发现56种本疾病相关突变。本研究新报道了18种新突变，经过生物信息学分析认定为致病性变异，丰富了疾病突变谱。16个家系的单倍型分析显示ITGA2B基因中的c.2333A>C突变可能起“建立者效应”的作用，即这些家系可能来自于同一祖先。

Introduction: Glanzmann’s thrombasthenia (GT) is a rare inherited bleeding disease, incidence being approximately one in one million worldwide. GT is characterized by the impaired platelet aggregation to multiple physiologic agonists such as collagen, adenosine diphosphate (ADP), arachidonic acid (AA), but normal reaction to ristocetin. Platelet counts and sizes are within normal range in most patients. Patients presented with bleeding of varing severity, sometimes life-threatening. Bleeding severity varies from person to person, Bleeding symptoms of a single patient could even alter with age. The ITGA2B and ITGB3 gene, located at chromosome 17q21.31-32 closely, encode for αIIb and β3 unit respectively. Pathogenic variants of αIIb or β3 could cause the disease. The database of gene mutations is continuously updated on the Internet (http://www.hgmd.org, accessed 2020.4.6); it lists a total of 238 variants of the ITGA2B gene and 175 variants of the ITGB3 gene.

Objective: To analyze clinical and laboratory characteristics of GT patients，compare bleeding severity between different groups (based on gender, age and type of GT disease). And to investigate the gene mutations and founder effect of two variants in Chinese GT patients.

Methods: In total, 102 patients were recruited in our study. Information on baseline characteristics, laboratory tests, bleeding sites and severity were recorded. The severity of bleeding was evaluated by the WHO bleeding scale. The bleeding scores were compared between groups divided according to gender, age and type of GT. Next generation sequencing (NGS) was performed using a custom-made panel for the bleeding and platelet disorders. Prediction of variants were made using bioinformatics tools. The founder effect was assessed in patients with two recurrent mutations using haplotype analysis.

Results: Most patients presented with moderate-to-severe bleeding symptoms according to World Health Organization (WHO) bleeding scale, and the median age at diagnosis is 5 years (IQR, 2-9 years). The mean bleeding score in female patients is significantly higher than that of male patients (3.21 vs 2.59, p<0.001). There is no significant difference in bleeding scores between each types. Fifty-six different mutations were detected (ITGA2B: n=35; ITGB3: n=21), including 29 missense, 11 nonsense, 7 splice site and 9 frameshift mutations. Among 56 detected mutations, there were 18 novel pathogenic mutations, 12 in ITGA2B (c.2444_2445del, c.2915dupC, c.2758_2759delCA, c.1229C>G, c.2896dupC, c.432G>A, c.454G>A, c.989A>T, c.338_356del, c.1622T>A, c.1210+1G>A, c.2452_2454del) and 6 in ITGB3 (c.361+1G>A, c.1690G>C, c.756delC, c.1288C>T, c.1985G>A, c.1684T>G).  Haplotype analysis shows ITGA2B c.2333A>C locates in a strong linkage disequilibrium zone.

Conclusion: The clinical manifestations of patients with GT were heterogeneous, and bleeding symptoms varied from person to person. There is no significant difference in the severity of bleeding between different types of GT patients. In total, 56 different variants were detected in ITGA2B and ITGB3, 18 of which were newly identified. Theses novel findings expand the GT mutation spectrum. The haplotype analysis of several patients with ITGA2B c.2333A>C indicated that founder effect might be involved.