基于遗传性球形红细胞增多症膜蛋白缺陷类型的差异确定特定人群相关基因突变谱的研究

Hereditary spherocytosis (HS) is a hemolytic anemia caused by defects of erythrocyte membrane proteins deriving from specific genetic mutations. Although many screening tests, such as determination of mean sphered corpuscular volume and flow cytometric osmotic fragility from patient's erythrocytes can improve the diagnostic efficiency of the HS, misdiagnosis and missed diagnosis are still inevitable. our pilot study in patients from 20 HS pedigrees in our region confirmed that analysis of membrane protein defects and mutations of the HS is the key to the diagnosis, treatment and even prevention of the disease. However, the genes encoding membrane protein defects of erythrocytes are different from race and region. It is why establishing genetic pedigree of HS patients has to be done in our region if we want to solve the problems above. Therefore, the aim of our project is to establish genetic pedigree of HS patients in our region through identifying pathogenic gene mutations by using PCR and its derived techniques, high resolution melting analysis and DNA sequencing. Our project will contribute to improving analytical methods of erythrocyte membrane protein and gene diagnosis, exploring erythrocyte membrane protein defect types and the gene mutation spectrum of HS patients.

遗传性球形红细胞增多症（HS）是因基因突变导致红细胞膜蛋白缺陷而引起的溶血性贫血。虽然应用球形红细胞平均体积和流式细胞术渗透脆性试验等筛查试验，较好地提高了HS的诊断效率，但我们仍然发现一些患者被误诊、漏诊。在前期工作中，我们对本地区20个HS家系进行了初步研究，发现分析HS的膜蛋白缺陷，确定其基因突变类型是疾病诊治甚至预防的关键，但膜蛋白缺陷的编码基因存在种族和地区差异，因此，确定特定人群基因突变谱是解决此问题的核心。本课题拟应用Western Blot等方法在确定HS患者膜蛋白缺陷类型后，使用PCR及其衍生技术、高分辨熔解曲线分析和DNA测序等方法检测特定人群的相关基因突变谱。本课题的完成将有助于改进我国HS患者红细胞膜蛋白分析和基因诊断实验方法，探索本地区HS患者的红细胞膜蛋白缺陷类型与基因突变谱，为HS诊断、治疗及产前优生检查奠定基础。

遗传性球形红细胞增多症(HS)是因基因突变导致红细胞膜蛋白缺陷而引起的一种常见的溶血性贫血。临床上往往容易漏诊，误诊与误治。本课题通过收集HS、地中海贫血、G6PD缺乏症、自身免疫性溶血性贫血等患者的血液标本，进行血常规和网织红细胞分析、外周血红细胞形态学检查、血清胆红素测定、流式细胞术渗透脆性试验和伊红-5-马来酰亚胺(eosin-5-maleimide，EMA)结合试验等，探索快速、有效鉴别HS的实验诊断方法。对于HS患者及其家系成员，我们应用SDS-PAGE、Western Blot和real-time PCR技术对其红细胞膜蛋白进行定量分析，明确主要红细胞膜蛋白缺陷类型；应用PCR、高分辨熔解曲线分析、sanger测序、目标区域捕获技术和高通量测序等技术对其缺陷蛋白的相关基因进行检测，探索基因突变的类型。研究结果显示：(1)以MSCV<MCV作为诊断界值诊断HS时，灵敏度为89.28%，特异度为96.14%；(2)以流式细胞术渗透脆性试验剩余红细胞(%)<23.6%为HS诊断界值时，灵敏度为85.71%，特异度为97.24%；(3)当MRV≤95.77fl时，诊断HS的灵敏度为86.80%，特异性为91.20%。(4)本地区HS患者的主要红细胞膜蛋白缺陷类型以带3蛋白缺陷为主，其次为a、b-收缩蛋白和4.2蛋白缺陷。(5)不同家系的HS患者致病基因存在散在的突变位点，突变可出现于外显子和内含子中，其突变类型包括点突变、缺失突变、插入突变或无义突变。以上研究成果有利于改进HS实验诊断方案，同时丰富了我国HS的遗传学数据，明确了本地区HS患者的红细胞膜蛋白缺陷类型与相关基因突变谱，为HS诊断、治疗及产前优生检查奠定了基础。