ITGB2新发突变致白细胞黏附分子缺陷病的致病机制研究

Novel mutations of ITGB2 induced leukocyte adhesion defect type 1

目的 探讨ITGB2新发突变所致白细胞黏附分子缺陷1型的致病机制.方法 患儿女,11岁,因反复发热于2014年8月收入青岛大学附属医院.收集患儿临床资料,用流式细胞术检测患儿白细胞表面CD18的表达水平;以全外显子基因二代测序明确突变基因;软件预测突变致病性;构建含野生型及突变型ITGB2基因的载体,分别比较野生型蛋白与突变型蛋白的稳定性及其亚细胞定位.结果 患儿临床表现（反复感染发热、牙槽嵴低平、牙齿缺如等）与白细胞黏附分子缺陷高度相符;患儿外周血白细胞表面CD18表达水平（0.2％）较对照组（患儿父亲：99.0％,患儿母亲：99.1％）显著下降;经基因检测明确患儿ITGB2的2个等位基因各携带有1个新发现的突变,分别为chr21（46319021）c.954delG（新发突变）和chr21 （46309266）c.1802C＞A（来自父亲）;其中c.954delG突变为移码突变,确定为致病突变;c.1802C＞A经软件预测为有害突变;突变型蛋白在稳定性上与野生型蛋白无差别,但合成后无法定位于细胞膜.结论 该患儿可确诊为白细胞黏附分子缺陷1型,其致病突变为ITGB2基因c.954del G和c.1802C＞A突变;其中c.1802C＞A突变使CD18无法定位于细胞膜,从而导致白细胞黏附分子缺陷的发生.

Objective To investigate the pathogenic mechanism of two novel ITGB2 mutations in leukocyte adhesion defect type 1 （LAD1）.Methods The clinical history and blood sample of an 11 years old patient admitted to Affiliated Hospital of Qingdao University in August 2014 were collected.Expression of CD18 （encoded by ITGB2） was analyzed by flow cytometry.Novel ITGB2 mutations were identified by next-generation sequencing technology and confirmed by Sanger sequencing.The functional effect of ITGB2 mutations was detected by PolyPhen2.Expression vectors of both wild type and mutant ITGB2 were constructed and transfected into mammalian cells for analysis of protein stability and subcellular location.Results The symptoms of the patient （recurrent infections,lowered alveolar ridge and hypodontia） supported the diagnosis of LAD1.Expression of CD18 on the leukocytes was significantly decreased （0.2%） compared with the control samples from the parents （paternal：99.0%;maternal：99.1%）.The patient was identified to be compound heterozygous for ITBG2 c.954del G （novel mutation） and c.1802C〉A （paternal originated）.ITGB2 c.954 del G was confirmed to be a harmful frameshift mutation;ITGB2 c.1802C〉A was also predicted to be harmful.In terms of protein stability.There was no significant difference between mutant D18 and wild type.However,subcellular location analysis showed the mutant D18 could not locate on cell membrane.Conclusion The compound heterozygous of ITGB2 mutations （c.954del G and c.1802C〉A） decreases the expression and impairs the location of CD 18 on leukocytes,which leads to LAD 1.