背景:白细胞粘附缺陷III(LAD-III)是一种罕见的常染色体隐性综合征,其特征是由于编码kindlin-3的FERMT3基因突变而发生的血小板和白细胞功能缺陷。Kindlin-3是包含FERM结构域的衔接蛋白,其在整联蛋白激活中是必需的。我们先前已经证明kindlin-3的FERM结构域在结构上是紧凑的,并且在小鼠模型中支持整联蛋白激活中起重要作用。kindlin-3中紧凑的FERM结构域不稳定对人类LAD-III发育的影响仍不确定。
目的:使用来自LAD-III患者的原代细胞来验证紧凑型FERM结构域在血小板和白细胞kindlin-3功能中的作用。
方法:患者为4岁,从婴儿期起就表现出LAD-III的临床特征。对患者的血小板和白细胞进行了功能分析,并对kindlin-3变体进行了结构分析。
结果:我们在FERMT3中鉴定了一个新的纯合错义突变(c.412G>A,p.E138K)FERM域。在先证者的血小板和白细胞中检测到kindlin-3水平显著降低。功能评估证实整合素αIIbβ3介导的血小板活化,传播,聚集和β2-整合素介导的中性粒细胞粘附和扩散明显受损。结构分析表明,kindlin-3中这种新鉴定的E138K取代使压缩的FERM结构域不稳定,导致血细胞和随后的LAD-III中kindlin-3的表达不良。
结论:我们已经确定了一个新的错义突变,并验证了紧凑kindlin-3FERM结构域在支持血小板和白细胞中整合素功能方面的功能意义。
BACKGROUND: Leukocyte adhesion deficiency III (LAD-III) is a rare autosomal recessive syndrome characterized by functional deficiencies of platelets and leukocytes that occurs due to mutations in the FERMT3 gene encoding kindlin-3. Kindlin-3 is a FERM domain-containing adaptor protein that is essential in integrin activation. We have previously demonstrated that the FERM domain of kindlin-3 is structurally compact and plays an important role in supporting integrin activation in a mouse model. The impact of destabilizing the compact FERM domain in kindlin-3 on the development of LAD-III in humans remains uncertain.
OBJECTIVE: To use primary cells from a patient with LAD-III to validate the role of the compact FERM domain in kindlin-3 function in platelets and leukocytes.
METHODS: The patient is a 4-year-old girl who since infancy has displayed clinical features of LAD-III. Patient platelets and leukocytes were functionally analyzed, and structural analysis of the kindlin-3 variant was conducted.
RESULTS: We identified a novel homozygous missense mutation in the FERMT3 (c.412G>A, p.E138K) FERM domain. Substantially reduced levels of kindlin-3 were detected in the proband\'s platelets and leukocytes. Functional evaluation verified that integrin αIIbβ3-mediated platelet activation, spreading, and aggregation and β2-integrin-mediated neutrophil adhesion and spreading were significantly compromised. Structural analysis revealed that this newly identified E138K substitution in kindlin-3 destabilizes the compacted FERM domain, resulting in poor expression of kindlin-3 in blood cells and subsequent LAD-III.
CONCLUSIONS: We have identified a novel missense mutation and verified the functional significance of the compact kindlin-3 FERM domain in supporting integrin functions in platelets and leukocytes.