FERMT1基因新失功能突变通过干扰整合素活化引发Kindler综合征的分子致病机制研究

Kindler syndrome (KS; OMIM 173650) is a rare autosomal recessive disorder caused by loss-of-function mutations in the FERMT1 gene encoding Kindlin-1 (also known as fermitin family homolog-1, FFH1). The Kindlin protein family members have recently been implicated in β1 integrin activation by interrupting the protein-protein interaction. In our previous study, we have found a novel loss of function mutation in FERMT1 gene. However, the detailed pathogenic mechanism is not fully understood.. To date, over 73 distinct mutations of the FERMT1 gene have been identified, including deletions of parts or the entire gene, splice-site mutations, nonsense and frame shift mutations. Although the course of the disease varies extensively between the patients, it is not possible to correlate the manifestation of specific symptoms or the severity of disease to specific mutations in the FERMT1 gene. Thus, the pathogenic mechanism of the FERMT1 gene mutation needs to be elucidated. . This project aims to study the pathogenic mechanism of the novel mutation in the FERMT1 gene by in vivo and vitro tests. In this study, we firstly investigate the epidermal thickness and the distribution of several basement membrane proteins, including types IV, VII, and XVII collagens and laminin-332 in Kindler syndrome skin. Next, we would observe the effect of the loss of function mutation on the keratinocytes biology, such as cell adhesion and cell migration and proliferation ect. Finally, we analyze β1 integrin activation level and subcelluar location in keratinocytes, and also evaluate the binding of kindling-1 and other proteins, such as Kindlin-2、migfilin、integrin or integrin-linked-kinase (ILK). This research would provide the basic effect of the FERMT1 gene novel mutation on cellular phenotype by influencing β1 integrin activation, which might be helpful to reveal the pathogenic mechanism of the FERMT1 mutation.

FERMT1基因失活性功能突变是引起Kindler综合征（KS）发生的主要原因，最新研究表明由FERMT1 基因编码的kindlin-1蛋白可与多种结合蛋白，如Kindlin-2、migfilin以及整合素连接激酶 (ILK)相互作用，参与调控整合素活化，对于维持角质形成细胞的正常特性有重要作用。前期研究报告了一例新FERMT1基因失活性功能突变，然而其详细致病机制并不清楚。本研究拟结合体内、体外研究，从组织、细胞以及分子水平综合进行研究：①观察FERMT1基因失功能突变对表皮厚度及基底膜结构的影响；②探讨其对角质形成细胞黏附、迁移及增殖能力的影响；③分析Kindlin-1突变蛋白与其结合蛋白的相互作用，并联合ß1整合素定位和表达情况，最终明确该突变引发KS的致病机制。本研究期望从整合素活化角度探索新突变在KS疾病发生中的作用机制，为基于整合素活化调控的药物研究提供一定理论依据。

FERMT1基因失活性功能突变是引起Kindler综合征（KS）发生的主要原因。本项目研究观察了Kindler综合征患儿的皮肤临床外观，分析了KS患儿携带的FERMT1基因失活性功能突变对患者皮肤厚度、表皮结构的影响作用，探索了KS患儿皮肤基底膜蛋白（层粘连蛋白-γ2、ß1整合素、IV型胶原蛋白、VII型胶原蛋白）的表达情况。利用构建的FERMT1基因RNA干扰慢病毒载体进行HaCaT细胞病毒感染，测定了靶基因表达产物多水平（mRNA和蛋白）的表达情况，为揭示KS致病机制提供了数据支持。研究结果表明，①KS患儿的腰腹部皮肤萎缩呈皱缩样外观，散在数十个绿豆大小褐色斑片。②KS患儿病损处皮肤组织切片后HE染色，显微镜下可见表皮轻度网篮状角化过度，表皮突消失变平，失去正常的皮突结构，真表皮交界处散在小裂隙，真皮浅层可见色素失禁现象。真皮浅中层毛细血管扩张，管周可见散在淋巴细胞浸润。③KS患儿的腹部表皮厚度为61.06±10.10μm，显著低于同年龄同部位的正常儿童相比（P＜0.01），提示患儿表皮存在萎缩变薄. ④与正常皮肤相比，KS患儿皮肤基底膜蛋白（层粘连蛋白-γ2和 ß1整合素、IV型胶原蛋白、VII型胶原蛋白）表达均显著减弱，基底膜带结构异常，缺乏连续性，提示基底膜蛋白的表达异常和该病的发病相关。⑤KS患儿存在真皮弹力纤维结构的异常：基于真皮弹性纤维为皮肤提供弹性和适应性，考虑患儿的皮肤呈萎缩起皱外观可能和弹力纤维异常有关。⑥构建了FERMT1基因RNA干扰慢病毒载体，并进行慢病毒包装。结果显示，I. 利用阴性对照组(感染含Control-shRNA的病毒液，MOI=50)和干扰组(感染含FERMT1-shRNA的病毒液，MOI=50)感染HaCaT细胞的感染率均可达80%左右。II.与阴性对照组相比，干扰组HaCaT细胞内的FERMT1 mRNA表达量降低约51.3％。该结果表明合成的FERMT1 shRNA慢病毒可以有效敲低靶基因的转录表达。III.与Control-shRNA阴性对照组相比，FERMT1-shRNA干扰组细胞内的FERMT1蛋白水平明显下降，提示合成的FERMT1 shRNA慢病毒可以有效敲低靶蛋白表达。本研究为深入理解KS疾病的致病机制提供了理论数据。