Olmsted综合征临床表型与基因型关系研究

Olmsted syndrome (OS) is an autosomal dominant inherited disease characterized by severe periorificial and palmoplantar keratoderma with intolerant pruritus. We have recently identified mutations in TRPV3 as a cause of OS by exome sequencing. The mutations found in OS exert a gain-of-function effect, which was demonstrated in our previous electrophysiological studies. However, the exact pathogenesis of OS is not definitely understood, and the relationship betweent the phenotype of OS and TRPV3 genotype is unclear. Based on our previous studies, a correlation between the severity of clinical manifestations of OS and the extent of mutation effect on TRPV3 channel activity was assumed. The predilection sites of OS lesions were postulated to be related either with the variability of TRPV3 expression in different sites of skin tissue, or with the altered temperature properties of the TRPV3 mutants. In the present studies, the mutations detected in OS patients will be co-expressed with wild type TRPV3 in HEK293 cells, followed by electrophysiological studies and dynamic calcium imaging for intracellular calcium ion measurement. The cytotoxic effect and the temperature properties of the HEK293 cells co-expressing mutants and wild type TRPV3 will also be evaluated. Meanwhile, the expression variability of TRPV3 in different sites of skin tissues from normal controls and OS patients will be studied by real-time PCR and immunofluorescence. Our studies will further reveal the pathogenesis of OS and the relationship between the phenotype and TRPV3 genotype. The　function of several cirtical amino acids of TRPV3 will be unraveled, which may shed a light in the development of blocking drugs specially targeting TRPV3.

Olmsted综合征（OS）是一种以掌跖及腔口周围皮肤严重角化伴剧烈瘙痒为特征的常显遗传病。我们研究组近期通过外显子组测序及电生理学方法确定OS为TRPV3基因功能增强型突变所致。然而，OS的临床表现严重程度是否与基因突变位点相关？OS皮疹为何仅限于掌跖及腔口周围？这些都是亟待解决的问题。我们在前期工作的基础上，提出假设：OS临床严重程度可能与TRPV3突变后通道活性改变程度相关；OS皮损部位特异性可能与TRPV3在不同部位皮肤中差异性表达，或者与突变体通道的温度特性改变相关。本研究拟采用突变体与野生型共表达方法比较不同TRPV3突变体通道活性及通道温度特性改变情况，同时研究TRPV3在OS患者以及正常人不同部位皮肤组织中的表达情况，深入阐明OS的发病机制及临床表型与基因型的关系，从而揭示部分关键性氨基酸位点在TRPV3通道功能中的作用，为开发靶点特异性TRPV3阻断剂奠定基础。

负责人在三年内共完成了7例新确诊的Olmsted综合征的基因突变研究，并且检测到了7个TRPV3基因的不同突变位点（c.C2017T，p.L673F；c.G2075C，p.W692S；c.G1247A，p.R416Q；c.C1246T，p.R416W；c.G1703T，p.G568V；c.G1702T，p.G568C；c.1964T>C，p.L655P）；其中4个突变位点为既往未报道过的。由于Olmsted综合征的患者临床严重程度差异较大，从严重残毁性掌跖角化到轻度局灶性足底皮肤增厚，我们采用了电生理学方法研究部分TRPV3基因突变位点的电生理学改变情况，以期确定Olmsted综合征临床表型与TRPV3突变基因型之间的关系。.负责人及其成员利用膜片钳方法评估了我们新发现的致病性突变体以及既往报道过数个代表性突变体的电生理学性质的改变。通过研究发现，所有导致Olmsted综合征的TRPV3突变位点电生理学都出现了功能增强性的改变，然而功能增强情况存在程度差异：G568C、R416Q、 R416W和W521S四个位点功能增强的程度低于G573S、G573A、G573V、L673F及W692S突变位点。为了进一步模拟患者皮肤中TRPV3的真实表达情况（杂合突变），我们通过同转染等比例野生型TRPV3和突变体质粒，发现共转染后不同突变位点的电生理学改变发生了不同改变，即L673F、R416Q和W521S电生理学增加程度依次得到小到大的降低，而G573A、G573V和W692S几乎无任何改变。这些不同突变体的电生理学改变程度与其引起的Olmsted综合征临床表型呈正向相关，较准确地解释了患者临床严重程度。最终我们发现，若基因突变发生在TRPV3的S4-S5连接区或TRP区域，则临床表现将相对较重；若基因突变发生在N端区域，则临床表现将相对较轻。本研究基本完成Olmsted综合征的基因型和临床表现之间的关系研究。.在课题资助下，负责人还对一系列遗传性皮肤病进行研究，代表性成果包括在国际上首先命名了PLACK综合征并确定其致病基因为CAST，确定了掌跖角化-少毛-白甲综合征及Bothnia掌跖角化症的致病基因为GJA1和AQP5基因，确立了中国人最常见的遗传性掌跖角化——长岛型掌跖角化及其致病基因突变热点。在此基础上负责人获得了本年度国家自然科学基金委优秀青年科学基金项目的资助。