【作者】 熊志敏；

【导师】 夏昆； 胡正茂；

【作者基本信息】 中南大学 ， 遗传学， 2013， 博士

【摘要】 寻常型鱼鳞病(Ichthyosis Vulgaris, IV, OMIM#146700)是一种常见的遗传性角化障碍性皮肤病,发病率在1：5300到1：250之间,通常在出生后一年内发病,好发于四肢伸侧及背部,以小腿最为明显。Ⅳ临床异质性较大,病情轻者仅表现为冬季皮肤干燥、粗糙,表面有细碎的糠样鳞屑；典型的皮损表现为灰褐色或深褐色菱形或多角形鳞屑,鳞屑中央固着,边缘游离,如鱼鳞状；其他临床特征还表现为掌跖线状裂、掌纹加深、遗传性过敏症和热耐受不良。FLG失去功能的突变被公认为是Ⅳ的致病因素。到目前为止,在寻常型鱼鳞病患者中报道的FLG突变种类已接近30种。Filaggrin基因(OMIM#135940)位于1q21.3,由3个外显子组成,3号外显子长12.7-14.7kb,由10-12个几乎完全一样的重复区组成,编码几乎整个profilaggrin,是人类基因组外最大的外显子之一；Filaggrin蛋白由S100钙离子结合区、B结构域、2个FLG不完全重复区、10-12个FLG重复区和C-端组成。C-末端结构域是profilaggrin水解成filaggrin单体所必需的结构,FLG失去功能的突变使profilaggrin缺失C-末端结构域而无法正常水解,最终使表皮颗粒层filaggrin单体减少或缺失。为了鉴定3个寻常型鱼鳞病家系的致病变异,本研究首先对家系的先证者开展了FLG突变研究,结果在家系1的先证者中鉴定了2个未见报道的变异(c.477-478insA, c.6218-6219delAA)和一个外来的已知致病变异c.3321delA。随后对家系1进行疾病共分离研究,发现c.477-478insA与家系1疾病共分离；c.6218-6219delAA是一个新发突变；c.3321delA与家系中外来的一支共分离。为了进一步验证上述结果,我们将变异c.477-478insA与家系进行了连锁分析,结果得到LOD值大于1的结果,支持该位点与疾病连锁；同时使用TA克隆测序方法对c.6218-6219delAA进行了验证,结果证实了该位点是家系1先证者新发的变异；这2个突变在200个正常对照中均未检测到,进一步证明了它们是致病变异。此外,在家系2和3中均鉴定到了已知致病变异c.3321delA。这些结果丰富了中国人群FLG突变谱。第二部分中国人群寻常型银屑病Filaggrin突变研究银屑病(Psoriasis, OMIM#177900),是一种以红斑、鳞屑为主要表现的慢性炎症性、免疫介导的皮肤疾病。临床可分为四种亚型：寻常型银屑病、关节型银屑病、脓疱型银屑病及红皮病型银屑病,其中寻常型银屑病为最常见的类型。因人种遗传背景不同该病的人群患病率为0.1%～3%,中国汉族人发病率为0.123%。银屑病的具体发病机制尚未完全阐明,已有研究表明：环境、遗传因素和免疫炎症反应均在其发生和病理过程中起到十分重要的作用。通过全基因组扫描和连锁分析至少已鉴定了9个银屑病易感位点(PSORS1-9)。Filaggrin (FLG)是位于易感区间1q21的一个重要候选基因。FLG自发突变体小鼠(5303delA)皮损部位出现类似银屑病的炎性特征,其他研究小组在银屑病患者皮肤中检测到filaggrin表达量和表达模式都发生了显著变化,提示FLG可能与银屑病的发生相关。为了证明FLG与银屑病的相关性,研究小组分别在德国、爱尔兰和英国人群银屑病患者中开展的关联研究,结果显示FLG突变与银屑病的发生无关。到目前为止,还没有遗传学证据表明FLG与银屑病的发生有关。本课题组许晓娟博士2009年采集到1个来自湖南岳阳的家系,这个家系的特点是银屑病与鱼鳞病患者共存：先证者表现为典型的银屑病症状,而先证者的外婆和姑姑为鱼鳞病患者。这个家系提示银屑病与鱼鳞病可能存在共同的发病机制。许晓娟随后对这个特殊的银屑病家系进行了FLG突变研究,发现先证者携带FLG p.K4022X纯合突变,而家系的鱼鳞病患者携带p.K4022X杂合突变,这个结果提示我们p.K4022X的纯合突变可能导致银屑病的发生。本研究为了进一步明确FLG与银屑病易感性的关系,我们首先在441例患者和500个正常对照中针对p.K4022X进行了突变检测,结果检测到了2例患者携带p.K4022X的纯合改变,而在正常人中未发现纯合改变,提示纯合突变可能导致银屑病的发生。另外分别在29例患者(6.6%)和15个正常对照(3%)中检测到了p.K4022X的杂合改变。随后我们开展了p.K4022X病例-对照关联研究,结果显示p.K4022X在441例银屑病患者和500个对照样本间的基因型频率和等位基因频率比较的P值分别为0.011和0.002(p<0.05),说明差异有显著统计学意义。同时相对危险度(OR)大于1,且95%可信区间下限大于1(OR=2.552(95%CI=1.377-4.731)),提示p.K4022X是中国人群银屑病发生的危险因素。其次,为了进一步研究FLG突变与银屑病的关系,我们分析了441例患者的整个FLG编码区序列,发现了5个无义或移码突变：一个未见报道的无义突变(p.W2583X)和4个在寻常型鱼鳞病或特异性皮炎中报道过的突变(p.R826X, c.3321delA, p.Q2417X, c.7945delA)。此外,我们在银屑病患者中还鉴定了18个错义突变。其中p.E2652D在2个银屑病／鱼鳞病共存的家系中都鉴定到,2个家系中3个银屑病患者均携带该纯合突变,而2个鱼鳞病患者和3个表型正常的个体携带该杂合变异,提示p.E2652D的纯合突变可能导致银屑病的发生。这些结果提示使FLG失去功能的纯合突变导致银屑病的可能性大。同时,纯合的错义突变也可能导致银屑病的发生。这是首次在遗传学上证明了FLG与银屑病的相关性,为中国人群银屑病发生机制的深入研究奠定了科学基础。更多还原

【Abstract】 Ichthyosis vulgaris (IV, OMIM#146700) is a common genetic skin cornification disorder with a prevalence of one in250-1000. The onset of IV is usually within one year after birth. The patients manifest with white scaling on the extensor surfaces of the extremities and occasionally the trunk. Ichthyosis vulgaris, involves symmetric scaling of the skin, which ranges from minor roughness and dryness to the desquamation of large plates Their color varies from white to dirty gray to brown,which are usually small, irregular, and fine, with curled up edges that give the skin a rough feel. Clinical features also include hyperinearity of the palms and soles, atopy and heat intolerance or keratosis pilaris.Loss-of-function mutations in FLG have been known to be a major predisposing factor for ichthyosis vulgaris, about30FLG variants have been identified in IV patients. The FLG gene (OMIM#135940), which encodes the protein filaggrin, is located on chromosome1q21.3, and consists of three exons,exon3(12-14kb) contains the N-terminal domain and10-12copies of a sequence about1kb in length, which encodes the proteolytically cleaved parts of filaggrin. Filaggrin plays a key role in facilitating epidermal differentiation, and in maintaining normal skin-barrier function and hydration. To identify FLG mutations in three Chinese pedigrees with ichthyosis vulgaris, we first sequenced the entire coding region of FLG in the proband of each pedigree. We found two novel FLG null mutations (c.477-478insA and c.6218-6219delAA) and a known mutation (c.3321delA). Both novel mutations were identified in the proband of pedigree1; Then, we perfermed disease cosegregate study in pedegree1, the results suggesting c.477-478insA was cosegragate with IV while c.6218-6219delAA was a de novo mutation. To further confirm the above results, we performed linkage analysis with the site c.477-478insA in pedegree1, and we abtain a LOD greater than1which surport the above disease cosegragate study result; In addition, we used another sequencing methord--TA clone proved the mutation c.6218-6219delAA was a de novo one. Neither of these two mutations was found in200unrelated controls. These findings extend the spectrum of functional FLG variants possibly causing ichthyosis vulgaris. Section2Mutation Analysis in Filaggrin Associated with Psorasis Vulgaris in Chinese PopulationPsoriasis (OMIM#177900) is a chronic inflammatory immune-mediated skin disorder characterized by red lesions with silver white scales, affects approximately3%of Caucasian population and0.123%of the Chinese population. Psoriasis can be differentiated into four subgroups:psoriasis vulgaris, psoriaticarthritis, erythrodermic psoriasis, and annular pustular psoriasis. Of these, Psoriasis vulgaris is the most common one.The pathogenesis of psoriasis involves both genetic and environmental risk factors. At least nine susceptibility loci for psoriasis have been identified through classic genome-wide linkage, which are called psoriasis susceptibility1through9(PSORS1-9). FLG, encoding a keratin filament associated protein (filaggrin), has been reported to be major causative genes for two skin diseases, ichthyosis vulgaris and atopic dermatitis. FLG spontaneous mutated mouse (5303de1A) showed a phenotype of inflammatory infiltration in skin lesions similarity to psoriasis, and Huffineier et al. showed that the filaggrin expression altered markedly in psoriatic skins, all these suggest a relation between psoriasis and FLG. However, another two groups failed to identify the association between psoriasis and mutations of FLG in German, Irish and United Kingdom population.Our aim is to identify FLG causative mutations in Chinese Psoriasis patients. Our previous study collected a Chinese psoriasis/IV coexisting family. And through mutation analysis on FLG with this family, our laboratory colleagues Xu Xiaojuan identified a non-sense mutation of FLG (p.K4022X) in this psoriasis/Ⅳ coexisting family. The homozygous p.K4022X mutation was detected in a psoriasis patient, whereas the heterozygous p.K4022X mutation was identified in two IV patients and four apparently normal family members, It is the first time reported FLG mutation in psoriasis patients.This study aims to confirm the association between FLG and psoriasis, we first genotyped p.K4022X variant in441sporadic Chinese psoriasis patients and found homozygous mutation in two patients(0.45%), while no homozygous variant was found in500control individuals (0%). Moreover, p.K4022X heterozygous mutation was identified in29psoriasis patients (6.6%) and15control individuals (3%), respectively. Mutation p.K4022X showed a significant association with psoriasis. The P values of genotypic frequency and allelic frequency for p.K4022X in case-control analysis were0.011and0.002, respectively. The odds ratio (OR) for the dominant model (T vs. A) was2.552(95%CI,1.377-4.731; P=0.002). Thus our data implied an association of p.K4022X variant in FLG gene with the psoriasis in Chinese population. Second, in order to further investigate FLG mutations in psoriasis patients, we sequenced the entire coding region of FLG in441psoriasis patients, and identified another five mutations, including one novel mutation (p.W2583X) and four mutations (p.R826X, c.3321delA, p.Q2417X and c.7945de1A) that have been reported in IV or AD patients.Additionally, we identified18missense mutations.One of the missense mutation p.E2652D was identified in two psoriasis/Ichthyosis co-exiting pedigrees.Interestingly, three psoriasis patients of the two pedigree carry the homozygous p.E2652D while two IV patients carry heterozygous one, suggesting that the homozygous variant of p.E2652D may lead to psoriasis.These results suggest that loss-of-function homozygous mutations of FLG much more likely lead to psoriasis. As well, the homozygous missense mutation may lead to psoriasis. This is the first time revealed the relation between FLG and psoriasis, and it will provide evidence for further understanding the pathogenisis of psoriasis.更多还原