【作者】 许晓娟；

【导师】 夏昆；

【作者基本信息】 中南大学 ， 遗传学， 2010， 硕士

【摘要】 一、银屑病患者的FLG基因突变筛查银屑病(Psoriasis, PS, OMIM 177900)是一种常见的慢性炎症性皮肤病,临床特征为皮肤上出现红斑,并覆有银白色鳞屑,可累及皮肤任何部位,好发于头皮、躯干及四肢伸侧。病理特征为角质增厚或角化不全、颗粒层变薄或消失、棘层增厚；细胞间隙增宽,炎症细胞向表皮和真皮浸润。银屑病在世界范围内发病率约为0.6-4.8%；据估计我国银屑病患者已接近1,000万。银屑病多发病于15岁至30岁之间,常伴随患者一生。银屑病被认为是一种多基因遗传病,其发病机制复杂、至今尚未完全阐明。各种遗传或环境因素主要通过影响免疫系统、炎症反应通路或表皮功能而致病,吸烟、饮酒、精神紧张和物理损伤等其他原因也是常见促发因素。FLG(filaggrin)基因,位于1q21.3,编码丝聚蛋白(filaggrin, Filament Aggregating Protein)的无活性不溶性前体丝聚蛋白原(profilaggrin)。丝聚蛋白是透明角质颗粒(Keratohyalin Granules,KHG)中的主要成份。丝聚蛋白的终产物在皮肤表面形成保湿因子。FLG基因的突变可导致表皮角化异常,从而引发相关皮肤病。FLG基因最早被证明是寻常性鱼鳞病的致病基因,最近又有报导FLG基因的突变也会导致特异性皮炎的发生。免疫组化的结果显示银屑病患者的表皮颗粒层中丝聚蛋白明显减少,提示FLG基因可能参与了银屑病的发生。在本研究中,我们收集了2个中国汉族银屑病／鱼鳞病家系,328例中国汉族散发银屑病患者和500例正常对照,采用长片断PCR扩增和直接测序的方法分析患者与正常人的FLG基因序列,寻找与银屑病相关的突变。我们在家系1中检测到FLG基因的无义突变K4022X,其中先证者携带有纯合突变K4022X,鱼鳞病患者(Ⅰ：4,Ⅱ：6)携带有杂合突变K4022X,另有4个表型正常个体(Ⅰ：2,Ⅱ：1,Ⅱ：4,Ⅱ：5)携带有杂合突变K4022X。在328例散发病人中检测到2个纯合突变K4022X,22个杂合突变K4022X。在500例正常对照中检测到15个杂合突变K4022X。等位基因频率(χ2=12.56,P<0.005)；基因型频率(χ2=16.76,P＜0.005)。Logistic回归中,OR=2.855,95%CIs=2.275-9.974。家系2中我们没有检测到FLG基因的任何突变。上述研究提示,FLG基因的无义突变K4022X导致银屑病家系1银屑病的发生。银屑病家系2可能存在其它未知基因的突变。FLG基因的K4022X突变在中国汉族人群中的等位基因频率为1.5%,可能是一种皮肤角化异常有关的常见突变。二、孤独症患者的NLGN3/4/Y基因的突变筛查孤独症(Autism, OMIM 209850),又称为自闭症,是一种严重的儿童精神发育障碍疾病。通常起病于3岁之前,目前国外报道儿童孤独症的患病率为6%0,国内报道学龄前儿童患病率为1‰,美国约为4-10万,欧洲约10万,中国约2.8-10万男女患病率之比为4：1。主要临床表现为不同程度的社会交往能力缺陷、语言交流技巧障碍、重复刻板的动作和兴趣狭窄等。孤独症的发病机制至今尚未阐明,已有研究表明,孤独症属于复杂性疾病,且遗传度较高。细胞遗传学研究、同胞、家系研究以及病例对照关联分析、全基因组关联分析等研究已经发现了很多与孤独症相关的位点,克隆了多个易感基因。研究表明,神经突触异常可能会导致孤独症的发生。NLGN家族属于突触后细胞粘附分子,通过与突触前细胞粘附因子α-neurexin或β-neurexin形成异四聚体,调控神经元的突触兴奋或突触抑制。NLGN家族共包括5个成员：NLGN1 (3q26.31), NLGN2 (17p13.1), NLGN3 (Xq13.1), NLGN4 (Xp22.3)和NLGNY (Yq11.221)。已有研究发现,NLGN3/4/Y的突变可导致孤独症的发生,但在中国人群中还没有NLGN家族基因与孤独症的相关研究。在本研究中,我们收集了中国汉族人群318例散发孤独症患者及278例正常对照,采用PCR扩增和直接测序的方法分析患者的NLGN3/4/Y基因序列,确定中国孤独症患者的NLGN3/4/Y基因突变。318例散发孤独症患者中我们检测到NLGN3基因的1个同义突变c.1638G>A和1个错义突变G406S以及NLGN4基因的1个同义突变c.1194C>T和3个错义突变G84R、Q162K、A283T。在278例正常对照中没有发现这6个突变。NLGN3/4的同义突变c.1638G>A(NLN3)、c.1194C＞T(NLGN4)和错义突变G406S (NLGN3)、G84R (NLGN4)、Q162K (NLGN4)、A283T (NLGN4)有可能会导致孤独症的发生；NLGN3/4基因的突变在不同人群中都可导致孤独症的发生；其他孤独症患者可能存在其它基因的突变。更多还原

【Abstract】 Chapter 1. Mutation analysis of FLG gene in Chinese Han psoriasis patientsPsoriasis [OMIM 177900] is a common chronic inflammatory dermatosis, clinically characterized by red raised patches covering with white scale on the skin and pathologically characterized by abnormal differentiation in epidermal layers(hyperkeratosis, parakeratosis, stratum granulosum attenuation or disappearance, and acanthosis) and inflammation(intercellular space widen, inflammatory cell infiltration of the dermis and dermal vessel dilatation). By investigating, current prevalence are 0.6-4.8% worldwidely, and 10 million psoriasis patients exist in China. The usual age of onset of psoriasis is between 15 and 30 years and then usually persists for life. Psoriasis is a kind of polygenic disease, and the pathogenesy of psoriasis is so complicated that is still elusive. By affecting immune system, inflammation pathway,the epidermal barrier,various of genetic and environmental factors cause psoriasis. Smoking, drinking, psychentonia and physical injuries and so on are also the triggering factors.FLG gene, which encodes filaggrin, is located within the epidermal differentiation complex (EDC) on 1q21.3 and expressed late in epidermal differentiation. Keratohyalin granules in the granular layer are predominantly composed of phosphorylated and insoluble protein profilaggrin. Keratohyalin granules in the granular layer are predominantly composed of the phosphorylated and insoluble protein profilaggrin. Loss-of-function FLG mutation leads to abnormal differentiation (hyperkeratosis, parakeratosis) in epidermal layers and cause relative dermatosis。FLG gene is identitied to cause ichthyosis vulgaris first. Resently, it is reported that FLG gene also cause Atopic Dermatitis. Immunohistochemical analysis revealed a markedly reduction of filaggrin expression in psoriatic skin.2 psoriasis Chinese Han psoriasis/ ichthyosis vulgaris families,328 sporadic unrelated Chinese Han patients with psoriasis and 500 controls were involved in our study. By long range PCR and direct sequencing we analyze the FLG gene of the psoriasis patients and the health individuals,we identified a nonsense mutation, K4022X, in the family 1. The proband(Ⅲ:2) carries homozygous nonsense mutation, K4022X. TheⅣpatients (Ⅰ:4,Ⅱ:6)carry heterozygous nonsense mutation, K4022X. Besides, four health members (Ⅰ:2,Ⅱ:1,Ⅱ:4,Ⅱ:5)carry heterozygous nonsense mutation, K4022X. We identified 2 of 328 cases carry homozygous nonsense mutation, K4022X, and 22 of 328 cases carry heterozygous nonsense mutation, K4022X. We identified 15 of 500 controls carry heterozygous nonsense mutation, K4022X. By statistical analysis, we obtain allele frequencies:χ2=12.56,ν=1, P<0.005; genotype frequencies:χ2=16.76,ν=2, P<0.005; the OR(odds ratio)=2.855,95% CIs(Confidence Intervals)=2.275-9.974. We did not found any FLG mutation in the family 2.According to the studies aboved, the nonsense mutation, K4022X, may lead to the onset of the psoriasis in the family 1. May any other gene mutation exists in the family 2 to cause psoriasis. The allele frequency of the nonsense mutation, K4022X, is 1.5% in Chinese Han People. K4022X is a kind of common FLG mutation related to abnormal differentiation in epidermal layers.Chapter 2. Mutation analysis of NLGN3/4/Y gene in Chinese Han autism patientsAutism [OMIM 209850] is a severe childhood neurodevelopmental disorder. The onset of autism is before 3 years of age. The prevalence reported is increasing year by year. By investigating, current prevalence are 0.6% worldwidely and 0.1% in China. Males are affected by autism approximately 4 times more frequently than females. The main clinical character is impairment of social and communication skills, repetitive and stereotypical behaviors, restricted range of interests.The etiology of autism is still unknown. However, by investigating, autism is a complex disease with high heritability. Cytogenetics study showed that it has a high incidence of chromosome abnormality. Many related loci and several predisposing genes have been identified by sibling, family study, case-control association study, Genome-wide association study(GWAS) and so on.It is demonstrated that the abnormal neuronal synapse is related to autism. Neuroligins are postsynaptic cell adhesion molecules, by forming heterotetramer with presynaptic cell adhesion molecules neurexins, regulate the fine balance between excitation and inhibition of the synapse. In humans, the neuroligin protein family comtain five genes, NLGN1,2, 3,4 and 4Y. It is reported the NLGN3/4 mutations cause autism. However, no study on NLGNs and autism have done in Chinese Han people.318 sporadic unrelated Chinese Han patients with autism were involved in our study. By routine PCR and direct sequencing we analyze the NLGN3/4/Y genes of the autism patients.we identified a samesense mutation c.1638G>A and 1 missense mutations G406S in NLGN3 and a samesense mutation c.1194C>T and 3 missense mutations G84R、Q162K and A283T in NLGN4. All the 6 mutations have not been identified in 278 controls.These mutations in NLGN3/4 may cause autism. The NLGN3/4 mutations can cause autism in different race of people. Maybe other gene mutations exist in the patients.更多还原