【作者】 刘勇；

【导师】 何春涤；

【作者基本信息】 中国医科大学 ， 皮肤病与性病学， 2007， 博士

【摘要】 中国汉族瘢痕疙瘩致病基因初步连锁分析和相关基因多态性的研究前言瘢痕疙瘩（Keloid）是易感个体在皮肤损伤修复过程中成纤维细胞大量增殖，胶原基质大量分泌形成的良性真皮肿瘤，临床特征为瘢痕组织超越原有损伤范围，侵袭周围外观正常皮肤，手术切除后易复发。虽然国内外许多学者对其进行了大量的研究，但发病机制目前尚不清楚。随着遗传学特别是分子遗传学的发展，遗传因素在疾病发生和发展中所起的作用被越来越多的学者重视。大量的研究显示，遗传在本病的发生中起着至关重要的作用，且存在着明显的遗传异质性。Marneros等通过对一个瘢痕疙瘩日本家系和一个非洲裔美国人家系的家族成员进行全基因组扫描，将瘢痕疙瘩易感基因分别定位于2q23和7p11，为下一步寻找和克隆致病基因，揭示瘢痕疙瘩发生的遗传本质打下了坚实的基础。由于两个家系的易感基因定位在不同的染色体区域，提示瘢痕疙瘩的发生存在明显的遗传异质性。目前国内还没有关于瘢痕疙瘩易感基因成功定位的报告。研究证实瘢痕疙瘩成纤维细胞大量增生和凋亡相对减少与p53基因的功能状态密切相关。p53基因是一种重要的肿瘤抑制基因，在凋亡调控过程中起着关键性作用。人p53基因第72密码子存在CGC／CCC单核苷酸多态性（Single nucleotide polymorphisms，SNP），其编码的氨基酸分别为精氨酸（Arg）和脯氨酸（Pro），两种p53蛋白的结构和活性存在一定差异，p53_Pro蛋白诱导凋亡的能力比p53_Arg明显减弱。p73基因是p53家族成员之一，p73蛋白在结构与p53蛋白具有同源性，可以激活一些p53应答基因的转录，从而参与细胞周期调控、DNA修复和凋亡，并且以与p53同样的方式通过诱导凋亡或G1期细胞停滞来抑制细胞增殖。有研究发现在p73基因第二外显子非编码区存在两个连锁的单核苷酸多态性（G4C14-A4T14），两个SNP可形成茎环结构而影响基因的表达。有研究表明p53和p73的这两个功能性SNP可能存在相互作用，能够影响一些肿瘤发生的易感性。超强的炎症反应可能会促进瘢痕疙瘩的发生和发展。单核细胞趋化蛋白-1（Monocyte chemoattractant protein-1，MCP-1）是一种重要的细胞趋化因子，由创伤部位的成纤维细胞、上皮细胞、粒细胞等细胞合成，能特异性的募集单核／巨噬细胞游走到创伤部位参与炎症过程，在创伤愈合中起着关键作用。研究发现MCP-1基因启动子区-2518位点存在G／A多态性，具有重要的生物学意义，进一步的研究发现，此多态性可能与炎症及纤维化疾病发生的易感性相关。本研究从分子遗传学角度入手，对瘢痕疙瘩发病机制的分子基础进行研究。首先利用两个瘢痕疙瘩大家系，对2q23和7p11两个区域分别进行连锁分析，以确定中国汉族瘢痕疙瘩的易感基因位点是否与染色体2q23或7p11区域的微卫星标记存在连锁关系。同时应用聚合酶链反应—限制性片段长度多态性（Polymerase chain reaction-restriction fragment length polymorphism，PCR-RFLP）方法和相对的两对引物—聚合酶链反应（Polymerase chain reaction with confronting two-pair primers，PCR-CTPP）方法对92例辽宁籍汉族瘢痕疙瘩患者和180例种族与地域相匹配的正常对照者的p53、p73和MCP-1基因的多态性位点进行检测，以探讨这些功能性SNP位点与瘢痕疙瘩的遗传易感性的关系，为揭示瘢痕疙瘩遗传学发病机理提供重要线索。材料和方法一、研究对象1、两个辽宁汉族瘢痕疙瘩家系。2、病例组：92例瘢痕疙瘩患者为中国医科大学附属第一医院皮肤科门诊及住院患者，符合临床诊断标准：增生瘢痕组织超过原损伤范围，病程大于1年。所有患者均为彼此间无血缘关系的辽宁籍汉族人，其中男性44例，女性48例，发病年龄12～52岁，平均年龄26.4（±5.9）岁。3、正常对照组：180例，为随机抽取的健康体检志愿者，其中男84例，女96例，平均年龄24.3（±2.7）岁，彼此间无血缘关系，排除瘢痕疙瘩病史及家族史。二、实验材料10％SDS、EDTA、蛋白酶K、Tris饱和酚、氯仿、醋酸钠、无水乙醇、TE缓冲液、琼脂糖、8％变性聚丙烯酰胺溶液、LA Taq酶、限制性内切酶styⅠ、限制性内切酶AccⅡ、限制性内切酶PvuⅡ。三、实验方法1、应用聚合酶链式反应（PCR），变性聚丙烯酰胺凝胶电泳（PAGE）和银染技术，对微卫星标记D2S1328、D2S1334、D2S349、D2S1399、D2S356、D7S473、D7S499、D7S1830进行等位基因分型，判断连锁关系。2、应用聚合酶链反应—限制性片段长度多态性（PCR-RFLP）方法对p53 Arg／Pro、p73 C14T以及MCP-1 G／A等位基因进行分型。3、应用相对的两对引物—聚合酶链反应（PCR-CTPP）方法对p73 G4A等位基因进行分型。四、统计学处理用LINKAGE（Version 5.2）软件包中的MLINK软件，对所采用的微卫星位点进行两点连锁分析，计算LOD值。根据Hardy-Weinberg平衡定律计算各基因型个体数的期望值。Hardy-Weinberg平衡吻合性检验、组间基因型频率及等位基因频率的差异性比较采用x²检验，并以优势比（odds ratio，OR）及其95％可信区间（confidence interval，CI）表示相对风险度。使用SPSS 13.0软件分析数据，认为P＜0.05有统计学意义。结果1、瘢痕疙瘩致病基因位点连锁分析结果（1）两个家系均符合常染色体显性遗传。（2）两个家系的所有微卫星标记LOD值均为负值。2、瘢痕疙瘩与p53、p73基因多态性的相关研究（1）瘢痕疙瘩组和正常对照组人群p53 Arg／Pro、p73 G4C14-A4T14基因型及等位基因频率分布符合Hardy-Weinberg平衡定律。（2）瘢痕疙瘩组p53基因Arg／Pro等位基因的频率与正常对照组比较，差异无统计学意义（P＞0.05）。瘢痕疙瘩组p73基因GC／AT等位基因的频率与正常对照组比较，差异无统计学意义（P＞0.05）。（3）瘢痕疙瘩组中Arg／Arg基因型和GC／AT基因型同时存在的频率较正常对照组明显升高，差异有统计学意义（16.3％vs 6.7％，x²=6.325，P=0.012，OR=2.727，95％CI：1.219～6.104）。3、瘢痕疙瘩与MCP-1基因多态性的相关研究（1）瘢痕疙瘩组和正常对照组人群MCP-1基因启动子区-2518基因型分布符合Hardy-Weinberg平衡定律。（2）瘢痕疙瘩组MCP-1基因-2518G／A等位基因的频率与正常对照组比较，差异无统计学意义（P＞0.05）。（3）瘢痕疙瘩多发组AA和AG基因型（A等位基因携带者）频率（83.8％）明显高于单发组（63.6％，x²=4.429，P=0.035，OR=2.952，95％CI：1.051～8.290）。结论1、两个瘢痕疙瘩家系的易感位点可能不在染色体2q23和7p11区域。2、瘢痕疙瘩存在遗传异质性。3、瘢痕疙瘩组和正常对照组人群p53、p73和MCP-1基因频率已达到Hardy-Weinberg遗传平衡。4、单独比较p53基因Pro／Arg等位基因在瘢痕疙瘩人群和正常人群中的分布频率时，差异无统计学意义。单独比较p73基因GC／AT等位基因在两组人群中的分布差异时，结果亦无统计学意义。5、同时携带p53基因Arg／Arg基因型和p73基因GC／AT基因型的个体，较携带其他基因型组合的个体，发生瘢痕疙瘩的风险增高了约2.7倍。6、MCP-1基因启动子区-2518位A等位基因的存在可能与多发性瘢痕疙瘩的形成和发展有一定的相关性。更多还原

【Abstract】 Preliminary linkage analysis of keloid susceptibility loci and polymorphisms of correlation genes in Chinese Han populationIntroductionKeloids are fibrous overgrowths that develop at sites of cutaneous injury and are characterized by the deposition of excessive extracellular matrix collagen, synthesized by the increased number of fibroblasts. The excessive scar tissue proliferates beyond the boundaries of the original wound. Keloids are considered to be benign tumors and, unlike normal scars and hypertrophic scars, do not regress spontaneously, commonly recurring following excision. Although the cause of keloids is unknown, it is thought that they are due to a failure to tum off the healing process needed to repair broken skin and genetic factors influence susceptibility and modify progression.Mameros studied two families with an autosomal dominant inheritance pattern of keloids. One African-American family showed a high degree of variability in the extent of keloid formation between family members, whereas the second family from Japan showed a pattem of full penetrance and the formation of only small keloids. They performed a genomewide linkage search for genes predisposing to keloid formation in these two families and identified linkage to chromosome 2q23 for the Japanese family. The African-American family showed evidence for a keloid susceptibility locus on chromosome 7p11. The observed locus heterogeneity in autosomal dominant keloid disease is consistent with the clinical heterogeneity of this scarring disorder. This study provides the first genetic evidence for keloid susceptibility loci and serves as a basis for the identification of responsible genes. It is now accepted that alteration of apoptosis and cell proliferation has been implicated in the etiology of keloids and, probably due to dysfunctional p53 molecule. The p53 tumor suppressor acts as a transcription factor and has a central function in controlling apoptosis. The most frequent p53 polymorphism is that of codon 72, which codifies either for a praline(CCC) or an arginine(CGC). This polymorphism lies in a proline-rich region, which is important for its ability to induce apoptosis. The two alleles of p53 differ biologically, p53 Arg is able to induce 5-10 times more apoptosis than p53pro, p73, a novel p53 homolog, activates transcription of p21 and p53-responsive genes and inhibits cell growth in a p53-like manner by inducing apoptosis or G1 cell-cycle arrest. The two polymorphisms in the non-coding region of the exon 2 of p73 gene, named G4C14-A4T14, can form a stem-loop structure, and may influence p73 expression. A lot of studies suggest that interaction of these two functional SNPs of p53 and p73 gene may can increase the risk of development of some tumors.Several lines of evidence suggest that excessive inflammatory play an important role in the development and progression of keloids. Monocyte chemoattractant protein-1 (MCP-1) is a key molecule for monocyte chemotaxis and tissue extravasation and for the modulation of leukocyte function during inflammation. MCP-1 was expressed by fibroblasts, endothelial cells and infiltrating inflammatory cells of skin at sites of cutaneous injury. Recently, a biallelic A/G polymorphism in the MCP-1 promoter at position -2518 has been found, influencing the level of MCP-1 expression in response to an inflammatory stimulus. A nucleotide substitution from adenosine to guanosine at position -2518 of the MCP-1 promoter was shown to be associated with susceptibility of fibrosis-related and inflammatory disease.To investigate whether the keloid susceptibility loci occur on chromosome 2q23 and 7p11 in two Chinese pedigrees, five microsatellites on chromosome 2q23 and three microsatellites on chromosome 7p11 were selected as the genetic markers according to a recent report of the similar study. These markers were subsequently amplified by PCR, and all the PCR products were genotyped and linkage analyses were conducted. To evaluate the potential association between polymorphisms of p53, p73, MCP-1 gene and keloid development in Chinese, the distribution of allelic and genotypic frequencies of these genes were analyzed from the 92 keloid patients and 180 unrelated healthy controls of Chinese with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and polymerase chain reaction with confronting two-pair primers(PCR-CTPP).Material and method1. Subjects(1) Two keloids families in Liaoning Han population.(2) keloid patient group: 92 keloid patients were diagnosed in Department of Dermatology, First Hospital of China Medical University, conforms to the clinical diagnosis standard: excessive scar tissue proliferates beyond the boundaries of the original wound, the course of disease is more than 1 year. All patients were unrelated Liaoning Han person, in which male 44 examples, feminine 48 examples, age of onset is 12-52 year old, average age is 26.4(±5.9) year old.(3) Normal control group: 180 examples, a random selection of volunteers for health examination, 84 male and 96 female. The average age is 24.3 (+2.7) year old, no blood relationship between them, and the keloid medical history and family history were excluded.2. Materials10% SDS, EDTA, proteinase K, Tris saturated phenol, chloroform, sodium acetate, alcohol, TE buffer, agarose, 8% denatured polyacrylamide solution, LA Taq enzyme, restrictive enzyme StyⅠ, AccⅡand PvuⅡ.3. Methods(1) Polymerase chain reaction (PCR), denatured polyacrylamide gel electrophoresis (PAGE) and the silver dye technology, genotyping markers D2S1328, D2S 1334, D2S349, D2S1399, D2S356, D7S473, D7S499, D7S 1830, linkage analysis.(2) The polymerase chain reaction - restrictive fragment length polymorphism (PCR-RFLP) method to genotyping the allele type of p53 Arg/Pro, p73 C14T and MCP-1 G/A.(3) Two pairs of primers corresponding application-polymerase chain reaction (PCR-CTPP), detect the polymorphism of p73 G4A.4. Statistical AnalysisThe two-point LOD scores were calculated by MLINK program of LINKAGE(Ver.5.2). To determine whether genotype was in Hardy-Weinberg equilibrium, ax~2 was performed. The genotype frequency and the allele frequency of three genes compared by x~2, and odds ratio (OR) and 95% confidence interval (CI) indicate the relative risk. Statistical analyses were performed in SPSS 13.0.Result1. Results of linkage analysis(1) Two keloids families conform to an autosomal dominant inheritance pattern(2) The two-point LOD scores for these markers were negative values.2. The keloid susceptibility and p53, p73 gene polymorphisms(1) The allele frequencies and genotype distributions of p53 Arg/Pro, p73 GC/AT were in Hardy-Weinberg equilibrium in keloid cases and in controls.(2) There was no significant difference in the allele frequencies and genotype distributions of p53 gene between the keloid patients and the controls (p＞0.05). No significant difference was observed between the keloid patients and healthy subjects in the distribution allelic and genotypic frequencies of the p73 gene polymorphism (p＞0.05).(3) When the p53 and p73 variant alleles were combined and analyzed, there was a increase (2.727, 95% CI: 1.219～6.104) in keloid risk for individuals with Arg/Arg and GC/AT genetype simultaneously. 3. The keloid susceptibility and the MCP-1 gene polymorphism(1) The allele frequencies and genotype distributions of MCP-1 G/A was in Hardy-Weinberg equilibrium in keloid cases and in controls.(2) No significant difference was observed between the keloid patients and healthy subjects in the distribution allelic and genotypic frequencies of the MCP-1 gene polymorphism (p＞0.05).(3) A significant increase in the frequency of the carriers of the A allele was found among patients with multiple site scars with respect to those with single site scar (p=0.035,OR=2.952, 95% CI: 1.051～8.290).Conclusion1. Keloid susceptibility loci of two Chinese pedigrees were not on chromosome 2q23 and 7p11.2. Locus heterogeneity and clinical heterogeneity of keloid is obviously.3. The allele frequencies of p53, p73 and MCP-1 gene were in Hardy-Weinberg equilibrium in keloid cases and in controls.4. No significant difference was observed between the keloid patients and healthy subjects in the distribution allelic and genotypic frequencies of the alone p53 and the p73 gene polymorphism (p＞0.05).5. There was a increase (about 2.7 times) in keloid risk for individuals with Arg/Arg and GC/AT genetype simultaneously.6. The A allele of MCP-1 gene promoter -2518 may associate with the formation and development of multiple site keloid scars.更多还原