Preliminary Linkage Analysis Of Keloid Susceptibility Loci And Polymorphisms Of Correlation Genes In Chinese Han Population

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.