| Background and Objective:Keloids are the results of irregular wound healing process. They are characterized by the formation of excess scar tissues that proliferate beyond the boundaries of the original wound. Keloids are locally aggressive, and in contrast to hypertrophic scars, they invade healthy tissue, therefore, they are much more likely to recur after treatment of removal or reduction and can actively persist for many years. In this manner, keloids behave clinically as benign tumors that form as results of inherited defects in wound healing. Fibroblasts are testified to be the functional cells which contribute to the pathogenesis of keloid. The unbalance of proliferation and apoptosis in fibroblasts may directly lead to the accumulation of fibroblasts in keloid tissue.Apoptosis is one of the most important regulatory mechanisms of tissue homeostasis in organisms. Many human pathologies are associated with aberrant apoptosis. It is now believed that clonal expansion and tumor growth are the results of the deregulation of intrinsic proliferation and apoptosis. Failure of apoptosis could allow the survival of transformed cells that are prone to undergo further genetic damage and play an important role in the pathogenesis of tumors. In recent years, studies of occurrence and regulation of apoptosis suggest that many genes are implicated in the process. Now it is considered that p53 gene and Fas gene arefamily to apoptosis.The p53 gene on chromosome 17pl3.1 encodes a 53kd phosphoprotein that acts as a transcription factor and has tumor-suppressor function. Evidences suggest that p53 controls a cell cycle checkpoint responsible for maintaining the integrity of the genome. It has been shown that the wide-type p53 induces cell cycle arrest and/or apoptosis in response to DNA damage.The cell-surface Fas receptor (Fas) on chromosome 10p24.1, also termed Apo-1 or CD95, is a member of the tumor necrosis factor and nerve growth factor family of receptors. Fas is widely distributed in skin components and it has been shown that Fas receptor stimulation can induce apoptosis or proliferation in human dermal fibroblasts.As a complex disease, the evolution of Keloid is a multi-step, multi-phase and multi-factor process. Study on polymorphisms of caspase family genes is an important content of disease genomics in Keloid. Single Nucleotide Polymorphisms (SNPs), are single base pair positions in genomic DNA at which different sequence alternative (alleles) exist in normal individuals in some population(s), wherein the least frequent allele has an abundance of 1% or greater. Due to the wide distribution through whole genome and comparable genetic stability, Many properties of SNPs make them powerful markers for studying complex diseases, drug susceptibility and even evolutionary history of human populations.Single nucleotide polymorphisms (SNPs) in regulatory regions could affect expression of mRNA and proteins. SNPS in coding regions could change structures and functions of proteins. They greatly decide phenotype variation of human. A tremendous amount of SNPS exists in the human genome with high density, but their patterns of distribution differ considerably among populations. To date, there is no report on polymorphisms of p53n Fas genes in healthy people of Han nationalityor on association analysis between SNPs in p53^ Fas genes and complex disease. In this study, we performed polymerase chain reaction-reverse dot blot(PCR-RDB) and DNA direct sequencing to analyse the entire coding region and their flanking sequences of p53^ Fas genes in healthy people and Keloid patients of Han nationality and to determine whether SNPs and mutations of these genes could be involved in Keloid development. The aim of the present study was, therefore, to provide some evidence to finding susceptibility gene of keloid and to explore the mechanism of keloid further. Finally to study the p53 gene detection kit which can be used to predicate high-risk individuals for Keloid. Subject and Methods:1. SubjectThe patients with keloid and unrelated healthy controls were collected from GuangDong province. 65 patients with keloid were approved with clinic and pathology in Nanfang Hospital, of which males account for 36 and female account for 29, the age was in range from 10 to 56 years, their average age was 28.4 years.The position of keloids were in prothoraxN deltoid and earlap, the medical record were in range from 5 months to 12 years. 80 unrelated healthy controls who did not suffer from keloid after traumatic record and had not familial keloid disease, of which males account for 42 and female account for 38, the age was in range from 13 to 58 years, their average age was 29.3 years. Genomic DNA was prepared from 2ml peripheral blood, the samples of blood used Sodium Citrate anticoagulant,which were deposited in -20 °C refrigeratory.2. Methods(1) Genomic DNA was distilled from the kit which was offered from Beijin Saibaisheng company.(2) DNA direct sequencing was used to detect the polymorphisms in the exon4 ofp53 gene^ promoter and intron7 of Fas gene from 18 samples ot GuangDong province.(3) The p53 code72 and Fas-670 genotypes were determined by polymerase chain reaction-reverse dot blot(PCR-RDB) and DNA direct sequencing in 65 patients with keloid and 80 unrelated healthy controls. The distribution of genotypes and alleles in keloid group and in controls were compared to examine the association between gene polymorphism and keloid. The aim to the present study was, therefore, to provide some evidence for finding susceptibility gene of keloid and to explore the mechanism of keloid further.(4) The models of gene detection kit was produced which adopted PCR-RDB(5) The gene counting method was used to estimate the allele and genotype frequencies in patients group and normal subject. The Hardy-Weinberg equilibrium for the frequencies of genotypes was tested by x2 analysis, x2 analysis was used to compare differences in the distribution of genotypes between patients and the normal subjects and other risk factors in patients.The odds ratio(OR) and 95% confidence intervals(CI) were calculated to estimate the relative risk of keloid disease associated . Statistic analyses were performed with the SPSS for windows 10.0 statistic program package. Values were considered statistically significant at P<0.05.Results:1.Three SNPs were identified in coding exons and promoter sequences of p53 andFas gene in a southern Chinese population.They were located in exon4 of p53 gene>intron7 and promoter of Fas gene respectively, of which the SNP of Fas geneintron7 was a novel SNP.2.The distribution of p53 code72 and Fas-670 in the unrelated healthy controls andkeloid patients were in the Hardy-Weinberg equilibrium, which indicated that thefrequency of gene was provided with representation.3. p53 gene code72: The frequency of the p53 C allele among keloid patients wassignificantly higher than that among healthy controls(x2=6.334,P=0.012, P<0.05 ). The CG and GG genotype distribution among keloid patients was not significantly different from that among healthy controls(P=0.275 and 0.150, respectively). However, the CC genotype frequency among keloid patients was significantly higher than that among healthy controls(x2=6.115,P=0.013,P<0.05 ). The p53 CC genotype significantly increased the risk of developing keloid, compared to the combination of CG and GG genotypes, with the odds ratio(OR) of 2.430(95%CI: 1.193—4.949). 4.Fas-670: the study indicated that the frequency of the allele and genotype distribution among keloid patients was not significantly different from that among healthy controls. However, compared to the healthy controls, the frequency of AA genotype distribution among keloid patients has a significantly increased trend(x2=4.375,P=0.055).5.The p53 gene detection kit which can be used to predicate high-risk individuals for keloid was produced successful. Conclusion:l.In this study a novel SNP of Fas gene intron7 was detected in population from Guangdong Province, and the SNPs of p53 code72 and Fas-670 were approved. 2.The p53 gene codon 72 polymorphism may play a role in susceptibility to detect keloid, The relative risk suffered from keloid carrying CC genotype was 2.43 times of the CG and GG genotype(OR=2.430,95%CI: 1.193—4.949).3. Determination of the p53 codon 72 genotype may be used as a stratification marker to predicate high-risk individuals for keloid.4. The p53 gene detection kit can be used to predicate high-risk individuals for keloid, therefore, it is useful to clinical diagnosis and therapy. |
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