OBJECTIVETo explore the significance and the role of the p53 gene mutation in the exon 4 to 8 in keloid fibroblasts.

METHODSTissue samples from twelve patients with keloid and twelve hyperplastic scar respectively were harvested for in vitro culture of fibroblasts, and normal skin samples from the same patients were employed as the control. Polymerase chain reaction-based single-strained conformational polymorphism (PCR-SSCP) and DNA sequencing were employed to detect p53 gene mutations of the fibroblasts.

RESULTSThe points and frameshift mutations in the exon 4, 5, 6, 7 of p53 gene were identified in 9 of the 12 keloid tissue samples. No p53 gene mutation was detected in all hyperplastic scar and normal skin samples.

CONCLUSIONp53 gene mutation might play an important role in the formation and development of keloids.

Female ; Fibroblasts ; metabolism ; Genes, p53 ; Humans ; Keloid ; genetics ; Male ; Mutation

Keloids are benign skin tumors resulting from the excessive proliferation of connective tissue in wound skin. Precise prediction of keloid risk in trauma patients and timely early diagnosis are of paramount importance for in-depth keloid management and control of its progression. This study analyzed four keloid datasets in the high-throughput gene expression omnibus (GEO) database, identified diagnostic markers for keloids, and established a nomogram prediction model. Initially, 37 core protein-encoding genes were selected through weighted gene co-expression network analysis (WGCNA), differential expression analysis, and the centrality algorithm of the protein-protein interaction network. Subsequently, two machine learning algorithms including the least absolute shrinkage and selection operator (LASSO) and the support vector machine-recursive feature elimination (SVM-RFE) were used to further screen out four diagnostic markers with the highest predictive power for keloids, which included hepatocyte growth factor (HGF), syndecan-4 (SDC4), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), and Rho family guanosine triphophatase 3 (RND3). Potential biological pathways involved were explored through gene set enrichment analysis (GSEA) of single-gene. Finally, univariate and multivariate logistic regression analyses of diagnostic markers were performed, and a nomogram prediction model was constructed. Internal and external validations revealed that the calibration curve of this model closely approximates the ideal curve, the decision curve is superior to other strategies, and the area under the receiver operating characteristic curve is higher than the control model (with optimal cutoff value of 0.588). This indicates that the model possesses high calibration, clinical benefit rate, and predictive power, and is promising to provide effective early means for clinical diagnosis.
Humans ; Keloid/genetics* ; Nomograms ; Algorithms ; Calibration ; Machine Learning

OBJECTIVETo investigate the genome structure variation (SV) related with keloid using the whole-gene resequencing technology.

METHODSWe studied a keloid pedigree containing 4 generation of 27 people. 5 people (4 cases of keloid patients, and 1 case of normal) were selected to extract the genomic DNA. Then the whole-gene resequencing technique was used to check the variations.

RESULTSThrough database comparison and variation annotation analysis, we obtained 2 SVs associated with keloid formation. We used DAVID software to do the gene ontology and pathway analysis. We found a 168 bp inversion in gene tetraspanin 8 (TSPAN8) in all keloid patients, which contained the forth exon of TSPAN8.

CONCLUSIONSThere was no report about SVs related to keloid. In this study, we found 2 SVs associated with keloid, especially TSPAN8. The tumor cells express the TSPAN8 can up-regulate the vascular endothelial growth factor and its receptors, promote the adjacent fibroblasts secrete matrix metalloproteinases and uridylyl phosphate adenosine. So we hypothesis that the inversion of the forth exon in TSPAN8 may lead to the signal transduction disorder in the keloid patients. This study was a preliminary research. It needs a further study containing large sample to confirm.

Base Sequence ; Female ; Humans ; Keloid ; genetics ; Male ; Molecular Sequence Data ; Pedigree ; Sequence Analysis ; methods ; Tetraspanins ; genetics

OBJECTIVETo analyze the polymorphism at -509C/T site of TGF-β1 gene in patients with keloids, and to explore its relationship with the occurrence of keloid and its influence on the plasma level of TGF-β1.

METHODSOne hundred and sixty-nine patients with keloids and hospitalized from June 2011 to April 2014 were included as keloid group, and 119 healthy blood donors were enrolled as healthy control group. Venous blood of study subjects was collected. The -509C/T genotype of TGF-β1 gene was determined with PCR-restriction fragment length polymorphism technique combined with DNA sequence analysis, and the frequency of allele C or T was calculated. The theoretical frequency of the 3 genotypes CC, CT, and TT was calculated according to the theory of Hardy-Weinberg equilibrium to determine whether the gene frequency of the study subject was group representative or not. The distribution of -509C/T genotype of TGF-β1 gene among patients in keloid group was analyzed by grouping in gender, age, with or without family history of keloid, and the number of keloid respectively. Plasma level of TGF-β1 of all study subjects was determined with ELISA, and the plasma level TGF-β1 of patients with various -509C/T genotypes of TGF-β1 gene in keloid group was analyzed. The relative risk of allele frequency of patients in keloid group was analyzed by Logistic regression analysis, and the other data were processed with chi-square test and t test.

RESULTSAmong the -509C/T genotypes of TGF-β1 gene between subjects in the two groups, the distribution of genotypes CC, CT, and TT was quite similar, and they were respectively 38 cases (22.5%), 79 cases (46.7%), and 52 cases (30.8%) in keloid group and 39 persons (32.8%), 52 persons (43.7%), and 28 persons (23.5%) in healthy control group (χ² = 4.225, P>0.05). The distribution frequency of alleles C and T in the two groups were obviously different, and they were respectively 45.9% and 54.1% in keloid group and 54.6% and 45.4% in healthy control group (χ² = 4.291, P<0.05). The theoretical frequency values of the three kinds of genotypes of CC, CT, and TT were respectively 35 cases (21.0%), 84 cases (49.7%), and 50 cases (29.3%) in keloid group, and 35 persons (29.8%), 59 persons (49.6%), and 25 persons (20.6%) in healthy control group. There were no statistically significant differences between the actual genotype frequency and the theoretical values (with χ² values respectively 0.581 and 1.672, P values above 0.05), showing that the research group reached Hardy-Weinberg equilibrium. Relative risk analysis of allele frequency showed that the risk of suffering from keloid of patients carrying allele C was 1.421 times of that of patients without carrying allele C (odds ratio = 1.421, with 95% confidence interval 1.109-1.983, P < 0.05). The distribution of -509C/T genotypes of TGF-β1 gene among patients in keloid group was similar by grouping in gender, age, and number of keloid (with χ² values 0.895-5.008, P values above 0.05). Between patients with or without family history of keloid, the differences of distribution frequencies of genotypes CC and CT were significantly different, which were respectively 61.8% (21/34) and 37.8% (51/135) in patients with family history of keloid and 14.7% (5/34) and 34.1% (46/135) in patients without a family history of keloid, with χ² values respectively 6.391 and 4.835, P values below 0.05; the distribution frequency of genotype TT was close (χ² = 0.292, P > 0.05). The plasma level of TGF-β1 of patients in keloid group was (42 ± 9) µg/L, which was significantly higher than that of people in healthy control group \[(34 ± 8) µg/L, t = 4.408, P < 0.05\]. In keloid group, the plasma level of TGF-β1 in patients with genotype CC and that of patients with genotype CT was quite similar, which were respectively (43 ± 9) and (40 ± 9) µg/L (t = 0.680, P > 0.05), and they were significantly higher than that of patients with genotype TT \[(34 ± 8) µg/L, with t values respectively 2.676 and 2.137, P values below 0.05\].

CONCLUSIONSTGF-β1 gene -509C/T polymorphism was shown to be present in patients with keloids who were admitted to our hospital. It was shown to influence the plasma level of TGF-β1 in patients. The individuals who carry TGF-β1 allele C may increase the risk of developing keloid by promoting the expression of TGF-β1.

Alleles ; Gene Frequency ; Genotype ; Humans ; Keloid ; genetics ; Polymorphism, Genetic ; Transforming Growth Factor beta1 ; blood ; genetics

OBJECTIVETo screen SNP information of keloid pedigrees through whole genome sequencing.

METHODSWe Collected information and clinical data of the keloid pedigree and constructed charts of the pedigree. The DNA was extracted from peripheral venous blood samples of the pedigree to sequence the whole genome.

RESULTS27 SNP and 8 disease-associated genes were screened out.

CONCLUSIONSWhole genome sequencing technology can select new genetic mutations associated with keloid, and provide a new way for the research of keloid.

China ; ethnology ; Genome, Human ; Humans ; Keloid ; genetics ; Mutation ; Pedigree ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

OBJECTIVETo investigate the expression of Tenascin-C mRNA in keloids and hypertrophic Total RNA was isolated from normal adult skin. A cDNA fragment (base 5941-6481bp) of the scars.

METHODSfull-length human Tenascin-C cDNA was synthesized by polymerase chain reaction and subcloned in pGEM-T-easy. Dioxygen-labeled anti-sense and sense probes were synthesized by using a Sp6/T7 RNA synthesis kit in the present of Dig-UTP in vitro. The samples were taken from keloids in 10, hypertrophic scars in 10 and normal adult skin in 5. The hybridization was performed with 4% paraformaldehyde-fixed and wax-embedded sections to detect the Tenascin-C mRNA.

RESULTSThe Tenascin-C mRNA was negative in the normal adult epidermis and weakly located in the fibroblasts of the papillary dermis and the epidermal adnexa. In all of the 10 keloid specimens, the Tenascin-C mRNA was positive throughout the epidermis and widely distributed in the dermis included in the fibroblasts, endothelial cells and epidermal adnexa. In the specimens of the 3 hypertrophic scars,the Tenascin-C mRNA was also positive in the epidermis, but in the other 7 cases, it became negative. In the dermis of the hypertrophic scar,the Tenascin-C mRNA was weaker than that in the keloid, but stronger than that in the normal skin.

CONCLUSIONSThe expression of Tenascin-C mRNA is markedly enhanced in the keloids.

Cicatrix, Hypertrophic ; genetics ; metabolism ; pathology ; Female ; Humans ; Keloid ; genetics ; metabolism ; pathology ; Male ; RNA, Messenger ; metabolism ; Tenascin ; genetics ; metabolism

OBJECTIVETo explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid.

METHODSCTGF antisense oligonucleotides (ASODN) conjugated with isothiocyanate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblast (HKF) culturing media. The intracellular distribution of CTGF ASODN was observed with fluorescence microscopy in the fixed HKF. The proliferation of HKF was measured by MIT test. The apoptosis of HKF was measured with a flow cytometer. The collagen synthesis of HKF was measured by using H3-proline incorporation method.

RESULTSThe CTGF ASODN inhibited the proliferation and collagen synthesis of the HKF, compared with the control, but it increased the apoptosis after the transfection (P < 0.01).

CONCLUSIONCTGF ASODN may has anti-fibrotic effects on human keloid in vitro, and the CTGF may play an important role in promoting the fibrosis of human keloid.

Apoptosis ; Cell Differentiation ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; Fibroblasts ; cytology ; Humans ; Keloid ; metabolism ; pathology ; Oligonucleotides, Antisense ; genetics ; Transfection

OBJECTIVETo investigate the relationship between p53 gene codon 72 polymorphism and genetic predisposition to keloid in Chinese population.

METHODSPCR-based restriction fragment length polymorphism (PCR-RFLP) analysis was used to detect p53 gene codon 72 genotypes of 60 keloid samples and 102 whole blood samples from healthy controls in China.

RESULTSThere was no significant difference in the distribution of p53 gene codon 72 polymorphism between the keloid patients and the healthy controls (X2 = 2.910, P = 0.233), nor did the frequencies for Pro and Arg alleles (X2 = 0.882, P = 0.348), and there was no significant difference in the distribution of p53 gene codon 72 polymorphism in keloid patients and normal controls from China and Japan respectively (X2 = 3.942, P = 0.139; X2 = 3.260, P = 0.196). But the Arg/Arg genotype was significantly higher than the Pro/Pro genotype among the patients with keloid in shoulder and back (P < 0.01).

CONCLUSIONSThere was no significant association between the distribution of p53 gene codon 72 polymorphism and keloid in Chinese population, but Arg/Arg genotype may affect the formation of keloids in shoulder and back compared to others. Further research should be done to investigate the relationship between p53 gene codon 72 polymorphism and keloids in different sites.

Asian Continental Ancestry Group ; genetics ; Codon ; genetics ; Female ; Humans ; Keloid ; genetics ; Male ; Polymorphism, Restriction Fragment Length ; Tumor Suppressor Protein p53 ; genetics

OBJECTIVETo investigate the effects of wild type p16 gene on the proliferation and metabolism of human keloid fibroblasts.

METHODSEukaryotic expression vector pcDNA3-p16 was constructed and imported into KFb by gene transfection mediated by liposome. And the positive clones were screened by G418. The transfected and untransfected KFbs were stained by Immunocytochemical method. The expression of p16 protein was observed. The changes of the proliferation and DNA synthesis of KFb before and after transfection were observed and compared by drafting cell growth curve and by (3)H-TdR incorporation method.

RESULTSThe recombinant vector pcDNA3-p16 was successfully constructed and identified by enzyme digestion. The positive clones were identified by G418 selection for 10 days from transfected KFb and with p16 protein expression. The growth rate of transfected KFb slowed down obviously and its DNA synthesis decreased significantly (P < 0.05) when compared with those of normal KFb.

CONCLUSIONp16 gene might inhibit the growth and DNA synthesis of KFb.

Cell Proliferation ; Cells, Cultured ; DNA ; biosynthesis ; Fibroblasts ; metabolism ; pathology ; Genes, p16 ; Genetic Therapy ; Humans ; Keloid ; genetics ; pathology ; Transfection

BACKGROUNDKeloid is an intricate lesion that is probably regulated by many genes. In this study, the authors used the technique of complementary DNA (cDNA) microarray to analyse abnormal gene expression in keloids and normal control skins.

METHODSThe polymerase chain reaction (PCR) products of 8400 genes were spotted in an array on chemical-material-coated-glass plates. The DNAs were fixed on the glass plates. The total RNAs were isolated from freshly excised human keloid and normal control skins, and the mRNAs were then purified. The mRNA from both keloid and normal control skins were reversely transcribed to cDNAs, with the incorporation of fluorescent dUTP, for preparing the hybridisation probes. The mixed probes were then hybridised to the cDNA microarray. After thorough washing, the cDNA microarray was scanned for differing fluorescent signals from two types of tissues. Gene expression of tissue growth factor-beta1 (TGF-beta1) and of c-myc was detected with both RT-PCR and Northern blot hybridisation to confirm the effectiveness of cDNA microarray.

RESULTSAmong the 8400 human genes, 402 were detected with different expression levels between keloid and normal control skins. Two hundred and fifty genes, including TGF-beta1 and c-myc, were up-regulated and 152 genes were down-regulated. Higher expressions of TGF-beta1 and c-myc in keloid were also revealed using RT-PCR and Northern blot methods.

CONCLUSIONcDNA microarray analysis provides a powerful tool for investigating differential gene expression in keloid and normal control skins. Keloid is a complicated lesion with many genes involved.

DNA, Complementary ; analysis ; Humans ; Keloid ; genetics ; Oligonucleotide Array Sequence Analysis ; methods ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; Skin