TGF-(beta)1 polymorphisms and Dupuytren’s disease

Genetic susceptibility in Dupuytren’s disease: TGF-(beta)1 polymorphisms and Dupuytren’s disease

Bayat, A

Dupuytren’s disease is a benign fibroproliferative disease of unknown aetiology. It is often familial

and commonly affects Northern European Caucasian men, but genetic studies have yet to identify the relevant genes.

Transforming growth factor beta one (TGF-beta1) is a multifunctional cytokine which plays a central role in wound healing and fibrosis. It stimulates the proliferation of fibroblasts and the deposition of extracellular matrix. Previous studies have implicated TGF-beta1 in Dupuytren’s disease, suggesting that it may represent a candidate susceptibility gene for this condition.

We have investigated the association of four common single nucleotide polymorphisms in TGF-beta1 with the risk of developing Dupuytren’s disease. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping TGF-beta1 polymorphisms. DNA samples from 135 patients with Dupuytren’s disease and 200 control subjects were examined.

There was no statistically significant difference in TGF-beta1 genotype or allele frequency distributions between the patients and controls for the codons 10, 25, -509 and -800 polymorphisms.

Our observations suggest that common TGF-beta1 polymorphisms are not associated with a risk of developing Dupuytren’s disease. These data should be

interpreted with caution since the lack of association was shown in only one series of patients with only known, common polymorphisms of TGF-beta1. To our knowledge, this is the first report of a case-control association study in Dupuytren’s disease using single nucleotide polymorphisms in TGF-beta1.

J Bone Joint Surg [Br] 2002;84-B:211-5.

Received 23 January 2001; Accepted after revision 16 July 2001

Dupuytren’s disease (DD) is a nodular palmar fibromatosis causing progressive and permanent contracture of the digits. It is often familial and is common in individuals of Northern European extraction.1 More than 25% of men of Celtic origins over 60 years of age have evidence of DD,2 and it is one of the most common inheritable disorders of connective tissue in Caucasians.3

Autosomal dominance with variable penetrance has been proposed as the likely mode of inheritance,4 although no single gene has so far been identified. It is, however, unclear whether DD is a complex oligogenic condition or a simple monogenic Mendelian disorder. The identification of susceptible genetic loci would provide an ideal approach to unravelling the hereditary component of this common disease.

The myofibroblast has been shown to be a key cell responsible for the tissue contraction in DD.5,6 Iwasaki et al7 studied the histopathological changes in 43 patients and concluded that growth factors may induce proliferation of genetically abnormal myofibroblasts.

Transforming growth factor beta one (TGF-beta1) is a multifunctional cytokine which has been implicated in the pathogenesis of DD.6,8-13 It modulates cellular growth and differentiation in a wide variety of cell types including fibroblasts. It also stimulates the proliferation and migration of fibroblasts and deposition of extracellular matrix (ECM) and inhibits degradation of the latter.14,15 The precursor to TGF-beta1 is a latent protein composed of 390 amino acids,16 while the active form consists of two identical linked peptide chains of 112 amino acids, which are highly conserved between species.

Variability in the TGF-beta1 gene resulting in the induction of different levels of protein expression of ECM is a possible cause of DD,17 which would result in different levels of deposition and cellular growth, proliferation and differentiation of ECM.18

Recently, pathological dysregulation of the TGF-beta pathway has been implicated in the development of fibrotic disease.19 The TGF-beta1 gene is polymorphic and is associated with increased production of TGF-beta1 in fibrotic conditions. Several polymorphisms of the TGF-beta1 gene have been reported.18,20 One of these at codon position 25 has been associated with increased production of TGF-beta1 and fibrosis.20,21

TGF-beta1 promotes the development of a myofibroblast phenotype in normal fibroblasts.22 Several experiments have suggested that it could be involved in the pathogenesis of DD.8-11 For example, TGF-beta1 is widespread in fibroblasts in all stages (proliferative, involutional and residual) of the disease,10 and significantly stimulates proliferation of myofibroblasts in DD.13 By contrast, normal palmar fascia contains only an occasional cell staining positively for TGF-beta1. We have tested the hypothesis that there is an association between four known common TGF-beta1 polymorphisms and the development of DD.

TGF-beta1 genotyping was undertaken in Caucasian individuals with DD and compared with a control Caucasian population.

Results

Four known SNPs, two in the promoter region and two in exon 1, were genotyped using the PCR-RFLP method. The minor differences between the numbers of patients and control subjects for different SNPs were due to technical problems in genotyping some of the samples. The genotype distributions in both groups were in Hardy-Weinberg equilibrium for all SNPs examined. Allele and genotype frequencies of all four SNPs were compared using chi-squared analysis (Table II). The frequency of the genotypes (p = 0.293) and alleles (p = 0.455) for codon 10 polymorphism was similar for patients and controls as were the frequency of the genotypes (p=0.281) and alleles (p = 0.687) for codon 25 polymorphism. The genotype frequency (p = 0.498) and allele frequency (p = 0.597) for -800 polymorphism and genotype frequency (p = 0.573) and allele frequency (p = 0.357) for -509 polymorphism were both similar for patients and controls.

Codon 10 and -509 SNPs in TGF-beta1 gene show a similar allele frequency of approximately 65% and 35% for both patients and controls. By comparison, frequency of alleles of approximately 90% and 10% were observed in TGF-beta1 gene SNPs at codon 25 and -800 for both patients and controls. These ratios are of interest in determining the appropriate sample sizes for such studies. The genotype and allele frequencies of all TGF-beta1 gene SNPs examined were not significantly different (p > 0.05 for both genotype and allele frequency) between patients and controls.

Discussion

Since its description by Dupuytren in 1833,24 the exact pathogenesis of DD has remained an enigma. Various risk factors such as age, gender, smoking, diabetes, anticonvulsant medication, alcohol abuse, cirrhosis of the liver and manual labour have been implicated in its pathology.25 The relevance of some of these factors has been questioned and none so far has been proven to be of significant value in understanding the pathology.26

There are, however, two elements in the aetiology of DD which stand out. One is its common occurrence in Caucasians and the other is its familial nature.1 No genetic study has been carried out to identify a gene or genes involved in the pathogenesis. The identification of candidate loci in the development of DD is now possible using polymorphism association.

Of the growth factors which have been studied for a possible role in the development of DD, TGF-beta appears to be the most likely candidate.27 In view of the pathogenic role of TGF-beta1 in the formation of Dupuytren’s tissue which has been shown in numerous experiments,6 the TGF– beta1 gene was selected for the purpose of identifying the genetic regulation of this condition.

One results show that there is no statistically significant associations between the development of DD in Caucasian patients for known polymorphisms of TGF-beta1. In our investigation we only undertook genotypic analysis in patients with the disease. Patients suffering from other fibrotic conditions of the skin, such as scleroderma and keloid scarring were excluded.

There are a number of possible explanations for the observed lack of association between DD and the polymorphisms investigation in our study. A difference may have been present, but undetectable because of the sample size. Larger numbers may have to be studied. For an SNP with allele frequencies of approximately 65%:35% in the population, to detect an odds ratio of 2.0 as being significant at the 5% level with 80% power, would require a sample size of approximately 142 individuals. These approximate percentages do not represent averages of allele frequencies derived in our study. These values serve only as guides for calculating sample sizes. Codon 10 and -509 SNPs of TGF-beta1 gene have an approximately similar allele frequency in both patients and controls. Therefore, it would seem that our sample size is adequate to detect an association of that strength. By contrast, allele frequencies of approximately 90%:10% for any SNP in the general population would require a sample size of 283 to detect an odds ratio of 2.0 with an 80% power and p value of 0.05. Codon 25 and -800 SNPs in the TGF-31 gene have an approximately similar frequency of alleles. It is therefore possible that our present sample size would need to be increased to demonstrate any significant association.

Another possibility is that an association does not exist with the SNPs examined in this study, but is present in other as yet unidentified SNPs in the TGF-beta1 gene. It may be necessary to identify new SNPs within the TGF-beta1 gene. The common SNPs used in our experiments have been identified as lying within a specific region of the gene, between position -1321 and +966 relative to the first major transcription start site.20 It is possible that unknown TGF– beta1 polymorphisms located outside this region may be associated with DD. The technique used by previous groups18,20 for the detection and characterisation of mutations (SNPs) within the TFG-beta1 gene has been the single– stranded conformational polymorphism method. This technique is not 100% sensitive. Better techniques for detecting mutations are emerging, such as denaturing high– performance liquid chromatography using transgenomic wave nucleic acid fragment analysis. Furthermore, the association of TGF-beta1 in the pathogenesis of DD described by a number of previous authors may result from polymorphisms in other members of the TGF-beta regulatory system such as other superfamily members.

Presently, we are increasing our sample size and looking for new polymorphisms in the TGF-beta1 gene and other members of the TGF-beta signalling system. The detailed genetic basis of DD is essential to provide prognostic and diagnostic advice to patients and to develop new regimes of treatment.

The Medical Research Council (MRC), UK, has supported this study.

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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A. Bayat, J. S. Watson, J. K. Stanley, A. Alansari, M. Shah,

M. W. J. Ferguson, W. E. R. Ollier

From the Wrightington Hospital, Wigan, Withington Hospital, Manchester and the University of Manchester, England

A. Bayat, BSc, MRCS, AFRCS Ed, MRC Clinical Training Fellow

A. Alansari, BSc, MSc, Research Scientist

W. E. R. Ollier, PhD, FRCPath, Professor of Immunogenetics

Centre for Integrated Genomic Medical Research

M. W. J. Ferguson, CBE, PhD, FDS, Professor

School of Biological Sciences

University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK.

M. Shah, PhD, FRCS (Plast), Consultant Plastic and Reconstructive Surgeon J. S. Watson, MRCP, FRCS, Consultant Plastic and Reconstructive Surgeon Department of Plastic, Reconstructive and Hand Surgery, Withington Hospital, Nell Lane, West Didsbury, Manchester M20 2LR, UK.

J. K. Stanley, MCh Orth, FRCS, Professor of Hand Surgery Wrightington Hospital, Hall Lane, Wigan, Lancashire WN6 9EP, UK.

Correspondence should be sent to Dr A. Bayat.

Copyright British Editorial Society of Bone & Joint Surgery Mar 2002

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