Mark William James Ferguson

Genetic susceptibility to keloid disease: Transforming growth factor beta receptor gene polymorphisms are not associated with keloid disease

Abstract:  Keloid disease (KD) is an abnormal form of scarring with a familial predisposition. Genetic studies have yet to identify the genes involved in KD. Transforming growth factor β (TGF‐β) has multiple cellular activities including cellular proliferation, differentiation and extracellular matrix production. TGF‐β family members such as TGF‐β1 and TGF‐β2 are known to be involved in KD formation. However, we previously demonstrated a lack of association between common TGF‐β1 and TGF‐β2 polymorphisms and KD. Other studies have implicated TGF‐β receptors in KD pathogenesis. TGF‐β receptors were therefore selected as candidate‐susceptibility genes for this condition. Single‐nucleotide polymorphisms (SNPs) in TGF‐β receptors I, II and III (TGF‐βRI, TGF‐βRII and TGF‐βRIII) were identified and investigated for association with the risk of developing KD. A polymerase chain reaction‐restriction fragment length polymorphism method was used for genotyping novel and known TGF‐β receptor polymorphisms. DNA samples from 92 KD cases and 181 controls were examined. There were no statistically significant differences in genotype or allele frequency distributions between cases and controls for the TGF‐β receptor SNPs. Therefore, these TGF‐β receptor polymorphisms are unlikely to be associated with keloid scarring. It is possible that other SNPs in other TGF‐β family members are associated with KD. To our knowledge, this is the first report of a case‐control association study with KD and TGF‐β receptor gene polymorphisms.

Genetic susceptibility to Dupuytren's disease: transforming growth factor beta receptor (TGFβR) gene polymorphisms and Dupuytren's disease

Dupuytrens disease (DD) is a benign fibroproliferative disease of unknown cause. It is a familial condition that commonly affects Caucasians. Genetic studies have yet to identify the genes involved in DD. Transforming growth factor beta (TGFbeta) family members are multifunctional; some play a central role in wound healing and fibrosis. Previous studies have implicated TGFbeta cytokines and receptors in DD. In the light of this evidence, TGFbeta receptors represent candidate susceptibility genes for this condition. In this study, we investigated the association of single nucleotide polymorphisms (SNPs) in TGFbeta receptors one, two and three (TGFbetaRI, RII and RIII) with the risk of DD formation. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping novel and known TGFbeta receptor polymorphisms. DNA samples from 183 DD patients and 181 controls were examined. There was a statistically significant difference (p<0.05) in genotype frequency distributions between cases and controls for TGFbetaRI polymorphisms in the recessive model. However, there were no significant difference in genotype or allele frequency distributions between cases and controls for the TGFbetaRII and TGFbetaRIII SNPs.

Genetic susceptibility in Dupuytren's disease: TGF-β1 Polymorphisms and Dupuytren's disease

Dupuytrens 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 Dupuytrens 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 Dupuytrens disease. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping TGF-beta1 polymorphisms. DNA samples from 135 patients with Dupuytrens 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 Dupuytrens 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 Dupuytrens disease using single nucleotide polymorphisms in TGF-beta1.

The cell biology of suturing tendons

Trauma by suturing tendon form areas devoid of cells termed “acellular zones” in the matrix. This study aimed to characterise the cellular insult of suturing and acellular zone formation in mouse tendon. Acellular zone formation was evaluated using single grasping sutures placed using flexor tendons with time lapse cell viability imaging for a period of 12 h. Both tension and injury were required to induce cell death and cell movement in the formation of the acellular zone. DNA fragmentation studies and transmission electron microscopy indicated that cells necrosed. Parallel in vivo studies showed that cell-to-cell contacts were disrupted following grasping by the suture in tensioned tendon. Without tension, cell death was lessened and cell-to-cell contacts remained intact. Quantitative immunohistochemistry and 3D cellular profile mapping of wound healing markers over a one year time course showed that acellular zones arise rapidly and showed no evidence of healing whilst the wound healing response occurred in the surrounding tissues. The acellular zones were also evident in a standard modified “Kessler” clinical repair. In conclusion, the suture repair of injured tendons produces acellular zones, which may potentially cause early tendon failure.

Mitochondrial mutation detection using enhanced multiplex denaturing high-performance liquid chromatography

In this study, we investigated the presence of mutations within the mitochondrial genome in 40 Caucasian subjects using an enhanced multiplex denaturing high‐performance liquid chromatography (DHPLC) approach. The enhanced DHPLC approach has increased sensitivity and throughput, and reduced analysis time per individual sample compared to conventional methods. This technique involved amplifying the mitochondrial genome in 18 fragments ranging in size from 300 to 2000 bp using a novel proofreading polymerase (OptimaseTM, Transgenomic Inc., Omaha, NE) with a low misincorporation rate. Fourteen of these fragments underwent subsequent restriction digestion using a combination of five restriction enzymes to enable multiplex DHPLC analysis; the remaining four underwent conventional DHPLC. Using this complete mitochondrial genome‐screening approach, we confirmed a number of previously reported mutations and additionally identified a large number of novel mutations using an enhanced DHPLC technique.

The discovery and development of new therapeutic treatments for the improvement of scarring

Abstract: Scarring is a major cause of physical and psychological morbidity and, whilst a variety of model systems exist that have increased our understanding of the pathways and processes underlying scar formation, they have typically not translated to the development of effective therapeutic approaches for scar management. In this chapter we describe the basic science underlying both scar-free and scar-forming healing, as well as the utility and translation of pre-clinical model systems to humans. Finally we outline our pioneering approach to the discovery and development of therapeutic approaches for the prophylactic improvement of scarring in man.