Identification of biomarkers and whole genome scanning in Dupuytren's Disease

Background: Dupuytren’s Disease (DD) is a common fibroproliferative disease of unknown origin affecting the hand. The hypotheses of this thesis are; i) DD is a complex polygenic disease and ii) the cells that comprise DD tissue may be derived not only from fascia but also from fat and dermis. On this basis, the overall aims of this thesis were to: ia) estimate the level of penetrance in a UK DD population; ib) present the largest DD family available to date (from Iceland) and attempt linkage analysis following whole genomic scanning; ii) observe stem cell markers as potential biomarkers in DD fascia, fat and skin compared to appropriate controls. Methods: Patients diagnosed with DD (n = 135) were randomly selected from hospitals in the UK. One family was identified in Reykjavik, Iceland. Family pedigrees were compiled for each patient and subsequently analysed to calculate a revised sibling recurrence risk ratio (λs) and estimate the level of penetrance. Members of the Icelandic family from whom DNA was available were genotyped using the Affymetrix 6.0 SNP chip and linkage analysis performed to identify susceptible genetic regions for DD. Biopsies of DD patients (n=9) with digital fixed flexion deformity were taken at operation from the diseased cord, nodule, peri-nodular fat, distant palmar fat and skin. Fluorescence Activated Cell Sorting (FACS), immunohistochemistry and Quantitative Real Time Polymerase Chain reaction (QRT-PCR) were used to identify expression of five mesenchymal (CD’s 13, 29, 44, 90, 166) and two haematopoietic (CD’s 34,117) stem cell markers. Results: ia) The DD status of 1156 relatives of the 135 probands was established. Patients with a family history had a greater severity of disease than those who did not (p<0.05). Despite published studies pointing towards DD being a polygenic disease, DD appears to show a near autosomal pattern of inheritance with variable penetrance (estimated penetrance level: 18%). The revised sibling recurrence risk ratio for the UK, λs=6.2. ib) The Icelandic family had 25 affected members. The pedigree approximated autosomal dominance. Results identified 8 candidate genes with susceptibility loci over 3 chromosomes (GRK4, ADD1, SH3BP2 on chromosome 4; SGCZ on chromosome 8; NAV2, MRGPRX1, SAAL1, MYOD1 on chromosome 11).ii) There was a significantly higher expression of selected stem cell markers in DD tissue over control: CD13 protein expression was increased in all DD tissue compared to controls (p=0.02), while CD44 was significantly over expressed in the cord and nodule (p=0.02). CD34 in the skin was also significantly enhanced (p=0.008). The mean number of positive cells expressing the stem cell markers was significantly greater in DD cord tissue compared to healthy carpal tunnel fascia (p= 0.003).Conclusions: ia) DD appears to show a near autosomal pattern of inheritance with variable penetrance (estimated at 18%), and the sibling recurrence risk ratio was revised. These calculations provide a more up to date evaluation of familial aggregation and more accurate estimates for complex bio-informatic DD models. ib) A large DD pedigree has been identified and a whole genome scan has found 8 potential candidate genes within susceptibility loci on chromosomes 4, 8 and 11. ii) The analysis of stem cell markers in various DD tissue components questions the previously proposed origin of abnormal DD fibroblasts from the fascia alone and suggests that the surrounding palmar fat and skin are also involved in DD pathobiology. Future work may involve functional analysis of these stem cell markers and potential use as biomarkers.