Dominic Furniss

Wnt Signaling and Dupuytren's Disease

BACKGROUND Dupuytrens disease is a benign fibromatosis of the hands and fingers that leads to flexion contractures. We hypothesized that multiple genetic and environmental factors influence susceptibility to this disease and sought to identify susceptibility genes to better understand its pathogenesis. METHODS We conducted a genomewide association study of 960 Dutch persons with Dupuytrens disease and 3117 controls (the discovery set) to test for association between the disease and genetic markers. We tested the 35 single-nucleotide polymorphisms (SNPs) most strongly associated with Dupuytrens disease (P<1×10(-4)) in the discovery set in three additional, independent case series comprising a total of 1365 affected persons and 8445 controls from Germany, the United Kingdom, and The Netherlands. RESULTS Initially, we observed a significant genomewide association between Dupuytrens disease and 8 SNPs at three loci. Tests of replication and joint analysis of all data from 2325 patients with Dupuytrens disease and 11,562 controls yielded an association with 11 SNPs from nine different loci (P<5.0×10(-8)). Six of these loci contain genes known to be involved in the Wnt-signaling pathway: WNT4 (rs7524102) (P=2.8×10(-9); odds ratio, 1.28), SFRP4 (rs16879765) (P=5.6×10(-39); odds ratio, 1.98), WNT2 (rs4730775) (P=3.0×10(-8); odds ratio, 0.83), RSPO2 (rs611744) (P=7.9×10(-15); odds ratio, 0.75), SULF1 (rs2912522) (P=2.0×10(-13); odds ratio, 0.72), and WNT7B (rs6519955) (P=3.2×10(-33); odds ratio, 1.54). CONCLUSIONS This study implicates nine different loci involved in genetic susceptibility to Dupuytrens disease. The fact that six of these nine loci harbor genes encoding proteins in the Wnt-signaling pathway suggests that aberrations in this pathway are key to the process of fibromatosis in Dupuytrens disease.

A variant in the sonic hedgehog regulatory sequence (ZRS) is associated with triphalangeal thumb and deregulates expression in the developing limb

A locus for triphalangeal thumb, variably associated with pre-axial polydactyly, was previously identified in the zone of polarizing activity regulatory sequence (ZRS), a long range limb-specific enhancer of the Sonic Hedgehog (SHH) gene at human chromosome 7q36.3. Here, we demonstrate that a 295T>C variant in the human ZRS, previously thought to represent a neutral polymorphism, acts as a dominant allele with reduced penetrance. We found this variant in three independently ascertained probands from southern England with triphalangeal thumb, demonstrated significant linkage of the phenotype to the variant (LOD = 4.1), and identified a shared microsatellite haplotype around the ZRS, suggesting that the probands share a common ancestor. An individual homozygous for the 295C allele presented with isolated bilateral triphalangeal thumb resembling the heterozygous phenotype, suggesting that the variant is largely dominant to the wild-type allele. As a functional test of the pathogenicity of the 295C allele, we utilized a mutated ZRS construct to demonstrate that it can drive ectopic anterior expression of a reporter gene in the developing mouse forelimb. We conclude that the 295T>C variant is in fact pathogenic and, in southern England, appears to be the most common cause of triphalangeal thumb. Depending on the dispersal of the founding mutation, it may play a wider role in the aetiology of this disorder.

Genetic screening of 202 individuals with congenital limb malformations and requiring reconstructive surgery

Background: Congenital limb malformations (CLMs) are common and present to a variety of specialties, notably plastic and orthopaedic surgeons, and clinical geneticists. The authors aimed to characterise causative mutations in an unselected cohort of patients with CLMs requiring reconstructive surgery. Methods: 202 patients presenting with CLM were recruited. The authors obtained G-banded karyotypes and screened EN1, GLI3, HAND2, HOXD13, ROR2, SALL1, SALL4, ZRS of SHH, SPRY4, TBX5, TWIST1 and WNT7A for point mutations using denaturing high performance liquid chromatography (DHPLC) and direct sequencing. Multiplex ligation dependent probe amplification (MLPA) kits were developed and used to measure copy number in GLI3, HOXD13, ROR2, SALL1, SALL4, TBX5 and the ZRS of SHH. Results: Within the cohort, causative genetic alterations were identified in 23 patients (11%): mutations in GLI3 (n = 5), HOXD13 (n = 5), the ZRS of SHH (n = 4), and chromosome abnormalities (n = 4) were the most common lesions found. Clinical features that predicted the discovery of a genetic cause included a bilateral malformation, positive family history, and having increasing numbers of limbs affected (all p<0.01). Additionally, specific patterns of malformation predicted mutations in specific genes. Conclusions: Based on higher mutation prevalence the authors propose that GLI3, HOXD13 and the ZRS of SHH should be prioritised for introduction into molecular genetic testing programmes for CLM. The authors have developed simple criteria that can refine the selection of patients by surgeons for referral to clinical geneticists. The cohort also represents an excellent resource to test for mutations in novel candidate genes.

A 10-year review of benign and malignant peripheral nerve sheath tumors in a single center: clinical and radiographic features can help to differentiate benign from malignant lesions.

Background: Malignant peripheral nerve sheath tumors are rare, and their aggressive nature mandates treatment in specialist centers. In contrast, benign peripheral nerve sheath tumors are common and are treated by a variety of specialist surgeons, including plastic surgeons. The authors aimed to detect features in the clinical presentation of peripheral nerve sheath tumors that point toward a diagnosis of malignant peripheral nerve sheath tumor and therefore prompt referral to a specialist center. Methods: All histologically diagnosed primary peripheral nerve sheath tumors from January of 1995 to December of 2004 were identified from histopathology records. Notes were reviewed and analyzed with regard to symptoms, signs, radiology, electrophysiology, surgery, and pathology. Statistical comparisons used Fishers exact test and the Mann-Whitney test. Results: During the study period, 32 cases of malignant peripheral nerve sheath tumor in 30 patients were treated. Factors in the clinical evaluation that significantly predicted the presence of malignant peripheral nerve sheath tumor included site, large size, depth in relation to the deep fascia, short duration of symptoms, and pain. Magnetic resonance imaging and computed tomography were sensitive and specific ways of confirming the clinical diagnosis. Interestingly, schwannomata were harder to distinguish from malignant peripheral nerve sheath tumors both clinically and radiologically. Conclusions: The authors have reviewed their institutional experience of peripheral nerve sheath tumors over a 10-year period. Their results will help to focus clinical and radiologic investigation of patients presenting with these tumors.

Lymphaticovenular anastomosis to prevent cellulitis associated with lymphoedema

One of the complications of lymphoedema is recurrent cellulitis. The aim was to determine whether lymphaticovenous anastomosis (LVA) was effective at reducing cellulitis in patients with lymphoedema.

Nonsense-mediated decay and the molecular pathogenesis of mutations in SALL1 and GLI3.

Mutations in SALL1 and GLI3 are responsible for human limb malformation syndromes. The molecular pathophysiology of these mutations is incompletely understood, and many conclusions have been drawn from studies performed in the mouse. We identified truncating mutations in SALL1 and GLI3 in patients with limb malformation and studied the contribution of nonsense‐mediated decay (NMD) to the expression of mutant mRNA in patient‐derived fibroblasts. Quantification of the relative proportions of mutant and wild‐type alleles was performed by pyrosequencing. In SALL1, a mutant allele causing Townes–Brocks syndrome was unexpectedly resistant to NMD, whereas a different mutation causing a much milder phenotype was susceptible to NMD. In GLI3, all three mutant alleles tested were susceptible to NMD. This work provides novel insights into the molecular pathophysiology of SALL1 and GLI3 mutations, extends the phenotypic spectrum of SALL1 mutations, and provides an example of a human mutation which does not follow the usual accepted positional rules governing mammalian NMD.

Polydactyly in the mouse mutant Doublefoot involves altered Gli3 processing and is caused by a large deletion in cis to Indian hedgehog

The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly with 6–9 triphalangeal digits in all four limbs and additional abnormalities including a broadened skull, hydrocephalus, and a thickened, kinked tail. The autopod undergoes a characteristic expansion between late embryonic day (E) 10.5 and E11.5, following the onset of ectopic Indian hedgehog (Ihh) expression in the entire distal mesenchyme, except for the zone of polarising activity (ZPA), at E10.5. We show here that limb prepattern, as indicated by expression of Gli3 and Hand2 at E9.5 is unaffected by the mutation. As both Sonic hedgehog (Shh) and Ihh expression are present in Dbf limb buds at E10.5, we generated Dbf/+;Shh−/− mutants to analyse the effects of different patterns of Hedgehog activity on the limb phenotype and molecular differentiation. Dbf/+ embryos lacking Shh showed postaxial as well as preaxial polydactyly, and the Ihh expression domain extended posteriorly into the domain in which Shh is normally expressed, indicating loss of ZPA identity. Differences in gene expression patterns in wild type, single and compound mutants were associated with differences in Gli3 processing: an increased ratio of Gli3 activator to Gli3 repressor was observed in the anterior half of Dbf/+ limb buds and in both anterior and posterior halves of compound mutant limb buds at E10.5. To identify the cause of Ihh misregulation in Dbf/+ mutants, we sequenced ∼20 kb of genomic DNA around Ihh but found no pathogenic changes. However, Southern blot analysis revealed a ∼600 kb deletion disrupting or deleting 25 transcripts, starting 50 kb 5′ of Ihh and extending away from the gene. The large deletion interval may explain the wide range of abnormalities in Dbf/+ mutants. However, we did not detect anologous deletions in cases of Laurin–Sandrow syndrome, a human disorder that shows phenotypic similarities to Dbf.

Modern surgical management of breast cancer therapy related upper limb and breast lymphoedema.

Breast cancer is the commonest cancer in the UK. Advances in breast cancer treatment means that the sequelae of treatment are affecting more women and for a longer duration. Lymphoedema is one such sequela, with wide-ranging implications, from serious functional and psychological effects at the individual level to wider economic burdens to society. Breast cancer-related lymphoedema is principally managed by conservative therapy comprising compression garments and manual decongestive massage. This approach is effective for early stages of lymphoedema, but it is not curative and the effectiveness depends on patient compliance. Early surgical approaches were ablative, gave significant morbidity and hence, reserved for the most severe cases of refractory lymphoedema. However, recent non-ablative reconstructive surgical approaches have seen a revival of interest in the prevention or surgical management of breast cancer-related lymphoedema. This review examines the modern surgical techniques for the treatment of breast cancer-related lymphoedema. Liposuction reduces the volume and symptoms of lymphedema, but requires continual compressive therapy to avoid recurrence. Lymphatic reconstruction or bypass techniques including lymph node transfer (inguinal nodes are transferred to the affected limb), lymphatico-lymphatic bypass (lymphatics bypass the axilla using a lymph vessel graft reconstructing lymphatic flow from arm to neck) and lymphaticovenous anastomoses (lymphatics in the arm are joined to the venous system aiding lymph drainage) show promise in reducing lymphedema significantly. Further research is required, including into the role of primary lymphaticovenous anastomoses in the prevention of lymphedema at the time of axillary dissection.

Linear regression analysis of Hospital Episode Statistics predicts a large increase in demand for elective hand surgery in England.

Summary Introduction We integrated two factors, demographic population shifts and changes in prevalence of disease, to predict future trends in demand for hand surgery in England, to facilitate workforce planning. Methods We analysed Hospital Episode Statistics data for Dupuytrens disease, carpal tunnel syndrome, cubital tunnel syndrome, and trigger finger from 1998 to 2011. Using linear regression, we estimated trends in both diagnosis and surgery until 2030. We integrated this regression with age specific population data from the Office for National Statistics in order to estimate how this will contribute to a change in workload over time. Results There has been a significant increase in both absolute numbers of diagnoses and surgery for all four conditions. Combined with future population data, we calculate that the total operative burden for these four conditions will increase from 87,582 in 2011 to 170,166 (95% confidence interval 144,517–195,353) in 2030. Discussion The prevalence of these diseases in the ageing population, and increasing prevalence of predisposing factors such as obesity and diabetes, may account for the predicted increase in workload. The most cost effective treatments must be sought, which requires high quality clinical trials. Our methodology can be applied to other sub-specialties to help anticipate the need for future service provision.

Effective treatment of pelvic lymphocele by lymphaticovenular anastomosis.

OBJECTIVE Pelvic lymphocele can be a severe complication associated with surgical procedures such as pelvic lymphadenectomy. Lymphaticovenular anastomosis (LVA) is increasing in popularity as a surgical treatment for lymphedema. The aim of this study was to evaluate whether LVA is an effective treatment for lymphocele, which is caused by an obstruction of the lymphatic flow in a manner similar to the development of lymphedema. METHODS Eleven female patients, who presented with lymphocele, were treated with LVA. Before the operation, 3 of them were treated with a percutaneous catheter. Lymphocele size and the volume of daily drainage were measured before and after LVA. RESULTS The lymphocele was completely resolved in 6 patients and partially resolved in the remaining 5 patients. The mean size of the pelvic lymphocele changed from 400 ml (range 50-1050 ml) to 43 ml (range 0-120 ml) (P<0.01). In the 3 patients who had percutaneous drainage catheters, the volume of fluid drained decreased from 340 ml/day to 20 ml/day after LVA. CONCLUSIONS Our technique is minimally invasive and is performed under local anesthesia. LVA is effective regardless of the size of the lymphocele. Therefore, LVA should be considered as a therapy for lymphocele because of its low invasiveness and its effectiveness in re-establishing circulation of lymphatic flow. Further studies should be performed to compare LVA with other minimally invasive techniques, such as percutaneous catheter and sclerotherapy.