Henk Giele

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.

Skin closure after acute shortening

Z-plasty is used to lengthen scars and wounds. We describe the use of a modified technique to shorten wounds in ten consecutive patients undergoing acute shortening of a limb as part of an Ilizarov procedure. The modified technique gave good exposure, easy closure of the wound and fewer problems with healing than standard incisions.

The role of manual occupation in the aetiology of Dupuytren's disease in men in England and Wales.

We compared the incidence of significant Dupuytren’s disease in men across occupational social classes in England and Wales, using data from the National Morbidity Survey. We found that manual occupational social class was not associated with an increased incidence of Dupuytren’s disease. In fact, the incidence rates of Dupuytren’s disease in the elderly were higher in non-manual than in manual social classes.

Cellular and humoral mechanisms of osteoclast formation in Ewing's sarcoma.

Cellular mechanisms that account for tumour osteolysis associated with Ewings sarcoma are uncertain. Osteoclasts are marrow-derived multinucleated cells (MNCs) that effect tumour osteolysis. Osteoclasts are known to form from macrophages by both receptor activator for nuclear factor-κB (RANK) ligand (RANKL)-dependent and -independent mechanisms. In this study, our aim has been to determine whether tumour-associated macrophages (TAMs) isolated from Ewings sarcoma are capable of differentiating into osteoclasts and to characterise the cellular and humoral mechanisms whereby this occurs. Tumour-associated macrophages were isolated from two Ewings sarcomas and cultured on both coverslips and dentine slices for up to 21 days with soluble RANKL and macrophage colony stimulating factor (M-CSF). Osteoclast formation from TAMs (CD14+) was evidenced by the formation of tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR)-positive MNCs, which were capable of carrying out lacunar resorption. This osteoclast formation was inhibited by the addition of bisphosphonates. Both Ewings sarcoma-derived fibroblasts and some bone stromal cells expressed RANKL and supported osteoclast formation by a contact-dependent mechanism. We also found that osteoclast differentiation occurred when Ewings TAMs were cultured with tumour necrosis factor (TNF)-α in the presence of M-CSF and that TC71 Ewings sarcoma cells stimulated osteoclast formation through the release of a soluble factor, the action of which was abolished by an antibody to TNF-α. These results indicate that TAMs in Ewings sarcoma are capable of osteoclast differentiation by both RANKL-dependent and TNF-α-dependent mechanisms and that Ewings sarcoma cells produce osteoclastogenic factor(s). Our findings suggest that anti-resorptive and anti-osteoclastogenic therapies may be useful in inhibiting the osteolysis of Ewings sarcoma.

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.

An acceptor splice site mutation in HOXD13 results in variable hand, but consistent foot malformations

HOXD13 is the most 5′ of the HOXD cluster of homeobox genes in chromosome band 2q31.1. Heterozygous expansions of a polyalanine tract in HOXD13 are typically associated with synpolydactyly characterized by insertional digit duplication associated with syndactyly. We screened for mutations of HOXD13 in patients with a variety of limb malformations and identified a novel heterozygous mutation (758‐2delA) in a three‐generation family without the typical synpolydactyly phenotype in the hands, but with bilateral partial duplication of the 2nd metatarsals within the first web space of the feet. This mutation locates in the acceptor splice site of exon 2 and is predicted to cause failure of normal splicing of HOXD13. The foot abnormality in this family is similar to that described in two families by Goodman et al. [ 1998 : Am. J. Hum. Genet. 63: 992–1000] in which different deletions of HOXD13 were reported. These findings together lend support to a distinct phenotype resulting from haploinsufficiency of HOXD13.

Malignant melanoma and bone resorption.

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51−) differentiated into osteoclasts (CD14−/CD51+) in the presence of receptor activator for nuclear factor κB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-α and interleukin (IL)-1α. RT–PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively.

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.