Variation in WNT7A is unlikely to be a cause of familial Congenital Talipes Equinovarus
© Liu et al; licensee BioMed Central Ltd. 2008
Received: 28 September 2007
Accepted: 06 June 2008
Published: 06 June 2008
Genetic factors make an important contribution to the aetiology of congenital talipes equinovarus (CTEV), the most common developmental disorder of the lower limb. WNT7A was suggested as a candidate gene for CTEV on the basis of a genome-wide scan for linkage in a large multi-case family. WNT7A is a plausible candidate gene for CTEV as it provides a signal for pattern formation during limb development, and mutation in WNT7A has been reported in a number of limb malformation syndromes.
We investigated the role of WNT7A using a family-based linkage approach in our large series of European multi-case CTEV families. Three microsatellite markers were used, of which one (D3S2385) is intragenic, and the other two (D3S2403, D3S1252) are 700 kb 5' to the start and 20 kb from the 3' end of the gene, respectively. Ninety-one CTEV families, comprising 476 individuals of whom 211 were affected, were genotyped. LOD scores using recessive and incomplete-dominant inheritance models, and non-parametric linkage scores, excluded linkage.
No significant evidence for linkage was observed using either parametric or non-parametric models. LOD scores for the parametric models remained strongly negative in the regions between the markers, and in the 0.5 cM intervals outside the marker map. No significant lod scores were obtained when the data were analysed allowing for heterogeneity.
Our evidence suggests that the WNT7A gene is unlikely to be a major contributor to the aetiology of familial CTEV.
Congenital talipes equinovarus (CTEV), colloquially known as "clubfoot", is a common developmental disorder of the lower limb, with an incidence of 1 – 7 per 1000 births in various populations . In the UK 1–2 births per 1000 are affected . CTEV is a three dimensional malformation immediately recognisable at birth; the ankle is in the plantar flexed (equinus) position, the heel is inverted (varus) and the midfoot and forefoot are inverted and adducted (varus). Epidemiological studies implicate multifactorial inheritance. Pedigree analyses have suggested a major role of a single gene, with both variably penetrant autosomal dominant and recessive patterns fitting the data [3–5]. In a systematic review of the literature we found that a family history of CTEV was present in 24–50% of cases depending on the population studied . Identification and characterization of the causative gene(s) will contribute to our understanding and treatment of clubfoot as well as the determinants of normal limb growth and development.
Wnt genes encode a family of highly conserved cysteine rich glycoproteins that play an important role in the normal developmental processes during embryogenesis [6, 7] and in carcinogenesis . The Wnt family has at least 19 members; several of them are expressed in the limb, where they control patterning, outgrowth and/or differentiation . WNT7A is known to be involved in limb development [10–13]. In mouse  and chicken , Wnt-7a provides a signal for pattern formation during limb development. In human, mutations in WNT7A cause a range of limb malformations including Fuhrmann syndrome and Al-Awadi/Raas-Rothschild/Schinzel Phocomelia syndrome, indicating the specific and conserved importance of WNT7A in multiple aspects of vertebrate limb development . WNT7A is thus an excellent candidate gene for CTEV. In 2005, Dietz and co-workers reported a genome wide scan for linkage in a four generation CTEV family, comprising 13 individuals with clubfoot and 41 unaffected members . The highest LOD score of 2.18 was obtained for markers on chromosome 3, close to the WNT7A gene. We therefore performed a linkage study of the WNT7A locus in our large series of European multiplex CTEV families.
Recruitment of families
The study was approved by the Grampian Research Ethics Committee and the Medical Ethical Committee of the Academic Medical Center in Amsterdam.
Details of STS markers linked to the WNT7A gene used in this study
Gen Map* (cM)
Physical location (bp)**
Genotypes were analysed for linkage using the package GENEHUNTER . Parametric and non-parametric models were used, and inter-marker genetic distances were estimated from physical map distances (1 cM/Mb). The parametric models were autosomal recessive and autosomal dominant with partial penetrance. The non-parametric model (NPL, non-parametric linkage) implemented by GENEHUNTER is described in Kruglyak et al , and is a development of previous affected-pedigree-member methods, allowing efficient use of multipoint data.
The marker allele frequencies were estimated from one randomly selected individual per pedigree. The frequencies listed in order of increasing allele size were as follows (frequencies set to 0.0001 are for alleles not observed in our sample, since a frequency of 0 is not allowed by GENEHUNTER):
D3S2403: 0.0001, 0.0001, 0.0173, 0.0878, 0.0216, 0.0001, 0.0043, 0.0288, 0.1685, 0.0894, 0.5763, 0.0057;
D3S2385: 0.0155, 0.2795, 0.472, 0.1988, 0.0342;
D3S1252: 0.69, 0.0001, 0.3099.
For the heterogeneity analysis, we used the facility of GENEHUNTER to optimise the value of alpha (fraction of linked families) to maximise the LOD score.
Results of linkage analysis
Recessive Model LOD
Incomplete penetrance LOD**
WNT7A encodes a secreted protein that stimulates LMX-1 to confer dorsal patterning in the developing limb ectoderm . The linkage findings of Dietz et al.  suggested WNT7A as a highly plausible candidate gene for CTEV. They used seven markers around WNT7A on chromosome 3 in a linkage study of a single large family. Marker D3S3608, about 0.16 Mb away from WNT7A, gave the highest LOD score of 2.18. We used one marker intragenic to WNT7A, one downstream, and one D3S2403 upstream of D3S3608 to ensure that the region surrounding D3S3608 was excluded. This marker also allowed us to exclude linkage to a gene upstream of WNT7A expressed in skeleton, FIBULIN 2. We found no evidence for linkage to any of these markers in this large study, and thus we have shown that variation in either WNT7A or FIBULIN 2 is very unlikely to be significant causes of familial CTEV.
Samples from the 91 families studied represented 168 affected, and 92 unaffected meioses. It is very unlikely that our ability to detect linkage was comprised by inadequate power, as if inheritance were autosomal dominant with perfectly informative markers, these meioses, could yield a lod of 50 or so at theta = 0. Analyses were performed using both plausible models of inheritance. Heterogeneity analysis gave no evidence that the linkage might be present in a sub-set of families. Such a linkage study cannot exclude genetic variation in WNT7A as a low penetrance risk factor for CTEV, nor can it exclude linkage in rare families or in populations other than those European populations studied. However, this candidate gene was proposed on the basis of such linkage analysis, and this study does exclude it as a major contributor to familial congenital talipes equinovarus.
To date, a small number of genes have been implicated in a small proportion of CTEV families. A mutation (R279W) in the diastrophic dysplasia sulphate transporter gene (DTDST) was reported as the aetiology of CTEV in two sets of siblings of western French ancestry , but this finding was not confirmed in a large series studied by transmission disequilibrium test for linkage and association . A single missense mutation was once identified (M319K 956 T > A) in the homeodomain recognition helix of the HoxD 10 gene that segregated with disease in one large British family . But after sequencing the HoxD 10 coding and 5' and 3' untranslated regions in 190 patients and linkage analysis in one large family, this gene was suggested not responsible for idiopathic clubfoot . Heck et al.  identified a variant allele in the CASP10 gene that displays evidence of linkage and association with simplex CTEV cases. They further genotyped 40 SNPs spanning seven apoptotic genes including CASP10 in 210 simplex trios and 139 multiplex families confirming that the variation in these genes may play a role in development of clubfoot . We recently found that polymorphism of the methylenetetrahydrofolate reductase gene (MTHFR) was associated with clubfoot . Also recently N-acetylation genes, NAT1 and NAT2 were reportedly associated with CTEV . Despite rapid recent progress in the molecular basis of CTEV, a "major gene" as suggested by the segregation analyses remains elusive. The best way forward for CTEV research may be collaborative studies to perform high density genome-wide scans for linkage and association.
Our evidence suggests that the WNT7A gene is unlikely to be a major contributor to the aetiology of familial CTEV.
This research was funded by Sports Action Research for Kids (SPARKs). The authors are grateful to STEPS (National Association for Children with Lower Limb Abnormalities) and VOK (Netherlands clubfoot support group) for facilitating the recruitment of families affected by clubfoot, to Martine Barnes for administrative support, and Hazel Hailey and Anne Marie Fegen for contacting the families, and to Mr Simon Barker for helpful discussions.
- Miedzybrodzka Z: Congenital talipes equinovarus (clubfoot): A disorder of the foot but not the hand. J Anat. 2003, 202: 37-42. 10.1046/j.1469-7580.2003.00147.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Porter RW: Clubfoot: congenital talipes equinovarus. Journal of the Royal College of Surgeons of Edinburgh. 1995, 40: 66-71.PubMedGoogle Scholar
- Wang J, Palmer RM, Chung CS: The role of major gene in clubfoot. Am J Hum Genet. 1988, 42: 772-776.PubMedPubMed CentralGoogle Scholar
- Rebbeck TR, Dietz FR, Murray JC, Buetow KH: A single-gene explanation for the probability of having idiopathic talipes equinovarus. Am J Hum Genet. 1993, 53: 1051-1063.PubMedPubMed CentralGoogle Scholar
- de Andrade M, Barnholtz JS, Amos CI, Lochmiller C, Scott A, Risman M, Hecht JT: Segregation analysis of idiopathic talipes equinovarus in Texan population. Am J Med Genet. 1998, 79: 97-102. 10.1002/(SICI)1096-8628(19980901)79:2<97::AID-AJMG4>3.0.CO;2-K.View ArticlePubMedGoogle Scholar
- McMahon AP, Joyner AL, Bradley A, McMahon JA: The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell. 1992, 69: 581-595. 10.1016/0092-8674(92)90222-X.View ArticlePubMedGoogle Scholar
- Peifer M, Polakis P: Wnt signaling in oncogenesis and embryogenesis-a look outside the nucleus. Science. 2000, 287: 1606-1609. 10.1126/science.287.5458.1606.View ArticlePubMedGoogle Scholar
- Nusse R, Varmus HE: Wnt genes. Cell. 1992, 69: 1073-1087. 10.1016/0092-8674(92)90630-U.View ArticlePubMedGoogle Scholar
- Church VL, Francis-West P: Wnt signalling during limb development. Int J Dev Biol. 2002, 46: 927-936.PubMedGoogle Scholar
- Parr BA, McMahon AP: Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb. Nature. 1995, 374: 350-353. 10.1038/374350a0.View ArticlePubMedGoogle Scholar
- Riddle RD, Ensini M, Nelson C, Tsuchida T, Jessell TM, Tabin C: Induction of the LIM homeobox gene Lmx I by WNT7a establishes dorsoventral pattern in the vertebrate limb. Cell. 1995, 83: 631-640. 10.1016/0092-8674(95)90103-5.View ArticlePubMedGoogle Scholar
- Tufan AC, Tuan RS: Wnt regulation of limb mesenchymal chondrogenesis is accompanied by altered N-cadherin-related functions. FASEB J. 2001, 15: 1436-1438.PubMedGoogle Scholar
- Woods CG, Stricker S, Seemann P, Stern R, Cox J, Sherridan E, Roberts E, Springell K, Scott S, Karbani G, Sharif SM, Toomes C, Bond J, Kumar D, Al-Gazali L, Mundlos S: Mutations in WNT7A cause a range of limb malformations, including Fuhrmann syndrome and Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome. Am J Hum Genet. 2006, 79: 402-408. 10.1086/506332.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang Y, Niswander L: Interaction between the signaling molecules WNT7a and SHH during vertebrate limb development: Dorsal signals regulate anteroposterior patterning. Cell. 1995, 80: 939-947. 10.1016/0092-8674(95)90297-X.View ArticlePubMedGoogle Scholar
- Dietz FR, Cole WG, Tosi LL, Carroll NC, Wemer RD, Comstock D, Murray JC: A search for the gene(s) predisposing to idiopathic clubfoot. Clin Genet. 2005, 67: 361-362. 10.1111/j.1399-0004.2005.00407.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES: Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet. 1996, 58: 1347-63.PubMedPubMed CentralGoogle Scholar
- Chapman C, Stott NS, Port RV, Nicol RO: Genetics of club foot in Maori and Pacific people. J Med Genet. 2000, 37: 680-683. 10.1136/jmg.37.9.680.View ArticlePubMedPubMed CentralGoogle Scholar
- Tickle C: Genetics and limb development. Dev Genet. 1996, 19: 1-8. 10.1002/(SICI)1520-6408(1996)19:1<1::AID-DVG1>3.0.CO;2-D.View ArticlePubMedGoogle Scholar
- Superti-Furga A, Neumann L, Riebel T, Eich G, Steinmann B, Spranger J, Kunze J: Recessively inherited multiple epiphyseal dysplasia with normal stature, club foot, and double layered patella caused by a DTDST mutation. J Med Genet. 1999, 36: 621-624.PubMedPubMed CentralGoogle Scholar
- Bonafe L, Blanton SH, Scott A, Broussard S, Wise CA, Superti-Furga A, Hecht JT: DTDST mutations are not a frequent cause of idiopathic talipes equinovarus (club foot). J Med Genet. 2002, 39: e20-10.1136/jmg.39.4.e20.View ArticlePubMedPubMed CentralGoogle Scholar
- Dobbs MB, Gurnett CA, Pierce B, Exner GU, Robarge J, Morcuende JA, Cole WG, Templeton PA, Foster B, Bowcock AM: HOXD10 M319K mutation in a family with isolated congenital vertical talus. J Orthop Res. 2006, 24: 448-453. 10.1002/jor.20052.View ArticlePubMedGoogle Scholar
- Gurnett CA, Keppel C, Bick J, Bowcock AM, Dobbs MB: Absence of HOXD10 mutations in idiopathic clubfoot and sporadic vertical talus. Clin Orthop Relat Res. 2007, 462: 27-31. 10.1097/BLO.0b013e31805d8649.View ArticlePubMedGoogle Scholar
- Heck AL, Bray MS, Scott A, Blanton SH, Hecht JT: Variation in CASP10 gene is associated with idiopathic talipes equinovarus. J Pediatr Orthop. 2005, 25: 598-602. 10.1097/01.bpo.0000173248.96936.90.View ArticlePubMedGoogle Scholar
- Ester AR, Tyerman G, Wise CA, Blanton SH, Hecht JT: Apoptotic gene analysis in idiopathic talipes equinovarus (Clubfoot). Clin Orthop Relat Res. 2007, 462: 32-37. 10.1097/BLO.0b013e318073c2d9.View ArticlePubMedGoogle Scholar
- Sharp L, Miedzybrodzka Z, Cardy AH, Inglis J, Madrigal L, Barker S, Chesney D, Clark C, Maffulli N: The C677T polymorphism in the methylenetetrahydrofolate reductase gene (MTHFR), maternal use of folic acid supplements, and risk of isolated clubfoot: A case-parent-triad analysis. Am J Epidemiology. 2006, 164: 852-861. 10.1093/aje/kwj285.View ArticleGoogle Scholar
- Hecht JT, Ester A, Scott A, Wise CA, Iovannisci DM, Lammer EJ, Langlois PH, Blanton SH: NAT2 variation and idiopathic talipes equinovarus (clubfoot). Am J Med Genet. 2007, 143: 2285-2291. 10.1002/ajmg.a.31927.View ArticleGoogle Scholar
- Broman KW, Murray JC, Sheffield VC, White RL, Weber JL: Comprehensive human genetic maps: individual and sex-specific variation in recombination. Am J Hum Genet. 1998, 63: 861-869. 10.1086/302011.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/9/50/prepub