Genetics (clinical); Genetics; Molecular Biology; General Medicine; Human Genetics; cell signalling; Wnt Pathway; early-onset osteoporosis; osteoblastogenesis; Bone Mineral Density
Abstract :
[en] Monogenic early-onset osteoporosis (EOOP) is a rare disease defined by low bone mineral density (BMD) that results in increased risk of fracture in children and young adults. Although several causative genes have been identified, some of the EOOP causation remains unresolved. Whole-exome sequencing revealed a de novo heterozygous loss-of-function mutation in WNT11 (NM_004626.2:c.677_678dup p.Leu227Glyfs*22) in a 4-year-old boy with low BMD and fractures. We identified two heterozygous WNT11 missense variants (NM_004626.2:c.217G > A p.Ala73Thr) and (NM_004626.2:c.865G > A p.Val289Met) in a 51-year-old woman and in a 61-year-old woman respectively, both with bone fragility. U2OS cells with heterozygous WNT11 mutation (NM_004626.2:c.690_721delfs*40) generated by CRISPR-Cas9 showed reduced cell proliferation (30%) and osteoblast differentiation (80%) as compared with wild-type U2OS cells. The expression of genes in the Wnt canonical and non-canonical pathways was inhibited in these mutant cells, but recombinant WNT11 treatment rescued the expression of Wnt pathway target genes. Furthermore, the expression of RSPO2, a WNT11 target involved in bone cell differentiation, and its receptor LGR5, was decreased in WNT11 mutant cells. Treatment with WNT5A and WNT11 recombinant proteins reversed LGR5 expression, but WNT3A recombinant protein treatment had no effect on LGR5 expression in mutant cells. Moreover, treatment with recombinant RSPO2 but not WNT11 or WNT3A activated the canonical pathway in mutant cells. In conclusion, we have identified WNT11 as a new gene responsible for EOOP, with loss-of-function variant inhibiting bone formation via Wnt canonical and non-canonical pathways. WNT11 may activate Wnt signaling by inducing the RSPO2-LGR5 complex via the non-canonical Wnt pathway.
Disciplines :
Genetics & genetic processes
Author, co-author :
Caetano Da Silva, Caroline ; INSERM U1132 and Université de Paris, Reference Centre for Rare Bone Diseases, Hospital Lariboisière, F-75010 Paris, France
Edouard, Thomas ; Endocrine Bone Diseases and Genetics Unit, Reference Centre for Rare Diseases of Calcium and Phosphate Metabolism, ERN BOND, OSCAR Network, Pediatric Clinical Research Unit, Children's Hospital, RESTORE INSERM U1301, Toulouse University Hospital, Toulouse, 31300, France
Fradin, Melanie; Service de génétique clinique, centre de référence des anomalies du développement de l'Ouest, hôpital Sud de Rennes, F-35033 Rennes, France
Aubert-Mucca, Marion; Endocrine Bone Diseases and Genetics Unit, Reference Centre for Rare Diseases of Calcium and Phosphate Metabolism, ERN BOND, OSCAR Network, Pediatric Clinical Research Unit, Children's Hospital, RESTORE INSERM U1301, Toulouse University Hospital, Toulouse, 31300, France
Ricquebourg, Manon; INSERM U1132 and Université de Paris, Reference Centre for Rare Bone Diseases, Hospital Lariboisière, F-75010 Paris, France
Raman, Ratish ; Université de Liège - ULiège > GIGA > GIGA I3 - Laboratory for Organogenesis and Regeneration
Salles, Jean Pierre; Endocrine Bone Diseases and Genetics Unit, Reference Centre for Rare Diseases of Calcium and Phosphate Metabolism, ERN BOND, OSCAR Network, Pediatric Clinical Research Unit, Children's Hospital, RESTORE INSERM U1301, Toulouse University Hospital, Toulouse, 31300, France
Charon, Valérie; Department of Radiology, CHU de Rennes, F-35000 Rennes, France
Guggenbuhl, Pascal; Department of Rheumatology, CHU de Rennes, F-35000 Rennes, France
Muller, Marc ; Université de Liège - ULiège > GIGA > GIGA I3 - Laboratory for Organogenesis and Regeneration
Cohen-Solal, Martine; INSERM U1132 and Université de Paris, Reference Centre for Rare Bone Diseases, Hospital Lariboisière, F-75010 Paris, France
Collet, Corinne; INSERM U1132 and Université de Paris, Reference Centre for Rare Bone Diseases, Hospital Lariboisière, F-75010 Paris, France ; UF de Génétique Moléculaire, Hôpital Robert Debré, APHP, F-75019 Paris, France
Language :
English
Title :
WNT11, a new gene associated with early-onset osteoporosis, is required for osteoblastogenesis.
Slemenda, C.W., Turner, C.H., Peacock, M., Christian, J.C., Sorbel,J., Hui, S.L. and Johnston, C.C. (1996) The genetics of proximalfemur geometry, distribution of bone mass and bone mineraldensity. Osteoporos. Int., 6, 178-182.
Gu(c)guen, R., Jouanny, P., Guillemin, F., Kuntz, C., Pourel, J. andSiest, G. (1995) Segregation analysis and variance componentsanalysis of bone mineral density in healthy families. J. Bone Miner.Res., 10, 2017-2022.
Koromani, F., Trajanoska, K., Rivadeneira, F. and Oei, L.(2019) Recent advances in the genetics of fractures inosteoporosis. Front. Endocrinol., 10, 337. https://doi.org/10.3389/fendo.2019.00337.
Gregson, C.L., Newell, F., Leo, P.J., Clark, G.R., Paternoster, L.,Marshall, M., Forgetta, V., Morris, J.A., Ge, B., Bao, X. et al. (2018)Genome-wide association study of extreme high bone mass:contribution of common genetic variation to extreme BMD phenotypes and potential novel BMD-associated genes. Bone, 114,62-71.
Morris, J.A., Kemp, J.P., Youlten, S.E., Laurent, L., Logan, J.G., Chai,R.C., Vulpescu, N.A., Forgetta, V., Kleinman, A., Mohanty, S.T. et al.(2019) An atlas of genetic inf luences on osteoporosis in humansand mice. Nat. Genet., 51, 258-266.
Forgetta, V., Keller-Baruch, J., Forest, M., Durand, A., Bhatnagar,S., Kemp, J.P., Nethander, M., Evans, D., Morris, J.A., Kiel, D.P. et al.(2020) Development of a polygenic risk score to improve screening for fracture risk: a genetic risk prediction study. PLoS Med.,17, e1003152. https://doi.org/10.1371/journal.pmed.1003152.
Collet, C., Ostertag, A., Ricquebourg, M., Delecourt, M., Tueur, G.,Isidor, B., Guillot, P., Schaefer, E., Javier, R.M., Funck-Brentano, T.et al. (2017) Primary osteoporosis in young adults: genetic basisand identification of novel variants in causal genes. JBMR Plus, 2,12-21.
Mäkitie, R.E., Kämpe, A., Costantini, A., Alm, J.J., Magnusson, P.and Mäkitie, O. (2020) Biomarkers in WNT1 and PLS3 osteoporosis: altered concentrations of DKK1 and FGF23. J. Bone Miner. Res.,35, 901-912.
Hartikka, H., Mäkitie, O., Männikkö, M., Doria, A.S., Daneman, A.,Cole, W.G., Ala-Kokko, L. and Sochett, E.B. (2005) Heterozygousmutations in the LDL receptor-related protein 5 (LRP5) gene areassociated with primary osteoporosis in children. J. Bone Miner.Res., 20, 783-789.
Korvala, J., Löija, M., Mäkitie, O., Sochett, E., J1/4ppner, H., Schnabel,D., Mora, S., Cole, W.G., Ala-Kokko, L. and Männikkö, M. (2012)Rare variations in WNT3A and DKK1 may predispose carriers toprimary osteoporosis. Eur. J. Med. Genet., 55, 515-519.
St1/4rznickel, J., Rolvien, T., Delsmann, A., Butscheidt, S., Barvencik, F., Mundlos, S., Schinke, T., Kornak, U., Amling, M. and Oheim,R. (2021) Clinical phenotype and relevance of LRP5 and LRP6variants in patients with early-onset osteoporosis (EOOP). J. BoneMiner. Res., 36, 271-282.
Baron, R. and Kneissel, M. (2013) WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat.Med., 19, 179-192.
Huybrechts, Y., Mortier, G., Boudin, E. and Van Hul, W. (2020)WNT Signaling and bone: lessons from skeletal dysplasiasand disorders. Front. Endocrinol., 11, 165. https://doi.org/10.3389/fendo.2020.00165.
Krishnan, V., Bryant, H.U. and Macdougald, O.A. (2006) Regulation of bone mass by Wnt signaling. J. Clin. Invest., 116, 1202-1209.
Kubota, T., Michigami, T. and Ozono, K. (2009) Wnt signaling inbone metabolism. J. Bone Miner. Metab., 27, 265-271.
Maeda, K., Kobayashi, Y., Udagawa, N., Uehara, S., Ishihara, A.,Mizoguchi, T., Kikuchi, Y., Takada, I., Kato, S., Kani, S. et al.(2012) Wnt5a-Ror2 signaling between osteoblast-lineage cellsand osteoclast precursors enhances osteoclastogenesis. Nat.Med., 18, 405-412.
Albers, J., Schulze, J., Beil, F.T., Gebauer, M., Baranowsky, A., Keller,J., Marshall, R.P., Wintges, K., Friedrich, F.W., Priemel, M. et al.(2011) Control of bone formation by the serpentine receptorFrizzled-9. J. Cell Biol., 192, 1057-1072.
Zhang, Z., Rankin, S.A. and Zorn, A.M. (2013) Different thresholdsof Wnt-frizzled 7 signaling coordinate proliferation, morphogenesis and fate of endoderm progenitor cells. Dev. Biol., 378,1-12.
Häusler, K.D., Horwood, N.J., Chuman, Y., Fisher, J.L., Ellis, J.,Martin, T.J., Rubin, J.S. and Gillespie, M.T. (2004) Secreted frizzledrelated protein-1 inhibits RANKL-dependent osteoclast formation. J. Bone Miner. Res., 19, 1873-1881.
de Lau, W., Peng, W.C., Gros, P. and Clevers, H. (2014) The Rspondin/Lgr5/Rnf43 module: regulator of Wnt signal strength.Genes Dev., 28, 305-316.
Miller, J.R. (2002) The Wnts. Genome Biol., 3, REVIEWS3001.https://doi.org/10.1186/gb-2001-3-1-reviews3001.
Caetano da Silva, C., Ricquebourg, M., Orcel, P., Fabre, S., FunckBrentano, T., Cohen-Solal, M. and Collet, C. (2021) More severephenotype of early-onset osteoporosis associated with recessiveform of LRP5 and combination with DKK1 or WNT3A. Mol. Genet.Genomic Med., 9, e1681. https://doi.org/10.1002/mgg3.1681.
Turin, C.G., Joeng, K.S., Kallish, S., Raper, A., Asher, S., Campeau,P.M., Khan, A.N. and Al Mukaddam, M. (2021) Heterozygous variant in WNT1 gene in two brothers with early onset osteoporosis.Bone Rep., 15, 101118. https://doi.org/10.1016/j.bonr.2021.101118.
Rocha-Braz, M., França, M.M., Fernandes, A.M., Lerario, A.M.,Zanardo, E.A., de Santana, L.S., Kulikowski, L.D., Martin, R.M.,Mendonca, B.B. and Ferraz-de-Souza, B. (2020) Comprehensivegenetic analysis of 128 candidate genes in a cohort with idiopathic, severe, or familial osteoporosis. JES, 4, 1-13.
Mäkitie, O. and Zillikens, M.C. (2021) Early-onset osteoporosis.Calcif. Tissue Int. https://doi.org/10.1007/s00223-021-00885-6.
Uysal-Onganer, P. and Kypta, R.M. (2012) Wnt11 in 2011 -theregulation and function of a non-canonical Wnt. Acta Physiol.,204, 52-64.
Boyan, B.D., Olivares-Navarrete, R., Berger, M.B., Hyzy, S.L. andSchwartz, Z. (2018) Role of Wnt11 during osteogenic differentiation of human mesenchymal stem cells on microstructuredtitanium surfaces. Sci. Rep., 8, 8588. https://doi.org/10.1038/s41598-018-26901-8.
Friedman, M.S., Oyserman, S.M. and Hankenson, K.D. (2009)Wnt11 promotes osteoblast maturation and mineralizationthrough R-spondin 2. J. Biol. Chem., 284, 14117-14125.
Lako, M., Strachan, T., Bullen, P., Wilson, D.I., Robson, S.C. andLindsay, S. (1998) Isolation, characterisation and embryonicexpression of WNT11, a gene which maps to 11q13.5 and haspossible roles in the development of skeleton, kidney and lung.Gene, 219, 101-110.
Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J.,Grody, W.W., Hegde, M., Lyon, E., Spector, E. et al. (2015) Standardsand guidelines for the interpretation of sequence variants: ajoint consensus recommendation of the American College ofMedical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med., 17, 405-424.
Touma, M., Kang, X., Gao, F., Zhao, Y., Cass, A.A., Biniwale, R., Xiao,X., Eghbali, M., Coppola, G., Reemtsen, B. and Wang, Y. (2017)Wnt11 regulates cardiac chamber development and diseaseduring perinatal maturation. JCI Insight., 2, e94904. https://doi.org/10.1172/jci.insight.94904.
Sinha, T., Lin, L., Li, D., Davis, J., Evans, S., Wynshaw-Boris, A. andWang, J. (2015) Mapping the dynamic expression of Wnt11 andthe lineage contribution of Wnt11-expressing cells during earlymouse development. Dev. Biol., 398, 177-192.
Garriock, R.J., D(tm)Agostino, S.L., Pilcher, K.C. and Krieg, P.A. (2005)Wnt11-R, a protein closely related to mammalian Wnt11, isrequired for heart morphogenesis in Xenopus. Dev. Biol., 279,179-192. https://doi.org/10.1016/j.ydbio.2004.12.013 Erratum in:Dev. Biol. 2008 Oct 322, 235.
Heisenberg, C.P., Tada, M., Rauch, G.J., Saúde, L., Concha, M.L.,Geisler, R., Stemple, D.L., Smith, J.C. and Wilson, S.W. (2000)Silberblick/Wnt11 mediates convergent extension movementsduring zebrafish gastrulation. Nature, 405, 76-81.
Heisenberg, C.P. and N1/4sslein-Volhard, C. (1997) The function ofsilberblick in the positioning of the eye anlage in the zebrafishembryo. Dev. Biol., 184, 85-94.
Laine, C.M., Joeng, K.S., Campeau, P.M., Kiviranta, R., Tarkkonen,K., Grover, M., Lu, J.T., Pekkinen, M., Wessman, M., Heino, T.J.et al. (2013) WNT1 mutations in early-onset osteoporosis andosteogenesis imperfecta. N. Engl. J. Med., 368, 1809-1816.
Zhu, J.H., Liao, Y.P., Li, F.S., Hu, Y., Li, Q., Ma, Y., Wang, H., Zhou,Y., He, B.C. and Su, Y.X. (2018) Wnt11 promotes BMP9-inducedosteogenic differentiation through BMPs/Smads and p38 MAPKin mesenchymal stem cells. J. Cell. Biochem., 119, 9462-9473.
Mori, H., Yao, Y., Learman, B.S., Kurozumi, K., Ishida, J., Ramakrishnan, S.K., Overmyer, K.A., Xue, X., Cawthorn, W.P., Reid,M.A. et al. (2016) Induction of WNT11 by hypoxia and hypoxiainducible factor-1α regulates cell proliferation, migration andinvasion. Sci. Rep., 6, 21520. https://doi.org/10.1038/srep21520.
Tenjin, Y., Kudoh, S., Kubota, S., Yamada, T., Matsuo, A., Sato, Y.,Ichimura, T., Kohrogi, H., Sashida, G., Sakagami, T. and Ito, T.(2019) Ascl1-induced Wnt11 regulates neuroendocrine differentiation, cell proliferation, and E-cadherin expression in smallcell lung cancer and Wnt11 regulates small-cell lung cancerbiology. Lab. Investig., 99, 1622-1635.
Wilkesmann, S., Fellenberg, J., Nawaz, Q., Reible, B., Moghaddam, A., Boccaccini, A.R. and Westhauser, F. (2020) Primaryosteoblasts, osteoblast precursor cells or osteoblast-like celllines: which human cell types are (most) suitable for characterizing 45S5-bioactive glass? J. Biomed. Mater. Res. A, 108, 663-674.
Zhou, Y., Lin, J., Shao, J., Zuo, Q., Wang, S., Wolff, A., Nguyen, D.T.,Rintoul, L., Du, Z., Gu, Y. et al. (2019) Aberrant activation of Wntsignaling pathway altered osteocyte mineralization. Bone, 127,324-333.
Maye, P., Zheng, J., Li, L. and Wu, D. (2004) Multiple mechanismsfor Wnt11-mediated repression of the canonical Wnt signalingpathway. J. Biol. Chem., 279, 24659-24665.
Bisson, J.A., Mills, B., Paul Helt, J.C., Zwaka, T.P. and Cohen, E.D.(2015) Wnt5a and Wnt11 inhibit the canonical Wnt pathwayand promote cardiac progenitor development via the caspasedependent degradation of AKT. Dev. Biol., 398, 80-96.
van Amerongen, R., Fuerer, C., Mizutani, M. and Nusse, R.(2012) Wnt5a can both activate and repress Wnt/β-catenin signaling during mouse embryonic development. Dev. Biol., 369,101-114.
Qin, L., Yin, Y.T., Zheng, F.J., Peng, L.X., Yang, C.F., Bao, Y.N.,Liang, Y.Y., Li, X.J., Xiang, Y.Q., Sun, R. et al. (2015) WNT5A promotes stemness characteristics in nasopharyngeal carcinomacells leading to metastasis and tumorigenesis. Oncotarget, 6,10239-10252.
Kumawat, K. and Gosens, R. (2016) WNT-5A: signaling and functions in health and disease. Cell. Mol. Life Sci., 73, 567-587.
Jho, E.H., Zhang, T., Domon, C., Joo, C.K., Freund, J.N. and Costantini, F. (2002) Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway.Mol. Cell. Biol., 22, 1172-1183.
Mov(c)rare-Skrtic, S., Henning, P., Liu, X., Nagano, K., Saito, H.,Börjesson, A.E., Sjögren, K., Windahl, S.H., Farman, H., Kindlund,B. et al. (2014) Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures. Nat. Med.,20, 1279-1288.
Glinka, A., Dolde, C., Kirsch, N., Huang, Y.L., Kazanskaya, O.,Ingelfinger, D., Boutros, M., Cruciat, C.M. and Niehrs, C. (2011)LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling. EMBO Rep., 12, 1055-1061.
de Lau, W., Barker, N., Low, T.Y., Koo, B.K., Li, V.S., Teunissen,H., Kujala, P., Haegebarth, A., Peters, P.J., van de Wetering, M.et al. (2011) Lgr5 homologues associate with Wnt receptors andmediate R-spondin signalling. Nature, 476, 293-297.
Gong, X., Carmon, K.S., Lin, Q., Thomas, A., Yi, J. and Liu, Q. (2012)LGR6 is a high affinity receptor of R-spondins and potentiallyfunctions as a tumor suppressor. PLoS One, 7, e37137. https://doi.org/10.1371/journal.pone.0037137.
Knight, M.N., Karuppaiah, K., Lowe, M., Mohanty, S., Zondervan,R.L., Bell, S., Ahn, J. and Hankenson, K.D. (2018) R-spondin-2 is aWnt agonist that regulates osteoblast activity and bone mass.Bone Res., 6, 24. https://doi.org/10.1038/s41413-018-0026-7.
Jin, Y.R. and Yoon, J.K. (2012) The R-spondin family of proteins:emerging regulators of WNT signaling. Int. J. Biochem. Cell Biol., 44,2278-2287.
Martineau, X., Abed, É., Martel-Pelletier, J., Pelletier, J.P. and Lajeunesse, D. (2017) Alteration of Wnt5a expression and of the noncanonical Wnt/PCP and Wnt/PKC-Ca2+ pathways in humanosteoarthritis osteoblasts. PLoS One, 12, e0180711. https://doi.org/10.1371/journal.pone.0180711.
Manousaki, D., Rauch, F., Chabot, G., Dubois, J., Fiscaletti, M. andAlos, N. (2016) Pediatric data for dual X-ray absorptiometricmeasures of normal lumbar bone mineral density in childrenunder 5 years of age using the lunar prodigy densitometer. J.Musculoskelet. Neuronal Interact., 16, 247-255.
Shpaer, E.G., Robinson, M., Yee, D., Candlin, J.D., Mines, R. andHunkapiller, T. (1996) Sensitivity and selectivity in protein similarity searches: a comparison of Smith-waterman in hardwareto BLAST and FASTA. Genomics, 38, 179-191.
Li, H. and Durbin, R. (2009) Fast and accurate short readalignment with burrows-wheeler transform. Bioinformatics, 25,1754-1760.
Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relativegene expression data using real-time quantitative PCR and the2' CT method. Methods, 25, 402-408.
Zhang, W., Zhuang, Y., Zhang, Y., Yang, X., Zhang, H., Wang, G.,Yin, W., Wang, R., Zhang, Z. and Xiao, W. (2017) Uev1A facilitatesosteosarcoma differentiation by promoting Smurf1-mediatedSmad1 ubiquitination and degradation. Cell Death Dis., 8, e2974.https://doi.org/10.1038/cddis.2017.366.