Genetic Variation in RIN3 in the Belgian Population Supports Its Involvement in the Pathogenesis of Paget’s Disease of Bone and Modifies the Age of Onset

  • Raphaël De Ridder
  • Eveline Boudin
  • Geert Vandeweyer
  • Jean-Pierre Devogelaer
  • Erik Fransen
  • Geert Mortier
  • Wim Van HulEmail author
Original Research


Paget’s disease of bone (PDB) is a common, late-onset bone disorder characterized by focal increase of bone turnover. Mutations in the SQSTM1 gene are found in up to 40% of patients and recent GWAS have led to novel associations with several loci. RIN3, the candidate gene located at the associated 14q32 locus, has recently been studied in a British cohort to elucidate its contribution to the pathogenesis. In this study, we performed a genetic screening of RIN3 in an unrelated cohort to validate these findings and to further explore genetic variation in this gene in the context of PDB. In our screening, we examined the 5′ untranslated region (UTR), the exonic regions and the intron–exon boundaries of the gene in a control cohort and a patient cohort. Our findings show clustering of variation similar to the British cohort and support a protective role for common genetic variation (rs117068593, p.R279C) in the proline-rich region and a functionally relevant role for rare genetic variation in the domains that mediate binding and activation of its interaction partner, Rab5. Additive regression models, fitted for the common variants, validated the association of the rs117068593 variant with the disease (OR+/+ 0.315; OR+/− 0.562). In addition, our analyses revealed a potentially modifying effect of this variant on the age of onset of the disease. In conclusion, our findings support the involvement of genetic variation in RIN3 in PDB and suggest a role for RIN3 as a potential modifier of the age of onset of the disease.


Paget’s disease of bone Pathogenesis Targeted sequencing Molecular inversion probes RIN3 Modifier 



The authors would like to thank Alex Hoischen (Radboudumc Nijmegen) for his help in the probe design used in our targeted sequencing approach.


This work was supported by grants of the ‘Fonds voor Wetenschappelijk Onderzoek Vlaanderen’ (FWO Grant G019712N and G031915N) and the European Community’s Seventh Framework Programme (FP7/2007–2013) under Grant Agreement No. 602300 (SYBIL). RDR holds a doctoral grant with the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (1S07717N). EB and GV hold postdoctoral Grants (12A3814N and 12D1717N) with the Fonds voor Wetenschappelijk Onderzoek Vlaanderen.

Compliance with Ethical Standards

Conflict of interest

None to declare.

Ethical Approval

All patients were obtained prior to the study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.


  1. 1.
    Alonso N, Calero-Paniagua I, Del Pino-Montes J (2017) Clinical and genetic advances in Paget’s disease of bone: a review. Clin Rev Bone Miner Metab 15(1):37–48. CrossRefGoogle Scholar
  2. 2.
    Hocking LJ, Lucas GJ, Daroszewska A, Mangion J, Olavesen M, Cundy T, Nicholson GC, Ward L, Bennett ST, Wuyts W, Van Hul W, Ralston SH (2002) Domain-specific mutations in sequestosome 1 (SQSTM1) cause familial and sporadic Paget’s disease. Hum Mol Genet 11(22):2735–2739CrossRefGoogle Scholar
  3. 3.
    Laurin N, Brown JP, Morissette J, Raymond V (2002) Recurrent mutation of the gene encoding sequestosome 1 (SQSTM1/p62) in Paget disease of bone. Am J Hum Genet 70(6):1582–1588. CrossRefGoogle Scholar
  4. 4.
    Albagha OM, Wani SE, Visconti MR, Alonso N, Goodman K, Brandi ML, Cundy T, Chung PY, Dargie R, Devogelaer JP, Falchetti A, Fraser WD, Gennari L, Gianfrancesco F, Hooper MJ, Van Hul W, Isaia G, Nicholson GC, Nuti R, Papapoulos S, Montes Jdel P, Ratajczak T, Rea SL, Rendina D, Gonzalez-Sarmiento R, Di Stefano M, Ward LC, Walsh JP, Ralston SH, Genetic Determinants of Paget’s Disease C (2011) Genome-wide association identifies three new susceptibility loci for Paget’s disease of bone. Nat Genet 43(7):685–689. CrossRefGoogle Scholar
  5. 5.
    Albagha OME, Visconti MR, Alonso N, Langston AL, Cundy T, Dargie R, Dunlop MG, Fraser WD, Hooper MJ, Isaia G, Nicholson GC, Montes JD, Gonzalez-Sarmiento R, di Stefano M, Tenesa A, Walsh JP, Ralston SH (2010) Genome-wide association study identifies variants at CSF1, OPTN and TNFRSF11A as genetic risk factors for Paget’s disease of bone. Nat Genet 42(6):520–526. CrossRefGoogle Scholar
  6. 6.
    Kajiho H, Saito K, Tsujita K, Kontani K, Araki Y, Kurosu H, Katada T (2003) RIN3: a novel Rab5 GEF interacting with amphiphysin II involved in the early endocytic pathway. J Cell Sci 116(Pt 20):4159–4168. CrossRefGoogle Scholar
  7. 7.
    Kajiho H, Sakurai K, Minoda T, Yoshikawa M, Nakagawa S, Fukushima S, Kontani K, Katada T (2011) Characterization of RIN3 as a guanine nucleotide exchange factor for the Rab5 subfamily GTPase Rab31. J Biol Chem 286(27):24364–24373. CrossRefGoogle Scholar
  8. 8.
    Stenmark H, Olkkonen VM (2001) The Rab GTPase family. Genome Biol. Google Scholar
  9. 9.
    Gorvel JP, Chavrier P, Zerial M, Gruenberg J (1991) rab5 controls early endosome fusion in vitro. Cell 64(5):915–925CrossRefGoogle Scholar
  10. 10.
    Bucci C, Wandingerness A, Lutcke A, Chiariello M, Bruni CB, Zerial M (1994) Rab5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial-cells. Proc Natl Acad Sci USA 91(11):5061–5065. doi: CrossRefGoogle Scholar
  11. 11.
    Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M (1992) The small gtpase Rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70(5):715–728. doi: CrossRefGoogle Scholar
  12. 12.
    Yoshikawa M, Kajiho H, Sakurai K, Minoda T, Nakagawa S, Kontani K, Katada T (2008) Tyr-phosphorylation signals translocate RIN3, the small GTPase Rab5-GEF, to early endocytic vesicles. Biochem Biophys Res Commun 372(1):168–172. CrossRefGoogle Scholar
  13. 13.
    Vallet M, Soares DC, Wani S, Sophocleous A, Warner J, Salter DM, Ralston SH, Albagha OME (2015) Targeted sequencing of the Paget’s disease associated 14q32 locus identifies several missense coding variants in RIN3 that predispose to Paget’s disease of bone. Hum Mol Genet 24(11):3286–3295. CrossRefGoogle Scholar
  14. 14.
    Boyle EA, O’Roak BJ, Martin BK, Kumar A, Shendure J (2014) MIPgen: optimized modeling and design of molecular inversion probes for targeted resequencing. Bioinformatics 30(18):2670–2672. CrossRefGoogle Scholar
  15. 15.
    O’Roak BJ, Vives L, Fu W, Egertson JD, Stanaway IB, Phelps IG, Carvill G, Kumar A, Lee C, Ankenman K, Munson J, Hiatt JB, Turner EH, Levy R, O’Day DR, Krumm N, Coe BP, Martin BK, Borenstein E, Nickerson DA, Mefford HC, Doherty D, Akey JM, Bernier R, Eichler EE, Shendure J (2012) Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders. Science 338(6114):1619–1622. CrossRefGoogle Scholar
  16. 16.
    Jansen S, Hoischen A, Coe BP, Carvill GL, Van Esch H, Bosch DGM, Andersen UA, Baker C, Bauters M, Bernier RA, van Bon BW, Claahsen-van der Grinten HL, Gecz J, Gilissen C, Grillo L, Hackett A, Kleefstra T, Koolen D, Kvarnung M, Larsen MJ, Marcelis C, McKenzie F, Monin ML, Nava C, Schuurs-Hoeijmakers JH, Pfundt R, Steehouwer M, Stevens SJC, Stumpel CT, Vansenne F, Vinci M, van de Vorst M, Vries P, Witherspoon K, Veltman JA, Brunner HG, Mefford HC, Romano C, Vissers L, Eichler EE, de Vries BBA (2018) A genotype-first approach identifies an intellectual disability-overweight syndrome caused by PHIP haploinsufficiency. Eur J Hum Genet EJHG 26(1):54–63. CrossRefGoogle Scholar
  17. 17.
    Neveling K, Mensenkamp AR, Derks R, Kwint M, Ouchene H, Steehouwer M, van Lier B, Bosgoed E, Rikken A, Tychon M, Zafeiropoulou D, Castelein S, Hehir-Kwa J, Tjwan Thung D, Hofste T, Lelieveld SH, Bertens SM, Adan IB, Eijkelenboom A, Tops BB, Yntema H, Stokowy T, Knappskog PM, Hoberg-Vetti H, Steen VM, Boyle E, Martin B, Ligtenberg MJ, Shendure J, Nelen MR, Hoischen A (2017) BRCA testing by single-molecule molecular inversion probes. Clin Chem 63(2):503–512. CrossRefGoogle Scholar
  18. 18.
    Vandeweyer G, Van Laer L, Loeys B, Van den Bulcke T, Kooy RF (2014) VariantDB: a flexible annotation and filtering portal for next generation sequencing data. Genome Med 6(10):74. CrossRefGoogle Scholar
  19. 19.
    Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M (2018) CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res:gky1016–gky1016.
  20. 20.
    Ioannidis NM, Rothstein JH, Pejaver V, Middha S, McDonnell SK, Baheti S, Musolf A, Li Q, Holzinger E, Karyadi D, Cannon-Albright LA, Teerlink CC, Stanford JL, Isaacs WB, Xu J, Cooney KA, Lange EM, Schleutker J, Carpten JD, Powell IJ, Cussenot O, Cancel-Tassin G, Giles GG, MacInnis RJ, Maier C, Hsieh C-L, Wiklund F, Catalona WJ, Foulkes WD, Mandal D, Eeles RA, Kote-Jarai Z, Bustamante CD, Schaid DJ, Hastie T, Ostrander EA, Bailey-Wilson JE, Radivojac P, Thibodeau SN, Whittemore AS, Sieh W (2016) REVEL: an ensemble method for predicting the pathogenicity of rare missense variants. Am J Hum Genet 99(4):877–885. CrossRefGoogle Scholar
  21. 21.
    Kemp JP, Medina-Gomez C, Estrada K, St Pourcain B, Heppe DH, Warrington NM, Oei L, Ring SM, Kruithof CJ, Timpson NJ, Wolber LE, Reppe S, Gautvik K, Grundberg E, Ge B, van der Eerden B, van de Peppel J, Hibbs MA, Ackert-Bicknell CL, Choi K, Koller DL, Econs MJ, Williams FM, Foroud T, Zillikens MC, Ohlsson C, Hofman A, Uitterlinden AG, Davey Smith G, Jaddoe VW, Tobias JH, Rivadeneira F, Evans DM (2014) Phenotypic dissection of bone mineral density reveals skeletal site specificity and facilitates the identification of novel loci in the genetic regulation of bone mass attainment. PLoS Genet 10(6):e1004423. CrossRefGoogle Scholar
  22. 22.
    Itzstein C, Coxon FP, Rogers MJ (2011) The regulation of osteoclast function and bone resorption by small GTPases. Small GTPases 2(3):117–130. CrossRefGoogle Scholar
  23. 23.
    Weivoda MM, Oursler MJ (2014) The roles of small GTPases in osteoclast biology. Orthop Muscul Syst Curr Res. Google Scholar
  24. 24.
    Lambert JC, Ibrahim-Verbaas CA, Harold D, Naj AC, Sims R, Bellenguez C, DeStafano AL, Bis JC, Beecham GW, Grenier-Boley B, Russo G, Thorton-Wells TA, Jones N, Smith AV, Chouraki V, Thomas C, Ikram MA, Zelenika D, Vardarajan BN, Kamatani Y, Lin CF,Gerrish A, Schmidt H, Kunkle B, Dunstan ML, Ruiz A, Bihoreau MT, Choi SH, Reitz C,Pasquier F, Cruchaga C, Craig D, Amin N, Berr C, Lopez OL, De Jager PL, Deramecourt V, Johnston JA, Evans D, Lovestone S, Letenneur L, Moron FJ, Rubinsztein DC, Eiriksdottir G, Sleegers K, Goate AM, Fievet N, Huentelman MW, Gill M, Brown K, Kamboh MI, Keller L, Barberger-Gateau P, McGuiness B, Larson EB, Green R, Myers AJ, Dufouil C, Todd S, Wallon D, Love S, Rogaeva E, Gallacher J, St George-Hyslop P, Clarimon J, Lleo A, Bayer A, Tsuang DW, Yu L, Tsolaki M, Bossu P, Spalletta G, Proitsi P, Collinge J, Sorbi S, Sanchez-Garcia F, Fox NC, Hardy J, Deniz Naranjo MC, Bosco P, Clarke R,Brayne C, Galimberti D, Mancuso M, Matthews F, European Alzheimer’s Disease I, Genetic,Environmental Risk in Alzheimer’s D, Alzheimer’s Disease Genetic C, Cohorts for H,Aging Research in Genomic E, Moebus S, Mecocci P, Del Zompo M, Maier W, Hampel H,Pilotto A, Bullido M, Panza F, Caffarra P, Nacmias B, Gilbert JR, Mayhaus M, Lannefelt L, Hakonarson H, Pichler S, Carrasquillo MM, Ingelsson M, Beekly D, Alvarez V, Zou F, Valladares O, Younkin SG, Coto E, Hamilton-Nelson KL, Gu W, Razquin C, Pastor P,Mateo I, Owen MJ, Faber KM, Jonsson PV, Combarros O, O’Donovan MC, Cantwell LB, Soininen H, Blacker D, Mead S, Mosley TH, Jr., Bennett DA, Harris TB, Fratiglioni L, Holmes C, de Bruijn RF, Passmore P, Montine TJ, Bettens K, Rotter JI, Brice A, Morgan K,Foroud TM, Kukull WA, Hannequin D, Powell JF, Nalls MA, Ritchie K, Lunetta KL, Kauwe JS, Boerwinkle E, Riemenschneider M, Boada M, Hiltuenen M, Martin ER, Schmidt R, Rujescu D, Wang LS, Dartigues JF, Mayeux R, Tzourio C, Hofman A, Nothen MM, Graff C, Psaty BM, Jones L, Haines JL, Holmans PA, Lathrop M, Pericak-Vance MA, Launer LJ, Farrer LA, van Duijn CM, Van Broeckhoven C, Moskvina V, Seshadri S, Williams J, Schellenberg GD, Amouyel P (2013) Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer’s disease. Nat Genet 45(12):1452–1458.
  25. 25.
    Fecto F, Yan J, Vemula SP, Liu E, Yang Y, Chen W, Zheng JG, Shi Y, Siddique N, Arrat H, Donkervoort S, Ajroud-Driss S, Sufit RL, Heller SL, Deng HX, Siddique T (2011) SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis. Arch Neurol 68(11):1440–1446. CrossRefGoogle Scholar
  26. 26.
    Jain N, Ganesh S (2016) Emerging nexus between RAB GTPases, autophagy and neurodegeneration. Autophagy 12(5):900–904. CrossRefGoogle Scholar
  27. 27.
    Xu W, Fang F, Ding J, Wu C (2018) Dysregulation of Rab5-mediated endocytic pathways in Alzheimer’s disease. Traffic 19(4):253–262. CrossRefGoogle Scholar
  28. 28.
    Grantham R (1974) Amino acid difference formula to help explain protein evolution. Science 185(4154):862–864CrossRefGoogle Scholar
  29. 29.
    Barbieri MA, Kong C, Chen PI, Horazdovsky BF, Stahl PD (2003) The Src homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis. J Biol Chem 278(34):32027–32036. CrossRefGoogle Scholar
  30. 30.
    Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46(W1):W296–W303. CrossRefGoogle Scholar
  31. 31.
    Delprato A, Merithew E, Lambright DG (2004) Structure, exchange determinants, and family-wide rab specificity of the tandem helical bundle and Vps9 domains of Rabex-5. Cell 118(5):607–617. CrossRefGoogle Scholar
  32. 32.
    Tall GG, Barbieri MA, Stahl PD, Horazdovsky BF (2001) Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1. Dev Cell 1(1):73–82. doi: CrossRefGoogle Scholar
  33. 33.
    Babu MM (2016) The contribution of intrinsically disordered regions to protein function, cellular complexity, and human disease. Biochem Soc Trans 44(5):1185–1200. CrossRefGoogle Scholar
  34. 34.
    Vacic V, Markwick PR, Oldfield CJ, Zhao X, Haynes C, Uversky VN, Iakoucheva LM (2012) Disease-associated mutations disrupt functionally important regions of intrinsic protein disorder. PLoS Comput Biol 8(10):e1002709. CrossRefGoogle Scholar
  35. 35.
    Kumar P, Henikoff S, Ng PC (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4(7):1073–1082. CrossRefGoogle Scholar
  36. 36.
    Davydov EV, Goode DL, Sirota M, Cooper GM, Sidow A, Batzoglou S (2010) Identifying a High Fraction of the Human Genome to be under Selective Constraint Using GERP plus. PLoS Comput Biol 6(12):e1001025. CrossRefGoogle Scholar
  37. 37.
    Adzhubei I, Jordan DM, Sunyaev SR (2013) Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet. Google Scholar
  38. 38.
    Lek M, Karczewski KJ, Minikel EV, Samocha KE, Banks E, Fennell T, O’Donnell-Luria AH, Ware JS, Hill AJ, Cummings BB, Tukiainen T, Birnbaum DP, Kosmicki JA, Duncan LE, Estrada K, Zhao F, Zou J, Pierce-Hoffman E, Berghout J, Cooper DN, Deflaux N, DePristo M, Do R, Flannick J, Fromer M, Gauthier L, Goldstein J, Gupta N, Howrigan D, Kiezun A, Kurki MI, Moonshine AL, Natarajan P, Orozco L, Peloso GM, Poplin R, Rivas MA, Ruano-Rubio V, Rose SA, Ruderfer DM, Shakir K, Stenson PD, Stevens C, Thomas BP, Tiao G, Tusie-Luna MT, Weisburd B, Won HH, Yu D, Altshuler DM, Ardissino D, Boehnke M, Danesh J, Donnelly S, Elosua R, Florez JC, Gabriel SB, Getz G, Glatt SJ, Hultman CM, Kathiresan S, Laakso M, McCarroll S, McCarthy MI, McGovern D, McPherson R, Neale BM, Palotie A, Purcell SM, Saleheen D, Scharf JM, Sklar P, Sullivan PF, Tuomilehto J, Tsuang MT, Watkins HC, Wilson JG, Daly MJ, MacArthur DG, Exome Aggregation C (2016) Analysis of protein-coding genetic variation in 60,706 humans. Nature 536(7616):285–291. CrossRefGoogle Scholar
  39. 39.
    Kajiho H, Fukushima S, Kontani K, Katada T (2012) RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin. PLoS ONE 7(1):e30575. CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Center of Medical GeneticsUniversity of Antwerp & Antwerp University HospitalAntwerpBelgium
  2. 2.Department of Rheumatology, Saint-Luc University HospitalUniversité Catholique de LouvainBrusselsBelgium

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