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Novel candidate genes putatively involved in stress fracture predisposition detected by whole-exome sequencing

  • EITAN FRIEDMAN (a1) (a2), DANIEL S. MORAN (a3) (a4), DANNY BEN-AVRAHAM (a5), RAN YANOVICH (a4) and GIL ATZMON (a5)...


While genetic factors in all likelihood contribute to stress fracture (SF) pathogenesis, a few studies focusing on candidate genes have previously been reported. The objective of this study is to gain better understanding on the genetic basis of SF in a gene-naive manner. Exome sequence capture followed by massive parallel sequencing of two pooled DNA samples from Israeli combat soldiers was employed: cases with high grade SF and ethnically matched healthy controls. The resulting sequence variants were individually verified using the Sequenom™ platform and the contribution of the genetic alterations was validated in a second cohort of cases and controls. In the discovery set that included DNA pool of cases (n = 34) and controls (n = 60), a total of 1174 variants with >600 reads/variant/DNA pool were identified, and 146 (in 127 genes) of these exhibited statistically significant (P < 0·05) different rates between SF cases and controls after multiple comparisons correction. Subsequent validation of these 146 sequence variants individually in a total of 136 SF cases and 127 controls using the Sequenom™ platform validated 20/146 variants. Of these, three missense mutations (rs7426114, rs4073918, rs3752135 in the NEB, SLC6A18 and SIGLEC12 genes, respectively) and three synonymous mutations (rs2071856, rs2515941, rs716745 in the ELFN2, GRK4, LRRC55 genes) displayed significant different rates in SF cases compared with controls. Exome sequencing seemingly unravelled novel candidate genes as involved in SF pathogenesis and predisposition.

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Corresponding author

* Corresponding author: Head, the Susanne Levy Gertner Oncogenetics Unit, The Danek Gertner Institute of Human Genetics, Chaim Sheba Medical Center, 52621 Tel-Hashomer, Israel. Tel.: 972 3 530 3173. Fax: 972 3 535 7308. E-mail:;


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Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, P., Kondrashov, A. S. & Sunayev, S. R. (2010). A method and server for predicting damaging missense mutations. Nature Methods 7, 248249.
Albisetti, W., Perugia, D., De Bartolomeo, O., Tagliabue, L., Camerucci, E. & Calori, G. M. (2010). Stress fractures of the base of the metatarsal bones in young trainee ballet dancers. International Orthopaedics 34, 5155.
Allayee, H., de Bruin, T. W., Dominguez, M. K., Cheng, L. S., Ipp, E., Cantor, R. M., Krass, K. L., Keulen, E. T., Aouizerat, B. E., Lusis, A. J. & Rotter, J. I. (2001). Genome scan for blood pressure in Dutch dyslipidemic families reveals linkage to a locus on chromosome 4p. Hypertension 38, 773778.
Altarac, M., Gardner, J. W., Popovich, R. M., Potter, R., Knapik, J. J. & Jones, B. H. (2000). Cigarette smoking and exercise-related injuries among young men and women. American Journal of Preventive Medicine 18, 96102.
Bachmann, M., Gaston, M. S. & Hefti, F. (2011). Supracondylar stress fracture of the femur in a child. Journal of Pediatric Orthopaedics B 20, 7073.
Boudin, E., Steenackers, E., de Freitas, F., Nielsen, T. L., Andersen, M., Brixen, K., Van Hul, W. & Piters, E. (2013). A common LRP4 haplotype is associated with bone mineral density and hip geometry in men – data from the Odense androgen study (OAS). Bone 53, 414420.
Chatzipapas, C., Boikos, S., Drosos, G. I., Kazakos, K., Tripsianis, G., Serbis, A., Stergiopoulos, S., Tilkeridis, C., Verettas, D. A. & Stratakis, C. A. (2009). Polymorphisms of the vitamin D receptor gene and stress fractures. Hormone and Metabolic Research 41, 635640.
Cosman, F., Ruffing, J., Zion, M., Uhorchak, J., Ralston, S., Tendy, S., McGuigan, F. E., Lindsay, R. & Nieves, J. (2013). Determinants of stress fracture risk in United States Military Academy cadets. Bone 55, 359366.
DePristo, M. A., Banks, E., Poplin, R., Garimella, K. V., Maguire, J. R., Hartl, C., Philippakis, A. A., del Angel, G., Rivas, M. A., Hanna, M., McKenna, A., Fennell, T. J., Kernytsky, A. M., Sivachenko, A. Y., Cibulskis, K., Gabriel, S. B., Altshuler, D. & Daly, M. J. (2011). A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature Genetics 43, 491498.
Dubin, R., Jing, Q., O'Broin, P., Calder, B., McLellan, A., Moskowitz, D., Suzuki, M., & Greally, J. M. (2010). WASP: Wiki-based automated sequence processor for epigenomics and genomics applications. Journal of Biomolecular Techniques 21(3 Suppl), S11.
Duncan, E. L., Danoy, P., Kemp, J. P., Leo, P. J., McCloskey, E., Nicholson, G. C., Eastell, R., Prince, R. L., Eisman, J. A., Jones, G., Sambrook, P. N., Reid, I. R., Dennison, E. M., Wark, J., Richards, J. B., Uitterlinden, A. G., Spector, T. D., Esapa, C., Cox, R. D., Brown, S. D., Thakker, R. V., Addison, K. A., Bradbury, L. A., Center, J. R., Cooper, C., Cremin, C., Estrada, K., Felsenberg, D., Gluer, C. C., Hadler, J., Henry, M. J., Hofman, A., Kotowicz, M. A., Makovey, J., Nguyen, S. C., Nguyen, T. V., Pasco, J. A., Pryce, K., Reid, D. M., Rivadeneira, F., Roux, C., Stefansson, K., Styrkarsdottir, U., Thorleifsson, G., Tichawangana, R., Evans, D. M., & Brown, M. A. (2011). Genome-wide association study using extreme truncate selection identifies novel genes affecting bone mineral density and fracture risk. PLoS Genetics 7, e1001372.
Eddy, S. R. (2004). Where did the BLOSUM62 alignment score matrix come from? Nature Biotechnology 22, 10351036.
Estrada, K., Styrkarsdottir, U., Evangelou, E., Hsu, Y. H., Duncan, E. L., Ntzani, E. E., Oei, L., Albagha, O. M., Amin, N., Kemp, J. P., Koller, D. L., Li, G., Liu, C. T., Minster, R. L., Moayyeri, A., Vandenput, L., Willner, D., Xiao, S. M., Yerges-Armstrong, L. M., Zheng, H. F., Alonso, N., Eriksson, J., Kammerer, C. M., Kaptoge, S. K., Leo, P. J., Thorleifsson, G., Wilson, S. G., Wilson, J. F., Aalto, V., Alen, M., Aragaki, A. K., Aspelund, T., Center, J. R., Dailiana, Z., Duggan, D. J., Garcia, M., Garcia-Giralt, N., Giroux, S., Hallmans, G., Hocking, L. J., Husted, L. B., Jameson, K. A., Khusainovam, R., Kim, G. S., Kooperberg, C., Koromila, T., Kruk, M., Laaksonen, M., Lacroix, A. Z., Lee, S. H., Leung, P. C., Lewis, J. R., Masi, L., Mencej-Bedrac, S., Nguyen, T. V., Nogues, X., Patel, M. S., Prezelj, J., Rose, L. M., Scollen, S., Siggeirsdottir, K., Smith, A. V., Svensson, O., Trompet, S., Trummer, O., van Schoor, N. M., Woo, J., Zhu, K., Balcells, S., Brandi, M. L., Buckley, B. M., Cheng, S., Christiansen, C., Cooper, C., Dedoussis, G., Ford, I., Frost, M., Goltzman, D., Gonzalez-Macias, J., Kahonen, M., Karlsson, M., Khusnutdinova, E., Koh, J. M., Kollia, P., Langdahl, B. L., Leslie, W. D., Lips, P., Ljunggren, O., Lorenc, R. S., Marc, J., Mellstrom, D., Obermayer-Pietsch, B., Olmos, J. M., Pettersson-Kymmer, U., Reid, D. M., Riancho, J. A., Ridker, P. M., Rousseau, F., Slagboom, P. E., Tang, N. L., Urreizti, R., Van Hul, W., Viikari, J., Zarrabeitia, M. T., Aulchenko, Y. S., Castano-Betancourt, M., Grundberg, E., Herrera, L., Ingvarsson, T., Johannsdottir, H., Kwan, T., Li, R., Luben, R., Medina-Gomez, C., Palsson, S. T., Reppe, S., Rotter, J. I., Sigurdsson, G., van Meurs, J. B., Verlaan, D., Williams, F. M., Wood, A. R., Zhou, Y., Gautvik, K. M., Pastinen, T., Raychaudhuri, S., Cauley, J. A., Chasman, D. I., Clarkm, G. R., Cummings, S. R., Danoy, P., Dennison, E. M., Eastell, R., Eisman, J. A., Gudnason, V., Hofman, A., Jackson, R. D., Jones, G., Jukema, J. W., Khaw, K. T., Lehtimaki, T., Liu, Y., Lorentzon, M., McCloskey, E., Mitchell, B. D., Nandakumar, K., Nicholson, G. C., Oostra, B. A., Peacock, M., Pols, H. A., Prince, R. L., Raitakari, O., Reid, I. R., Robbins, J., Sambrook, P. N., Sham, P. C., Shuldiner, A. R., Tylavsky, F. A., van Duijn, C. M., Wareham, N. J., Cupples, L. A., Econs, M. J., Evans, D. M., Harris, T. B., Kung, A. W., Psaty, B. M., Reeve, J., Spector, T. D., Streeten, E. A., Zillikens, M. C., Thorsteinsdottir, U., Ohlsson, C., Karasik, D., Richards, J. B., Brown, M. A., Stefansson, K., Uitterlinden, A. G., Ralston, S. H., Ioannidis, J. P., Kiel, D. P. & Rivadeneira, F. (2012). Genome-wide meta-analysis identifies 56 bone mineral density loci and reveals 14 loci associated with risk of fracture. Nature Genetics 44, 491501.
Flanagan, J. M., Sheehan, V., Linder, H., Howard, T. A., Wang, Y. D., Hoppe, C. C., Aygun, B., Adams, R. J., Neale, G. A. & Ware, R. E. (2013). Genetic mapping and exome sequencing identify 2 mutations associated with stroke protection in pediatric patients with sickle cell anemia. Blood 121, 32373245.
Friedl, K. E., Nuovo, J. A., Patience, T. H. & Dettori, J. R. (1992). Factors associated with stress fracture in young army women: indications for further research. Military Medicine 157, 334338.
Frusztajer, N. T., Dhuper, S., Warren, M. P., Brooks-Gunn, J. & Fox, R. P. (1990). Nutrition and the incidence of stress fractures in ballet dancers. American Journal of Clinical Nutrition 51, 779783.
Garcia-Ibarbia, C., Perez-Nunez, M. I., Olmos, J. M., Valero, C., Perez-Aguilar, M. D., Hernandez, J. L., Zarrabeitia, M. T., González-Macías, J. & Riancho, J. A. (2013). Missense polymorphisms of the WNT16 gene are associated with bone mass, hip geometry and fractures. Osteoporosis International 24, 24492454.
Giladi, M., Milgrom, C., Kashtan, H., Stein, M., Chisin, R. & Dizian, R. (1986). Recurrent stress fractures in military recruits. One-year follow-up of 66 recruits. Journal of Bone and Joint Surgery. British Volume 68, 439441.
Giladi, M., Milgrom, C., Simkin, A., Stein, M., Kashtan, H., Margulies, J., Rand, N., Chisin, R., Steinberg, R., Aharonson, Z., Kedem, R. & Frankel, V. H. (1987). Stress fractures and tibial bone width. A risk factor. Journal of Bone and Joint Surgery. British Volume 69, 326329.
Giladi, M., Milgrom, C., Simkin, A. & Danon, Y. (1991). Stress fractures. Identifiable risk factors. American Journal of Sports Medicine 19, 647652.
Givon, U., Friedman, E., Reiner, A., Vered, I., Finestone, A. & Shemer, J. (2000). Stress fractures in the Israeli Defense Forces from 1995 to 1996. Clinical Orthopaedics and Related Research 373, 227232.
Hallel, T., Amit, S. & Segal, D. (1976). Fatigue fractures of tibial and femoral shaft in soldiers. Clinical Orthopaedics and Related Research 118, 3543.
Hod, N., Ashkenazi, I., Levi, Y., Fire, G., Drori, M., Cohen, I., Bernstine, H. & Horne, T. (2006). Characteristics of skeletal stress fractures in female military recruits of the Israel defense forces on bone scintigraphy. Clinical Nuclear Medicine 31, 742749.
Jones, B. H., Bovee, M. W., Harris, J. M. 3rd & Cowan, D. N. (1993). Intrinsic risk factors for exercise-related injuries among male and female army trainees. American Journal of Sports Medicine 21, 705710.
Jurinke, C., van den Boom, D., Cantor, C. R. & Koster, H. (2002). The use of MassARRAY technology for high throughput genotyping. Advance in Biochemical Engineering/Biotechnology 77, 5774.
Karasik, D., Shimabuku, N. A., Zhou, Y., Zhang, Y., Cupples, L. A., Kiel, D. P. & Demissie, S. (2008). A genome wide linkage scan of metacarpal size and geometry in the Framingham Study. American Journal of Human Biology 20, 663670.
Karasik, D., Cheung, C. L., Zhou, Y., Cupples, L. A., Kiel, D. P. & Demissie, S. (2012). Genome-wide association of an integrated osteoporosis-related phenotype: is there evidence for pleiotropic genes? Journal of Bone Mineral Research 27, 319330.
Knapik, J., Montain, S. J., McGraw, S., Grier, T., Ely, M. & Jones, B. H. (2012). Stress fracture risk factors in basic combat training. International Journal of Sports Medicine 33, 940946.
Korvala, J., Hartikka, H., Pihlajamaki, H., Solovieva, S., Ruohola, J. P., Sahi, T., Barral, S., Ott, J., Ala-Kokko, L. & Mannikko, M. (2010). Genetic predisposition for femoral neck stress fractures in military conscripts. BMC Genetics 11, 95.
Lee, S. H., Baek, J. R., Han, S. B. & Park, S. W. (2005). Stress fractures of the femoral diaphysis in children: a report of 5 cases and review of literature. Journal of Pediatric Orthopaedics 25, 734738.
Li, H. & Durbin, R. (2009). Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 17541760.
Li, H. & Durbin, R. (2010). Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589595.
Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G. & Durbin, R. (2009). The sequence alignment/map format and SAMtools. Bioinformatics 25, 20782079.
Mattila, V. M., Niva, M., Kiuru, M. & Pihlajamaki, H. (2007). Risk factors for bone stress injuries: a follow-up study of 102,515 person-years. Medicine and Science in Sports and Exercise 39, 10611066.
McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M. & DePristo, M. A. (2010). The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Research 20, 12971303.
Mirina, A., Atzmon, G., Ye, K. & Bergman, A. (2012). Gene size matters. PloS ONE 7, e49093.
Moran, D. S., Evans, R. K. & Hadad, E. (2008). Imaging of lower extremity stress fracture injuries. Sports Medicine 38, 345356.
Murray, S. R., Reeder, M. T., Udermann, B. E. & Pettitt, R. W. (2006). High-risk stress fractures: pathogenesis, evaluation, and treatment. Comprehensive Therapy 32, 2025.
Ng, P. C. & Henikoff, S. (2002). Accounting for human polymorphisms predicted to affect protein function. Genome Research 12, 436446.
Paternoster, L., Ohlsson, C., Sayers, A., Vandenput, L., Lorentzon, M., Evans, D. M. & Tobias, J. H. (2010). OPG and RANK polymorphisms are both associated with cortical bone mineral density: findings from a meta-analysis of the Avon longitudinal study of parents and children and Gothenburg osteoporosis and obesity determinants cohorts. Journal of Clinical Endocrinology and Metabolism 95, 39403948.
Paternoster, L., Lorentzon, M., Lehtimaki, T., Eriksson, J., Kahonen, M., Raitakari, O., Laaksonen, M., Sievanen, H., Viikari, J., Lyytikainen, L. P., Mellstrom, D., Karlsson, M., Ljunggren, O., Grundberg, E., Kemp, J. P., Sayers, A., Nethander, M., Evans, D. M., Vandenput, L., Tobias, J. H. & Ohlsson, C. (2013). Genetic determinants of trabecular and cortical volumetric bone mineral densities and bone microstructure. PLoS Genetics 9, e1003247.
Ralston, S. H. & Uitterlinden, A. G. (2010). Genetics of osteoporosis. Endocrine Reviews 31, 629662.
Schwellnus, M. P., Jordaan, G. & Noakes, T. D. (1990). Prevention of common overuse injuries by the use of shock absorbing insoles. A prospective study. American Journal of Sports Medicine 18, 636641.
Singer, A., Ben-Yehuda, O., Ben-Ezra, Z. & Zaltzman, S. (1990). Multiple identical stress fractures in monozygotic twins. Case report. Journal of Bone and Joint Surgery. American Volume 72, 444445.
Sunyaev, S., Ramensky, V., Koch, I., Lathe, W. 3rd, Kondrashov, A. S. & Bork, P. (2001). Prediction of deleterious human alleles. Human Molecular Genetics 10, 591597.
Takahata, M., Iwasaki, N., Nakagawa, H., Abe, Y., Watanabe, T., Ito, M., Majima, T. & Minami, A. (2007). Sialylation of cell surface glycoconjugates is essential for osteoclastogenesis. Bone 41, 7786.
Valimaki, V. V., Alfthan, H., Lehmuskallio, E., Loyttyniemi, E., Sahi, T., Suominen, H. & Valimaki, M. J. (2005). Risk factors for clinical stress fractures in male military recruits: a prospective cohort study. Bone 37, 267273.
Villar, V. A., Jones, J. E., Armando, I., Palmes-Saloma, C., Yu, P., Pascua, A. M., Keever, L., Arnaldo, F. B., Wang, Z., Luo, Y., Felder, R. A. & Jose, P. A. (2009). G protein-coupled receptor kinase 4 (GRK4) regulates the phosphorylation and function of the dopamine D3 receptor. Journal of Biological Chemistry 284, 2142521434.
Yan, J. & Aldrich, R. W. (2012). BK potassium channel modulation by leucine-rich repeat-containing proteins. Proceedings of the National Academy of Science of the United States of America 109, 79177922.
Yanovich, R., Milgrom, R., Friedman, E. & Moran, D. S. (2011). Androgen receptor CAG repeat size is associated with stress fracture risk: a pilot study. Clinical Orthopaedics and Related Research 469, 29252931.
Yanovich, R., Friedman, E., Milgrom, R., Oberman, B., Freedman, L. S. & Moran, D. S. (2012). Candidate gene analysis in Israeli soldiers with stress fractures. Journal of Sport Science and Medicine 11, 147155.
Zhang, F., Qiu, T., Wu, X., Wan, C., Shi, W., Wang, Y., Chen, J. G., Wan, M., Clemens, T. L. & Cao, X. (2009). Sustained BMP signaling in osteoblasts stimulates bone formation by promoting angiogenesis and osteoblast differentiation. Journal of Bone and Mineral Research 24, 12241233.
Zhao, L. J., Liu, X. G., Liu, Y. Z., Liu, Y. J., Papasian, C. J., Sha, B. Y., Pan, F., Guo, Y. F., Wang, L., Yan, H., Xiong, D. H., Tang, Z. H., Yang, T. L., Chen, X. D., Guo, Y., Li, J., Shen, H., Zhang, F., Lei, S. F., Recker, R. R. & Deng, H. W. (2010). Genome-wide association study for femoral neck bone geometry. Journal of Bone and Mineral Research 25, 320329.
Zheng, H. F., Tobias, J. H., Duncan, E., Evans, D. M., Eriksson, J., Paternoster, L., Yerges-Armstrong, L. M., Lehtimaki, T., Bergstrom, U., Kahonen, M., Leo, P. J., Raitakari, O., Laaksonen, M., Nicholson, G. C., Viikari, J., Ladouceur, M., Lyytikainen, L. P., Medina-Gomez, C., Rivadeneira, F., Prince, R. L., Sievanen, H., Leslie, W. D., Mellstrom, D., Eisman, J. A., Moverare-Skrtic, S., Goltzman, D., Hanley, D. A., Jones, G., St Pourcain, B., Xiao, Y., Timpson, N. J., Smith, G. D., Reid, I. R., Ring, S. M., Sambrook, P. N., Karlsson, M., Dennison, E. M., Kemp, J. P., Danoy, P., Sayers, A., Wilson, S. G., Nethander, M., McCloskey, E., Vandenput, L., Eastell, R., Liu, J., Spector, T., Mitchell, B. D., Streeten, E. A., Brommage, R., Pettersson-Kymmer, U., Brown, M. A., Ohlsson, C., Richards, J. B. & Lorentzon, M. (2012). WNT16 influences bone mineral density, cortical bone thickness, bone strength, and osteoporotic fracture risk. PLoS Genetics 8, e1002745.
Zwas, S. T., Elkanovitch, R. & Frank, G. (1987). Interpretation and classification of bone scintigraphic findings in stress fractures. Journal of Nuclear Medicine 28, 452457.
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