Targeted Sequencing of a Pediatric Metabolic Bone Gene Panel Using a Desktop Semiconductor Next-Generation Sequencer
Metabolic bone disorders in children frequently are heritable, but the expanding number of genes associated with these conditions makes it difficult to perform molecular diagnosis. In the present study, we therefore evaluated a semiconductor (SC)-based sequencing system for this purpose. A total of 65 DNA samples were analyzed comprising 24 samples from patients with 27 known pathogenic mutations, 6 samples from patients with prior negative Sanger sequencing, and 35 consecutive samples from patients with suspected heritable metabolic bone disorders who had not had prior molecular diagnosis. In the samples with known pathogenic mutations, 26 of 27 mutations were identified by SC sequencing. All single nucleotide variants were correctly identified, but a 7-nucleotide duplication in CYP27B1 was not detected. SC sequencing revealed two pathogenic mutations in the six samples where prior Sanger sequencing had failed to identify a mutation. Finally, pathogenic mutations were found in 27 samples of patients with unknown mutation status (15 in COL1A1, 9 in COL1A2, 1 in LEPRE1, 1 in LRP5, 1 in PHEX). Subsequent Sanger sequencing confirmed the mutations in all 27 samples. In conclusion, we found that SC sequencing is suitable for the diagnosis of heritable metabolic bone disorders in children.
KeywordsBone mineral density Fractures Juvenile osteoporosis Osteogenesis imperfecta Rickets Sequencing
We thank Ghalib Bardai for assistance with sequencing data analysis and Mark Lepik for preparation of the figures. This study was supported by the Shriners of North America and the Fonds de recherche Québec - Santé.
Conflict of interests
Frank Rauch received support from the Chercheur-Boursier Clinicien program of the Fonds de Recherche du Québec – Santé and has received consultancy fees from Genzyme Inc and Alexion Inc. Francis H Glorieux has received consultancy fees from Novartis Inc., Amgen Inc. and Alexion Inc. Liljana Lalic, Pierre Moffatt, and Peter Roughley declare no conflict of interest.
Human and Animal Rights and Informed Consent
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from study participants or the legal guardians.
- 6.Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, Leamon JH, Johnson K, Milgrew MJ, Edwards M, Hoon J, Simons JF, Marran D, Myers JW, Davidson JF, Branting A, Nobile JR, Puc BP, Light D, Clark TA, Huber M, Branciforte JT, Stoner IB, Cawley SE, Lyons M, Fu Y, Homer N, Sedova M, Miao X, Reed B, Sabina J, Feierstein E, Schorn M, Alanjary M, Dimalanta E, Dressman D, Kasinskas R, Sokolsky T, Fidanza JA, Namsaraev E, McKernan KJ, Williams A, Roth GT, Bustillo J (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475:348–352PubMedCrossRefGoogle Scholar
- 10.Tarabeux J, Zeitouni B, Moncoutier V, Tenreiro H, Abidallah K, Lair S, Legoix-Ne P, Leroy Q, Rouleau E, Golmard L, Barillot E, Stern MH, Rio-Frio T, Stoppa-Lyonnet D, Houdayer C (2014) Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model. Eur J Hum Genet 22:535–541PubMedCrossRefGoogle Scholar
- 11.Beck J, Pittman A, Adamson G, Campbell T, Kenny J, Houlden H, Rohrer JD, de Silva R, Shoai M, Uphill J, Poulter M, Hardy J, Mummery CJ, Warren JD, Schott JM, Fox NC, Rossor MN, Collinge J, Mead S (2014) Validation of next-generation sequencing technologies in genetic diagnosis of dementia. Neurobiol Aging 35:261–265PubMedCrossRefGoogle Scholar
- 16.Morello R, Bertin TK, Chen Y, Hicks J, Tonachini L, Monticone M, Castagnola P, Rauch F, Glorieux FH, Vranka J, Bachinger HP, Pace JM, Schwarze U, Byers PH, Weis M, Fernandes RJ, Eyre DR, Yao Z, Boyce BF, Lee B (2006) CRTAP is required for prolyl 3-hydroxylation and mutations cause recessive osteogenesis imperfecta. Cell 127:291–304PubMedCrossRefGoogle Scholar
- 23.Korvala J, Juppner H, Makitie O, Sochett E, Schnabel D, Mora S, Bartels CF, Warman ML, Deraska D, Cole WG, Hartikka H, Ala-Kokko L, Mannikko M (2012) Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity. BMC Med Genet 13:26PubMedCrossRefPubMedCentralGoogle Scholar
- 26.van Dijk FS, Zillikens MC, Micha D, Riessland M, Marcelis CL, de Die-Smulders CE, Milbradt J, Franken AA, Harsevoort AJ, Lichtenbelt KD, Pruijs HE, Rubio-Gozalbo ME, Zwertbroek R, Moutaouakil Y, Egthuijsen J, Hammerschmidt M, Bijman R, Semeins CM, Bakker AD, Everts V, Klein-Nulend J, Campos-Obando N, Hofman A, te Meerman GJ, Verkerk AJ, Uitterlinden AG, Maugeri A, Sistermans EA, Waisfisz Q, Meijers-Heijboer H, Wirth B, Simon ME, Pals G (2013) PLS3 mutations in X-linked osteoporosis with fractures. N Engl J Med 369:1529–1536PubMedCrossRefGoogle Scholar
- 27.Fahiminiya S, Majewski J, Al-Jallad H, Moffatt P, Mort J, Glorieux FH, Roschger P, Klaushofer K, Rauch F (2014) Osteoporosis caused by mutations in PLS3—clinical and bone tissue characteristics. J Bone Miner Res 29:1805–1814Google Scholar
- 29.Keupp K, Beleggia F, Kayserili H, Barnes AM, Steiner M, Semler O, Fischer B, Yigit G, Janda CY, Becker J, Breer S, Altunoglu U, Grunhagen J, Krawitz P, Hecht J, Schinke T, Makareeva E, Lausch E, Cankaya T, Caparros-Martin JA, Lapunzina P, Temtamy S, Aglan M, Zabel B, Eysel P, Koerber F, Leikin S, Garcia KC, Netzer C, Schonau E, Ruiz-Perez VL, Mundlos S, Amling M, Kornak U, Marini J, Wollnik B (2013) Mutations in WNT1 cause different forms of bone fragility. Am J Hum Genet 92:565–574PubMedCrossRefPubMedCentralGoogle Scholar
- 30.Pyott SM, Tran TT, Leistritz DF, Pepin MG, Mendelsohn NJ, Temme RT, Fernandez BA, Elsayed SM, Elsobky E, Verma I, Nair S, Turner EH, Smith JD, Jarvik GP, Byers PH (2013) WNT1 mutations in families affected by moderately severe and progressive recessive osteogenesis imperfecta. Am J Hum Genet 92:590–597PubMedCrossRefPubMedCentralGoogle Scholar
- 31.Laine CM, Joeng KS, Campeau PM, Kiviranta R, Tarkkonen K, Grover M, Lu JT, Pekkinen M, Wessman M, Heino TJ, Nieminen-Pihala V, Aronen M, Laine T, Kroger H, Cole WG, Lehesjoki AE, Nevarez L, Krakow D, Curry CJ, Cohn DH, Gibbs RA, Lee BH, Makitie O (2013) WNT1 mutations in early-onset osteoporosis and osteogenesis imperfecta. N Engl J Med 368:1809–1816PubMedCrossRefPubMedCentralGoogle Scholar