REPORT Exome Sequencing Identifies Truncating Mutations in Human SERPINF1 in Autosomal-Recessive Osteogenesis Imperfecta Jutta Becker, 1,11 Oliver Semler, 2,11 Christian Gilissen, 3,11 Yun Li, 1,4 Hanno Jo ¨rn Bolz, 1,5 Cecilia Giunta, 6 Carsten Bergmann, 5,7 Marianne Rohrbach, 6 Friederike Koerber, 8 Katharina Zimmermann, 1 Petra de Vries, 3 Brunhilde Wirth, 1,4,9 Eckhard Schoenau, 2 Bernd Wollnik, 1,4,10 Joris A. Veltman, 3 Alexander Hoischen, 3 and Christian Netzer 1, * Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. After mutations in all known OI genes had been excluded by Sanger sequencing, we applied next-generation sequencing to analyze the exome of a single individual who has a severe form of the disease and whose parents are second cousins. A total of 26,922 variations from the human reference genome sequence were subjected to several filtering steps. In addition, we ex- tracted the genotypes of all dbSNP130-annotated SNPs from the exome sequencing data and used these 299,494 genotypes as markers for the genome-wide identification of homozygous regions. A single homozygous truncating mutation, affecting SERPINF1 on chromo- some 17p13.3, that was embedded into a homozygous stretch of 2.99 Mb remained. The mutation was also homozygous in the affected brother of the index patient. Subsequently, we identified homozygosity for two different truncating SERPINF1 mutations in two unre- lated patients with OI and parental consanguinity. All four individuals with SERPINF1 mutations have severe OI. Fractures of long bones and severe vertebral compression fractures with resulting deformities were observed as early as the first year of life in these individuals. Collagen analyses with cultured dermal fibroblasts displayed no evidence for impaired collagen folding, posttranslational modification, or secretion. SERPINF1 encodes pigment epithelium-derived factor (PEDF), a secreted glycoprotein of the serpin superfamily. PEDF is a multifunctional protein and one of the strongest inhibitors of angiogenesis currently known in humans. Our data provide genetic evidence for PEDF involvement in human bone homeostasis. Osteogenesis imperfecta (OI; MIM 166200, 166210, 610854, 259420, 166220, 610967, 610968, 610682, 610915, and 259440 for type I to IX of the disease) is a genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. Disease severity ranges from very mild forms without fractures to intrauterine fractures and perinatal lethality. 1,2 Typical ex- traskeletal manifestations, which affect a variable number of OI patients, are dentinogenesis imperfecta, hearing loss, and blue sclera. Most individuals with OI have autosomal- dominantly inherited mutations in COL1A1 (MIM 120150) or COL1A2 (MIM 120160), the two genes that encode the a chains of the major bone matrix protein collagen type 1. Mutations in these genes result in quanti- tative and/or qualitative defects in type 1 collagen produc- tion by osteoblasts. 3–6 In a minority of OI patients, the disorder is inherited in an autosomal-recessive manner. Recently, mutations in six different genes have been associated with these OI forms. CRTAP (MIM 605497), LEPRE1 (MIM 610339), and PPIB (MIM 123841) encode components of the prolyl 3-hydroxylation complex. 7–14 This protein complex is located in the rough endoplasmatic reticulum and, among other functions, modifies the Pro986 residue of the type 1 collagen a1 chains. 12,15 SERPINH1 (MIM 600943) and FKBP10 (MIM 607063) encode collagen chaperones. 16–20 HSP47, the gene product of SERPINH1, may monitor the final integrity of the triple helix that is formed out of two a1 chains and one a2 chain. 17 SP7 or OSX (MIM 606633) encodes a transcription factor that, based on the function of its murine homolog, is assumed to regulate the differentiation of preosteoblasts to osteo- blasts. 21,22 In a substantial number of COL1A1 and COL1A2 mutation-negative OI cases, the underlying molecular defect is unknown. 17 The current standard therapy for moderate and severe forms of OI (types II, III, and IV in the Sillence classifica- tion 23 ) is cyclic intravenous application of bisphopho- nates. 1 These compounds are synthetic pyrophosphate analogs that deposit on the bone surface, where they inhibit osteoclasts. 24,25 Bisphosphonate treatment, ideally starting in early childhood, increases bone mineral density. There is evidence that it also reduces fracture rates, chronic bone pain, and immobility in OI patients. 26 1 Institute of Human Genetics, University of Cologne, 50931 Cologne, Germany; 2 Children’s Hospital, University of Cologne, 50931 Cologne, Germany; 3 Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences and Institute for Genetic and Metabolic Disorders, Radboud University Nij- megen Medical Centre, 6500 HB Nijmegen, The Netherlands; 4 Center of Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany; 5 Center for Human Genetics, Bioscientia, 55218 Ingelheim, Germany; 6 Division of Metabolism, Connective Tissue Unit, University Children’s Hospital and Pediatric Research Center, 8032 Zurich, Switzerland; 7 Department of Human Genetics, RWTH Aachen University, 52074 Aachen, Germany; 8 Depart- ment of Radiology, University of Cologne, 50931 Cologne, Germany; 9 Institute of Genetics, University of Cologne, 50674 Cologne, Germany; 10 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50674 Cologne, Germany 11 These authors contributed equally to this work *Correspondence: christian.netzer@uk-koeln.de DOI 10.1016/j.ajhg.2011.01.015. Ó2011 by The American Society of Human Genetics. All rights reserved. 362 The American Journal of Human Genetics 88, 362–371, March 11, 2011