title: Osteoblastic differentiation of bone marrow mesenchymal stromal cells in Bruck Syndrome.
abstract: Osteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus.
  
  
  
  We analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System.
  
  
  
  No mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different.
  
  
  
  Our work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.
authors.affiliation: Department of Genetics, Medical School of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil. cmkaneto@uesc.br.|Department of Biological Science, Universidade Estadual de Santa Cruz, Ilheus, BA, Brazil. cmkaneto@uesc.br.
authors.forename: Carla M
authors.initials: CM
authors.lastname: Kaneto
authors.affiliation: Department of Natural Science, Universidade Estadual do Sudoeste da Bahia, Vitória da Conquista, Bahia, Brazil.
authors.forename: Patrícia S P
authors.initials: PS
authors.lastname: Lima
authors.affiliation: Department of Genetics, Medical School of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.|Regional Blood Center of Ribeirão Preto and National Institute of Science and Technology in Cell Therapy, Ribeirão Preto, Brazil.
authors.forename: Dalila Lucíola
authors.initials: DL
authors.lastname: Zanette
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, NY, USA.
authors.forename: Thiago Yukio Kikuchi
authors.initials: TY
authors.lastname: Oliveira
authors.affiliation: Department of Internal Medicine and Pathology, Universidade Federal de Sergipe, Aracaju, SE, Brazil.
authors.forename: Francisco
authors.initials: F
authors.lastname: de Assis Pereira
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, NY, USA.
authors.forename: Julio Cesar Cetrulo
authors.initials: JC
authors.lastname: Lorenzi
authors.affiliation: Department of Biological Science, Universidade Estadual de Santa Cruz, Ilheus, BA, Brazil.
authors.forename: Jane Lima
authors.initials: JL
authors.lastname: Dos Santos
authors.affiliation: Regional Blood Center of Ribeirão Preto and National Institute of Science and Technology in Cell Therapy, Ribeirão Preto, Brazil.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Prata
authors.affiliation: Department of Genetics, Medical School of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: João M Pina
authors.initials: JM
authors.lastname: Neto
authors.affiliation: Department of Clinical Medicine, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Francisco J A
authors.initials: FJ
authors.lastname: de Paula
authors.affiliation: Department of Genetics, Medical School of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.|Regional Blood Center of Ribeirão Preto and National Institute of Science and Technology in Cell Therapy, Ribeirão Preto, Brazil.
authors.forename: Wilson A
authors.initials: WA
authors.lastname: Silva
mesh_terms: D000293:Adolescent; D000328:Adult; D001176:Arthrogryposis / Q000235:genetics*; D001853:Bone Marrow / Q000473:pathology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D002648:Child; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D005819:Genetic Markers / Q000235:genetics*; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis; D010013:Osteogenesis Imperfecta / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D055815:Young Adult
chemical_list: D005819:Genetic Markers
keywords: Bone marrow mesenchymal stromal cell; Bruck syndrome; Gene expression; Osteogenesis Imperfecta; Osteogenic differentiation