Kaneto Carla M, Lima Patrícia S P, Zanette Dalila Lucíola, Oliveira Thiago Yukio Kikuchi, de Assis Pereira Francisco, Lorenzi Julio Cesar Cetrulo, Dos Santos Jane Lima, Prata Karen L, Neto João M Pina, de Paula Francisco J A, Silva Wilson A
Department of Genetics, Medical School of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
Department of Biological Science, Universidade Estadual de Santa Cruz, Ilheus, BA, Brazil.
BMC Med Genet. 2016 May 4;17(1):38. doi: 10.1186/s12881-016-0301-7.
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.
成骨不全症（OI）（OMIM %259450）是一组遗传性疾病，其特征是骨脆性增加，临床严重程度从轻度到致命不等。大多数OI病例是由COL1A1或COL1A2基因突变引起的。布鲁克综合征（BS）是一种进一步的隐性遗传的类似OI的表型，其中骨脆性与大关节翼状胬肉和挛缩的异常发现有关。值得注意的是，几项研究未能在BS患者中发现I型胶原生物合成的任何异常。已获得证据表明BS中前胶原端肽赖氨酸羟基化存在特定缺陷，而PLOD2基因中的突变已被鉴定出来。最近，几项研究描述了类似OI和BS患者中的FKBP10突变，表明FKBP10是一个真正的BS基因座。
我们使用ABI PRISM 3130自动测序仪和Big Dye Terminator测序方案，通过序列分析来分析COL1A1、COL1A2、PLOD2和FKBP10基因的编码区以及内含子/外显子边界。从BS、OI患者和健康供体的骨髓中获取单核细胞进行培养，并诱导成骨分化。在间充质干细胞（MSC）向成骨细胞分化过程中，还使用ABI7500序列检测系统通过qRT-PCR评估成骨细胞特异性标志物的基因表达。
在BS患者中未发现COL1A1、COL1A2或PLOD2基因的突变。我们发现了一个纯合的1个碱基对重复（c.831dupC），预计会产生翻译移码突变，并在下游17个氨基酸处产生过早的蛋白质截短（p.Gly278ArgfsX95）。在向成骨细胞分化过程中，通过实时RT-PCR评估成骨细胞特异性标志物BGLAP、COL1A1、MSX2、SPARC和VDR的基因表达，结果显示BS患者和成骨细胞特异性标志物表达模式在BS和健康对照中相似。另一方面，与OI患者相比，发现这些基因的表达模式有所不同。
我们的研究表明，与OI患者相比，BS患者间充质基质细胞向成骨细胞分化过程中观察到的基因表达谱是不同的。本研究首次表明，参与骨生成的基因在BS和OI患者中差异表达。
