Laboratory of Veterinary Physiology, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180–8602, Japan.
Bone. 2011 Nov;49(5):1027-36. doi: 10.1016/j.bone.2011.08.001. Epub 2011 Aug 7.
Homozygous rats (ocd/ocd) of a mutant inbred strain, OCD (osteochondrodysplasia), show osteochondrodysplasia, systemic edema, cleft palate, protruding tongue, disproportionate dwarfism, and lethality immediately after birth. Their epiphyses show decreased levels of glycosaminoglycans and weak staining for extracellular matrix proteins. The epiphyseal chondrocytes have large vesicles and expanded endoplasmic reticulum and Golgi apparatus. These phenotypic features are inherited in an autosomal recessive manner, and the ocd locus responsible for these phenotypes has been mapped close to D11Mgh3 on rat chromosome 11. In the present study, we characterized the embryonic pathogenesis of ocd/ocd rats and identified the mutant gene. Subcutaneous edema in the dorsal portion was found at embryonic day (E) 16.5, and the other anomalies described above were apparent after E18.5 in ocd/ocd. Whole mount immunohistochemistry for Sox9 revealed that mesenchymal condensation was delayed in limb bud in ocd/ocd, and skeletal preparation showed that the progression of whole-body chondrogenesis was delayed in ocd/ocd. Histological and immunohistological analyses of the femur showed that cell proliferations of resting and proliferative zones of growth plate were significantly reduced in ocd/ocd embryos. Fine linkage mapping localized the ocd locus within 84kb of positions 65,584-65,668kb containing a part of Golgb1 gene on chromosome 11. Expression of Golgb1 mRNA was found in limb buds, somite derivatives and calvaria. Sequence analysis identified a 10-bp insertion in exon 13 of the Golgb1 gene in ocd/ocd rats. The Golgb1 gene encodes the COPI vesicle tethering factor, giantin. This insertion mutation causes a frame shift, and introduces a premature termination codon at codon 1082, leading to truncation of the C-terminal two thirds of giantin. By in-gel Western analysis using anti-giantin antibody that recognizes an epitope within 200 aa of the C-terminus, the expression of giantin was not detected in ocd/ocd embryos. As the C-terminal region of giantin is required for localization to the Golgi apparatus, these results strongly suggested that giantin is functionally defective in ocd/ocd rats. Therefore, we concluded that mutation of the Golgb1 gene is responsible for the phenotypic characteristics including osteochondrodysplasia of ocd/ocd, and that giantin plays a pivotal role in multiple aspects of chondrogenesis.
纯合子 OCD（骨软骨发育不良）突变近交系大鼠（ocd/ocd）表现出骨软骨发育不良、全身性水肿、腭裂、舌突出、不成比例的侏儒症和出生后立即死亡。它们的骺板显示糖胺聚糖水平降低，细胞外基质蛋白染色较弱。骺板软骨细胞有大泡和扩张的内质网和高尔基器。这些表型特征以常染色体隐性方式遗传，负责这些表型的 ocd 基因座已被定位在大鼠 11 号染色体上的 D11Mgh3 附近。在本研究中，我们描述了 ocd/ocd 大鼠的胚胎发病机制并鉴定了突变基因。在胚胎第 16.5 天（E）时，背部出现皮下水肿，在 ocd/ocd 大鼠中，上述其他异常在 E18.5 后出现。Sox9 的全骨免疫组织化学显示，肢芽中的间充质凝聚在 ocd/ocd 中延迟，骨骼准备显示 ocd/ocd 中全身软骨发生的进展延迟。股骨的组织学和免疫组织学分析表明，ocd/ocd 胚胎的生长板静止和增殖区的细胞增殖明显减少。精细连锁图谱将 ocd 基因座定位在包含 11 号染色体上 Golgb1 基因一部分的 65,584-65,668kb 范围内的 84kb 内。在肢芽、体节衍生物和颅骨中发现 Golgb1mRNA 的表达。序列分析鉴定出 ocd/ocd 大鼠 Golgb1 基因外显子 13 中的 10bp 插入。Golgb1 基因编码 COPI 囊泡连接因子 giantin。该插入突变导致移码，并在密码子 1082 处引入一个过早终止密码子，导致 giantin 的 C 端三分之二截断。通过使用抗 giantin 抗体的胶内 Western 分析，该抗体识别 C 端 200 个氨基酸内的表位，在 ocd/ocd 胚胎中未检测到 giantin 的表达。由于 giantin 的 C 端区域对于定位于高尔基器是必需的，因此这些结果强烈表明 giantin 在 ocd/ocd 大鼠中功能缺陷。因此，我们得出结论，Golgb1 基因突变负责包括 ocd/ocd 大鼠的骨软骨发育不良在内的表型特征，并且 giantin 在软骨发生的多个方面发挥关键作用。
