Genetic Disorders of Bone and Extracellular Matrix

在实验室和临床的综合研究中，我们研究了遗传性结缔组织病成骨不全(OI)的分子生物学。我们的目标是阐明初级基因缺陷导致骨骼脆性的机制，然后将我们从研究中获得的知识应用于患有这些疾病的儿童的治疗。众所周知，I型胶原分子的结构缺陷是导致OI的主要骨病。十几年前，我们发现胶原蛋白3-羟基化复合体的两个组分CRTAP和P3H1(由LEPRE1编码)的缺陷是隐性OI的原因。我们的工作产生了一种新的基质胶原相关疾病的范式，其中胶原的结构缺陷导致主要的OI，而与胶原相互作用的蛋白质的缺陷导致罕见的OI。 隐性OI现在是BEMB的一个主要调查领域。VII型和Oi的表型与经典显性Oi有所不同，但很难相互区分。我们证明CRTAP和P3H1的相互稳定是III型和III型OI型表型和生化相似性的基础。最近，我们与Boltzman骨学研究所的合作者一起，重点研究了III型OI患者的非致命性亚群的骨骼。骨组织学与III型OI型相似，但具有极薄的骨小梁和骨样斑块增多的显著特征，提示VIII型OI的矿化速度慢于III型OI型。BMDD导致VIII型骨矿化增加，与经典OI和VIII型OI相同，但VIII型骨的低矿化比例高于VIII型。 类型IX OI具有独特的表型，没有根茎，具有独特的生物化学。我们培育了一只CyPB KO小鼠，它降低了骨密度和强度，但增加了脆性。在没有CyPB的情况下，胶原的折叠速度较慢，但CsA治疗使另一种胶原PPIase的潜在存在变得欣喜。CyPB支持LH1的活性，并且它的缺失强烈地调节了LH伴侣复合体。KO骨显著减少了交联残基K87的羟基化，从而改变了纤维结构，降低了骨强度。与北卡罗来纳大学的合作者一起，我们证明了CyPB与所有的黄体生成素形式(LH1-3)相互作用。CyPB通过以特定的方式影响赖氨酸羟化来调节交联度。在皮肤中，尽管LH1和LH2蛋白表达上调，但胶原赖氨酸87也是低羟化和低糖化的。此外，缺乏赖氨酸羟化导致两个不寻常的胶原交联物，来自端肽。KO小鼠的皮肤降低了对纳米压痕的抵抗力，增加了PPIB功能在皮肤力学中的作用。 我们描述了编码跨膜蛋白BRIL的IFITM5的一个突变，它在V型和VI型之间建立了联系。BRIL S40L替换导致突变的FB和成骨细胞表达和分泌最少的PEDF。与导致VOI型的功能获得突变BRIL相反，BRIL S40L导致骨标记物矿化和表达减少。只有I型胶原在两个突变中表现出相似的表达模式，表达、分泌和基质掺入减少。我们已经为这种突变建立了一个小鼠模型，其中杂合子和纯合子都是可行的，这一点目前正在研究中。 XIV OI是2013年发现的一种中度严重OI。这是由编码内质网阳离子通道tre-B的TMEM38B的隐性缺陷引起的。在trc-B隐性零缺陷的先证者中，我们发现内质网钙流动受损，导致沿ATF4途径的内质网应激。铁缺乏被证明与胶原相关，在多个步骤中损害胶原的合成和组装。LH1蛋白水平升高，但胶原螺旋赖氨酸羟化水平降低。我们与英国和维也纳的同事合作，研究了8名XIV OI患者TMEM38B缺乏症的独特方面。即使在兄弟姐妹之间，OI的严重程度也有显着的差异。与其他OI类型一样，XIV型患者的DXA骨密度较低。然而，与其他OI类型不同的是，骨矿化不增加，纳米孔隙度低。XIV型OI的低骨转换可能是破骨细胞固有缺陷所致，破骨细胞通常表达TMEM38B。 最近，我们描述了第一个X连锁的OI隐性形式，由于其新颖的骨机制而变得更加令人兴奋。X-连锁OI中度严重，出生前和产后肋骨和长骨骨折，发育不良的骨骼伴有弯曲和皱缩。它是由编码2位点蛋白酶(S2P)的MBTPS2错义突变引起的。S2P是调节膜内蛋白分解(RIP)的重要组成部分，在RIP过程中，位于高尔基膜上的S1P和S2P在细胞应激或甾醇代谢物缺乏时，依次切割内质网膜上运输的调节蛋白。突变的S2P转录本和蛋白水平正常，但底物OASIS、ATF6和SREBP上的RIP功能受损。在骨组织水平上，I型胶原K87残基的羟化作用是一半正常的，改变了骨中胶原的交联性。S2P缺陷的成骨细胞存在分化缺陷。

In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and then apply the knowledge gained from our studies to the treatment of children with these conditions. Structural defects of type I collagen molecule are well known to cause the dominant bone disorder OI. A dozen years ago, we identified defects in two components of the collagen prolyl 3-hydroxylation complex, CRTAP and P3H1 (encoded by LEPRE1) as the cause of recessive OI. Our work generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects in proteins that interact with collagen cause the rare forms of OI. Recessive OI is now a major area of investigation for the BEMB. The phenotypes of types VII and VIII OI are distinct from classical dominant OI, but difficult to distinguish from each other. We showed that mutual stabilization of CRTAP and P3H1 underlies the phenotypic and biochemical similarity of types VII and VIII OI. With collaborators at the Boltzman Osteology Institute, we recently focused on the bone of the non-lethal subset of type VIII OI patients. Bone histology was similar to type VII OI, although it had the distinctive feature of extremely thin trabeculae and patches of increased osteoid, suggesting mineralization is slower in type VIII than VII OI. BMDD yielded increased mineralization of type VIII bone, as in classical OI and type VIII OI, but the proportion of bone with low mineralization was increased in type VIII bone vs type VII. Type IX OI has a distinctive phenotype without rhizomelia, and distinctive biochemistry. We generated a CyPB KO mouse, which has reduced bone density and strength, but increased brittleness. Collagen folds more slowly in the absence of CyPB, but CsA treatment revels the potential existence of another collagen PPIase. CyPB supports LH1 activity and its absence strongly modulates LH chaperone complexes. KO bone has significant reduction of hydroxylation of crosslinking residue K87, which alters fibril structure and reduces bone strength. With collaborators at the University of North Carolina we showed that CyPB interacted with all LH forms (LH1-3). CyPB modulates crosslinking by affecting lysine hydroxylation in a site-specific manner. In skin, collagen lysine 87 is also underhydroxylated and underglycosylated, despite upregulation of LH1 and LH2 protein. In addition, lack of lysine hydroxylation leads to two unusual collagen crosslinks, derived from the telopeptides. The skin of KO mice has reduced resistance to nanoindentation, adding a role in skin mechanics to PPIB functions. We delineated a mutation in IFITM5, which encodes the transmembrane protein BRIL, that establishes a connection between types V and VI OI. The BRIL S40L substitution results in minimal expression and secretion of PEDF by mutant FB and osteoblasts. In contrast to the gain-of-function BRIL mutation that causes type V OI, the BRIL S40L causes decreased mineralization and expression of bone markers. Only type I collagen shows similar expression pattern in both mutations, with decreased expression, secretion and matrix incorporation. We have generated a murine model for this mutation, in which both heterozygotes and homozygotes are viable, which is currently being characterized. Type XIV OI is a moderately severe form of OI which was identified in 2013. It is caused by recessive defects in TMEM38B, which encodes TRIC-B, an ER cation channel. In probands with recessive null defects in TRIC-B, we demonstrated impaired ER calcium flux, resulting in ER-stress along the ATF4 pathway. TRIC-deficiency was shown to be collagen-related, impairing collagen synthesis and assembly at multiple steps. Collagen helical lysine hydroxylation was reduced, although the levels of LH1 protein were increased. We investigated the unique aspects of TMEM38B deficiency in 8 patients with type XIV OI in collaboration with colleagues in the UK and Vienna. There is striking variability of OI severity even between siblings. Like other OI types, type XIV patients have low DXA BMD. However, unlike other OI types, bone mineralization is not increased and nanoporosity is low. Low bone turnover in type XIV OI is likely explained by an intrinsic defect in osteoclasts, which normally express TMEM38B. Recently, we delineated the first X-linked recessive form of OI, made more exciting by its novel bone mechanism. X-linked OI is moderately severe with pre- and post-natal fractures of ribs and long bones, dysplastic bone with bowing and crumpling. It is caused by missense mutations in MBTPS2, which encodes Site-2 protease (S2P). S2P is a critical component of Regulated Intramembrane Proteolysis (RIP), a process in which S1P and S2P, located in the Golgi Membrane, sequentially cleave regulatory proteins transported from the ER membrane in times of cell stress or sterol metabolite deficiency. The levels of mutant S2P transcripts and protein are normal, but RIP function on substrates OASIS, ATF6 and SREBP are impaired. At the bone tissue level, hydroxylation of type I collagen K87 residues is half normal, altering collagen crosslinking in bone. Osteoblasts with S2P defects have a differentiation defect.