Genetic Bone Disorders-Autosomal Recessive OI

在一个综合的实验室和临床研究项目中，我们研究了遗传性结缔组织疾病成骨不全症（OI）和ehers - danlos综合征（EDS）的分子生物学。我们的目标是阐明主要基因缺陷导致骨骼脆弱和其他结缔组织症状的机制，然后将从我们的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，异三聚体I型胶原分子的结构缺陷是导致主要骨疾病成骨不全的原因。1979年首次提出了严重隐性成骨不全的假设。我们假设，伴有胶原生化异常和胶原基因序列正常的隐性成骨不全的原因可能与一个基因有关，该基因的产物与I型胶原相互作用。几年前，BEMB发现胶原脯氨酸3-羟基化复合物的两个组分CRTAP和P3H1（由LEPRE1编码）的缺陷是隐性成骨不全的原因。我们的工作为胶原相关的基质疾病创造了一个新的范例，其中胶原的结构缺陷导致显性成骨不全，而内质网中修饰胶原的复合物成分的缺陷导致隐性成骨不全。在OI的扩展分类中，CRTAP和LEPRE1中的缺陷分别被指定为VII型（OMIM #610682）和VIII型（OMIM #610915） OI。隐性成骨不全是目前BEMB研究的一个主要领域。VII型和VIII型成骨不全与经典显性成骨不全不同，但难以区分。两组儿童均有严重/致死性成骨不全，巩膜呈白色，头围正常或小，根状缺损，掌骨缩短和长骨严重管状不足。从生化角度看，两组的胶原蛋白序列均正常，Pro986残基没有3-羟基化，但螺旋型脯氨酸和赖氨酸被脯氨酸4-羟化酶和赖氨酸羟化酶完全过修饰。这种螺旋的过度修饰是出乎意料的，表明缺乏3-羟基化复合物的成分导致胶原螺旋的折叠延迟。我们现在已经证明，VII型和VIII型OI表型和胶原生化相似性的基础是CRTAP和P3H1在复合物中相互保护。我们观察到，尽管正常组分的转录水平没有降低，但在复合体的任何一个组分都有缺陷的细胞裂解物中，CRTAP严重减少，P3H1缺失。免疫荧光显微镜支持CRTAP和P3H1相互保护的解释；在任何一种基因发生突变的细胞中，检测到这两种蛋白质的水平降低。在抑制剂处理的CRTAP-null细胞中，P3H1部分获救。此外，在lepre1缺失的细胞中，CRTAP在条件培养基中的分泌量比对照细胞增加，占细胞中检测到的CRTP减少量的15-20%。最近，我们与一个意大利团队合作，研究了在一个非致死性先证中发现的由零插入/缺失突变纯合性引起的严重OI的CRTAP突变。CRTAP转录本和蛋白水平与存活无关，这可能与基质中分泌的CRTAP的功能有关。重要的是，该研究首次证明了CRTAP的缺失会导致沉积在基质中的胶原严重缺乏（占对照组的10-15%），并导致最小纤维网络的破坏。今年，BEMB发现了两个孩子的胶原3-羟基化复合物（CyPB）的第3组分突变，该成分由PPIB编码。这些兄弟姐妹有中度严重程度的隐性成骨不全，有白色巩膜，但没有根瘤。它们在肽脯氨酸异构酶基因的密码子开始处有纯合突变，这导致CyPB蛋白完全缺失，用3种不同的抗体和免疫荧光显微镜进行Western blots检测。令人惊讶的是，胶原蛋白Pro986的3-羟基化和螺旋赖氨酸和脯氨酸残基的羟基化都是正常的。首先，这意味着3-羟基化复合物的两个组分CRTAP和P3H1可以在缺少第三个组分的情况下完成胶原修饰。其次，正常的螺旋修饰表明胶原蛋白螺旋的折叠率正常。由于CyPB先前被认为是唯一的胶原顺式-反式脯氨酸异构酶，在缺乏CyPB的情况下，正常的胶原折叠意味着在人类细胞中必须有冗余的这一重要功能。

In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. <br><br>Structural defects of the heterotrimeric type I collagen molecule are well known to cause the dominant bone disorder osteogenesis imperfecta. A severe recessive form of OI was first postulated in 1979. We hypothesized that the cause of recessive OI with abnormal collagen biochemistry and normal collagen gene sequence would involve a gene(s) whose products interacted with type I collagen. Several years ago the BEMB 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 has generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects in the components of a complex in the endoplasmic reticulum that modifies collagen cause recessive OI. In the expanded nosology for OI, defects in CRTAP and LEPRE1 are designated as types VII (OMIM #610682) and VIII (OMIM #610915) OI, respectively. <br><br>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. Both groups of children have severe/lethal OI with white sclerae, normal or small head circumference, rhizomelia, metacarpal shortening and severe undertubulation of long bones. Biochemically, both groups have normal collagen sequences with absence of 3-hydroxylation of the Pro986 residue, but full overmodification of the helical prolines and lysines by prolyl 4-hydroxylase and lysly hydroxylase. This overmodification of the helix was unexpected and indicates that absence of the components of the 3-hydroxylation complex leads to delayed folding of the collagen helix. We have now shown that the basis of the phenotypic and collagen biochemical similarity of types VII and VIII OI is that CRTAP and P3H1 are mutually protectively in the complex. We observed that CRTAP is severely reduced and P3H1 is absent from cell lysates which have a defect in either component of the complex, although the transcript level of the normal component is not reduced. The interpretation of mutual protection of CRTAP and P3H1 is supported by immunofluorescence microscopy; reduced levels of both proteins are detected in cells with a mutation in either gene. P3H1 is partially rescued in CRTAP-null cells treated with inhibitors. Also, in LEPRE1-null cells, the secretion of CRTAP into conditioned media is increased compared with control cells and accounts for 15-20% of the decreased CRTP detected in cells. Recently, we have collaborated with an Italian team to study the CRTAP mutation found in a non-lethal proband with severe OI caused by homozygosity for a null insertion/deletion mutation. The levels of CRTAP transcripts and protein do not correlate with survival, which may be related to functions of the secreted CRTAP in matrix. Importantly, this study provided the first demonstration that absence of CRTAP results in a severe deficiency of collagen deposited into matrix (10-15% of control), with disorganization of the minimal fibrillar network. <br><br>This year the BEMB identified a two children with a mutation in the 3rd component of the collagen 3-hydroxylation complex, CyPB which is incoded by PPIB. These siblings have recessivvee OI of moderate severity with white sclerae but without rhizomelia. They have a homozygous mutation in the start of codon of the peptidly prolyl isomerase gene, which results in a total absence of CyPB protein as measured on Western blots with 3 different antibodies and by immunfluoresence microscopy. Surprisingly, the 3-hydroxylation of collagen Pro986 and the hydroxylation of helical lysine and proline residues were both normal. First of all, this means that the two component of the 3-hydroxylation complex, CRTAP and P3H1, can complete collagen modification in the absence of the 3rd component. Second, normal helical modification indicates that the folding rate of the collagen helix is normal. Since CyPB had been previously thought to be the unique collagen cis-trans prolyl isomerase, normal collagen folding in the absence of CyPB means that there must be redundancy for this important function in human cells. Among our LEPRE1-deficient patients, the BEMB identified a common mutant allele, IVS5+1G to T, which occurred in both African-Americans and West Africans. This so-called "West-African allele" has been found only in individuals of African descent. We determined a carrier frequency in Mid Atlantic USA of 1 in 200-300 African-Americans. In a collaboration which Charles Rotimi of NHGRI, we determined contemporary Ghanians and Nigerians had a carrier frequency for this lethal recessive mutations of 1.5%! This high carrier frequency makes the inheritance of severe OI in African distinct from the dominant form prevalent in North America and Europe, where recessive OI occurs in 5-7% of OI cases. The age of the mutation is calculated to be about 600 years old, consistent with a founder mutation that originated in West African and was introduced into North America by the Atlantic slave trade. Our studies have shown that the mutation is not found in a number of countries in Central and West Africa and hence is not pan-African SNP; the reasons for the limitation of this founder mutation to Ghana/Nigeria may reside in the use of languages with common roots spoken in this region. Finally, we have proposed that pathways common to dominant and recessive OI are likely to provide key insights into disease mechanism. These commonalities include alterations in collagen post-translational modification and folding, abnormalities in both cartilage and bone (osteochondrodystrophy), ER stress, collagen-protein binding, cell-matrix effects, increased bone turnover and hypermineralization of bone tissue.