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。异三聚体I型胶原蛋白分子的结构缺陷是导致主要骨疾病成骨不全症的原因。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，其中胶原结构缺陷导致显性成骨不全，而与胶原相互作用的缺陷蛋白导致隐性成骨不全。隐性成骨不全是目前BEMB研究的一个主要领域。VII型和VIII型成骨不全与典型的显性成骨不全不同，但难以区分，巩膜白色，头围正常，根状瘤，长骨严重的下管状。从生物化学角度看，这两组的胶原蛋白序列都是正常的，没有3-羟基化的Pro986残基，但螺旋型脯氨酸和赖氨酸完全过修饰。螺旋过修饰表明，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是相互保护的。此外，在lepre1缺失的细胞中，CRTAP分泌到条件培养基中的量增加，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。BEMB还与一个日本团队合作研究了LEPRE1的突变，该突变仅消除了P3H1中的KDEL er检索信号。这种突变发生在非致死性成骨不全的兄弟姐妹中。我们的报告显示，在内质网中保留P3H1的失败会导致Pro986 3-羟基化的适度减少，但会导致胶原螺旋的过度修饰。本研究表明，KDEl信号对P3H1功能至关重要。BEMB鉴定出两名患有胶原3-羟基化复合物CyPB第三组分突变的儿童，该成分由PPIB编码。这些兄弟姐妹有中等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，导致CyPB蛋白完全缺失。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在缺少第三个组分的情况下可以完成胶原修饰。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。在我们的lepre1缺陷患者中，BEMB发现了一种常见的突变等位基因IVS5+1G to T，这种等位基因发生在非洲裔美国人和西非人身上。这种所谓的“西非等位基因”只在非洲人后裔中发现。我们确定在美国大西洋中部，每200-300名非洲裔美国人中就有1人携带病毒。在与NHGRI的Charles Rotimi的合作中，我们确定当代加纳人和尼日利亚人携带这种致命隐性突变的频率为1.5%！这种高载体频率使得非洲严重成骨不全的遗传与北美和欧洲的主要形式不同，在北美和欧洲，5-7%的成骨不全病例发生隐性成骨不全。据估计，这种突变的年龄约为600岁，与起源于西非并通过大西洋奴隶贸易引入北美的创始突变一致。我们的研究表明，在中非和西非的一些国家没有发现这种突变，因此不是泛非SNP；这种创始人突变局限于加纳/尼日利亚的原因可能在于该地区使用具有共同根源的语言。最近，我们研究了XI型OI的发病机制，XI型OI是一种由亲免疫蛋白FKBP65缺失引起的隐性OI。1例中重度XI型OI患者FKBP65蛋白完全缺失。在缺乏FKBP65的细胞中，胶原折叠是正常的，这表明FKBP65的伴侣活性在胶原生物化学中不起主要作用。然而，我们证明了在正常胶原分泌的情况下，胶原沉积在基质中的显著减少。质谱分析表明，在缺乏FKBP65的情况下，参与交联的胶原端肽赖氨酸不会被羟基化，这将破坏胶原基质的结合。免疫荧光显示基质中胶原原纤维稀疏、无组织。最后，我们提出显性和隐性成骨不全共同的途径可能为疾病机制提供关键见解。这些共性包括胶原蛋白翻译后修饰和折叠的改变、软骨和骨的异常（骨软骨营养不良）、内质网应激、胶原蛋白结合、细胞基质效应、骨转换增加和骨组织的高矿化。

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. Structural defects of the heterotrimeric type I collagen molecule are well known to cause the dominant bone disorder osteogenesis imperfecta. 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 generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects proteins that interact with collagen cause recessive OI. <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, with white sclerae, normal head circumference, rhizomelia, 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. The helical overmodification 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 protective. Also, in LEPRE1-null cells, the secretion of CRTAP into conditioned media is increased 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. The BEMB also collaborated with a Japanese team to study a mutation in LEPRE1 that eliminates only the KDEL ER-retrieval signal from P3H1.This mutation occurs in siblings with non-lethal OI. Our report showed that failure to retain P3H1 in the ER leads to a modest reduction in Pro986 3-hydroxylation but causes overmodification of the collagen helix. This study demonstrated that the KDEl signal is essential for P3H1 function. The BEMB identified two children with a mutation in the 3rd component of the collagen 3-hydroxylation complex, CyPB, which is incoded by PPIB. These siblings have recessive 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. 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 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. More recently, we have investigated the mechanism of type XI OI, a recessive form of OI caused by absence of the immunophilin FKBP65. A case of moderately severe type XI OI has total absence of FKBP65 protein. Collagen folding is normal in the cells with absence of FKBP65, showing that the chaperone activity of FKBP65 does not play a major role in collagen biochemistry. However, we demonstrated a dramatic decrease in the collagen deposited into matrix in culture despite normal collagen secretion. On mass spectrometry, the collagen telopeptide lysine involved in cross-linking is not hydroxylated in the absence of FKBP65, which would undermine collagen matrix incorporation. Immunofluorescence shows sparse, disorganized collagen fibrils in matrix. 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.