Genetic Bone Disorders-Autosomal Recessive OI

遗传性骨病-常染色体隐性成骨不全

• 批准号: 8553840
• 负责人: Joan C Marini
• 金额: $ 90.96万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553840
• 关键词: Accounting; Adipocytes; Adverse effects; Affect; Africa; African; African American; Age; Age of Onset; Alleles; Apoptosis; Area; Biochemical; Biochemistry; Bone Diseases; Bone Tissue; Cartilage; Cell Transplants; Cell secretion; Cells; Child; Childhood; Clinical Trials; Collaborations; Collagen; Collagen Fibril; Collagen Type I; Complex; Conditioned Culture Media; Connective Tissue; Country; Defect; Deposition; Developmental Bone Diseases; Disease; Dose; Ehlers-Danlos Syndrome; Europe; Failure; Family; Fatty acid glycerol esters; Fibroblasts; Frequencies; Functional disorder; Genes; Genetic; Genotype; Ghana; Glycine; Goals; Growth; Head circumference; Human; Hydroxylation; Immunofluorescence Immunologic; Immunophilins; Individual; Initiator Codon; Investigation; Isomerase Gene; Japanese Population; Knock-in Mouse; Knowledge; Laboratories; Language; Location; Lung; Lysine; Mass Spectrum Analysis; Mediating; Metabolism; Modeling; Modification; Molecular Biology; Molecular Chaperones; Molecular Genetics; Morbidity - disease rate; Mucopolysaccharidosis IV; Mus; Mutation; National Human Genome Research Institute; Natural History; Nigeria; North America; Null Lymphocytes; Osteoblasts; Osteogenesis Imperfecta; Osteoporosis; Pathway interactions; Patients; Pattern; Peptidylprolyl Isomerase; Phenotype; Plant Roots; Play; Post-Translational Protein Processing; Procollagen; Proline; Protein Binding; Proteins; Reporting; Retrieval; Role; Sclera; Severities; Siblings; Signal Transduction; Skin; Slave; Somatotropin; Stress; Symptoms; Testing; Tissues; Transcript; Whole Organism; Work; base; bisphosphonate; bone; bone cell; bone quality; bone turnover; crosslink; founder mutation; hammerhead ribozyme; hearing impairment; heritable connective tissue disorder; improved; insertion/deletion mutation; insight; long bone; mouse model; mutant; novel; proband; programs; pulmonary function; response; scoliosis; skeletal; spine bone structure; translational study; treatment trial

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.

在实验室和临床研究的综合项目中，我们研究遗传性结缔组织疾病成骨不全症 (OI) 和埃勒斯-当洛斯综合征 (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，然后将我们研究中获得的知识应用于治疗患有这些疾病的儿童。众所周知，异源三聚体 I 型胶原蛋白分子的结构缺陷会导致显性骨病成骨不全。几年前，BEMB 发现胶原蛋白脯氨酰 3-羟基化复合物的两个成分 CRTAP 和 P3H1（由 LEPRE1 编码）的缺陷是隐性 OI 的原因。我们的工作为与胶原蛋白相关的基质疾病提供了一个新的范例，其中胶原蛋白的结构缺陷导致显性 OI，而与胶原蛋白相互作用的缺陷蛋白则导致隐性 OI。 <br><br>隐性 OI 现在是 BEMB 的主要研究领域。 VII 型和 VIII 型 OI 的表型与经典显性 OI 不同，但彼此难以区分，具有白色巩膜、正常头围、根茎和严重的长骨管状不足。从生化角度来看，两组都具有正常的胶原蛋白序列，不存在 Pro986 残基的 3-羟基化，但螺旋脯氨酸和赖氨酸完全过度修饰。 螺旋过度修饰表明 3-羟基化复合物成分的缺失会导致胶原螺旋折叠延迟。我们现在已经证明，VII 型和 VIII 型 OI 的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 缺失细胞中，条件培养基中 CRTAP 的分泌增加，占细胞中检测到的 CRTP 减少的 15-20%。最近，我们与一个意大利团队合作，研究了在一名患有严重 OI 的非致命先证者中发现的 CRTAP 突变，该先证者因空插入/缺失突变的纯合性而引起。 CRTAP转录本和蛋白的水平与生存无关，这可能与基质中分泌的CRTAP的功能有关。重要的是，这项研究首次证明，缺乏 CRTAP 会导致沉积到基质中的胶原蛋白严重缺乏（对照的 10-15%），并导致最小纤维网络的瓦解。 BEMB 还与一个日本团队合作研究 LEPRE1 的突变，该突变仅消除 P3H1 的 KDEL ER 检索信号。这种突变发生在患有非致命性 OI 的兄弟姐妹中。我们的报告表明，未能在 ER 中保留 P3H1 会导致 Pro986 3-羟基化适度减少，但会导致胶原蛋白螺旋过度修饰。该研究证明KDE1信号对于P3H1功能至关重要。 BEMB 发现两名儿童的胶原蛋白 3-羟基化复合物的第三个成分 CyPB 发生突变，该成分由 PPIB 编码。这些兄弟姐妹患有中等严重程度的隐性成骨不全症，有白色巩膜，但没有根瘤。它们的肽脯氨酰异构酶基因密码子起始处有纯合突变，导致 CyPB 蛋白完全缺失。令人惊讶的是，胶原蛋白Pro986的3-羟基化以及螺旋赖氨酸和脯氨酸残基的羟基化均正常。首先，这意味着3-羟基化复合物的两个组分CRTAP和P3H1可以在没有第三个组分的情况下完成胶原蛋白修饰。其次，正常的螺旋修饰表明胶原蛋白螺旋的折叠率是正常的。由于CyPB以前被认为是独特的胶原蛋白顺反脯氨酰异构酶，因此在没有CyPB的情况下正常的胶原蛋白折叠意味着人体细胞中的这一重要功能必定存在冗余。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，即 IVS5+1G 到 T，这种突变等位基因在非裔美国人和西非人中都存在。这种所谓的“西非等位基因”仅在非洲人后裔中发现。我们确定美国大西洋中部的载波频率为 200-300 个非洲裔美国人中就有 1 个。在 NHGRI 的 Charles Rotimi 的合作中，我们确定当代加纳人和尼日利亚人这种致命隐性突变的携带频率为 1.5%！这种高携带频率使得非洲严重成骨不全症的遗传与北美和欧洲流行的显性成骨不全症不同，在北美和欧洲，隐性成骨不全症发生在 5-7% 的成骨不全病例中。据计算，该突变的年龄约为 600 岁，与起源于西非并通过大西洋奴隶贸易引入北美的始祖突变一致。我们的研究表明，中非和西非的一些国家没有发现该突变，因此不是泛非洲的SNP；这种创始人突变仅限于加纳/尼日利亚的原因可能在于使用该地区使用的具有共同根源的语言。最近，我们研究了 XI 型 OI 的机制，这是一种由亲免素 FKBP65 缺失引起的隐性 OI 形式。中重度 XI 型 OI 病例完全缺乏 FKBP65 蛋白。在缺乏 FKBP65 的细胞中，胶原折叠是正常的，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，尽管胶原蛋白分泌正常，沉积到培养物基质中的胶原蛋白却急剧减少。质谱分析表明，在缺乏 FKBP65 的情况下，参与交联的胶原蛋白端肽赖氨酸不会被羟基化，这会破坏胶原蛋白基质的掺入。免疫荧光显示基质中稀疏、杂乱的胶原原纤维。最后，我们提出显性和隐性 OI 的共同途径可能为疾病机制提供重要见解。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过度矿化。