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计划中，我们研究了可遗传的结缔组织疾病的分子生物学成骨肌发生Imperfecta（OI）和Ehlers-Danlos综合征（EDS）。我们的目标是阐明主要基因缺陷会导致骨骼脆弱性和其他结缔组织症状，然后将我们从研究中获得的知识应用于患有这些条件的儿童的知识。众所周知，异三体I型胶原蛋白分子的结构缺陷会导致主要的骨骼疾病骨化不完美。几年前，BEMB在胶原​​蛋白蛋白3-羟基化复合物，CRTAP和P3H1（由Lepre1编码）的两个组成部分中鉴定出缺陷是造成隐性OI的原因。我们的工作为基质的胶原蛋白相关疾病产生了一个新的范式，其中胶原蛋白的结构缺陷导致OI显性OI，而与胶原蛋白相互作用的蛋白质引起了隐性OI。 <br> <br>隐性OI现在是BEMB的主要调查领域。 VII类型和VIII类型的表型与经典的主要OI不同，但很难彼此区分，白色巩膜，正常头圆周，根茎和长骨的严重插管。从生物化学上讲，两组的胶原蛋白序列不存在Pro986残基的3-羟基化，但对螺旋脯氨酸和赖氨酸的完全过度修饰。 螺旋过度修饰表明3-羟基化复合物的成分的不存在会导致胶原蛋白螺旋的延迟折叠。现在，我们已经表明，VII类型和VIII类型的表型和胶原蛋白生化相似性的基础是CRTAP和P3H1是互保护的。同样，在LEPRE1-NULL细胞中，CRTAP向条件培养基的分泌增加，占细胞中CRTP降低的15-20％。最近，我们与一支意大利团队合作研究了在非致命概率中发现的CRTAP突变，该突变是由纯合性引起的严重OI，这是无效的插入/缺失突变。 CRTAP转录物和蛋白质的水平与存活无关，这可能与矩阵中分泌的CRTAP的功能有关。重要的是，这项研究提供了首次证明，CRTAP的缺失导致沉积在基质中的胶原蛋白严重缺乏（占对照的10-15％），并且最小的原纤维网络混乱。 BEMB还与日本团队合作研究了Lepre1中的一个突变，该突变仅消除了P3H1中的KDEL ER-RETREREVAL信号。这种突变发生在非致命性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-羟基化的适度降低，但会导致胶原蛋白螺旋的过度修饰。这项研究表明，KDEL信号对于P3H1功能至关重要。 BEMB在胶原​​蛋白3-羟基化复合物CYPB的第三个成分中发现了两个患有突变的儿童，该复合物由PPIB构成。这些兄弟姐妹的隐性ei严重程度适中，但没有根茎。它们在肽蛋白酶异构酶基因的密码子开始时具有纯合突变，这导致总缺乏CYPB蛋白。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，CRTAP和P3H1的两个成分可以在没有第3个成分的情况下完成胶原蛋白的修饰。其次，正常的螺旋修饰表明胶原蛋白螺旋的折叠速率正常。由于以前被认为CYPB是独特的胶原蛋白顺式培养基异构酶，因此在没有CYPB的情况下，正常的胶原蛋白折叠意味着人类细胞中这种重要功能必须具有冗余性。在我们的LEPER1缺陷患者中，BEMB确定了一个常见的突变等位基因，即IVS5+1g T到T，这是在非裔美国人和西非人中发生的。这个所谓的“西非等位基因”仅在非洲血统的个体中被发现。我们在200-300名非裔美国人中确定美国中大西洋中部的载体频率为1。在NHGRI的Charles Rotimi的合作中，我们确定当代加纳人和尼日利亚人的载体频率为1.5％！这种高载体频率使非洲的严重OI继承与北美和欧洲普遍存在的主要形式不同，在5-7％的OI案件中发生隐性OI。该突变的年龄被计算出约600年的历史，与起源于西非的创始人突变一致，并被大西洋奴隶贸易引入北美。我们的研究表明，在中非和西非的许多国家没有发现突变，因此不是泛非SNP。该创始人突变对加纳/尼日利亚的限制的原因可能存在于使用该地区具有共同根源的语言。最近，我们研究了Xi oi型的机理，这是由于缺乏免疫光素FKBP65引起的一种隐性形式的OI。中度严重的XI OI的情况总缺少FKBP65蛋白质。胶原蛋白折叠在细胞中不存在FKBP65的情况下是正常的，表明FKBP65的伴侣活性在胶原蛋白生物化学中不起主要作用。但是，尽管胶原蛋白分泌正常，但我们证明了在培养物中沉积在基质中的胶原蛋白的急剧下降。在质谱上，在没有FKBP65的情况下，与交联的胶原蛋白蛋白赖氨酸未被羟基化，这会破坏胶原基质掺入。免疫荧光在基质中显示出稀疏，混乱的胶原纤维。最后，我们提出，占主导地位和隐性OI的途径可能会提供对疾病机制的关键见解。这些共同点包括胶原蛋白翻译后修饰和折叠的改变，软骨和骨骼（骨软骨植物）的异常（ER应激），胶原蛋白 - 蛋白质的结合，细胞矩阵作用，增加的骨转换和骨组织的高矿化。