Genetic Bone Disorders-Autosomal Recessive OI

遗传性骨病-常染色体隐性成骨不全症

• 批准号: 8941431
• 负责人: Joan C Marini
• 金额: $ 97.7万
• 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/8941431
• 关键词: 2-Oxoglutarate 5-Dioxygenase Procollagen-Lysine; Accounting; Adipocytes; Adverse effects; Affect; African; African American; Age of Onset; Alaska; Alleles; Apoptosis; Area; Biochemical; Biochemistry; Bone Density; Bone Diseases; Bone Tissue; Cell Transplants; Cells; Child; Childhood; Clinical; Clinical Trials; Collagen; Collagen Gene; Collagen Type I; Complex; Connective Tissue; Contracture; Cyclosporine; Defect; Developmental Bone Diseases; Disease; Dose; Ehlers-Danlos Syndrome; Endoplasmic Reticulum; Family; Fatty acid glycerol esters; Fibroblasts; Frequencies; Functional disorder; Genes; Genetic; Genotype; Glycine; Goals; Growth; Head circumference; Histology; Hydroxylation; Individual; Integral Membrane Protein; Investigation; Knock-in Mouse; Knockout Mice; Knowledge; LHX2 gene; Laboratories; Location; Lung; Lysine; Mediating; Metabolism; Metacarpal bone; Mixed Function Oxygenases; Modeling; Molecular Biology; Molecular Chaperones; Molecular Genetics; Morbidity - disease rate; Mus; Mutation; Natural History; Normal Range; Online Mendelian Inheritance In Man; Osteoblasts; Osteogenesis Imperfecta; Osteoporosis; Pathway interactions; Patients; Pattern; Peptidylprolyl Isomerase; Phenotype; Procollagen; Procollagen-Proline Dioxygenase; Proline; Proteins; Research Personnel; Residual state; Role; Sclera; Serum; Severities; Siblings; Site; Skin; Somatotropin; Stress; Structure; Symptoms; Syndrome; Testing; Tissues; Transcript; Whole Organism; Work; base; bisphosphonate; bone; bone cell; bone quality; bone strength; crosslink; disease classification; exome sequencing; fibrillogenesis; glycosylation; hammerhead ribozyme; hearing impairment; heritable connective tissue disorder; improved; long bone; migration; mouse model; mutant; novel; osteoblast differentiation; pigment epithelium-derived factor; 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. <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. More recently, investigators have noted that some patients with clinical OI do not have defects detected in the type I collagen genes during sequencing. These patients without mutations in collagen can be divided into those who have abnormal collagen biochemistry and those with normal electrophoretic migration of the collagen chains. 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. Seven 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. Among our LEPRE1-deficient patients, we identified a common mutant allele, IVS5+1G to T, which occurred in both African-Americans and West African families. This so-called "West-African allele" accounts for a third of the known LEPRE1 mutations, and has been found only in individuals of African descent. To our surprise, contemporary West Africans have a carrier frequency for this lethal recessive mutation of 1.5%! 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. Type IX OI has a distinctive phenotype without rhizomelia, and distinctive biochemistry compared to types VII and VIII. We have generated a CyPB KO mouse to explore these distinctions further. Knock-out mice are small, with reduced bone density and strength, but increased brittleness. Only 1-2% 3-hydroxyltion is detected in KO cells, showing the importance of CyPB to complex function. Collagen folds more slowly in the absence of CyPB, but CsA treatment revels the potential existence of another collagen PPIase. CyPB supports collagen lysyl hydroxylase (LH1) activity and its absence allows site-specific alterations in helical lysine hydroxylation, in particular significant reduction of hydroxylation of crosslinking residue K87. The decreased crosslink ratio alters fibril structure and reduces bone strength. The effects of CyPB on collagen glycosylation crosslinking and fibrillogenesis are novel findings. Recessive mutations in FKBP10, which encodes FKBP65, cause type XI OI. Mutations in this gene also cause Bruck Syndrome, which is OI plus congenital contractures. These mutations are allelic, since siblings with the same mutation my have OI or Bruck Syndrome. Thus, contractures are shown to be a variable manifestation of FKBP10 mutations. We also identified an FKBP10 mutation in Kuskokwim syndrome (KS), a recessive congenital contracture disorder found among Yupil Eskinos in Alaska. The causative mutation is an in-frame deletion which removes the highly conserved p.Tyr293 residue in FKBP65s third PPIase domain. This mutation destabilizes the protein but leaves residual 5%. FKBP65 supports LH2 function, so it absence substantially decreases hydroxylation of the telopeptide lysine important for collagen crosslinking. Thus FKBP65 mutations affects collagen indirectly through loss of LH2 function. Most recently, we have delineated a muttion in IFITM5, which encodes the transmembrane protein BRIL, that establishes a connection between types V and VI OI. We identified a patient with severe OI whose fibroblasts and osteoblasts secreted minimal amounts of PEDF and whose bone histology was typical of type VI OI, but whose serum PEDF was in the normal range. Whole exome sequencing revealed a de novo mutation in IFITM5 in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL. Both IFITM5 transcripts and BRTIL protein levels were normal in proband cells. However, SERPINF1 expression was minimal. Expression of type I collagen was similarly decreased in proband osteoblasts, and the pattern of osteoblast markers was consistent with a primary PEDF defect. Since this mutation in IFITM5 was causing bone-specific type VI OI, we compared these osteoblasts to osteoblasts with the type V OI-causing IFITM5 mutation at the 5:-end of the gene. In these cells we demonstrated increased SERPINF1 expression and PEDF secretion during osteoblast differentiation, connecting the two OI-causing genes in an important pathway under delineation.

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。 <br> <br>异三体I型胶原蛋白分子的结构缺陷众所周知，会导致骨气障碍骨的骨化不完美。 1979年首次假设了一种严重的隐性OI。这些在胶原蛋白中没有突变的患者可以分为那些患有异常的胶原蛋白生物化学的患者，以及胶原蛋白链的正常电泳迁移的患者。我们假设具有异常胶原蛋白生物化学和正常胶原蛋白基因序列的隐性OI原因将涉及一个基因，其产物与I型胶原蛋白相互作用。七年前，BEMB在胶原​​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性疾病产生了新的范式，其中胶原蛋白的结构缺陷导致OI显性OI，而内质网中复合物的缺陷在内质网中的成分中会改变胶原蛋白会导致胶原蛋白会导致隐性oi。在OI的扩展疾病中，CRTAP和LEPRE1中的缺陷分别指定为VII型（OMIM＃610682）和VIII（OMIM＃610915）OI。在我们的LEPER1缺陷患者中，我们确定了一个常见的突变等位基因，即IVS5+1G到T，这是在非裔美国人和西非家庭中发生的。这个所谓的“西非等位基因”占已知LEPRE1突变的三分之一，仅在非洲血统中才发现。令我们惊讶的是，当代西非人的载体频率为1.5％！ 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。两组儿童都有严重/致命的OI，具有白色巩膜，正常或小的头围，根茎，掌骨缩短和长骨的严重插管。从生物化学上讲，两组的胶原蛋白序列均不含Pro986残基的3-羟基化，但通过脯氨酰4-羟化酶和lysly羟基酶对螺旋脯氨酸和赖氨酸的完全过度修饰。螺旋螺旋的这种过度修饰是出乎意料的，表明3-羟基化复合物的成分的缺失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在复合物中相互保护。与VII型和VIII相比，IX型OI具有具有独特的表型，并且具有独特的生物化学。我们已经生成了CYPB KO鼠标，以进一步探索这些区别。敲除小鼠很小，骨密度和强度降低，但昏昏欲睡。在KO细胞中仅检测到1-2％的3-羟基甲基甲基甲基苯基甲基苯基甲基甲基甲基甲基苯基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基甲基的重要性。在没有CYPB的情况下，胶原蛋白折叠较慢，但CSA治疗引起了另一种胶原蛋白PPIASE的潜在存在。 CYPB支持胶原蛋白羟化酶（LH1）活性及其缺失允许螺旋赖氨酸羟基化的位点特异性改变，特别是显着降低了交联残基K87的羟基化的显着降低。降低的交联比率改变了原纤维结构并降低了骨强度。 CYPB对胶原蛋白糖基化交联和原纤维发生的影响是新发现。 编码FKBP65的FKBP10中的隐性突变，导致Xi oi型。该基因中的突变还会导致布鲁克综合征，即OI加上先天性染色。这些突变是等位基因的，因为具有相同突变的兄弟姐妹患有OI或布鲁克综合征。因此，缔合显示是FKBP10突变的变量表现。我们还确定了Kuskokwim综合征（KS）的FKBP10突变，这是阿拉斯加Yupil Eskinos中发现的隐性先天性缔约障碍。 病因突变是一种框内缺失，可去除FKBP65S中高度保守的p.tyr293残基第三PPIASE结构域。这种突变破坏了蛋白质的稳定，但留下了5％。 FKBP65支持LH2功能，因此它没有大大降低端肽赖氨酸对胶原蛋白交联至关重要的羟基化。因此，FKBP65突变通过LH2功能的丧失间接影响胶原蛋白。 最近，我们描绘了编码跨膜蛋白质的iFITM5中的杂种，该跨膜蛋白质是在V型和VI类型之间建立联系的。 我们确定了一名患有严重OI的患者，其成纤维细胞和成骨细胞分泌最小的PEDF，并且其骨骼学是典型的VI型，但其血清PEDF在正常范围内。整个外显子组测序表明，在概率的一个等位基因中，IFITM5中的从头突变，导致在棕褐色的细胞内结构域中替换P.S40L。在概率和细胞中，IFITM5转录本和BRTIL蛋白水平均正常。但是，serpinf1表达最小。 I型胶原蛋白的表达在探针和成骨细胞中类似降低，成骨细胞标记的模式与主要的PEDF缺陷一致。由于IFITM5中的这种突变引起了骨特异性VI oi，因此我们将这些成骨细胞与成骨细胞进行了比较，并在5： - 末端的V oi引起的IFITM5突变中进行了比较： - 基因的末端。在这些细胞中，我们证明了在成骨细胞分化过程中SERPINF1表达和PEDF分泌增加，并在描述下的重要途径中连接了两个引起OI的基因。