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: 7594278
• 负责人: Joan C Marini
• 金额: $ 38.08万
• 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/7594278
• 关键词: 2-Oxoglutarate 5-Dioxygenase Procollagen-Lysine; 2-oxoglutarate 3-dioxygenase proline; Affect; Africa; African; African American; Age; Alleles; Biochemistry; Bone Development; Bone Diseases; Cartilage; Cells; Chest; Child; Clinical; Clinical Trials; Collagen; Collagen Gene; Collagen Type I; Complex; Condition; Connective Tissue; DEXA; DNA; Defect; Developmental Bone Diseases; Differential Scanning Calorimetry; Disease; Ehlers-Danlos Syndrome; Endoplasmic Reticulum; Exons; Fibroblasts; Fracture; Gene Proteins; Genes; Genetic; Genomics; Goals; Haplotypes; Heterozygote; Hydroxylation; Hydroxylysine; Knock-in Mouse; Knock-out; Knockout Mice; Knowledge; Laboratories; Life; Lysine; Mass Spectrum Analysis; Messenger RNA; Metabolic Bone Diseases; Modeling; Modification; Molecular Biology; Mutation; Nonsense Codon; Organ; Osteogenesis Imperfecta; Osteoid; Osteoporosis; Parents; Patients; Peptides; Post-Translational Protein Processing; Prevalence; Proline; Proteins; RNA Splicing; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Rib Fractures; Sclera; Score; Shapes; Skeletal system; Skin; Structure; Symptoms; Testing; Time; Transcript; Tubular formation; Western Blotting; Work; bone; bone cell; bone quality; cyclophilin B; design; glycosylation; heritable connective tissue disorder; long bone; migration; mouse Crtap protein; mouse model; mutant; novel; null mutation; prenatal; proband; programs; repository

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. This past year the BEMB has identified the cause of recessive OI, which has been sought since 1979. 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. Post-translational modifications of collagen are required for efficient folding, secretion and fibril assembly, including 4-hydroxylation of proline residues, hydroxylation of lysine residues and subsequent glycosylation of some hydroxylysines. Collagen is also modified by 3-hydroxylation of a single residue in the alpha 1(I) chain at Pro986, although the function of this specific modification has yet to be elucidated. Morello and colleagues (Morello, et al. Cell. 127, 291-304 (2006)) have generated a knock-out mouse for Cartilage-Associated Protein (CRTAP) with recessive bone dysplasia, characterized by defective osteoid formation and severe osteoporosis. CRTAP was also shown by the Bachinger lab to form a complex in the endoplasmic reticulum with cyclophilin B and prolyl 3-hydroxylase 1 (P3H1)/leprecan (encoded by LEPRE1) which interacts with and 3-hydroxylates collagen. These discoveries made CRTAP a suitable candidate for recessive OI. The BEMB cell repository contained cells from 10 patients who have overmodified collagen biochemistry, yet lack a mutation in either of the type I collagen genes. Of these 10 cases, we have identified three patients with null mutations for CRTAP, designated as type VII OI. Their clinical finding overlap lethal type II OI but with distinctive features, including white sclerae. Patients were screened for null mutations by real-time RT-PCR using primary fibroblast mRNA. Sequencing of the exons and surrounding intronic regions of the CRTAP gene identified homozygous or compound heterozygous mutations in patient genomic DNA, resulting in altered splicing and introduction of premature termination codons in transcripts. All three of the CRTAP null patients have a severe recessive form of OI, which is lethal in the first year of life. Their long bones are extremely osteoporotic and deformed, with prenatal fractures and a tubular shape, due to abnormal modelling. Additionally, they have a narrow thorax with multiple prenatal rib fractures. The mutations identified in CRTAP include a homozygous IVS1+1G>C splicing mutation, a homozygous Gln276Stop in exon 4 and a compound heterozygote of a 16 nt duplication in exon 1 in one allele, and a Met1Ile in the other allele. Parents of CRTAP probands were shown to be heterozygous carriers. Probands had absence of CRTAP protein on Western blot and absence of Pro986 hydroxylation on mass spectrometry analysis. We identified null LEPRE1 mutations in the remaining 7 cases, which we classified as type VIII OI. These probands have bone dysplasia overlapping lethal type II OI and severe type III OI, but with distinctive features, including white sclerae, as found in CRTAP defects. Patients were screened for null mutations by real-time RT-PCR using primary fibroblast mRNA. Sequencing of the exons and surrounding intronic regions of the LEPRE1 gene identified homozygous or compound heterozygous mutations in patient genomic DNA, resulting in altered splicing and introduction of premature termination codons in transcripts. The seven null LEPRE1 patients have severe to lethal recessive bone dysplasia, characterized by shortened long bones, generalized bone disorganization and have extremely low BMD (L1-L4 DEXA z-score = -7). Five of these seven LEPRE1 patients have a common mutant allele, IVS5+1G>T, which apparently originated in West Africa and is also present in African-Americans. Other LEPRE1 mutations include IVS7+91G>A and IVS9+1G>T splicing mutations, a one nt deletion in exon 3, a seven nt insertion in exon 9, an 11 nt deletion in exon 14, and a homozygous Tyr552Stop in exon 11. Parents of LEPRE1 probands were shown to be heterozygous carriers. Furthermore, P3H1 protein was demonstrated to be absent in our patient fibroblasts on Western blots. Patient type I collagen has severely reduced or completely abolished 3-hydroxylation of the alpha 1(I) Pro986 residue, by mass spectrometry of collagen tryptic peptides. In contrast, the extent of overhydroxylation of proband collagens by lysyl hydroxylase is comparable to collagens containing a structural defect in the carboxyl terminus of the helical region, consistent with delayed folding of the molecule. Differential scanning calorimetry of patient collagens was consistent with an increased post-translational modification in the absence of a primary structural defect, with patient collagens showing a one degree centigrade increase in thermal stability compared to normal control collagen. Proband collagen secretion is moderately delayed but total collagen secretion is increased. These recessive null mutations of CRTAP and LEPRE1 result in a novel metabolic disorder of bone, and demonstrate that the 3-hydroxylation complex is crucial for normal bone development. 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.

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。过去一年，BEMB确定了自1979年以来一直在寻求隐性OI的原因。 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。 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。 有效的折叠，分泌和原纤维组装需要胶原蛋白的翻译后修饰，包括脯氨酸残基的4-羟基化，赖氨酸残基的羟基化以及随后的某些羟基酶的糖基化。胶原蛋白还通过在Pro986处的Alpha 1（i）链中的单个残基的3-羟基化来修饰，尽管该特定修饰的功能尚未阐明。 Morello及其同事（Morello等人Cell。127，291-304（2006））生成了一种用于软骨相关蛋白（CRTAP）的基因敲除小鼠，其隐性骨异常发育不良，其特征在于有缺陷的骨质骨质形成和严重的骨质疏松症。 Bachinger Lab还显示了CRTAP在内质网中与环蛋白B和脯氨酰3-羟化酶1（P3H1）/Leprecan（由LEPRE1编码）形成复合物，这些复合物与3-羟基蛋白相互作用。这些发现使CRTAP成为了隐性OI的合适候选者。 BEMB细胞存储库中包含来自10例胶原蛋白生物化学过度过度的患者的细胞，但在I型胶原蛋白基因中的任何一个中都缺乏突变。 在这10例病例中，我们已经确定了3例CRTAP无效突变患者，称为VII型OI。他们的临床发现重叠了致命的II型OI，但具有独特的特征，包括白巩膜。使用原发性成纤维细胞mRNA通过实时RT-PCR筛选患者的无效突变。 CRTAP基因的外显子和周围内含子区域的测序鉴定出患者基因组DNA中的纯合或复合杂合突变，从而改变了剪接和转录物中过早终止密码子的剪接和引入。所有三名CRTAP无效的患者均具有严重的隐性形式，这在生命的第一年都是致命的。由于异常建模，它们的长骨头非常骨质疏松和变形，带有产前骨折和管状形状。此外，它们具有狭窄的胸腔，有多个产前肋骨骨折。 在CRTAP中鉴定的突变包括纯合IVS1+1G> C剪接突变，外显子4中的纯合GLN276STOP和一个等位基因中外显子1中16 NT重复的化合物杂合子以及另一个等位基因的Met1ile。 CRTAP概率的父母被证明是杂合载体。在蛋白质印迹上没有CRTAP蛋白，并且在质谱分析中没有Pro986羟基化。 我们在其余7例中确定了Null Lepre1突变，我们列为VIII型OI。这些概率具有重叠的致死性II型OI和严重III型OI的骨骼发育异常，但具有独特的特征，包括白巩膜，如CRTAP缺陷所发现的那样。使用原发性成纤维细胞mRNA通过实时RT-PCR筛选患者的无效突变。 LEPRE1基因的外显子和周围内含子区域的测序鉴定出患者基因组DNA中的纯合或复合杂合突变，从而改变了转录本中的剪接和过早终止密码子的剪接和引入。七个NULL LEPER1患者患有严重至致命性隐性骨骼发育不良，其特征是长骨缩短，广泛的骨骼混乱，BMD极低（L1-L4 DEXA Z-SCORE = -7）。这七个LEPER1患者中有五个具有常见的突变等位基因IVS5+1G> T，显然起源于西非，也存在于非裔美国人。其他LEPRE1突变包括IVS7+91G> A和IVS9+1G> t剪接突变，外显子3中的一个NT删除，外显子9中的7 NT插入，外显子14中的11 nt删除，在外显子14中的11 nt删除，纯净的Tyr5552Stop in Exon552Stop in Exon52Stop in Exon52 shiper of Lepre1 probands Probands均为HETER。此外，在我们的患者成纤维细胞中，P3H1蛋白在蛋白质印迹中不存在。 I型I型胶原蛋白通过胶原蛋白酶肽的质谱法严重降低或完全消除了α1（I）Pro986残基的3-羟基化。相比之下，赖氨酸羟化酶对探针和胶原蛋白的过度羟基化程度可与胶原蛋白相媲美，该胶原蛋白在螺旋区域的羧基末端中含有结构缺陷，与分子的延迟折叠一致。患者胶原蛋白的差量扫描量量表与在没有原发性结构缺陷的情况下的翻译后修饰增加一致，与正常对照胶原蛋白相比，患者胶原蛋白表现出热稳定性一度摄影的增加。 概率和胶原蛋白分泌被适度延迟，但总胶原蛋白分泌增加。 这些隐性CRTAP和LEPRE1的无效无效突变导致一种新型的骨骼代谢疾病，并证明3-羟基化复合物对于正常的骨骼发育至关重要。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。