TRANSTHYRETIN VARIANTS IN FAMILIAL TTR AMYLOIDOSIS BY MASS SPECTROMETRY

通过质谱分析家族性 TTR 淀粉样变性中的转甲状腺素蛋白变异体

• 批准号: 8365507
• 负责人: MARTHA M SKINNER
• 金额: $ 0.77万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365507
• 关键词: Affect; African American; Algorithms; Amino Acid Substitution; Amino Acids; Amyloid; Amyloid Fibrils; Amyloidosis; Binding; Biochemical Genetics; Biology; Boston; Cardiac; Carrier Proteins; Clinical; Complex; Congo Red; DNA Sequence; DNA Sequence Analysis; Deposition; Detection; Development; Diagnosis; Diagnostic; Digestion; Electrospray Ionization; Exons; Funding; Gene Mutation; Genetic screening method; Grant; Heart; Heart Diseases; Histology; Hormones; Immunoglobulins; Inherited; Isoelectric Focusing; Light; Link; Mass Spectrum Analysis; Medical center; Medicine; Methods; Modification; Mutation; National Center for Research Resources; Organ; Paper; Patients; Peptide Hydrolases; Plasma; Polymerase Chain Reaction; Population; Prealbumin; Principal Investigator; Proteins; Publishing; Research; Research Infrastructure; Resources; Restriction fragment length polymorphism; Retinol Binding Proteins; Site; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thyroxine; Tissues; United States National Institutes of Health; Variant; Vitamin A; cost; disease diagnosis

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Transthyretin (TTR) is a transport protein consisting of 127 amino acid residues. TTR normally exists as a tetramer in the plasma and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR affect the stability of the tetramer and cause the TTR to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with the deposition of the TTR variants as amyloid fibrils in various tissues and organs. A definitive diagnosis of ATTR depends on the detection and characterization of TTR variants. Isoelectric focusing is initially used to screen for TTR variants. Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, in combination with enzymatic digestions, are used to determine the mass difference between the wild type and variant TTR and to locate the site(s) of the modification(s). Knowing the site of the modification in advance simplifies DNA sequence analysis because only the exon containing the mutation would need to be amplified by polymerase chain reaction. Genotypic and phenotypic expression in the inherited forms of transhyretin (TTR) associated amyloidosis (ATTR) are widely variable, however, and may obscure the accurate diagnosis of disease. Our multi-analyses approach for amyloid disease identification and type determination includes Congo red histology, isoelectric focusing (IEF), genetic mutation analyses (direct DNA sequencing, RFLP) and mass spectrometry of intact proteins and protease digests of immunoprecipitated TTR (MS). Using this diagnostic algorithm that combines histological, biochemical and genetic testing, we regularly assist in the diagnosis of patients referred to the Boston Medical Center Amyloid Treatment and Research Center. We have recently published a study of the occurrence of the Ile122 variant, which has been linked to heart disease, among the population of African-American patients (LH Connors et al., Cardiac amyloidosis in African Americans: comparison of clinical and laboratoryfeatures of transthyretin V122I amyloidosis and immunoglobulin light chainamyloidosis.Am Heart J. 2009, 158, 607-614. ) and a paper describing our development of top-down sequencing methods for direct and rapid analysis of the intact proteins.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 经硫代蛋白（TTR）是由127个氨基酸残基组成的转运蛋白。 TTR通常作为四聚体在血浆中存在，并结合激素甲状腺素和视黄醇结合蛋白 - 维生素A复合物。 TTR中的氨基酸取代会影响四聚体的稳定性，并导致TTR形成中间体，从而自我关联成淀粉样蛋白的原纤维。家族性经甲状腺素蛋白淀粉样变性（ATTR）与TTR变体作为淀粉样蛋白原纤维的沉积有关。对ATT的明确诊断取决于TTR变体的检测和表征。等电聚焦最初用于筛选TTR变体。电喷雾电离和基质辅助激光解吸/电离质谱法与酶消化相结合，用于确定野生型和变体TTR之间的质量差异并定位修饰的位点。事先了解修饰的位点可以简化DNA序列分析，因为只有包含突变的外显子才能通过聚合酶链反应进行扩增。但是，基因型和表型表达在透明素（TTR）相关淀粉样变性（ATTR）的遗传形式中是广泛的，并且可能掩盖了疾病的准确诊断。 我们用于淀粉样蛋白疾病鉴定和类型确定的多肛门方法包括刚果红色组织学，等电聚焦（IEF），遗传突变分析（直接DNA测序，RFLP）和完整蛋白质的质谱和免疫接种TTR的蛋白酶消化（MS）的质谱。使用这种结合了组织学，生化和基因检测的诊断算法，我们定期协助诊断为波士顿医疗中心淀粉样蛋白治疗和研究中心的患者。我们最近发表了一项关于非裔美国人患者人群中ILE122变体的发生的研究（LH Connors等人，非裔美国人的心脏淀粉样变性：临床和实验室的比较：trans蛋白的临床和实验室FEATIRES的比较。 607-614）和一篇论文，描述了我们开发自上而下的测序方法，以直接和快速分析完整的蛋白质。