TRANSTHYRETIN VARIANTS IN FAMILIAL TTR AMYLOIDOSIS BY MASS SPECTROMETRY

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 转甲状腺素(TTR)是一种由127个氨基酸残基组成的转运蛋白。TTR通常以四聚体的形式存在于血浆中，并与激素甲状腺素和视黄醇结合蛋白-维生素A复合体结合。TTR中的氨基酸取代影响四聚体的稳定性，并导致TTR形成中间产物，这些中间产物自结合成淀粉样纤维。家族性甲状腺激素转运蛋白淀粉样变性(ATTR)是一种以淀粉样纤维形式存在于各种组织和器官中的变异体。确定的诊断依赖于TTR变异体的检测和特征。等电聚焦最初用于筛选TTR型变异体。用电喷雾电离和基质辅助激光解吸电离质谱仪，结合酶消化，测定野生型和变异型TTR的质量差异，并定位修饰位点(S)(S)。事先知道修改的位置可以简化DNA序列分析，因为聚合酶链式反应只需要扩增包含突变的外显子。然而，遗传型反式维甲素相关淀粉样变性(ATTR)的基因和表型表达有很大的差异，可能会模糊疾病的准确诊断。我们的多分析方法用于淀粉样病的鉴定和类型确定，包括刚果红组织学、等电聚焦(IEF)、遗传突变分析(DNA直接测序，RFLP)以及完整蛋白质和免疫沉淀TTR(MS)的蛋白酶消化的质谱分析。使用这种结合了组织学、生化和基因检测的诊断算法，我们定期帮助波士顿医学中心淀粉样蛋白治疗和研究中心转诊的患者进行诊断。我们最近发表了一项关于Ile122变异在非裔美国人患者中发生的研究，该变异与心脏病有关(LH Connors等人，《非洲裔美国人的心脏淀粉样变性：转甲状腺素V122I淀粉样变性和免疫球蛋白轻链淀粉样变性的临床和实验室特征的比较》，心脏杂志，2009,158,607-614。)以及一篇论文，描述了我们开发的用于直接和快速分析完整蛋白质的自上而下测序方法。

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.