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: 7369228
• 负责人: MARTHA M SKINNER
• 金额: $ 0.71万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369228
• 关键词: TRANSTHYRETIN; VARIANTS; FAMILIAL; TTR; AMYLOIDOSIS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 and recently defined the amyloid disease type in two cases, each associated with a previously undescribed unique TTR variant protein. In the first case, a 55-year-old man with progressive cardiomyopathy and mild peripheral neuropathy had negative hematologic testing and no obvious family history. Congophilic deposits were demonstrated in both an endomyocardial biopsy and abdominal fat aspirate. Serum screening by IEF showed a variant TTR. DNA sequencing of TTR exons 1-4 demonstrated heterozygosity (GAG and GGG) in codon 61. This result was confirmed by RFLP using Bmr I. MS analysis verified the presence of TTR-E61G (-72 mass difference) in serum. The second case was a 57-year-old woman with vitreous opacities (a hallmark clinical feature for ATTR). Congo red staining was positive on a vitreous fluid biopsy and negative on an abdominal fat aspirate. A serum TTR variant was detected by IEF and a genetic abnormality in codon 35 (AAG and ACG) was noted. The mutation was confirmed by RFLP. Serum contained both the wild type protein and TTR-K35T (-27 mass difference) as characterized by MS. Two new pathologic TTR variants, TTR-E61G and TTR-K35T, are amyloidogenic and feature different clinical patterns of expression.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。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，最近在两种情况下定义了淀粉样蛋白疾病类型，每种淀粉样蛋白疾病类型与先前未描述的独特TTR变异蛋白相关联。 在第一种情况下，一名55岁的患有心肌病和轻度周围神经病的男性患有阴性血液学检测，没有明显的家族史。在心内膜活检和腹部脂肪吸入术中都证明了核沉积物。由IEF进行的血清筛选显示出变体TTR。 TTR外显子1-4的DNA测序在密码子61中表现出杂合性（GAG和GGG）。使用BMR I的RFLP证实了该结果。MS分析验证了血清中TTR-E61G（-72质量差）的存在。第二个案例是一名57岁的玻璃体渗透妇女（ATTR的标志性临床特征）。刚果红染色是玻璃体活检的阳性，腹部脂肪染色为阴性。 IEF检测到血清TTR变体，并注意到密码子35（AAG和ACG）中的遗传异常。 RFLP证实了该突变。血清包含MS的特征，既包含野生型蛋白质和TTR-K35T（-27质量差）。两个新的病理TTR变体TTR-E61G和TTR-K35T具有淀粉样蛋白生成，具有不同的临床表达模式。