TRANSTHYRETIN VARIANTS IN FAMILIAL TTR AMYLOIDOSIS BY MASS SPECTROMETRY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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疗中心淀粉样蛋白治疗和研究中心转诊的患者进行诊断，最近确定了两例淀粉样病类型，每个病例都与一种以前未描述的独特TTR变异蛋白有关。在第一个病例中，一位55岁的男性患者患有进行性心肌病和轻度周围神经病变，血液学检测阴性，没有明显的家族史。心内膜心肌活检和腹壁脂肪抽吸物均可见嗜中性沉着。IEF的血清筛查显示了一种变异型TTR。TTR1-4外显子的DNA测序证实了61密码子的杂合性(GAG和GGG)。这一结果得到了BMR I RFLP的证实。MS分析证实了血清中存在TTR-E61G(-72质量差)。第二个病例是一名57岁的女性，伴有玻璃体混浊(这是ATTR的一个显著临床特征)。玻璃体液活检刚果红染色阳性，腹部脂肪抽吸物染色阴性。IEF检测到血清TTR型变异体，发现35号密码子的遗传异常(AAG和ACG)。对该突变进行了RFLP分析。血清既含有野生型蛋白，又含有TTR-K35T(-27质量差)，这是MS的特征。两个新的病理TTR变异体，TTR-E61G和TTR-K35T，是淀粉样变性的，具有不同的临床表现模式。

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.