TRANSTHYRETIN VARIANTS IN FAMILIAL TTR AMYLOIDOSIS BY MASS SPECTROMETRY

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。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。甲状腺素运载蛋白（TTR）是由127个氨基酸残基组成的转运蛋白。TTR通常以四聚体形式存在于血浆中，并结合甲状腺素和视黄醇结合蛋白-维生素A复合物。TTR中的氨基酸取代影响四聚体的稳定性，并导致TTR形成自缔合成淀粉样蛋白原纤维的中间体。家族性甲状腺素运载蛋白淀粉样变性（ATTR）与TTR变体作为淀粉样纤维在各种组织和器官中的沉积有关。ATTR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法结合用于确定野生型和变体TTR之间的质量差异，并定位修饰位点。事先知道修饰的位点简化了DNA序列分析，因为只有含有突变的外显子需要通过聚合酶链反应扩增。然而，遗传形式的转黄蛋白（TTR）相关淀粉样变性（ATTR）的基因型和表型表达变化很大，可能会模糊疾病的准确诊断。我们用于淀粉样蛋白疾病鉴定和类型确定的多分析方法包括刚果红组织学、等电聚焦（IEF）、基因突变分析（直接DNA测序、RFLP）和完整蛋白质和免疫沉淀TTR（MS）的蛋白酶谱的质谱分析。使用这种结合了组织学、生化和基因检测的诊断算法，我们定期协助诊断转诊到波士顿医学中心淀粉样蛋白治疗和研究中心的患者，最近在两例病例中定义了淀粉样蛋白疾病类型，每例病例均与以前未描述的独特TTR变体蛋白相关。 在第一个病例中，一名55岁的男性患有进行性心肌病和轻度周围神经病变，血液学检查呈阴性，没有明显的家族史。在子宫内膜活检和腹部脂肪抽吸中均发现嗜充血性沉积物。IEF血清筛查显示TTR变异。TTR外显子1-4的DNA测序表明密码子61处存在杂合性（GAG和GGG）。这一结果通过使用Bmr I的RFLP得到证实。MS分析证实血清中存在TTR-E61 G（质量差约为72）。第二个病例是一名57岁的女性，患有玻璃体混浊（ATTR的标志性临床特征）。刚果红染色在玻璃体液活组织检查中呈阳性，在腹部脂肪抽吸物中呈阴性。通过IEF检测到血清TTR变异，并注意到密码子35（AAG和ACG）的遗传异常。经RFLP分析证实该突变。血清含有野生型蛋白质和TTR-K35 T（~ 27质量差异），如通过MS表征的。两种新的病理性TTR变体TTR-E61 G和TTR-K35 T是淀粉样蛋白生成的，并且具有不同的临床表达模式。

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.