TRANSTHYRETIN VARIANTS IN FAMILIAL TTR AMYLOIDOSIS BY MASS SPECTROMETRY

点击翻译按钮获取中文摘要

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.