Identification of transthyretin variants by sequential proteomic and genomic analysis

H. Robert Bergen, Steven R. Zeldenrust, Malinda L. Butz, Denise S. Snow, Peter J. Dyck, P. James B. Dyck, Christopher J. Klein, John F. O'Brien, Stephen N. Thibodeau, David C. Muddiman

Research output: Contribution to journalArticlepeer-review

50 Scopus citations


Background: Transthyretin-associated hereditary amyloidosis (ATTR) is an inherited disease in which variants in the primary structure of transthyretin (TTR; prealbumin) lead to the extracellular polymerization of insoluble protein fibrils, causing organ failure and ultimately death when major organs are involved. We have developed an integrated approach to molecular diagnosis with initial analysis of intact plasma TTR by electrospray ionization mass spectrometry (MS) and referral of positive samples for DNA sequence analysis and real-time PCR to confirm the common Gly6Ser polymorphism. Methods: Samples from 6 patients previously diagnosed with ATTR and from 25 controls with (n = 15) or without (n = 10) polyneuropathy were analyzed in a blinded fashion for the presence of variant TTR. TTR protein was extracted with an immunoaffinity resin from 20 μL of archived plasma samples. The purified TTR was reduced with tris(2-carboxyethyl)phosphine and analyzed by MS. The appearance of two peaks (or a single peak shifted in mass indicative of a homozygous variant), including the wild-type mass of 13 761 Da, was indicative of the presence of a variant, and the individual was referred for DNA sequence analysis. Results: MS analysis of intact reduced TTR correctly identified each of six samples known to contain variant TTR. These results were corroborated by subsequent DNA sequence analysis. Additionally, all Gly6Ser polymorphisms were correctly called based on the +30 mass shift and an equal relative abundance of the +30 polymorphism relative to wild-type TTR. No false-positive results were seen. Conclusions: This referral method eliminates the necessity of sequencing most samples and allows screening for the familial forms of amyloidosis in a broad patient population in a timely fashion. This method correctly identified all previously known variants and also identified a novel variant, Va194A1a.

Original languageEnglish (US)
Pages (from-to)1544-1552
Number of pages9
JournalClinical chemistry
Issue number9
StatePublished - Sep 2004

ASJC Scopus subject areas

  • General Medicine


Dive into the research topics of 'Identification of transthyretin variants by sequential proteomic and genomic analysis'. Together they form a unique fingerprint.

Cite this