A new transthyretin variant (Glu61Gly) associated with cardiomyopathy MICHAEL ROSENZWEIG 1,2 , MARTHA SKINNER 1,2 , TATIANA PROKAEVA 1,2 , ROGER THE ´ BERGE 1,3 , CATHERINE COSTELLO 1,3,4 , BRIAN M. DRACHMAN 5 ,& LAWREEN H. CONNORS 1,4 1 Amyloid Treatment and Research Program, 2 Department of Medicine, 3 Mass Spectrometry Resource, 4 Department of Biochemistry, Boston University School of Medicine, Boston, MA, and 5 Department of Medicine, Division of Cardiology, University of Pennsylvania Health System, Philadelphia, PA, USA Keywords: Transthyretin, cardiomyopathy, genetic mutation, familial amyloidosis Abbreviations: AL ¼ light chain amyloidosis; ATTR ¼ transthyretin amyloidosis; bp ¼ base pair; ESI ¼ electrospray ionization; IEF ¼ isoelectric focusing; MALDI ¼ matrix-assisted laser desorption/ionization; MS ¼ mass spectrometry; PCR ¼ polymerase chain reaction; PND ¼ paroxysmal nocturnal dyspnea; RFLP ¼ restriction fragment length polymorphism; SSCP ¼ single-strand conformation polymorphism; TOF ¼ time-of-flight; TTR ¼ transthyretin Abstract We report the identification of a new transthyretin (TTR) gene mutation and variant protein, Glu61Gly, in a 55-year-old man with progressive cardiomyopathy, mild peripheral neuropathy and bilateral carpal tunnel syndrome. A diagnosis of TTR-associated familial amyloidosis (ATTR) was considered after an endomyocardial biopsy revealed amyloid deposits in the heart of a patient who had no family history of amyloidosis and no evidence of a plasma cell dyscrasia. Serum screening for a TTR variant by isoelectric focusing (IEF) was positive and prompted further studies to identify the genetic abnormality and to characterize the amyloidogenic protein. Direct DNA sequence analysis of all four coding regions in the TTR gene demonstrated heterozygosity in exon 3. Near equal amounts of guanine (G) and adenine (A) were observed at the second base position of codon 61. The wild-type (GAG) and mutated (GGG) sequences found in codon 61 correspond to glutamic acid (Glu) and glycine (Gly) residues, amino acids which differ in mass by 772 Da. Mass spectrometric analyses of TTR immunoprecipitated from serum showed the presence of both wild-type and variant proteins. The observed mass results for the wild-type and variant proteins were consistent with the predicted values calculated from the genetic analysis data. Introduction The systemic amyloidoses are a broad category of diseases characterized by the deposition of fibrillar proteins in the extracellular matrix of tissues and organs [1]. Classification of the amyloidoses is dependent on the biochemical nature of the major protein present in the amyloid deposits. Primary or light chain amyloidosis (AL) is an acquired disease and the most frequently occurring form of systemic amyloidosis. In AL, amyloid fibrils are formed from clonal immunoglobulin light chains that are over- produced as a result of a plasma cell dyscrasia. The most commonly inherited type of systemic amyloi- dosis is caused by a pathologic form of the plasma protein transthyretin (TTR) and is termed familial TTR-associated amyloidosis (ATTR) [2]. Transthyretin circulates in the bloodstream as a tetramer composed of four identical polypeptide chains each composed of 127 amino acid residues. Synthesis occurs predominantly in the liver and TTR functions as a transport protein for thyroxine and the retinol-binding protein–vitamin A complex [2]. TTR is a single copy gene containing four coding regions and located on the long arm of chromosome 18 [2,3]. It has been hypothesized that genetic mutations leading to specific amino acid substitutions at key positions in the protein destabilize the TTR tetramer. Furthermore, variant residues may lead to partially unfolded or mis- folded intermediates that self-associate and ulti- mately organize to form amyloid fibrils [4,5]. As of 2003, over 100 TTR gene mutations and/or protein variants had been described, the majority of which have been found to be associated with systemic amyloidosis [6]. Although phenotypes vary among patients with ATTR, common clinical features are present among kindreds with the same TTR mutation. Correspondence: Dr. Lawreen H. Connors, Amyloid Treatment & Research Program, Boston University School of Medicine, 715 Albany Street, K-507, Boston, MA 02118, USA. Tel: 617-638-4313. Fax: 617-638-4493. E-mail: lconnors@bu.edu Amyloid, March 2007; 14(1): 65–71 ISSN 1350-6129 print/ISSN 1744-2818 online Ó 2007 Informa UK Ltd. DOI: 10.1080/13506120601116625