Amareth Lim

Cardiac amyloidosis in African Americans: Comparison of clinical and laboratory features of transthyretin V122I amyloidosis and immunoglobulin light chain amyloidosis

BACKGROUND Transthyretin (TTR) mutations known to cause cardiac amyloidosis include V122I, found almost exclusively in African Americans at a prevalence of 3-3.9%. This retrospective study describes TTR V122I-associated cardiac amyloid disease (ATTR) in a major amyloid referral clinic population. METHODS Self-identified African Americans with amyloidosis (n = 156) were screened for TTR V122I by serum isoelectric focusing; mutant TTR was confirmed by DNA sequencing or mass spectrometry. Cardiac findings in ATTR V122I and immunoglobulin light chain (AL) amyloidoses were compared. RESULTS TTR V122I was identified in 36/156 (23.1%) of evaluated patients and included 5 homozygotes; the allele frequency was 0.013. One compound heterozygote (F44L/V122I) and 4 patients who had AL and the mutant TTR allele were characterized. In patients negative for V122I, AL was the most frequent diagnosis (86/120). Cardiomyopathy was present in 100% of patients with ATTR and 84% of patients with AL (P = .01). In patients with dominant cardiac involvement, better survival occurred in ATTR (n = 30) compared to AL (n = 31), (27 vs 5 months, P < .01) although the mean age in ATTR was higher (70.3 vs 56.2 years, P < .01). Congestive heart failure symptoms and electrocardiographic findings were similar in ATTR and AL, but significant differences in echocardiographic measurements were observed. CONCLUSIONS ATTR V122I and AL are equally prevalent as the cause of cardiomyopathy in African Americans referred for a diagnosis of amyloidosis. Available therapy for AL underscores the need for early and accurate determination of amyloid type.

In Vitro and in Vivo Interactions of Homocysteine with Human Plasma Transthyretin

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and an emerging risk factor for cognitive dysfunction and Alzheimers disease. Greater than 70% of the homocysteine in plasma is disulfide-bonded to protein cysteine residues. The identity and functional consequences of protein homocysteinylation are just now emerging. The amyloidogenic protein transthyretin (prealbumin), as we now report, undergoes homocysteinylation at its single cysteine residue (Cys10) both in vitro and in vivo. Thus, when human plasma or highly purified transthyretin was incubated with 35S-l-homocysteine followed by SDS-PAGE and PhosphorImaging, two bands corresponding to transthyretin dimer and tetramer were observed. Treatment of the labeled samples with β-mercaptoethanol prior to SDS-PAGE removed the disulfide-bound homocysteine. Transthyretin-Cys10–S–S–homocysteine was then identified in vivo in plasma from normal donors, patients with end-stage renal disease, and homocystinurics by immunoprecipitation and high performance liquid chromatography/electrospray mass spectrometry. The ratios of transthyretin-Cys10–S–S–homocysteine and transthyretin-Cys10–S–S–sulfonate to that of unmodified transthyretin increased with increasing homocysteine plasma concentrations, whereas the ratio of transthyretin-Cys10–S–S–cysteine to that of unmodified transthyretin decreased. The hyperhomocysteinemic burden is thus reflected in the plasma levels of transthyretin-Cys10–S–S–homocysteine, which in turn may contribute to the pathological consequences of amyloid disease.

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild‐type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry‐based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix‐assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe → Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S‐sulfonated and S‐thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.

Molecular Organization of Amyloid Protofilament-Like Assembly of Betabellin 15D: Helical Array of β-Sandwiches

Betabellin is a 32-residue peptide engineered to fold into a four-stranded antiparallel beta-sheet protein. Upon air oxidation, the betabellin peptides can fold and assemble into a disulfide-bridged homodimer, or beta-sandwich, of 64 residues. Recent biophysical and ultrastructural studies indicate that betabellin 15D (B15D) (a homodimer of HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) forms unbranched, 35-A wide assemblies that resemble the protofilaments of amyloid fibers. In the present study, we have analyzed in detail the X-ray diffraction patterns of B15D prepared from acetonitrile. The fiber diffraction analysis indicated that the B15D fibril was composed of a double helix defined by the selection rule l = n + 7m (where l is even, and n and m are any integers), and having a 199-A period and pitch, 28-A rise per unit, and 10-A radius. This helical model is equivalent to a reverse-handed, single helix with half the period and defined by the selection rule l = -3n + 7m (where l is any integer). The asymmetric unit is the single B15D beta-sandwich molecule. These results suggest that the betabellin assembly that models the protofilaments of amyloid fibers is made up of discrete subunits on a helical array. Multiple intersheet hydrogen bonds in the axial direction and intersandwich polar interactions in the lateral direction stabilize the array.

Identification of a novel transthyretin Thr59Lys/Arg104His. A case of compound heterozygosity in a Chinese patient diagnosed with familial transthyretin amyloidosis.

Transthyretin (TTR) is a 127-amino acid residue protein synthesized mainly in the liver and in several minor sites, including the choroid plexus and the eye. in plasma, TTR circulates as a homotetramer and transports the hormone thyroxine and the retinol-binding protein-vitamin A complex. It is hypothesized that amino acid substitutions in TTR destabilize the tetramer by causing each subunit to form intermediates that may self-associate into amyloid fibrils. Deposition of wild type TTR, its variants and/or fragments as amyloid fibrils in tissues and organs is associated with familial transthyretin amyloidosis (A TTR). Reported herein is the characterization of a novel TTR Thr59Lys/Arg104His in a patient of Chinese ancestry, who was diagnosed with A TTR. The two variant proteins and the double gene mutations in this compound heterozygous case were detected and identified using a multifaceted approach consisting of isoelectric focusing, electrospray ionization mass spectrometry (MS), matrix-assisted laser desorption/ionization time-of-flight MS in combination with enzymatic digestion, and direct DNA sequence analysis. Previous studies have shown that the TTR Arg104His variant is non-pathologic. It appeared to provide a protective effect in another compound heterozygous case (TTR VaBOMet/Arg104His). However. the TTR Arg104His variant when presented with the TTR Thr59Lys variant did not seem to have any protective role.

Characterization of betabellins 15D and 16D, designed beta-sandwich proteins that have amyloidogenic properties

Amareth Lim, Alexander M. Makhov, Lawreen H. Connors, Jeremy Bond, Hideyo Inouye, Jack D. Griffith, Daniel A. Kirschner, Catherine E. Costello, and Bruce W. Erickson Department of Chemistry and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 U.S.A.; Department of Biochemistry and Department of Biophysics, Boston University School of Medicine, Boston, MA 02118 U.S.A.; and Department of Biology, Boston College, Chestnut Hill, MA 02467 U.S.A.