Roger Théberge

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.

Amyloidogenic and Associated Proteins in Systemic Amyloidosis Proteome of Adipose Tissue

In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of αB-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration.

Sequence Communication: Tabulation of transthyretin (TTR) variants as of 1/1/2000

he past 10 years have been a remarkable period of discovery in the study of familial amyloidotic polyT neuropathy (FAP), a disease associated with the plasma protein transthyretin (TTR), whose 127-residue amino acid sequence is shown in Figure 1. Since TTR was frst identified in the extracellular deposits of patients with hereditary amyloidosis more than two decades ago, there have been an extraordinary number of reports detailing the existence of over 80 variant forms of the protein. It is quite remarkable that more than 30 of these variants have been identified within the past four years; the current rate of discovery suggests that many more mutations will be reported, as improved analytical methods are applied to the quest and as a wider patient population gains access to appropriate diagnostic services. While the vast majority of the reported TTR mutations are amyloidogenic, the precise cause(s) and mechanism(s) responsible for aberrant TTR subunit assembly (or misassembly) into amyloid fibrils remains undetermined. The genetic basis of the disease stems fiom single base nucleotide mutations that occur throughout the entire coding region of the TTR gene. The phenotypes of TTR-associated familial amyloidosis are varied, but common clinical features and ethnic origins have been noted among kinships afflicted with identical TTR mutations. This special article provides a compilation of the polymorphic forms of TTR, both pathologic and non-pathologic, which have been reported as of January 1,2000. This collection should facilitate the work of laboratory investigators and provide a guide to the literature for those who wish to gain familiarity with the field. TTR proteins are presented in tabular form, listed in ascending order according to the position ofthe variant amino acid. As is customary, the threeletter code for the residue present in the wild type protein appears on the left, followed by the position number and the three-letter code for the variant amino acid, e.g. , CyslOArg indicates that the residue cysteine (Cys) at position 10 is replaced by arginine (Arg) in the mutant protein. The corresponding genetic, biochemical, clinical and demographic information (as currently available) is summarized for each variant. Specifically, genetic mutation data (DNA base change) and resultant amino acid mass shifts (change in amino acid residue mass between wild type and variant at the position where the base substitution occurs) are listed. For the amyloidogenic variants, clinical features (phenotype) and world-wide distribution (geographic focusl ethnic origin) are detailed. Non-amyloidogenic proteins are shown in italicized print. The earliest report of the gene or protein mutation appears in Table 1. A comprehensive list of initial and subsequent references to each variant and

The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation

The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell-based, chemically-defined, serum and feeder cell-free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice-compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.

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.

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal DIRECT ANALYSIS BY FLUORESCENCE-DETECTED ANALYTICAL ULTRACENTRIFUGATION

Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys10) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescence-detected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.

Enhancing bottom-up and top-down proteomic measurements with ion mobility separations.

Proteomic measurements with greater throughput, sensitivity, and structural information are essential for improving both in‐depth characterization of complex mixtures and targeted studies. While LC separation coupled with MS (LC–MS) measurements have provided information on thousands of proteins in different sample types, the introduction of a separation stage that provides further component resolution and rapid structural information has many benefits in proteomic analyses. Technical advances in ion transmission and data acquisition have made ion mobility separations an opportune technology to be easily and effectively incorporated into LC–MS proteomic measurements for enhancing their information content. Herein, we report on applications illustrating increased sensitivity, throughput, and structural information by utilizing IMS–MS and LC–IMS–MS measurements for both bottom‐up and top‐down proteomics measurements.

Detection of transthyretin variants using immunoprecipitation and matrix-assisted laser desorption/ionization bioreactive probes: a clinical application of mass spectrometry

In our continuing efforts to develop mass spectrometry-based methods for transthyretin (TTR) variant detection and characterization, we have sought to use matrix-assisted laser desorption/ionization (MALDI) bioreactive probes incorporating immobilized trypsin for screening purposes. These devices show good diagnostic potential as a clinical screening tool to detect amino acid substitutions in TTR. MALDI probes allow the on-probe generation of tryptic digests. The subsequent mass analysis of the on-probe digest yields the peptide map. The inherent advantages of this method include considerably reduced digestion times (minutes vs. hours), absence of autolysis products, minimized sample handling, and hence minimal sample loss. A further advantage is that the opportunity for loss of hydrophobic peptides is reduced because no sample transfer occurs. The method can be applied as a preliminary screen for TTR variants where TTR is isolated from patient serum through immunoprecipitation. This method should also be applicable to other proteins and suitable for automation.

Highly efficient and selective enrichment of peptide subsets combining fluorous chemistry with reversed‐phase chromatography

The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies for selective enrichment, problems are still encountered due to sample loss or poor selectivity of enrichment. Here we show that the recently introduced fluorous chemistry approach has attractive properties as an alternative method for selective enrichment. Through appending a perfluorine group to the target peptide, it is possible to dramatically increase the peptides hydrophobicity and thus enable facile separation of labeled from non-labeled peptides. Use of reversed-phase chromatography allowed for improved peptide recovery in comparison with results obtained using the formerly reported fluorous bonded phase methods. Furthermore, this approach also allowed for on-line separation and identification of both labeled and unlabeled peptides in a single experiment. The net result is an increase in the confidence of protein identification by tandem mass spectrometry (MS2) as all peptides and subsequent information are retained. Successful off-line and on-line enrichment of cysteine-containing peptides was obtained, and high quality MS2 spectra were obtained by tandem mass spectrometry due to the stability of the tag, allowing for facile identification via standard database searching. We believe that this strategy holds great promise for selective enrichment and identification of low abundance target proteins or peptides.

A new transthyretin variant (Ser2 sn) associated with familial amyloidosis in a Portuguese patient

The detection and characterization of a new transthyretin (ATTR) variant, Ser23Asn, associated with cardiomyopathy in a Portuguese patient with familial amyloidosis is described. Isoelectric focusing (IEF) of serum from the propositus demonstrated heterozygosity for the presence of wild type and variant ATTR. A combination of mass spectrometric (MS) analyses, including electrospray ionization mass spectrometry (ESI MS), high performance liquid chromatography (HPLC)/ESI MS and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) performed on the serum-derived TTR were used to identify and locate the amino acid replacement in the variant protein. Genetic mutation analysis by DNA sequencing and allele-specific PCR confirmed this finding.