Analyzing serum biomarkers in cardiac transthyretin amyloidosis: implications for diagnosis and future outcomes.Joanna Waś1, Monika Gawor-Prokopczyk2, Agnieszka Sioma2, Rafał Szewczyk3, Aleksandra Pel3, Jolanta Krzysztoń-Russjan1, Magdalena Niedolistek1, Dorota Sokołowska1, Jacek Grzybowski2, Łukasz Mazurkiewicz2 1 Department of Medical Biology, National Institute of Cardiology, Warsaw, Poland 2 Department of Cardiomyopathy, National Institute of Cardiology, Warsaw, Poland 3 LabExperts sp. z o.o., Gdańsk, Poland IntroductionCardiac transthyretin amyloidosis (ATTR-CA) is a leading cause of cardiac amyloidosis (CA). It occurs in two forms: hereditary (hATTR), caused by a variation of the transthyretin (TTR) gene, and wild-type (wtATTR), in which the TTR protein folds improperly. A readily available matrix, such as serum, that might serve as a source of biomarkers of cardiac transthyretin amyloidosis (ATTR-CA), would be beneficial in the development of future diagnostic and prognostic tools. This preliminary investigation aims to identify potential protein biomarkers in serum for ATTR-CA and select potential prognostic biomarkers to assess the success of tafamidis treatment in patients. MethodsThe serum sample preparation included several steps: albumin and IgG depletion, denaturation, reduction, alkylation, tryptic digestion, and desalting with solid-phase extraction (SPE), followed by evaporation and reconstitution of the sample. The samples were analyzed in reversed phase trap-elute mode on M5 MicroLC-TE system (SCIEX) coupled with ZenoTOF 7600 mass spectrometer (SCIEX) equipped with an OptiFlow ESI ion source. The MS and MS/MS analysis was performed using the DDA (IDA) method. The identification of proteins and localization of selected PTMs was performed using PEAKS 12 software (Bioinformatics Solutions Inc.) with UniProtKB (204,500, Human) sequence database. PCA analysis and T-Welch test on peptide areas from identified proteins in the entire set of samples was done in MarkerView 1.4 software (SCIEX). Preliminary dataThe study comprised 23 individuals with ATTR-CA and 13 patients with increased myocardial thickness, with CA omitted (control group). In the ATTR-CA group, 15 patients had not received specific treatment. The ATTR-CA group of patients treated with tafamidis for at least 6 months included 11 patients, three of whom were recruited in the tafamidis-treated group after 6 months of medication. All patients underwent blood tests, routine 12-lead electrocardiography, transthoracic echocardiography, and [99mTc]Tc-DPD scintigraphy. Although many proteins did not alter levels across all tested groups, the analysis indicated statistically significant changes in selected protein levels, with t-values ranging from -6.69 to 4.01 and p-values less than 0.01 in group-by-group comparisons. Key discoveries include an increase in the abundance of various proteins in ATTR-CA patients before specific treatment, including ceruloplasmin, apolipoprotein E, serpin family A 1, and cDNA FLJ54111, which is highly similar to serotransferrin. Tafamidis treatment of the ATTR-CA group resulted in positive correlations with SERPINA1, paraoxonase 1, and complement C2, among other proteins. Notably, in the ATTR-CA group treated with tafamidis, levels of transthyretin, cDNA FLJ54111, which is very similar to serotransferrin, serpin family A member 3, and apolipoprotein A-I were reduced. The putative protein biomarkers found in our investigation are engaged in a variety of biological processes. The major mechanisms that are affected are inflammation regulation, lipid and steroid metabolism, iron metabolism, immune system modulation, and the oxidative stress response. ATTR-CA serum biomarkers, which are readily available, might be useful as an initial screening tool in diagnostics of individuals suspected of having cardiac amyloidosis. Furthermore, using ATTR-CA biomarkers in additional tests to supplement already available approaches would help to improve diagnostic precision. Additionally, prognostic biomarkers could assist anticipate the impact of certain ATTR-CA treatments and provide more information about the disease’s pathophysiology. Novel aspectSelected serum proteins may serve as diagnostic tool for monitoring of tafamidis treatment efficacy of patients with ATTR-CA. |
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