Analysis of Friedreich’s Ataxia Blood and Tissue Protein Biomarkers in Cases and Gene Therapy Studies Using MRM/MS
Friedreich’s ataxia (FRDA) is a cardio-neurodegenerative disease caused by mutations in the frataxin (FXN) gene. GAA repeat expansion in intron 1 is the most prevalent mutation, resulting in decreased of frataxin protein expression, which results in progressive ataxia and hypertrophic cardiomyopathy. The mitochondrial form of frataxin arises through expression of full-length frataxin in the cytosol followed translocation to the mitochondria where it is converted to frataxin-M a 130-amino acid protein that is not secreted into the circulation. However, it is present in platelets and peripheral blood mononuclear blood cells.
We have identified and characterized an N-terminally acetylated 136-amino acid isoform of frataxin (frataxin-E) that is present in human erythrocytes. Frataxin-E lacks a mitochondrial sequence, and so it is distributed to both cytosol and the nucleus when expressed in cultured cells. The expression of frataxin-E is also down-regulated in FRDA patients; therefore, we have developed quantitative methods for both isoforms in whole blood as biomarkers for FRDA.
Whole blood is a challenging matrix for validated quantitative assays using MRM/MS because of the endogenous levels of the protein. A surrogate matrix has been utilized that satisfies the FDA requirements and provides an approach that will be useful for other blood protein biomarkers. The validated immuno-UHPLC-MRM/MS assay using stable isotopically labeled protein internal standards has specificity and sensitivity comparable with a high-resolution nano-UHPLC-parallel reaction monitoring/MS assay. However, it is much faster and so is useful for monitoring the natural history of the disease as well as pharmacologically-based approaches to therapy.
Unfortunately, gene therapy does not increase frataxin levels in blood cells and so it must be quantified in the affected tissues of pre-clinical rodent and non-human primate models as well as in future human studies. The UHPLC-MRM/MS assay distinguished human and monkey frataxin-M in even though the proteins are 99 % identical. This revealed an unexpected non-linear increase in protein levels and facilitated the development of less toxic gene therapy approaches.
Presenter: Ian A. Blair, PhD (A.N. Richards Professor of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania)
Dr. Blair received his Ph.D. in Organic Chemistry in 1971 from Imperial College of Science and Technology, London, under mentorship of the 1969 Nobel Laureate, Sir Derek H.R. Barton. He was appointed as the A.N. Richards Professor of Systems Pharmacology and Translational Therapeutics at the University of Pennsylvania in 1997 and Director of the Center for Cancer Pharmacology, a position he held until 2014. In 2002, Dr. Blair became the Vice-Chair of the Department of Systems Pharmacology and Translational Therapeutics, a position he held for 18-years. He is an internationally renowned expert in the use of mass spectrometric methods for the quantitation and elucidation of structures of proteins, DNA-adducts, protein-adducts, steroids, as well as the analysis of drugs and their metabolites. He has published over 430-refereed manuscripts that have been cited over 28,000 times, and he has an h-index of 84. Dr. Blair is currently a Senior Editor of Future Science Open and has been on the editorial boards of Journal of Biological Chemistry, Chemical Research in Toxicology, Molecular and Cellular Proteomics, Journal of Lipid Research, and Steroids. He regularly serves on NIH study sections, including as a charter member of the Cancer Biomarkers Study Section (2017-2021). Dr. Blair was awarded the RCM Beynon Prize and in 2005 was elected as a Fellow of the American Association for the Advancement of Science. He was cited for “distinguished contributions to the field of mass spectrometry and its applications to pharmaceutical medicine and for moving autacoid biology forward with sensitive bioanalytical techniques”. In 2006, he received the Dean's award for graduate student training, and he was elected as a fellow of the American Association of Pharmaceutical Scientists. He received the 2011 Eastern Analytical Award for Outstanding Achievements in Mass Spectrometry and in 2017, the Founder’s award from the American Chemical Society for his contributions to drug metabolism and mechanistic toxicology.
