# Analytical validation of frataxin proteoforms in blood as biomarkers of Friedreich's ataxia

> **NIH NIH U01** · UNIVERSITY OF PENNSYLVANIA · 2020 · $781,493

## Abstract

ABSTRACT: ANALYTICAL VALIDATION OF FRATAXIN PROTEOFORMS IN BLOOD AS BIOMARKERS
OF FRIEDREICH’S ATAXIA
Friedreich’s ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the
FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in
50,000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA,
although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being
tested. Frataxin is undetectable in serum or plasma, and whole blood could not be used because frataxin was
thought to be present in long-lived erythrocytes. An assay for analyzing frataxin in platelets, which have a half-
life of 10 days, which would allow therapeutic interventions to be tested, was developed by the Blair and Lynch
labs. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high
performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (nano-
UPLC-MS/HRMS) and is monitoring three tryptic peptides from frataxin. The assay had 100 % sensitivity and
specificity for discriminating between controls and FA cases but analyzing platelets on a routine basis is very
challenging in many clinical settings. We have now discovered that in erythrocyte is in fact a novel proteoform
of frataxin (isoform E) with 135-amino acids (76-210) and an N-terminally acetylated methionine residue. It
arises through an alternative splice-site form that is used for the canonical full-length form of frataxin (1-210).
There is three times as much isoform E in erythrocytes (26.7 ± 6.4 ng/mL) from the blood of healthy volunteers
(n=10) when compared with the mature mitochondrial frataxin present in other blood cells (7.1 ± 1.0 ng/mL).
We recently found that both isoform E (8.5 ± 1.1 ng/mL) and mature mitochondrial frataxin (2.1 ± 1.1 ng/mL)
are both reduced by > 70 % in blood from FA patients (n=29) when compared with healthy control subjects.
Isoform E lacks a mitochondrial targeting sequence and so it is distributed to both cytosol and the nucleus
when expressed in cultured cells. The ability to specifically quantify extra-mitochondrial and mitochondrial
isoforms of frataxin in whole blood would make it possible to follow the progress of the disease and monitor the
efficacy of therapeutic interventions. This will require the development of rigorously validated assays that
address the pre-analytical and analytical issues that are relevant to the use of blood as a biological matrix for
biomarker analysis rather than the more common use of serum or plasma. The present proposal addresses
these issues under following specific aims:
Aim 1: To conduct pre-analytical validation for collection and storage of mature frataxin and isoform E protein
in whole blood. Aim 2: To conduct analytical validation of the method for quantifying mature mitochondrial
frataxin and frataxin...

## Key facts

- **NIH application ID:** 9873659
- **Project number:** 1U01NS114143-01
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Ian Alexander Blair
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $781,493
- **Award type:** 1
- **Project period:** 2020-03-15 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9873659

## Citation

> US National Institutes of Health, RePORTER application 9873659, Analytical validation of frataxin proteoforms in blood as biomarkers of Friedreich's ataxia (1U01NS114143-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9873659. Licensed CC0.

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