Assay determines presence of FA by measuring frataxin protein in blood
Technique could also assess treatment effectiveness in early clinical trials
A new assay to measure levels of the frataxin protein in blood could be used to assess the effectiveness of treatments in early clinical trials.
The researchers showed that measuring frataxin with this method, which uses pig blood as part of the standardization procedure, could distinguish between people with or without Friedreich’s ataxia (FA). Frataxin measured using this method also correlated with age at onset among patients.
The study, “Liquid Chromatography–Mass Spectrometry Analysis of Frataxin Proteoforms in Whole Blood as Biomarkers of the Genetic Disease Friedreich’s Ataxia,” was published in Analytical Chemistry.
Friedreich’s ataxia is caused by mutations in the gene that provides instructions for making frataxin, resulting in reduced levels of it. Most cells make a form of frataxin called mitochondrial frataxin, or frataxin-M, which helps support the function of mitochondria (the so-called powerhouses of the cell). Red blood cells are only type of cell that doesn’t possess mitochondria and make a slightly different version — frataxin-E.
In order to assess the severity of FA and the effectiveness of potential therapies, measuring frataxin in a biological sample that can be collected easily, such as blood, would be useful. Blood-based analyses usually look only at noncellular parts of blood, but neither form of frataxin is found outside cells, however.
Measuring frataxin levels inside blood cells
Frataxin could theoretically be measured inside blood cells, but complications persist. Cells are full of other proteins present at much higher levels than frataxin, which will stick to many of these other proteins.
To measure frataxin inside blood cells reliably, there must be a way to standardize these interactions. Ideally, this would involve a surrogate matrix — a combination that essentially mimics all the proteins normally found in blood cells, but without human frataxin, which could then be added at known quantities, allowing for standardizations that could be used to quantify levels in patient samples.
In this study, researchers demonstrated that pig blood is well suited to this purpose. Since the pig version of frataxin is only 64% identical to the human form, it’s possible to isolate the human version with specific antibodies that won’t stick to the pig form. Frataxin can then be measured using a chemical analysis platform called liquid chromatography-mass spectrometry.
“The impact of surrogate matrix selection on successful protein biomarker qualification is sometimes overlooked but probably accounts, in part, for the relatively few protein biomarkers that have been approved by the [U.S. Food and Drug Administration],” the researchers wrote, noting their technique “met the criteria stipulated by the FDA” for biomarker tests.
Using this analytical technique, the researchers measured frataxin in 50 FA patients, 25 disease carriers, and 25 people with no FA-associated mutations, who acted as controls. Carriers had the typical FXN gene alterations in only one gene copy. Mutations in both copies are required for the disease to develop.
As expected, average frataxin levels were lowest in FA patients and highest in controls. In fact, there was a complete separation between the two, which the researchers noted is rare in biomarker studies. Frataxin levels in carriers were between patients and controls, overlapping some with both groups.
The researchers also showed that frataxin measured with this technique correlated significantly with age at onset among the FA patients. Frataxin levels also correlated with the length of the GAA repeat, as expected. A longer repeat is associated with less production of the protein.
Based on the data, the researchers concluded that this method could be used to accurately measure frataxin in blood samples.
“It should be possible to quantify modulation of dysregulated protein expression in these blood cells during relatively short-term phase I clinical trials that could serve as a proof of concept that the therapeutic approach is effective and nontoxic,” the researchers said.
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