The clinical utility of functional testing in fibroblasts to diagnose primary mitochondrial disease

Doctors often find genetic mutations in mitochondrial diseases that are incredibly difficult to interpret. While next-generation sequencing can pinpoint a change in the DNA, it cannot always tell if that change actually breaks the cell's machinery. This leaves clinicians with "variants of uncertain significance" (VUS)—genetic typos that might be harmless or might be the cause of a devastating disease.

A new study involving 308 fibroblast cell lines proposes a solution. It suggests doctors stop looking only at the blueprint and start testing the engine. By using a battery of four different functional tests on skin cells, the authors report they can confirm or rule out primary mitochondrial disease with much higher accuracy than using traditional enzyme assays alone.

The limits of reading the blueprint

Current methods for diagnosing primary mitochondrial disease (PMD) rely heavily on next-generation sequencing of mitochondrial and nuclear DNA. As shown in, this molecular approach provides a putative diagnosis for roughly 50% to 65% of patients.

However, a massive diagnostic gap remains. Many patients either return no identifiable variants or return variants of uncertain significance.

Historically, clinicians used respiratory chain enzyme (RCE) assays. These tests measure the activity of specific protein complexes in muscle or liver biopsies. But these traditional methods have significant drawbacks. They are invasive, requiring painful tissue samples. They also offer a narrow view of mitochondrial health. The authors note that retrospective analyses of RCE activity in fibroblasts (skin cells used as a proxy) showed a sensitivity of only about 50%. Relying solely on these single-point measurements is like trying to diagnose a car's engine failure by checking only the fuel flow. You might miss a broken transmission or a faulty spark plug.

A multi-layered diagnostic battery

To solve this, the researchers implemented a tiered testing strategy. This moves from simple activity measurements to structural inspections of the mitochondrial complexes. Their approach relies on four distinct methodologies, as outlined in :

1. Respiratory Chain Enzyme (RCE) Assays: These spectrophotometric tests measure the raw speed of specific complexes. This is the "throughput" metric of the mitochondria.
2. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE): This technique separates intact mitochondrial complexes by size and charge. By using in-gel activity staining, researchers can visually confirm if a complex is present and functioning. This checks if the "parts" are correctly assembled.
3. Complex I Assembly Blot: This is a specialized Western blot—a technique used to detect specific proteins—targeting the NDUFS2 subunit. It traces the assembly intermediates of Complex I. This shows exactly where the construction process stalls.
4. Targeted Protein Abundance (Western Blot): The authors found that measuring how much of a specific protein exists can be more informative than measuring how fast it works.

By combining these layers, the researchers move from asking "is the enzyme active?" to "is the protein present, is it assembled correctly, and is it functioning at scale?"

Increasing sensitivity through combination

The power of this approach lies in the cumulative effect of the tests. The authors report that individual assays have relatively modest performance. RCE assays achieved 46% sensitivity. BN-PAGE achieved 40%. The Complex I assembly blot achieved 49%. If a clinician relied on any single one of these, they would miss more than half of the affected patients.

However, the paper finds that using these tests as a collective battery changes the outcome. The overall sensitivity jumps to 76%. This means the combined tests catch significantly more cases than any single test could alone. Crucially, the specificity—the ability to correctly identify healthy individuals—remains excellent at 93%. This combination proved particularly potent for specific disease categories. In cases of isolated complex deficiencies, the combined sensitivity reached 85%. In mitochondrial protein synthesis disorders, it hit 82%.

The most striking result involves ARS2 disorders (defects in mitochondrial amino-acyl-tRNA synthetases). The authors report that while traditional enzymatic assays only caught 40% of these cases, adding a Western blot to measure protein abundance boosted the diagnostic rate to 78%. This demonstrates that for certain "silent" defects, looking at the quantity of the protein is far more effective than looking at its catalytic speed.

Identifying the blind spots

Despite the improved performance, the study highlights clear boundaries. The authors report that the battery performs poorly for two specific categories: mitochondrial DNA (mtDNA) maintenance disorders and certain mtDNA variants. For mtDNA maintenance disorders, the combined sensitivity was only 20%.

The reason for this failure is biological rather than technical. Maintenance disorders often involve the depletion or deletion of mtDNA. This is typically most detectable in "post-mitotic" cells (cells that no longer divide, like neurons). Because fibroblasts are actively dividing in a culture dish, they may lose the very genetic markers needed for a diagnosis.

Furthermore, the authors note that the study was retrospective. They used samples where the genetic cause was already known. This creates a potential bias that might overestimate the impact of functional tests in a real-world setting. Finally, the diagnostic rate was notably lower in adults (44%) than in children (72%). This is largely because adults are more likely to fall into these difficult-to-diagnose mtDNA categories.

The verdict: A new clinical template

Should clinicians adopt this multi-assay approach? The answer is yes, but with a caveat. It must be a targeted application rather than a random one.

The authors do not suggest running every test on every patient. Instead, they propose a rational template. If a patient has a suspected Complex I defect caused by nuclear DNA, the Complex I assembly blot is the superior tool. If the defect is suspected to be in the mtDNA, the RCE assay remains the most effective. The study proves that a "complete battery" is a robust diagnostic tool. It can turn ambiguous genetic data into a definitive clinical diagnosis. For the thousands of patients stuck in the "variant of uncertain significance" limbo, this layered approach offers a viable path toward clarity.

Figures from the paper