A newly developed lab method to study thymidine kinase 2 deficiency (TK2d) may not reliably detect disease-causing genetic changes, according to a study published recently in Genetics in Medicine Open. These results highlight ongoing challenges in diagnosing and understanding this ultra-rare condition.
TK2d is caused by variants in the TK2 gene, which plays a key role in maintaining mitochondrial DNA. Because the disease is so rare, doctors often struggle to interpret uncertain genetic findings, delaying diagnosis and treatment.
To address this, researchers developed an in vitro functional assay designed to mimic how TK2d affects cells. They used human cells engineered to lack the TK2 gene and introduced either normal or variant forms of TK2. The cells were exposed to ethidium bromide for seven days to reduce mitochondrial DNA, then allowed to recover over 21 days. Scientists measured mitochondrial DNA levels weekly to evaluate how well different TK2 variants restored function.
Read more about testing and diagnosis of TK2d
The team tested four known pathogenic TK2 variants previously linked to TK2d. One variant, c.604_606del, showed the slowest recovery of mitochondrial DNA after treatment. However, by day 21, all variants demonstrated mitochondrial DNA levels similar to normal TK2. In addition, differences between variants and normal cells before treatment were not significant. Overall, the variants produced only minor detectable effects in this system.
“Despite our efforts to develop an in vitro functional assay for TK2 VUS [variant of uncertain significance] screening, mtDNA [mitochondrial DNA] depletion phenotypes were mostly undetectable for the pathogenic variants tested, suggesting that patient-derived cells are likely the most appropriate source of information for investigating the effects of individual TK2 variants on mtDNA,” explained the authors of this research.
These findings suggest that another enzyme, thymidine kinase 1, may partially compensate for TK2d in laboratory conditions. This compensation could mask the true impact of harmful TK2 variants, making them harder to identify in standard cell-based assays. Researchers noted that this possibility requires further investigation.
For patients with TK2d and their families, the results mean that current lab-based testing methods may not always provide clear answers about whether a genetic variant is harmful. This uncertainty can delay diagnosis or complicate treatment decisions. The study also found that these experiments take weeks to complete, limiting their usefulness for rapid screening.
The researchers concluded that patient-derived cells may provide more accurate insights into how specific TK2 variants affect mitochondrial function. While the new assay represents progress, better tools are still needed to improve diagnosis and guide care for people living with TK2d.
Sign up here to get the latest news, perspectives, and information about TK2d sent directly to your inbox. Registration is free and only takes a minute.