Many people suffer for years from unexplained health problems and chronic symptoms but never receive a definitive medical diagnosis. These so-called "diagnostic refugees" are often left without answers. Some of them undergo whole-genome sequencing and mutations are found, yet the significance remains unclear and no diagnosis follows. This is largely because methods for understanding how genetic changes affect three-dimensional protein structure and function at the molecular level have been limited.
Professor Hiroshi Ishikita, Professor Shinichiro Kumagaya, Associate Professor Keisuke Saito, and their colleagues from the Research Center for Advanced Science and Technology at the University of Tokyo collaborated with Professor Kei Murayama of the Department of Pediatrics and Adolescent Medicine, Graduate School of Medicine, and Professor Yasushi Okazaki of Diagnostics and Therapeutics of Intractable Diseases/ Intractable Disease Research Center, Graduate School of Medicine at Juntendo University and others to analyze the case of a patient who had remained undiagnosed for decades despite visiting numerous medical institutions in Japan and abroad.
Whole-genome sequencing identified a genetic abnormality involving an amino acid substitution. If the three-dimensional structure of the protein encoded by the mutant gene can be determined, it becomes possible to estimate at the molecular level which functions are impaired and how symptoms ultimately manifest. However, conventional methods for determining structure, such as X-ray crystallography and cryo-electron microscopy, require considerable time and effort from sample preparation to structure determination, with no guarantee of success.
The research team therefore used AI-based structure prediction technology to rapidly estimate the three-dimensional structure of the mutant protein, revealing that the abnormality may affect a site essential for the activity of an enzyme involved in DNA repair. DNA repair enzymes function by binding ATP as an energy source. When ATP is not bound, the ATP-binding site remains in an open conformation. When ATP binds, it shifts to a closed conformation to use the energy. After the spent molecule is released, the structure returns to the open state. This open-close cycle is the normal mode of operation. However, in the genetic change identified in this study, the position of the "fingers" that grip ATP shifts slightly, causing parts that would not normally connect to form an unexpected bond, whereby the "lid" is kept closed and unlikely to return to the open state. As a result, ATP binding and the open-close cycle are disrupted, likely reducing the enzyme activity essential for DNA repair.
By utilizing the three-dimensional structure predicted by AI, the team was able to visually and through molecular science reveal structural distortions and mechanisms of functional decline that could not be assessed through conventional sequence analysis alone. This work presents a practical framework for linking genetic information with changes in protein structure to concretely interpret the causes of disease.
This opens a new path to uncovering hidden causes behind unexplained symptoms by examining structure. The spread of approaches that integrate genomics, structure, and molecular mechanisms is expected to accelerate the elucidation of disease mechanisms and support diagnosis in undiagnosed conditions, enabling provision of support for diagnostic refugees.
Furthermore, visualization-based understanding through protein structure, as demonstrated in this study, can help not only physicians and researchers but also patients themselves intuitively grasp "what is happening inside their own bodies." Even when no established treatment exists, clarifying the molecular-level mechanism may make it easier for patients to understand their own symptoms, potentially providing emotional support. The findings were published in Frontiers in Molecular Biosciences.
When ATP binds to the binding site (P-loop), the enzyme adopts a closed conformation and uses energy; after the spent products (ADP and phosphate) are released, it returns to the open conformation (a). In the enzyme carrying the amino-acid substitution caused by the genetic alteration (T652→R652), the ATP-gripping "fingers" (K654, T655) are displaced (b), and an unexpected interaction forms between regions that normally do not associate (R652 and D973; dashed line). This interaction makes it difficult for the enzyme to reopen, stabilizing the closed conformation (c).
Provided by the University of Tokyo
Journal Information
Publication: Frontiers in Molecular Biosciences
Title: A DNA2 mutation in the ATP-binding motif identified in a diagnostically unresolved individual
DOI: 10.3389/fmolb.2025.1706392
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