Assessment of HIF2α mutational pathogenicity using microscale thermophoresis

Area of Science:

• Biochemistry
• Genetics
• Endocrinology

Background:

• Pacak-Zhuang syndrome is a pseudohypoxic disorder characterized by neuroendocrine tumors and/or polycythemia.
• The condition arises from mutations in the EPAS1 gene, which encodes hypoxia-inducible factor 2α (HIF2α).
• Distinguishing disease-causing mutations from benign variants is crucial for patient care.

Purpose of the Study:

• To detail a protocol for assessing the binding affinities between HIF2α and prolyl-hydroxylase 2 (PHD2).
• To establish microscale thermophoresis (MST) as a method for evaluating the pathogenicity of novel HIF2α mutations.

Main Methods:

• Utilized microscale thermophoresis (MST) to determine binding affinities.
• Assessed interactions between HIF2α peptides or oxygen-dependent degradation domains and PHD2.
• Developed a protocol for standardized affinity measurements.

Main Results:

• Demonstrated that mutations in HIF2α reduce its binding affinity to PHD2, underlying Pacak-Zhuang syndrome.
• Established a reliable method using MST to quantify these affinity changes.
• Provided a framework for assessing the functional impact of new EPAS1 mutations.

Conclusions:

• Reduced HIF2α-PHD2 binding affinity is the mechanistic basis of Pacak-Zhuang syndrome.
• The described MST protocol offers a valuable tool for evaluating the pathogenicity of HIF2α mutations.
• This approach can guide clinical decisions and patient management for Pacak-Zhuang syndrome.