# Assessment of HIF2α mutational pathogenicity using microscale thermophoresis

## TL;DR

The paper introduces a method to assess the pathogenicity of HIF2α mutations using microscale thermophoresis, aiding in the diagnosis of Pacak–Zhuang syndrome.

## Contribution

A detailed protocol for using MST to evaluate HIF2α-PHD2 binding affinities is proposed for mutation pathogenicity assessment.

## Key findings

- Reduced PHD2 affinity for HIF2α is a key mechanism in Pacak–Zhuang syndrome.
- MST can effectively measure binding affinities of HIF2α mutations.
- The protocol can help distinguish pathogenic from benign mutations.

## Abstract

Pacak–Zhuang syndrome is an emerging pseudohypoxic disorder that causes defined but varied manifestations of neuroendocrine tumours with or without polycythemia or exclusively polycythemia. This disease is caused by mutations in the EPAS1 gene, which encodes for one of three hypoxia-inducible factor (HIF) α subunits, HIF2α. As new mutations in this gene are observed in individuals exhibiting the manifestations of Pacak–Zhuang syndrome, there is a need to distinguish bona-fide disease causing mutations from benign mutations, which could have a valuable impact on the direction of patient care. We recently showed that reductions in the affinity of prolyl-hydroxylase 2 (PHD2) for HIF2α due to mutations are at the root of the mechanism underlying Pacak–Zhuang syndrome. The determination of affinity was accomplished using microscale thermophoresis (MST). Here, we describe a detailed protocol for the assessment of binding affinities between HIF2α peptides or the entire oxygen-dependent degradation domains of HIFα proteins and PHD2 using MST and propose that this method can be used to assess the potential pathogenicity of novel mutations in HIF2α.