Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients

Abstract

Purpose: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype.

Methods: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach.

Results: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL.

Conclusion: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.

Keywords: Dominant-negative effect; MDH2; Molecular dynamics; Variants of unknown significance; pheochromocytoma and paraganglioma.

Fig. 1. Graphical representation of the variants identified in MDH2 and the residues of the protein affected.

The PV previously reported is indicated with a star. Missense variants are framed in continuous line; in-frame deletion is framed in discontinuous line; synonymous variant is shown in the upper part of the figure; intronic variants are shown in the lower part of the figure

Fig. 2. MDH2 activity measured in MDH2 KD cells transfected with different vectors containing the variant of unknown significance (VUS) in MDH2.

a Enzymatic activities at saturating concentrations of malate (25 mM) and NAD+ (2 mM) in p.R104G (p.Arg104Gly), p.Q130R (p.Gln130Arg), p.V160M (p.Val160Met), and p.S3F (p.Ser3Phe) variants, plus C (p.K301R-p.Lys301Arg) which is a polymorphism with minor allele frequency of 0.037, knockdown (KD) and wild-type (WT) as controls, are expressed as mean (nmol/min/mg) ± SD of 3 paired independent experiments in quadruplicate. Different shadings indicate the only variants tested in experiments in c–f. b Enzymatic activities at saturating concentrations of cells cotransfected with WT, empty vector (EV), and/or p.R104G plasmids expressed as mean of fold-change over activity in cells cotransfected with WT and EV (control) ± SD of 2 paired independent experiments at least in quadruplicate; μg of plasmid DNA transfected for each condition are showed in the figure. c–f Enzymatic activities in p.A256T, p.V160M, p.R104G, and control (p.K301R) expressed as mean of fold-change over control ± SD of at least 2 paired independent experiments in triplicate. Assays performed with reduced concentrations of malate or NAD+: c 5-fold reduction malate (5 mM), d 10-fold reduction malate (2.5 mM), e 4-fold reduction of NAD+ (0.5 mM), and f 20-fold reduction of NAD+ (0.1 mM). *****p < 0.0001, ****p< 0.001, ***p < 0.01, **p < 0.03, and *p < 0.05 based on a two-sided Mann–Whitney U test

Fig. 3. Molecular dynamics simulations in MDH2 p.Ala256Thr and p.Val160Met variants of unknown significance (VUS) versus wild-type (WT).

a Distance between the Cζ atom of F260 and the Cβ atom of A/T256 for both monomers of WT MDH2 (magenta, upper panels), Ala256Thr (blue, middle panels), and V160M (green, lower panels) during the simulations. A short distance corresponds to the F260 down conformation, and a long distance to the F260 up conformation (inset). b and c Distribution of the distance between the Cα atoms of K269 of the two monomers of the a Ala256Thr and b Val160Met variants from snapshots from the simulation trajectories, compared with WT