Figure 4.
Metabolite Assessment and Gene Expression Profiling of p.Gly374Glu-DLST Tumors
(A) α-ketoglutarate/fumarate ratios assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in p.Gly374Glu-DLST tumors (n = 5) compared with wild-type (WT)-DLST control PPGLs (n = 51). Black lines represent medians. A t test identified differences between means; ∗∗∗ p < 0.001.
(B) 2-Hydroxyglutarate/fumarate ratios assessed by LC-MS/MS in p.Gly374Glu-DLST tumors (n = 5) compared to WT-DLST control PPGLs (n = 6). The ratio of these metabolites in an IDH1-mutated tumor was included as a positive control of 2HG accumulation. Black lines represent medians. A t test identified differences between means; ∗ p < 0.05.
(C) A hierarchical clustering of 69 mutated tumors made on the basis of expression data for 451 genes reported as differentially expressed in PPGL-mutated samples.25 Control tumors (denoted with different colors depending on the gene mutated) were split up between the two main transcriptional clusters of PPGLs: cluster 1 (denoted in gray), which included VHL- (n = 12), SDHx- (n = 15), and EPAS1- (n = 8) mutated tumors, and cluster 2 (denoted in black), which included RET- (n = 14), HRAS- (n = 6), NF1- (n = 4), TMEM127- (n = 3), and MAX- (n = 3) mutated PPGLs. Two tumors carrying the p.Gly374Glu-DLST variant (#4 and #5A) were clustered within cluster 1 and grouped with EPAS1-mutated cases. City Block-uncentered and complete linkage characteristics were used for the analyses.
(D) HIF3A mRNA expression in p.Gly374Glu-DLST PPGLs (n = 4) versus WT-DLST control PPGLs (n = 18) by RT-qPCR. The expression level was normalized to β-actin (ACTB) and presented as a mean (n = 3). Significance was determined by a Mann-Whitney U non-parametric test; ∗∗ p < 0.01.