Recurrent Germline DLST Mutations in Individuals with Multiple Pheochromocytomas and Paragangliomas

Abstract

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and ¹³C₅-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.

Keywords: DLST; TCA cycle; cancer susceptibility gene; paraganglioma; pheochromocytoma.

Figure 1

DLST Protein Structure (A) A schematic representation of DLST (15 exons) indicating the main active sites and all variants found in this study. Vertical bars represent the number of individuals carrying each variant. (B) A multiple-sequence alignment and DLST residue conservation across different species determined with MultAlin software; high consensus is marked in red, neutral in black, and low consensus in blue. The blue arrows = predicted active sites in E. coli DLST. The black arrows indicate the different amino acids found mutated in our affected individuals. (C) A predicted structural model of the DLST catalytic domain. The catalytic residues are colored blue and indicated by blue arrows. The variants found in this study have been modeled, colored black, and are indicated by black arrows. The position of the putative substrate binding site is indicated with gray spheres for clarity.

Figure 2

LOH Analysis of p.Gly374Glu-DLST Tumors A schematic representation of chromosome 14 showing the uniparental disomy (UPD) identified by SNP array analysis in three PPGLs (#3C, #4, and #5A) that carry the p.Gly374Glu-DLST variant (indicated by a red asterisk) compared to a control tumor. Yellow bars represent the alleles with the lowest copy number (n = 0), and blue bars represent the alleles with the highest copy number (n = 2). MEG3, a maternally expressed imprinted gene, was used to determine the origin of the observed UPD. The presence in the three tumors of only the methylated allele of MEG3 (indicated by a red arrowhead) indicates that the UPD has a paternal origin.

Figure 3

Functional Analysis of DLST Variants (A) A schematic illustration of the tricarboxylic acid (TCA) cycle. Carbon-13 is denoted by green dots in the context of a ¹³C₅ –glutamate labeling experiment. The red arrow pointing at 2-hydroxyglutarate (2HG) represents the collective action of malate and lactate dehydrogenases. (B) An immunoblot analysis showing equivalent protein levels of wild-type (WT) and mutant DLST in DLST-KO cells. (C) Labeling patterns of TCA intermediates after ¹³C₅-glutamate labeling. (D) A pattern of ¹³C₅-2HG labeling after ¹³C₅-glutamate labeling. (C and D) Error bars represent the SD from a representative experiment. ^∗ p < 0.05 (Student’s t test; n = 3).

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. 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.

Figure 5

Immunohistochemistry of DLST in Different Tumors (A) Positive immunostaining (×20) with cytoplasmic aggregates was assessed in DLST- and TCA-cycle-mutated tumors (left and right columns, respectively) and compared to control tumors (middle column). (B) Representation of DLST immunohistochemistry (IHC) score (ranging from 0 to 3) for the 88 analyzed tumors, including tumors carrying mutations in TCA-cycle-related genes (n = 33; SDHA [1], SDHB [11], SDHC [1], SDHD [6], SDHAF2 [1], GOT2 [2], MDH2 [3], IDH1 [1], IDH3B [1], and DLST [6]), tumors carrying EPAS1 mutations (n = 9), and tumors carrying other mutations as controls (n = 46; RET [24], VHL [11], HRAS [1], NF1 [7], MAX [2], and TMEM127 [1]). Significance was determined by a Fisher’s exact test; ^∗∗∗ p < 0.001.