Co-occurrence of mutations in NF1 and other susceptibility genes in pheochromocytoma and paraganglioma

Abstract

Introduction: The percentage of patients diagnosed with pheochromocytoma and paraganglioma (altogether PPGL) carrying known germline mutations in one of the over fifteen susceptibility genes identified to date has dramatically increased during the last two decades, accounting for up to 35-40% of PPGL patients. Moreover, the application of NGS to the diagnosis of PPGL detects unexpected co-occurrences of pathogenic allelic variants in different susceptibility genes.

Methods: Herein we uncover several cases with dual mutations in NF1 and other PPGL genes by targeted sequencing. We studied the molecular characteristics of the tumours with co-occurrent mutations, using omic tools to gain insight into the role of these events in tumour development.

Results: Amongst 23 patients carrying germline NF1 mutations, targeted sequencing revealed additional pathogenic germline variants in DLST (n=1) and MDH2 (n=2), and two somatic mutations in H3-3A and PRKAR1A. Three additional patients, with somatic mutations in NF1 were found carrying germline pathogenic mutations in SDHB or DLST, and a somatic truncating mutation in ATRX. Two of the cases with dual germline mutations showed multiple pheochromocytomas or extra-adrenal paragangliomas - an extremely rare clinical finding in NF1 patients. Transcriptional and methylation profiling and metabolite assessment showed an "intermediate signature" to suggest that both variants had a pathological role in tumour development.

Discussion: In conclusion, mutations affecting genes involved in different pathways (pseudohypoxic and receptor tyrosine kinase signalling) co-occurring in the same patient could provide a selective advantage for the development of PPGL, and explain the variable expressivity and incomplete penetrance observed in some patients.

Keywords: DLST; MDH2; NF1; co-occurrent mutations; germline mutation; pheochromocytoma.

Figure 1

Mutational profile of the twenty-six samples included in the study. Each column represents a tumour, with coloured rectangles representing mutations that are germline (denoted with an asterisk) or somatic in each row. Missense variants that ClinVar does not classify as pathogenic mutations and/or that are absent in NF1 patients were categorized as variants of unknown significance. NA, not available.

Figure 2

(A) Hierarchical clustering performed on the basis of methylation data from PPGLs as follows: one of the two DLST/NF1 (indicated with a green and black rectangle), four SDH-mutated (indicated with blue rectangles), three DLST-mutated (indicated with green rectangles) and five cluster 2-mutated (denoted in black rectangles) PPGLs. The DLST/NF1 PPGL was grouped with DLST-mutated tumours, and separated from cluster 2-mutated PPGLs. City-block and complete linkage characteristics were used for the analyses. (B) α-ketoglutarate/fumarate ratios assessed by LC-MS/MS in DLST-mutated tumours (n = 6), NF1-mutated PPGLs (n = 4), and the two tumours carrying dual NF1/DLST mutations. Black lines represent medians. A t test identified significant differences between DLST-mutated and NF1-mutated tumours. (C) Immunostaining for DLST was conducted in the two tumours carrying DLST/NF1 dual mutations (upper panel) revealing the characteristic cytoplasmic aggregates (x20). A VHL-mutated tumor was used as a negative control (lower panel) (D) Hierarchical clustering of the two NF1/DLST mutated tumours, three DLST- and seven NF1-mutated PPGLs based on gene expression data from a previously reported list of genes differentially expressed in DLST-mutated PPGLs. The two tumours carrying the NF1/DLST mutations were clustered between PPGLs carrying mutations in NF1 and DLST. Uncentered correlation and complete linkage characteristics were used for the analysis.

Figure 3

(A) Positive 5-hmC IHC performed in case 1 (upper panel) compared to a negative control (lower panel) (x20). (B) 5-hmC IHC performed in case 24, showing patches of negative (upper panel) and positive (lower panel) staining (x20)(C) Positive SDHB IHC performed in the tumour carrying SDHB/NF1 dual germline mutations (upper panel) compared to a negative control (lower panel) (x20). Asterisks denote a somatic mutation.