Recurrent epimutation of SDHC in gastrointestinal stromal tumors

Abstract

Succinate dehydrogenase (SDH) is a conserved effector of cellular metabolism and energy production, and loss of SDH function is a driver mechanism in several cancers. SDH-deficient gastrointestinal stromal tumors (dSDH GISTs) collectively manifest similar phenotypes, including hypermethylated epigenomic signatures, tendency to occur in pediatric patients, and lack of KIT/PDGFRA mutations. dSDH GISTs often harbor deleterious mutations in SDH subunit genes (SDHA, SDHB, SDHC, and SDHD, termed SDHx), but some are SDHx wild type (WT). To further elucidate mechanisms of SDH deactivation in SDHx-WT GIST, we performed targeted exome sequencing on 59 dSDH GISTs to identify 43 SDHx-mutant and 16 SDHx-WT cases. Genome-wide DNA methylation and expression profiling exposed SDHC promoter-specific CpG island hypermethylation and gene silencing in SDHx-WT dSDH GISTs [15 of 16 cases (94%)]. Six of 15 SDHC-epimutant GISTs occurred in the setting of the multitumor syndrome Carney triad. We observed neither SDHB promoter hypermethylation nor large deletions on chromosome 1q in any SDHx-WT cases. Deep genome sequencing of a 130-kbp (kilo-base pair) window around SDHC revealed no recognizable sequence anomalies in SDHC-epimutant tumors. More than 2000 benign and tumor reference tissues, including stem cells and malignancies with a hypermethylator epigenotype, exhibit solely a non-epimutant SDHC promoter. Mosaic constitutional SDHC promoter hypermethylation in blood and saliva from patients with SDHC-epimutant GIST implicates a postzygotic mechanism in the establishment and maintenance of SDHC epimutation. The discovery of SDHC epimutation provides a unifying explanation for the pathogenesis of dSDH GIST, whereby loss of SDH function stems from either SDHx mutation or SDHC epimutation.

Fig. 1.. SDHC epimutation in SDHx-WT dSDH GIST.

(A) Janus plot showing upward facing CpG target hypermethylation (–log₁₀q) and downward facing gene probe set hypoexpression (log₁₀q) in SDHx-WT versus SDHx-mutant GIST, calculated from t-test comparison of genome-wide methylation and expression profiles, plotted on genome coordinates. q-value significance thresholds (q = 0.05, gray dotted lines) are indicated. (B) UCSC (University of California, Santa Cruz) genome browser display shows genomic position (hg19 coordinates), CGIs (green bars), SDHC promoter (red bar), and SDHC 5’UTR/exon 1 in relation to hypermethylated and hypoexpressed targets. The lower browser track shows the position of each 450K Infinium methylation CpG target in the region. (C) Heatmaps show methylation β values (legend at bottom right) of the 11 significant hypermethylated Infinium 450K CpG targets spanning the SDHC promoter (q < 0.05, red bar over upper heatmap) and upstream and downstream flanking regions. SDHCme-positive (upper heatmap) or SDHCme-negative (lower heatmap) GIST samples are ordered according to SDHx mutation. (D) 450K Infinium methylation data from 854 normal and tumor reference tissues from the GEO database (project IDs shown on the left of the heatmap). ETMR/PNET, embryonal tumor with multilayer rosettes/peripheral neuroectodermal tumor; PGL/Pheo, paraganglioma/pheochromocytoma; GBM, glioblastoma multiforme.

Fig. 2.. SDHC silencing in SDHx-WT dSDH GIST.

(A) Heatmap shows the expression (legend on the right) of all probe sets identified by Affymetrix U133P2 microarrays as being significantly different between SDHx-mutant GIST (n = 13) and SDHx-WT GIST (n = 7) (q < 0.05). The four probe sets map to SDHC. CTr, Carney triad. (B) Boxplots of SDHC probe set expression demonstrate an average 4.3-fold silencing in SDHx-WT GIST.

Fig. 3.. SDHC promoter methylation in blood.

Boxplot of SDHC promoter methylation in blood of SDHx-WT (n = 11) and SDHx-mutant (n = 14) GIST patients shows a 2.7% mean elevation in the SDHx-WT group (P = 0.003). SDHC promoter methylation in blood from individual patients is shown in fig. S9.