Germline mutations and variants in the succinate dehydrogenase genes in Cowden and Cowden-like syndromes

Abstract

Individuals with PTEN mutations have Cowden syndrome (CS), associated with breast, thyroid, and endometrial neoplasias. Many more patients with features of CS, not meeting diagnostic criteria (termed CS-like), are evaluated by clinicians for CS-related cancer risk. Germline mutations in succinate dehydrogenase subunits SDHB-D cause pheochromocytoma-paraganglioma syndrome. One to five percent of SDHB/SDHD mutation carriers have renal cell or papillary thyroid carcinomas, which are also CS-related features. SDHB-D may be candidate susceptibility genes for some PTEN mutation-negative individuals with CS-like cancers. To address this hypothesis, germline SDHB-D mutation analysis in 375 PTEN mutation-negative CS/CS-like individuals was performed, followed by functional analysis of identified SDH mutations/variants. Of 375 PTEN mutation-negative CS/CS-like individuals, 74 (20%) had increased manganese superoxide dismutase (MnSOD) expression, a manifestation of mitochondrial dysfunction. Among these, 10 (13.5%) had germline mutations/variants in SDHB (n = 3) or SDHD (7), not found in 700 controls (p < 0.001). Compared to PTEN mutation-positive CS/CS-like individuals, those with SDH mutations/variants were enriched for carcinomas of the female breast (6/9 SDH versus 30/107 PTEN, p < 0.001), thyroid (5/10 versus 15/106, p < 0.001), and kidney (2/10 versus 4/230, p = 0.026). In the absence of PTEN alteration, CS/CS-like-related SDH mutations/variants show increased phosphorylation of AKT and/or MAPK, downstream manifestations of PTEN dysfunction. Germline SDH mutations/variants occur in a subset of PTEN mutation-negative CS/CS-like individuals and are associated with increased frequencies of breast, thyroid, and renal cancers beyond those conferred by germline PTEN mutations. SDH testing should be considered for germline PTEN mutation-negative CS/CS-like individuals, especially in the setting of breast, thyroid, and/or renal cancers.

Figure 1

Experimental Design for SDH Mutation Testing and Functional Analysis Note PTEN gene testing encompasses intragenic PCR-based mutation analysis, promoter, and large deletion analysis. From the 2270 PTEN mutation-negative CS/CS-like individuals, the most proximal (i.e., most recent) consecutive 375 PTEN mutation-negative subjects were selected to proceed to MnSOD expression analysis. It is these 375 subjects that represent the series for this SDH study.

Figure 2

Genetic and Biochemical Analyses of CS/CS-like Patients without Germline PTEN Mutations Reveal a Subset with Germline SDH Mutations Resulting in Biochemical Dysfunction (A) Dot blots to screen for increased MnSOD protein levels. Boxed dots represent controls with low MnSOD levels. (B) Illustrative sequencing chromatograms of germline heterozygous mutations of SDH genes identified in patients with CS/CS-like phenotypes (mutations as noted above each chromatogram). The germline mutations/variants are heterozygous manifested by overlapping peaks (arrows). (C) Increased ROS in peripheral lymphoblasts from an individual with germline SDHD His50Arg. Increased ROS levels are measured by increased carboxy-H2DCFDA staining as seen in cultured lymphoblast cells from the patient with SDHD His50Arg mutation denoting 1.5-fold higher ROS levels (middle) compared to a lymphoblast cell line derived from a normal control individual (left; p < 0.001, Student's t test, 3 replicates). Finally, a control lymphoblast cell line treated with tert-butyl hydroperoxide for 90 min was used as a superpositive control and suprainduced ROS expression is noted by markedly increased carboxy-H2DCFDA staining (right). (D) Protein expression of P-AKT and P-MAPK (P-ERK44/42) in germline heterozygous PTEN mutation-positive individuals. Note different mutations result in varying activation of P-Akt and/or P-MAPK. (E) Germline protein expression of PTEN, actin (loading control), P-Akt, and P-MAPK (as labeled, from top to bottom). Fold change values beneath the P-Akt and P-MAPK blots represent the mean of normalized densitometrically obtained expressional levels of patient sample(s) relative to controls. In other words, (Patient P-Akt or P-MAPK intensity/corresponding patient actin intensity)/(Control P-Akt or P-MAPK intensity/corresponding control actin intensity). The ratio of control P-Akt or P-MAPK intensity to control actin intensity was normalized to 1.0. We have chosen to use this type of quantitation (taking the ratio of the ratios) because it results in the most conservative (i.e., underestimated) fold changes.

Figure 3

Proposed Model for the Final Common Pathway of Putative Mitochondrial Dysfunction Resulting from Either PTEN or SDHx Mutation in Cowden and Cowden-like Syndromes A simplified version of the signaling pathways involved in tumorigenesis in the setting of dysfunctional PTEN or SDH (represented by hatched colors). These pathways crosstalk, leading to the final common outcome of tumor angiogenesis, cell proliferation, and inhibition of apoptosis. Note that one of the functions of SDH is the conversion of succinate to fumarate as part of the Kreb's tricarboxylic acid cycle. SDH dysfunction will therefore lead to an accumulation of succinate, which inhibits prolyl hydroxylases (PHD) and subsequently leads to the stabilization of HIF-1a. The stabilization of the latter also occurs a number of steps downstream of dysfunctional PTEN signaling. It is interesting to note that activated Akt (P-Akt) can increase ATP levels that result in increased ROS, presumably via mitochondrial dysfunctional signaling. This is postulated to set up a double feedback loop linking both the PTEN and SDH pathways.

Figure 4

Suggested Algorithm for Clinical PTEN and SDH Testing for CS/CS-like Individuals See text for details.