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. 2016 May 1;22(9):2301-10.
doi: 10.1158/1078-0432.CCR-15-1841. Epub 2015 Dec 23.

Recurrent Mutations of Chromatin-Remodeling Genes and Kinase Receptors in Pheochromocytomas and Paragangliomas (VSports手机版)

Rodrigo A Toledo  1 , Yuejuan Qin  1 , Zi-Ming Cheng  1 , Qing Gao  1 , Shintaro Iwata  2 , Gustavo M Silva  3 , Manju L Prasad  4 , I Tolgay Ocal  5 , Sarika Rao  6 , Neil Aronin  6 , Marta Barontini  7 , Jan Bruder  1 , Robert L Reddick  8 , Yidong Chen  9 , Ricardo C T Aguiar  10 , Patricia L M Dahia  11
Affiliations

Recurrent Mutations of Chromatin-Remodeling Genes and Kinase Receptors in Pheochromocytomas and Paragangliomas

Rodrigo A Toledo et al. Clin Cancer Res. .

Abstract

Purpose: Pheochromocytomas and paragangliomas (PPGL) are genetically heterogeneous tumors of neural crest origin, but the molecular basis of most PPGLs is unknown VSports手机版. .

Experimental design: We performed exome or transcriptome sequencing of 43 samples from 41 patients. A validation set of 136 PPGLs was used for amplicon-specific resequencing. In addition, a subset of these tumors was subjected to microarray-based transcription, protein expression, and histone methylation analysis by Western blotting or immunohistochemistry. In vitro analysis of mutants was performed in cell lines. V体育安卓版.

Results: We detected mutations in chromatin-remodeling genes, including histone-methyltransferases, histone-demethylases, and histones in 11 samples from 8 patients (20%). In particular, we characterized a new cancer syndrome involving PPGLs and giant cell tumors of bone (GCT) caused by a postzygotic G34W mutation of the histone 3. 3 gene, H3F3A Furthermore, mutations in kinase genes were detected in samples from 15 patients (37%). Among those, a novel germline kinase domain mutation of MERTK detected in a patient with PPGL and medullary thyroid carcinoma was found to activate signaling downstream of this receptor V体育ios版. Recurrent germline and somatic mutations were also detected in MET, including a familial case and sporadic PPGLs. Importantly, in each of these three genes, mutations were also detected in the validation group. In addition, a somatic oncogenic hotspot FGFR1 mutation was found in a sporadic tumor. .

Conclusions: This study implicates chromatin-remodeling and kinase variants as frequent genetic events in PPGLs, many of which have no other known germline driver mutation. MERTK, MET, and H3F3A emerge as novel PPGL susceptibility genes. Clin Cancer Res; 22(9); 2301-10 VSports最新版本. ©2015 AACR. .

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Conflict of interest statement

Conflict of Interest: The authors have no conflict of interest to declare.

Figures

Fig.1
Fig.1
Overview of the pheochromocytoma and paraganglioma (PPGL) cohort including new genetic variants identified in chromatin-related and kinase genes, and mutations in known PPGL driver genes, along with their respective frequencies. Number of mutations per tumor is shown at the bottom of the diagram. Samples were sorted by inheritability likelihood. Samples with high inheritability carried one or more of the following features: young age at onset, familial history, multicentric tumors and/or co-occurrence of tumors associated with PPGLs), relative to the remaining samples (considered ‘likely sporadic”). Tumor location, malignancy status and type of sequencing (whole exome or transcriptome) are also indicated. Legends to the various mutation categories are shown below the image.
Fig.2
Fig.2
a) Sanger sequencing traces of two pheochromocytomas and one paraganglioma from the same patient displaying a H3F3A gene mutation (c.103G>T; p. G34W) alongside sequence of DNA isolated from formalin-fixed paraffin embedded (FFPE) tissue sections from normal tissue (gallbladder) and giant cell tumor (GCT) of the tibia, displaying variant (T) allele % representation quantified by Mutation Surveyor (Suppl.Methods). b) Structural impact of histone H3 G34W mutation on lysine 27 and 36 side-chain position. Predicted structures of the WT (blue) and the G34W (magenta) histones H3F3A were aligned and visualized as cartoons. Close up view depicts the side-chains of lysine (K) residues K27, K36 and the point mutation G34W. The angular change of the methylated nitrogen atom of both lysine residues was calculated using Pymol comparing the WT to the G34W mutant. c) Immunohistochemical staining of trimethylated H3 lysine 36 (H3K36me3) in samples from case 1: (pheo=pheochromocytoma, PGL-bladder= retroperitoneal, paraganglioma adjacent to the bladder; PGL-periaortic=periaortic paraganglioma; GCT=giant cell tumor of bone. Scale bars are 50 μm. d) Western blot of pheochromocytomas and paraganglioma lysates using an antibody that recognizes trimethylated forms of lysine 36 (H3K36me3) and 27 (H3K27me3) of histone 3. Total H3 was used as a loading control. Shown are lysates from two different regions of the retroperitoneal paraganglioma and the left pheochromocytoma with the H3F3A G34W mutation (H3F3A); unrelated pheochromocytomas/paragangliomas with wild-type H3F3A sequence and mutations in other susceptibility genes (SDHB VHL, HRAS mutation) or sporadic tumors. e) Western blot of pheochromocytoma lysates containing the indicated mutations, including two independent regions of the paraganglioma and the left pheochromocytoma from the same patient with a G34W-mutation (H3F3A), along with three tumors with intact H3F3A sequence (WT): one with a MAX mutation (arrow), one sporadic and one with a RET mutation, probed with an MYCN antibody. The neuroblastoma cell line Kelly, which has amplification of MYCN, was included as a positive control and loaded as 5% input lysate. Loading was verified with beta-actin.
Fig.3
Fig.3
a) Schematic of MERTK protein structure and functional domains (I-set=immunoglobulin set domain, Ig-2=immunoglobulin-like, FN3-fibronectin typeIII, TM=transmembrane; TK=tyrosine kinase; TAM=tyro3, axl, mertk domain). The two R758 mutations are indicated by the ‘lollipop—shaped marks. The magnified region displays the TAM KWIAIES kinase domain, which is conserved within TAM kinases but is highly homologous with other tyrosine kinase receptors and is here aligned to the corresponding region within the RET receptor. The R758 residue of MERTK and corresponding RET R921 are highlighted in red and orange, respectively. The RET M918 residue mutated in cancers is colored in blue. b) HEK293 cells expressing an empty vector (EV) or MERTK wild-type (WT) or mutants R758H, R758C, Y754F. The R758H/C variants were found in patients with pheochromocytoma, and Y754F is a dominant negative MERTK form used as a control. Cells were starved of nutrients for 3h and then exposed to MERKT ligand Gas6 for 10min. The Western blot shows expression of multiple MERTK isoforms [long (l.e.) and short (s.e.) exposures] and phosphorylation of the downstream kinase ERK (p-ERK);. c) HEK293 cells expressing the same constructs shown in b after nutrient starvation (3h); lysates were prepared and probed with MERTK and p-ERK. Beta-actin was used for loading in both b and c blots. d) Pedigree drawing of three generations of a family with pheochromocytoma (males indicated by squares and females, by circles). Affected individuals are represented by filled symbols. Genotype for the V806M MET mutation is indicated as mutant (MUT) or wild-type (WT). e) MET mutations identified in this study displayed along the protein structure. Length of vertical lines reflects the number of events. The V806M variant was found in a patient with familial pheochromocytoma and segregates with the phenotype in the family. The remaining variants were detected in tumor DNA. Plot was designed using the Mutation Mapper tool of the cBioPortal of Cancer Genomics(35, 36).
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References

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