Recurrent Germline Mutations of CHEK2 as a New Susceptibility Gene in Patients with Pheochromocytomas and Paragangliomas

Purpose Recently, pheochromocytomas and paragangliomas (PPGLs) have been strongly suspected as hereditary tumors, as approximately 40% of patients carry germline mutations. In the cancers where defects occur to corrupt DNA repair and facilitate tumorigenesis, a CHEK2 strong association has been observed. Therefore, the purpose of this study was to investigate the effect of CHEK2 mutations for its possible pathogenicity in PPGLs. Methods Four patients with CHEK2 mutations were recruited, as previously detected by the whole exome sequencing. Sanger sequencing was used to verify the germline mutations as well as the loss of heterozygosities (LOHs) in their somatic DNAs. Immunohistochemistry was used to analyze the expression of CHEK2 and its downstream target p53 Ser20 (phosphorylated p53). Results The average age of studied patients was 44.25 ± 11.18 years, at the time diagnosis. One patient had multiple tumors which recurred quickly, while two patients had distant metastasis. None of the patient had any relevant family history. Four germline CHEK2 mutations were identified (c.246_260del; c.715G > A; c.1008+3A > T; and c.1111C > T). All the patients were predicted to have either pathogenic or suspected pathogenic mutations. There was no LOH of CHEK2 gene in somatic DNAs found. Additionally, neither CHEK2 proteins nor its downstream target p53 Ser20 were expressed in the tumor tissues. The inactivation of CHEK2 leads to the decrease in the p53 phosphorylation, which might promote tumorigenesis. Conclusions For the first time, CHEK2 was identified as a susceptibility gene for PPGLs. However, the penetrance of CHEK2 gene with genotype-phenotype correlation needs to be investigated.


Introduction
e neuroendocrine tumors arising in the chromaffin cells of adrenal medulla are termed as pheochromocytomas (PCCs), whereas the extra-adrenal tumors originating in the chromaffin cells from the sympathetic and parasympathetic ganglia are known as paragangliomas (PGLs) [1]. PCCs and PGLs (PPGLs) affects around 2-5 patients/million/year, with the prevalence of about 1/300000 to 1/100000 for general population [2]. In the recent years, molecular pathogenesis of this group of lesions has advanced significantly. Almost 40% of PPGLs patients carry germline mutations in a growing list of genes including SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, RET, MAX, TEMEM127, FH, NF1, and KIF1B [3,4]. Besides, genes such as EGLN1, EGLN2, MDH2, SLC25A11, MERTK, DLST, and KMT2D are also shown to be related to PPGLs [5][6][7][8][9][10]. It is noteworthy that the majority of individuals with clinical features such as family history of PPGLs, multiple tumors, and an early age of onset might be indicative of a hereditary onset, but they lack mutations in any of the known PPGLs susceptibility genes.
Cell cycle checkpoint kinase 2 (CHEK2) is located in chromosome 22q12.1, which encodes multifunctional kinase crucial for cell cycle regulation, DNA repair, and apoptosis [11]. In response to DNA damage, CHEK2 is required for bridging between ataxia telangiectasia mutated (ATM) kinase with its downstream checkpoint effectors; therefore, CHEK2-deficient patients may have corrupt DNA repair and conserved mutations which ultimately facilitate tumorigenesis [12]. However, as candidate tumor suppressor, CHEK2 contributes to molecular pathogenesis in various human malignancy. ereby, heterozygous CHEK2 gene germline mutations have been observed in patients with the Li-Fraumeni cancer-predisposition syndrome (LFS), with other cancers such as breast cancer, colon cancer, thyroid cancer, bladder cancer, ovarian cancer, gastric cancer, renal cancer, and prostate cancer [13]. Hence, CHEK2 is speculated to be a low-penetrance, multiorgan cancer susceptibility gene.
Recently, whole exome sequencing (WES) technology has been employed to detect germline variations of 121 patients who did not have mutations on definite pathogenic genes. In our previous report, the use of next-generation sequencing (NGS) covering SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, RET, MAX, TEMEM127, FH, NF1, and KIF1B was analyzed in the cohort with 314 PPGL patients [3]. Among them, four patients showed CHEK2 gene heterozygous mutations. However, definitive validation of CHEK2 gene was required to ascertain it as a new candidate susceptibility gene in PPGLs and for the potential value for genetic risk assessment, prognosis, and surveillance. erefore, this present study aims to investigate the effect of CHEK2 mutations on DNA-damage pathway and to assess its possible pathogenicity in PPGLs.

Patients.
Out of PPGLs cohort, four patients (Patients 1, 2, 3, and 4) had variants of CHEK2 gene as detected by the WES.
e PPGLs cohort was recruited from the Peking Union Medical College Hospital between November 2007 and June 2013 (the detailed data of all 121 patients who received WES are not provided in this study). e collected blood samples and formalin fixed paraffin embedded (FFPE) tumor tissues and sections were collected after obtaining the written informed consent from the patients. e approval of the study was granted by the medical ethics committee of the hospital, and the results of this research were also agreed to be published. e DNAs from the peripheral blood leukocytes (Omega Blood DNA Midi Kit, Omega Bio-Tek, USA) and FFPE tumor tissues (Quick-DNATM FFPE Kit, ZYMO RESEARCH, USA) were obtained using a standard procedure from the patients having CHEK2 mutations.

Sanger Sequencing of CHEK2 Gene.
e four mutations of CHEK2 gene detected by WES were verified by the PCR amplification in combination with Sanger sequencing. e PCR primers and amplification methods are shown in Table 1. For distinguishing the sequence of CHEK2 with the highly homologous pseudogenes (CHEK2P1-5) from exon 11 to exon 15, we used nested PCR amplification for detecting mutation on exon 11 of Patient 4. All the sequences were studied for the mutations, by comparing them with the reference sequence of the CHEK2 gene (NM_007194.4 and NP_009125.1) through the NCBI website.

Loss of Heterozygosity (LOH) of CHEK2 in Tumor Tissue.
PCR amplification and Sanger sequencing were done to evaluate the LOH of corresponding sites in somatic DNAs of patients. However, one patient FFPE sample (Patient 4) was not sufficient; therefore, only three patients FFPE tumor tissues were subjected for studying the corresponding exons of CHEK2 with mutations in somatic DNA by using the sequencing method mentioned in Table 2. e homozygous mutant for supporting that LOH of CHEK2 was present, and for the heterozygote means, no LOH in the corresponding site in somatic DNA was found.

CHEK2 Immunohistochemistry.
e four patients with CHEK2 mutations were also evaluated for CHEK2 protein expression in the FFPE tumor sections by immunohistochemistry (IHC). Briefly, the sections were incubated with primary antibody of human Anti-Chk2 antibody (ab207446) (Abcam, England) at 1/100 dilution, followed by secondary incubation with the goat anti-rabbit IgG polymer (PV-9001, (ZSGB-BIO, China)) at 1/500 dilution. As a positive control, normal gland and PPGL tumor tissue with RET mutation were used.

Clinical Manifestation.
e detailed clinical symptoms of the four patients with CHEK2 mutations are shown in Table 3. Among the 4 patients, three were male and 1 was female patient. e average age of the patients at the time of diagnosis was 44.25 ± 11.18 years old, where Patient 2 was only 30 years old at the time of PPGLs onset. Patient 1 had adrenal and paraaortic multiple tumors, which recurred in situ after surgery. Two patients had distant metastasis (Patient 2: liver metastasis and Patient 4: bone metastasis); however, no patients had family history.  Table 4. Of note, these four patients had no other germline mutations of the confirmed susceptibility genes for PPGLs. e evidences of pathogenicity of ACMG mentioned in this table were as follows: PS1: the same amino acid change as a previously established pathogenic variant regardless of nucleotide change; PS3: well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product; PM1: located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation; PM2: absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium; PM4: protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants; PM6: assumed de novo, but without confirmation of paternity and maternity; PP3: multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.); PP5: reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

LOH of CHEK2 in Tumor Tissue.
In the FFPE tumor tissues, the sites of three mutations detected in peripheral blood DNA were heterozygous in somatic DNAs (Note: Patient 4 had insufficient FFPE tumor tissues) (Figure 2). e results confirm that there was no LOH of CHEK2 gene in the studied patients.

Immunohistochemistry of CHEK2 Protein and the
Downstream Target p53 Ser20. Compared with the normal adrenal or PPGL tumor tissue sections with RET mutation (the nucleus was positive for CHEK2 staining), the results of CHEK2 immunohistochemistry were negative in all patients except that the partial cytoplasm was weakly positive for Patient 4. is finding suggested that the CHEK2 proteins were either not expressing or inactivated in the tumor tissues ( Figure 3). e results of the downstream target p53 Ser20 immunohistochemical staining were nucleus negative for these patients (except for partial cytoplasm positivity in Patients 2 and 4), as compared with positive control from normal gland tissue. ese findings further confirm that the inactivation of CHEK2 could result in the decrease activity of phosphorylation of p53 protein (Figure 4). erefore, the abnormal phosphorylation of p53 protein might influence the biological function and can lead to tumorigenesis.

Discussion
We previously shown that the CHEK2 gene mutations accounted for 3.3% (4/121) of PPGLs patients, in which pathogenic mutations of the related genes were not detected, whereas in 1.3% (4/314) of PPGLs patients recruited cohort from Peking Union Medical College Hospital, a frequency equivalent to a few identified PPGLs susceptibility genes including SDHA, TMEM127, MAX, and FH was found [14][15][16][17]. It is noteworthy that CHEK2 gene mutations might be associated with the genetic background of PPGLS, as out of 4, three patients detected CHEK2 mutations were presented with the multiple tumors or malignant developments. Since checkpoint defects result in the accumulation of altered genetic information and a central feature of carcinogenesis, these DNA-damage checkpoint pathways have been of interest to the field of cancer biology [18]. Among the conserved DNA-damage activated kinases identified so far, the CHEK2 plays a central role in implementing many aspects of the checkpoint response, related to the occurrence of various cancers [19]. e CHEK2 protein contains three distinct functional domains: (1) the SQ/TQ-rich, (2) the forkhead-associated, and (3) and the serine/threonine kinase domain [20]. Figure 5 shows the pattern of CHEK2 gene and the four detected mutations location. However, except for the one mutation which was present next to the SQ/TQ-rich domain, all others were in the kinase domain.  e detected four germline variants of CHEK2 in this study were causing decreased expression of the CHEK2 protein, suggesting the alterations were resulting in loss-offunction pathogenicity. ough in PPGLs, the function of CHEK2 gene has not been well characterized; however, CHEK2 role in cell proliferation and tumor suppression has been confirmed by various reports. Hong et al. established a CHEK2-1100delC mutant, which promoted the gastric cancer cell proliferation, migration, and invasion, with downregulation of E-cadherin and upregulated vimentin expression, suggesting its possible role in altered biological behavior as epithelial mesenchymal transition (EMT) [21].

International Journal of Endocrinology
Another study reported the novel recurrent CHEK2-Y390 C mutant associated with increased breast cancer risk in Chinese population. e study further reported that the mutant protein's inability resulted in the lack of phosphorylation of CDC25 A Ser178 and p53 Ser20 after DNA damage, which was led to abnormal cell apoptosis and checkpoint repair [22]. In the present study, we also found that the p53 could not be phosphorylated due to CHEK2 mutations in the studied four patients, indicating the inability of variant CHEK2 proteins to efficiently bind and phosphorylate its substrates.
Among these four mutations found in the present study, two missense mutations were reported previously. In year 2003, the mutant CHEK2-E239 K was first mentioned for the prostate cancer [23]. e alteration of amino acid in the kinase activation domain significantly alter the phosphorylation of p53 in DNA-damage signaling, while the wild type CHEK2 completely retained CHEK2 kinase activity following ionizing radiation, and only 50% response was regained in the mutant group [24]. is studied mutation was later detected in patients with breast cancer and non-Hodgkin's lymphoma [25,26]. Another mutant CHEK2-H371Y detected in our study was confirmed as a breast cancer risk variant in 2011, for 4% of the total patients. Approximately 50% decrease was observed during functional analysis for the autophosphorylation, transphosphorylation, and CHEK2 activity of CHEK2-H371Y mutant [27]. e other two variants namely p.82_87del and c.1008 + 3A > T detected in our study were not reported in databases previously. Both had high pathogenicity as evaluated by the ACMG, suggesting that these CHEK2 mutations could be deleterious as they might influence the protein structure and kinase domain. Additionally, no LOHs were detected in these corresponding sites of the studied four patients with CHEK2 mutations. Moreover, haploinsufficiency caused by dominant negative effect, or the change in protein spatial structure with the mutant amino acid folding, can lead to the abnormal function by only one allele variant [28,29].
In the present study, lack of family history in four pedigrees was investigated for genes such as MDH2, BAP1, DLST, or SLC25A11 [6,9,10,30]. However, among the de novo mutation or low-penetrance inheritance, the latter is frequently associated with PPGLs [6,31]. On the other hand, due to the advancement in genetics, germline mutations and familial syndromes are known to be associated with 8-24% of sporadic PPGLs [2]. Germline testing is now generally recommended in PPGL, and besides the potential role played in PPGLs pathogenesis, the detection of germline variants in patients clinically defined as sporadic may be helpful in finding out the existence of unknown multineoplasia hereditary diseases [32,33]. e current study had the following limitations. First, due to the limitation of follow-up year, we did not observe the other multiple tumors in these patients with CHEK2 mutations or their family members. Second, the DNAs from blood leukocytes of patient's parents were not obtained; therefore, we could not identify if the mutations had de novo origin. irdly, all CHEK2 variants detected in somatic DNA Figure 4: Immunohistochemical staining of the phosphorylated p53 Ser20. (a) Staining for normal gland (positive control: the nucleus was positive for p53 Ser20), (b) for Patient 1 (negative for p53 Ser20), (c) for Patient 2 (the nucleus was negative but partial cytoplasm was positive for p53 Ser20), (d) for Patient 3 (negative for p53 Ser20), and (e) for Patient 4 (the nucleus was negative but partial cytoplasm was weak positive for p53 Ser20).  were heterozygous; therefore, the potential mechanisms leading to the abnormal function by only one allele variant should be further researched. Lastly, in the results of IHC for CHEK2 expression, tumor or normal adrenal tissue, stromal cells, such as vascular endothelial cells, were not stained positive. ese findings on CHEK2 staining were also discussed in previous studies [34][35][36][37]. erefore only positive or negative staining of tumor cells was compared and analyzed here.

Conclusions
In conclusion, we have identified four germline variants, which functionally compromises CHEK2, suggesting CHEK2 as a susceptibility gene for PPGLs. However, due to the limited number of patients and low prevalence of the CHEK2 mutations, more cases are required for the validation of its penetrance and genotype-phenotype correlation in PPGLs.

Data Availability
Data would be available upon request from corresponding author of this manuscript.

Conflicts of Interest
e authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.