Analysis of Somatic Variants in Growth Hormone Secreting Pituitary Adenomas by Whole Exome Sequencing

Background: Whole-genome and whole-exome sequencing (WES) studies of pituitary adenoma have reported on average three mutations per patient tumor and discovered calcium signaling pathway mediated role of tumorigenesis. The aim of this study is to analyze the somatic variants in growth hormone (GH) secreting pituitary adenoma using WES. Material and Methods: Whole-exome sequencing was used to identify somatic mutations in GH-secreting pituitary adenomas from 14 patients (7-45 years), including a sample from a patient with germline GPR101 duplication. A mean sequencing depth of 94x was achieved covering 98% of targeted exonic regions at a minimum depth of 10x. Results: A total of 30 somatic variants were identified from 14 tumor samples. A recurrent hotspot mutation in GNAS, a well-established gene implicated in acromegaly, were identified in four patients. In addition, missense variants in CACNA1H and WIPI1 were identified which have only been reported in single patients previously. We also observed variants in GNAQ, RASGEF1B and PARP4 (this latter one in the GPR101-positive sample) genes that are involved in cAMP and calcium signaling pathways but have not been previously reported to be altered in acromegaly. Large deletions in chromosomes 1, 6, 10, 11, 12, 16, 17, 19 and 20 were also observed in at least eight patients. Conclusion: By combining our findings with data from previous studies of GH-secreting adenomas, we conclude that pathways involving calcium and cAMP signaling are likely to be involved in the pathogenesis of somatotroph adenomas.


Identification of somatic genetic alternations in tumors is crit-
ical for understanding the molecular mechanism of tumorigenesis.
Whole-genome and whole-exome sequencing (WES) studies of GH-secreting pituitary adenoma have reported on average three mutations per patient tumor and discovered calcium signaling pathway mediated role of tumorigenesis. The low rate of somatic mutations in pituitary tumors also indicates the possible role of other mechanisms of tumorigenesis involving differential miRNA expression [18], epigenetic alterations [19,20] and copy number variations [9,21,22]. Recurrent copy number aberrations including the whole-chromosomal losses are also commonly observed in pituitary adenoma [9,13,23].
In this study, we report somatic variant profiles of 14 somatotropinomas by WES. We identified a recurrent hotspot mutation in GNAS (4/14) and describe a deleterious somatic variant in CAC-NA1H, and likely benign variants in WIPI1, GNAS, GNAQ, RASGEF1B and PARP4.

Patient subjects and tissue samples
Patients diagnosed with GH-secreting pituitary adenomas at the Neurosurgical Department of Sungkyunkwan University Samsung Changwon hospital were recruited to the study. We analyzed the formalin-fixed, paraffin-embedded (FFPE) samples at the pathological archives which were obtained for recent 5 years.
And the blood samples were routinely obtained before treatment including surgery and any medication at the department. All 14 patients provided written consent for use of their biological samples for diagnosis and research purposes. Pituitary tumors surgically resected at the time of endonasal endoscopic transsphenoidal surgery from the patients were collected, washed and frozen immediately. Peripheral blood from the patients was also collected and stored. Details of the patients including sex, age at diagnosis, pathological reports and levels of hormones for all patients are provided in Table 1. The median age of the patients at the time of diagnosis was 32 years (range 7-56).

Whole exome sequencing data analysis to identifying somatic mutations
Sequencing data analysis was carried out using an in-house computational pipeline established following the guidelines previously established for variant discovery [24]. In summary, the sequencing quality of data was assessed using FastQC (v0.10.1) and all low-quality bases with Phred score <20 from read ends were soft masked from mapping against human genome primary assembly (GRCh37) using BWA (v0.7.10) [25].  [32]. Further, functional effects of the mutations were predicted using SIFT and Polyphen-2 scores. The shortlisted variants were validated by visualization of aligned data using integrated genomics viewer (IGV) [33].

Results and Discussion
Whole exome sequencing data analysis A total of 14 tumor-blood sample pairs from acromegaly patients were selected for WES (Table 1)  and GNAQ genes ( Table 2).  Interestingly, the CACNA1H p.Y1511F change in the calcium voltage-gated channel subunit alpha1 H has not been previously described and is rendered deleterious by several prediction programs.

Somatic mutation profile of acromegaly
A different change affecting the same amino acid was seen in the COSMIC database (p.Y1511C). We found a novel missense change in WIPI1, also a calcium pathway protein playing a role in calcium-mediated autophagy. The RASGEF1B p.K109Q variant is rare and predicted to be deleterious by Polyphen, but not by REVEL. The RASGEF1B gene is a guanine-nucleotide exchange factor and known interactor of Ras family of proteins which are essential regulators of cell proliferation, survival, differentiation, actin organization, vesicular trafficking, and gene expression. The PARP4 p.L1080F somatic change was previously seen in the COSMIC database and predicted to be benign. Genetic changes seen in this study are listed in Table 2 and depicted in Figure 2A. and complex rearrangements [9][10][11][12][13]. In a recent unpublished study, increased somatic copy-number variation was associated with DNA damage resulting from enhanced cAMP activity in sporadic somatotroph adenomas [34]. Patient #4 with a benign somatic GNAQ variant (not seen in germline but seen in COSMIC previously) is a FIPA case as her mother was also affected by acromegaly. No tissue sample is available from the mother. Patient #6 was positive for GPR101 duplication and the benign PARP4 variant was observed in the tumor. Patient #7 with a recurrent tumor was identified with a germline variant p.I852M in the RET gene. He has normal calcitonin, calcium and parathormone levels, and normal urinary metanephrins. This RET variant has been described in medullary thyroid carcinoma [35] and Multiple Endocrine Neoplasia 2A patients [36] and most likely represents a benign variant, despite the prediction scores [37]. Acromegaly has been described in a few patients with RET mutations, but these probably represent coincidences, rather than linked pathogenetically to the RET mutations [38,39]. Mutation data in five independent cohorts provided an opportunity to compare our results to the genetic profiles of other somatotropinomas ( Figure 2C). A summary and a partial list of genes with variants reported by these recent publications are provided in Table 2

Global comparison of variants across various pituitary adenomas
Clinically non-functioning pituitary adenomas were analyzed in three studies [9,12,17] which did not identify any recurrent mu- RAS are almost never seen [5].     Figure 4 with the mutated genes highlighted in red. Identification of mutations across several of these genes strongly suggest the dysregulation of the calcium and cAMP signaling which are potentially involved in pituitary tumorigenesis (Table 5).