Analysis of Activating GCM2 Sequence Variants in Sporadic Parathyroid Adenomas
Aaliyah Riccardi  Tori Aspir  Lilia Shen  Chia-Ling Kuo  Taylor C Brown Reju Korah  Timothy D Murtha  Justin Bellizzi  Kourosh Parham Tobias Carling  ... Show more
The Journal of Clinical Endocrinology & Metabolism, Volume 104, Issue 6, June 2019, Pages 1948–1952, https://doi.org/10.1210/jc.2018-02517
Published: 08 January 2019  Article history
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Abstract
Context
Sporadic, solitary parathyroid adenoma is the most common cause of primary hyperparathyroidism (PHPT). Apart from germline variants in certain cyclin-dependent kinase inhibitor genes and occasionally in MEN1, CASR, or CDC73, little is known about possible genetic variants in the population that may confer increased risk for development of typical sporadic adenoma. Transcriptionally activating germline variants, especially within in the C-terminal conserved inhibitory domain (CCID) of glial cells missing 2 (GCM2), encoding a transcription factor required for parathyroid gland development, have recently been reported in association with familial and sporadic PHPT.

Objective
To evaluate the potential role of specific GCM2 activating variants in sporadic parathyroid adenoma.

Design and Patients
Regions encoding hyperparathyroidism-associated, activating GCM2 variants were PCR amplified and sequenced in genomic DNA from 396, otherwise unselected, cases of sporadic parathyroid adenoma.

Results
Activating GCM2 CCID variants (p.V382M and p.Y394S) were identified in six of 396 adenomas (1.52%), and a hyperparathyroidism-associated GCM2 non-CCID activating variant (p.Y282D) was found in 20 adenomas (5.05%). The overall frequency of tested activating GCM2 variants in this study was 6.57%, approximately threefold greater than their frequency in the general population.

Conclusions
The examined, rare CCID variants in GCM2 were enriched in our cohort of patients and appear to confer a moderately increased risk of developing sporadic solitary parathyroid adenoma compared with the general population. However, penetrance of these variants is low, suggesting that the large majority of individuals with such variants will not develop a sporadic parathyroid adenoma.

Issue Section: Parathyroid, Bone, and Mineral Metabolism
Primary hyperparathyroidism (PHPT) is a common endocrinopathy, with a prevalence of 1.12 to 3.6/1000 (1, 2). Whereas often minimally symptomatic in the most developed nations, hypercalcemia and accompanying inappropriately elevated PTH levels can lead to kidney stones, osteoporosis, and increased fracture risk. A solitary, sporadic (nonfamilial) parathyroid adenoma is the cause of PHPT in 85% of cases. MEN1 and CCND1 (encoding cyclin D1) are solidly established, somatically altered genetic drivers of parathyroid adenoma. Mutations in CDKN1B/p27 and certain other cyclin-dependent kinase inhibitor genes, EZH2, ZFX, CTNNB1/β-catenin, and others are seen in smaller subsets of sporadic adenomas (3). Rare germline mutations in MEN1, CASR, CDC73 (HRPT2), CDKN1B/p27, and other cyclin-dependent kinase inhibitor genes, which are each established causes of familial forms of PHPT, can occasionally be observed in otherwise typically presenting sporadic adenomas (4–6). Despite these significant advances, much remains unknown about the genetic underpinnings of sporadic parathyroid adenoma. Further insight into the genetic alterations that contribute to the risk of developing parathyroid adenomas could prove useful in early diagnostic detection, prevention of associated morbidity, and aid in the development of precision-targeted treatments for benign and malignant forms of PHPT.

Activating variants of the glial cells missing 2 (GCM2) gene have recently been reported in familial isolated hyperparathyroidism (FIHP) (7) and as potential predisposition alleles in sporadic parathyroid tumors (8, 9). The GCM2 (previously GCMB) gene encodes the GCM2 transcription factor, which is essential to the development of the parathyroid glands and subsequent PTH expression. Inactivating GCM2 mutations have been established to cause hypoparathyroidism in humans (10) and mice (11). GCM2 was reported to be upregulated in parathyroid adenoma, hyperplasia, and cancer (12), and a single missense GCM2 variant [c.1144G>A (p.V382M)] was identified in a series of 30 parathyroid adenomas (13). Increased prevalence of a variant of GCM2 [c.844T>G (p.Y282D)], which enhances the protein’s ability to transactivate transcription of a reporter construct in vitro, was described in three Italian cohorts with sporadic PHPT (9); variant prevalence was further elevated in patients with parathyroid carcinoma (14). Recurrent, in vitro activating variants within the C-terminal conserved inhibitory domain [CCID; c.1136T>A (p.L379Q) and c.1181A>C (p.Y394S)] of GCM2 were identified in seven of 40 kindreds with FIHP (7). The Y394S variant, which is 88 times more prevalent in Ashkenazi Jewish populations than in non-Ashkenazi populations (15), was particularly enriched within Ashkenazi Jewish patients with FIHP and in those with nonfamilial PHPT (8). Furthermore, both L379Q and Y394S were detected in non-Ashkenazi patients with sporadic PHPT (three of 275, or 1.09%), suggesting that such in vitro activating GCM2 variants, particularly those within the CCID domain, may play a role in predisposition to otherwise sporadic parathyroid tumors (8). Thus, we sought to examine this hypothesis more deeply and analyze the potential contribution of activating GCM2 CCID variants and the Y282D activating variant to the pathogenesis of typically presenting, sporadic parathyroid adenomas.

Materials and Methods
Patients and materials
We obtained 396 parathyroid tumor samples from patients who underwent parathyroidectomy as treatment of PHPT with typical presentations and no family history suggestive of inherited hyperparathyroidism. Disease was proven to be monoglandular and displayed no malignant and/or atypical features upon histological examination. All cases were apparently cured following parathyroidectomy, as indicated by the normalization of PTH and calcium levels. Nontumor tissue or blood obtained from the same patients served as a germline control and was available for 263 of the 396 cases. All samples were obtained with informed consent in accordance with Institutional Review Board-approved protocols. Genomic DNA was extracted from fresh, frozen tissue by proteinase K digestion, followed by phenol–chloroform extraction and ethanol precipitation. Genomic DNA was extracted from blood using the Gentra Puregene Blood kit (Qiagen Germantown, MD).

PCR and Sanger sequencing
The following PCR primers were designed to cover the CCID region of GCM2, i.e., amino acids 379 to 395 and the immediate flanking regions: GCM2-CCID-forward 5′-CAGCTATGAGAGAAGCTTTG-3′ and GCM2-CCID-reverse 5′-CTGCAATTTTCATAGGAGGTGG-3′. To examine the Y282D variant, the following primers, amplifying a 238-bp fragment, including c.844, were used: GCM2-Y282D-forward 5′-AAAGCTACCTGTGACCTAGC-3′ and GCM2-Y282D-reverse 5′-CATAGCTGCTGTATGAATTG-3′. These regions were PCR amplified from template genomic DNA of the 396 typical parathyroid adenomas and subsequently sequenced. The PCR reactions were carried out in 20 μL reaction volumes. Each reaction contained 25 ng tumor DNA, 12 μL H2O, 2μL 10× PCR buffer, 200 μM deoxynucleotide triphosphates, 1.2 μL MgCl2, 1 μM each primer, and 1 U AmpliTaq Gold (Thermo Fisher Scientific, Waltham, MA). PCR began with a single denaturation step of 95°C for 10 min and 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s and ended with a single extension step at 72°C for 10 min. PCR products were purified with ExoSAP-IT (Affymetrix, Santa Clara, CA) and sequenced using standard Sanger methodology with the same primers used for the PCR reactions (Genewiz, South Plainfield, NJ). The resulting sequencing data were aligned to the published reference sequence (ENST00000379491.4) using Sequencher (Gene Codes, Ann Arbor, MI). Variants were confirmed with an independent PCR/sequencing reaction. When available, nontumor, germline DNA from the same individual was also amplified and sequenced to confirm germline status of each variant.

Statistical analyses
Statistical analyses were performed using Prism 6 software (GraphPad Software, La Jolla, CA). Penetrance was calculated using the following formula:
Penetrance= disease prevalence in the general population× case allele frequency allele frequency in the general population
Population control allele frequency of each variant was obtained from the Genome Aggregation Database (15, 16).
Results
In this substantial cohort of 396 typically presenting sporadic parathyroid adenomas, one tumor (0.25%) was heterozygous for the c.1144G>A (p.V382M) variant, which was also present in the patient’s germline (Fig. 1A). The c.1181A>C (p.Y394) variant was found in a total of five adenomas (1.26%; Fig. 1B); this change was heterozygous in four cases and homozygous in one. We directly confirmed the positive germline status, as expected, of the Y394S variant in each of two cases in which germline DNA was available. The nonactivating CCID variant c.1158_1160dupCAC/p.T387dup was also seen as a heterozygous change in two typical sporadic parathyroid adenoma samples for which germline DNA was not available. The c.844T>G/Y282D variant was observed as a heterozygous allele in 20 adenomas (5.05%); again, as expected, its germline status was confirmed in all cases (8) for which germline DNA was available. No somatic mutations were identified in any instance where a variant’s germline/somatic status could be assessed, i.e., in which matched germline DNA was available. The activating CCID variant L379Q was not detected in any case in our series. No cases in our study exhibited both an activating CCID variant (V382M or Y394S) and the Y282D variant. Whereas clinical information was limited for the patients included in this study, no obvious differences in clinical presentation, including sex distribution and age of onset, were noted for the patients carrying these variants.

Figure 1.
Representative electropherograms showing identified GCM2-activating variants. (A) Wild-type GCM2 coding sequence (top). A heterozygous guanine-to-adenine transition was identified at position 1144 of the GCM2 coding sequence (c.1144G>A), resulting in a valine-to-methionine change at position 382 of the protein (V382M), in one sporadic parathyroid adenoma in this study (bottom). Position of the variant is indicated by an asterisk (*). (B) Wild-type GCM2 coding sequence (top). An adenine-to-cytosine transversion at position 1181 of the GCM2 coding sequence (c.1181A>C), resulting in a tyrosine-to-serine change at position 394 of the protein (Y394S), was identified in five tumors in this study; representative sequencing data from one tumor is shown (bottom). Position of the variant is indicated by an asterisk (*).
View largeDownload slide
Representative electropherograms showing identified GCM2-activating variants. (A) Wild-type GCM2 coding sequence (top). A heterozygous guanine-to-adenine transition was identified at position 1144 of the GCM2 coding sequence (c.1144G>A), resulting in a valine-to-methionine change at position 382 of the protein (V382M), in one sporadic parathyroid adenoma in this study (bottom). Position of the variant is indicated by an asterisk (*). (B) Wild-type GCM2 coding sequence (top). An adenine-to-cytosine transversion at position 1181 of the GCM2 coding sequence (c.1181A>C), resulting in a tyrosine-to-serine change at position 394 of the protein (Y394S), was identified in five tumors in this study; representative sequencing data from one tumor is shown (bottom). Position of the variant is indicated by an asterisk (*).


The penetrance of a given variant is dependent on a determination of disease prevalence. For PHPT, reasonable estimates of disease prevalence in the general population are between 0.112% (2) and 0.36% (1) from studies selected for their inclusion of a wide age range of adult patients and repeat measurements of serum calcium and PTH and/or confirmed surgical/histopathological diagnosis of one or more hypercellular parathyroid glands. The prevalence of 0.112% was obtained by the combination of the average prevalence values for males and females across all years of the study (2). We calculated penetrance separately for each variant using both values (Table 1). Given a disease prevalence of 0.112%, the penetrance of activating GCM2 CCID variants (V382M or Y394S) is 1.28%. At a disease prevalence of 0.36%, the penetrance is 4.13%. Thus, the risk of disease is >11-fold higher in carriers of activating GCM2 CCID variants than that in the general population, but nonetheless, the likelihood that an individual bearing a CCID variant will develop a sporadic parathyroid adenoma is quite small.


Table 1.
Frequency and Penetrance of Identified GCM2-Activating Variants

  V382M Y394S Y282D Activating CCID Variants All Activating Variants
Frequency in sporadic parathyroid adenoma 1/396 5/396 20/396 6/396 26/396
Frequency in general population 15/141,420 172/141,435 2523/141,448 187/141,435 2710/141,435
Percent penetrance at disease prevalence of 1.12 per 1000 2.67 1.16 0.32 1.28 0.38
Percent penetrance at disease prevalence of 3.6 per 1000 8.57 3.74 1.02 4.13 1.23
Fold increase in disease risk 23.8 10.4 2.8 11.5 3.4
View Large
Likewise, the penetrance of any of the known GCM2-activating variants (V382M, Y394S, or Y282D) is 0.38%, given a disease prevalence of 0.112%, and 1.23% when the disease prevalence is 0.36%. The risk of disease increases more than threefold higher in carriers of such variants than that in the general population, but again, the overwhelming majority of individuals with one of these variants would not be expected to develop a sporadic adenoma.

Discussion
Recent studies have suggested that germline GCM2 variants that accentuate its function in vitro as a transcriptional activator may play a role in predisposition to familial and sporadic PHPT (7, 8). GCM2 encodes a parathyroid-specific master regulatory transcription factor, previously reported to be overexpressed in parathyroid adenomas (6). We sought to assess the frequency of such variants in typically presenting sporadic parathyroid adenomas, the most common form of PHPT seen in clinical practice.

Our study determined an overall frequency of activating GCM2 CCID variants in typical, sporadic parathyroid adenomas of 1.52%. This frequency is significantly lower than previously reported for sporadic PHPT (5.19%; Fisher exact test P = 0.0055) (8). This difference may be a result of distinct selection/exclusion criteria and patient populations, as the prior study included many cases of multigland and persistent/recurrent disease, whereas the current study focused on typical solitary parathyroid adenomas. It is conceivable that the frequency of germline predisposition alleles could be higher in cases with multigland and/or persistent/recurrent disease. Indeed, Guan et al. noted a substantial difference in the number of glands resected with the comparison of a small number of sporadic PHPT patients with germline-activating GCM2 CCID variants vs wild-type GCM2 (8). Furthermore, this study included a substantial proportion of Ashkenazi Jewish patients (52 of 327) with sporadic PHPT; 26.9% of these carried the Y394S allele, which specifically is over-represented in the Ashkenazi Jewish population. Of the populations reported in the Genome Aggregation Database, 2.5% of the Ashkenazi Jewish population are variant carriers compared with 0.029% in the non-Ashkenazi population. The proportion of Ashkenazi Jewish patients in our study population is unknown but would not be expected to differ significantly from that of the general U.S. population (∼2%).

We also identified the activating Y282D variant in 20 cases (5.05%) in our series of typical sporadic parathyroid adenomas. This variant was previously observed in 11.8% of Italian patients with sporadic PHPT (9), 17.4% with parathyroid cancer, and 5.1% with atypical adenoma (14). However, as this variant was also reported in 5.26% of Italian patients without PHPT (9), the lower overall frequency observed in our study was likely a result of population differences.

The frequency of germline-activating GCM2 variants in our series of typical sporadic parathyroid adenomas is higher than that in the general population. The increase in frequency of these variants within our study population when compared with the general population suggests that germline-activating GCM2 variants confer a three- to 11-fold increased risk of developing sporadic parathyroid adenoma, depending on the specific variant. However, the penetrance of these variants and therefore, the overall likelihood of disease occurring in a carrier are low: 96% of individuals with the variants in the general population will not be expected to develop sporadic parathyroid adenomas. Our observations bear on a recent proposal that all individuals with an activating CCID variant undergo close monitoring of serum calcium and PTH. The benefit of such a “precision medicine” approach depends strongly on penetrance of the variants, and at least for the aim of improved detection/treatment of common sporadic parathyroid adenoma in the general community, this recommendation does not appear to be well supported by evidence.

It should be noted that because calculated penetrance is based, in part, on disease prevalence in the general community, any possible underestimation of the latter in the selected studies (1, 2) would have the effect of the underestimation of penetrance. Any such underestimation would, however, be directly proportional and thus, unlikely to alter our conclusions unless they were quite substantial. On the other hand, because these population studies did not uniformly separate parathyroid adenoma prevalence from the totality of PHPT (expected to be ∼85%), our calculated penetrance values for the variants may be slightly overestimated. If disease prevalence values changed in future reports based on use of different case-selection criteria, then it would be important to reexamine the GCM2 variant allele frequencies in affected individuals selected in the same fashion. Finally, it is also possible that penetrance of these variants in the predisposition to familial and/or multigland PHPT may be higher than what we observed for sporadic adenomas, or that it might be higher in specific subpopulations (e.g., Ashkenazi), and future studies to assess penetrance in those cohorts would be important in the clarification of the clinical use, if any, of genotyping for these variants.

Abbreviations:

CCID
C-terminal conserved inhibitory domain


FIHP
familial isolated hyperparathyroidism


GCM2
glial cells missing 2


PHPT
primary hyperparathyroidism

Acknowledgments
Financial Support: Funding for this study was provided by the University of Connecticut’s Young Innovative Investigator Program, Endocrine Society Summer Research Fellowship (to A.R.), and Murray-Heilig Fund in Molecular Medicine (to A.A.).

Disclosure Summary: The authors have nothing to disclose.

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