Primary hyperparathyroidism (PHPT) is a common endocrinological disorder with an estimated prevalence of one to seven per 1,000 adults (1). It is characterized by overproduction of the parathyroid hormone (PTH) and hypercalcemia, leading to complications such as osteoporosis and formation of renal stones. (2) The hereditary form of PHPT accounts for approximately 10% of all cases, including multiple endocrine neoplasia (MEN) 1, MEN2A, familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism, hyperparathyroidism jaw tumor syndrome (HPT-JT), and familial isolated hyperparathyroidism (FIHP). (3, 4) Eighty-five percent of PHPT cases are usually sporadic, and < 1% of PHPT cases present as parathyroid cancer, which is commonly associated with severe hypercalcemia and associated clinical manifestations (4).

Several genes have been established as containing disease-causing mutations for the familial PHPT: MEN1 gene for MEN1, RET gene for MEN2A, CASR gene for neonatal severe hyperparathyroidism, and CDC73 gene for HPT-JT. A few other genetic mutations in familial PHPT have been discovered relatively recently. FHH was initially known to be caused by a mutation in CASR. However, GNA11 and AP2S1 mutations were additionally identified as causes of FHH type 2 and type 3, respectively. (5, 6) In addition, GCM2 mutations were recently identified in FIHP, another form of familial PHPT. Several germline mutations in CDC73, MEN1, CASR, and PTH are associated with benign sporadic PHPT or parathyroid cancer. (7) However, data on genetic abnormalities in PHPT are limited. In this study, we identified genetic alterations that may be involved in the pathogenesis of PHPT, using next-generation sequencing (NGS) data.

The baseline characteristics of the study subjects are shown in Table 1 . The mean age of the study subjects was 47.6 ± 16.1 years and 73.8% of them were women. Of the 107 study patients, 12 (11.2%) patients were diagnosed with familial PHPT, comprised of five patients with MEN1, six with FHH, and one with FIHP. Other 95 patients were diagnosed with sporadic PHPT, and 13 of them had pathologically confirmed parathyroid cancer. Mean serum calcium and intact PTH levels were 11.3 ± 1.7 mg/dL and 264.6 ± 385.3 pg/mL, respectively. Seventy-seven subjects underwent gene sequencing with a targeted NGS panel, and 30 subjects underwent clinical exome sequencing. The baseline characteristics did not differ between the groups according to the NGS panel ( Supplementary Table 3 ). Targeted sequencing panel detected 41 variants (one pathogenic variant, seven likely pathogenic variants, and 33 VUSs) and the other panel for clinical exome sequencing identified 11 variants (two pathogenic variants, three likely pathogenic variants and six VUSs).

The variants identified by NGS are listed on Table 2 according to their clinical diagnosis. Patients with familial PHPT or parathyroid cancer had germline mutation in MEN1, CASR, and CDC73, classified as pathogenic or likely pathogenic variants. The specific criteria used for each pathogenic and likely pathogenic variants are shown in Supplementary Table 4 .

A total of 1,315 VUSs were detected in 484 genes, and we prioritized non-synonymous variants (missense variants, frameshift variants, and variants at canonical ± 1 or 2 splice sites), which were included in our candidate parathyroid gene panel ( Supplementary Table 2 ). Thirty-nine VUSs were selected and are listed in Table 2 . Among the 39 variants, three VUSs (WT1, ESR2, and CASR) were accompanied by pathogenic or likely pathogenic variants (CDC73, LRP5, and VHL, respectively). The clinical characteristics of the three cases are shown in Table 3 .

As APC and GCM2 variants were recently reported in PHPT and parathyroid cancer, (15, 17) we analyzed the data of subjects with variants in APC and GCM2 genes ( Table 3 ). One of the patients had a GCM2 variant, and was diagnosed with PHPT at the age of 27. Five subjects had APC variants, and their mean age at the time of diagnosis was 37.3 ± 12.0. Two out of five subjects with APC variants were diagnosed with parathyroid cancer.

We further analyzed the clinical characteristics of the study subjects according to their genetic tests ( Table 4 ). The median age of subjects with positive test results was not significantly different from that of subjects without positive test results. Among the patients with positive test results, 38.5% had the recurrent PHPT, which was significantly higher than those without positive test results (38.5% vs. 11.7%, p < 0.025) We had 11 patients with recurrent PHPT in this study, and 5 of them had positive genetic test results. Four out of these five patients had either MEN1 (n=2) or parathyroid cancer (n=2). In contrast, none of the five patients with persistent PHPT had positive test result. Of the subjects with positive test results, 76.9% met at least one of the following clinical features: 1) age < 40 years, 2) family history of PHPT, 3) multiglandular PHPT, or 4) recurrent PHPT. This proportion was significantly higher in those with positive results than in those without (76.9% vs. 38.3%, p = 0.010). Laboratory findings, such as serum calcium and intact PTH levels, were statistically similar between the groups.

PHPT is a common endocrinological disorder with relatively well-established diagnosis and management. (2, 10, 19) However, knowledge about the genetic background of PHPT is limited, and genetic testing of PHPT is often overlooked in clinical practice. The development of NGS has helped advance research into the genetics of various types of endocrinological disorders. In addition, NGS is being widely used in clinical settings to detect genetic abnormalities and provide genetic counseling. We analyzed NGS data of 107 patients with PHPT and identified 3 pathogenic and 10 likely pathogenic variants. We further assessed 39 VUSs that could be related to parathyroid disease.

Genetic variants associated with parathyroid cancer and familial PHPT have been reported, and MEN1, CDC73, and RET mutations are known to be associated with the pathogenesis of parathyroid cancer or familial PHPT. (20) Also, in this study, patients with familial PHPT or parathyroid cancer had variants in MEN1 and CDC73 genes reported as pathogenic or likely pathogenic variants. We included 13 patients with parathyroid cancer, and three of them had CDC73 mutations. Additionally, four patients with parathyroid cancer had VUSs: two APC variants (c.5378C>G [p.Ala1793Gly] and c.890C>T [p.Thr297Ile]), one WT1 variant (c.1139G>A [p.Arg380Gln]), and one PRKAR1A variant (c.567A>C [p.Glu189As]). However, since the number of the patients with parathyroid cancer was small, it was hard to conclude the causative role of these variants.

Several somatic mutations have been identified in benign sporadic PHPTs. Somatic MEN1 gene mutations occur in 12% to 35% of sporadic PHPT (21–24), and somatic mutations in the CCND1 gene are also observed in 20%–40% of sporadic PHPT. (25–27) However, as the studies on germline mutations in sporadic PHPT are limited, variants found in subjects with sporadic PHPT are usually classified as VUSs. In this study, 32 non-synonymous variants in genes that could be related to parathyroid disease were classified as VUSs in patients with benign sporadic PHPT.

One of the patients had a GCM2 variant (c.1162A>G [p.Lys388Glu]), classified as VUS. GCM2 is mainly expressed in the parathyroid gland and regulates its development. (15) Germline mutations in GCM2 have recently been described as causative genetic alterations in FIHP. The specific genetic cause of FIHP, one of the hereditary forms of PHPT, was unclear until 2016, when Guan et al. demonstrated that GCM2 mutation can cause FIHP. (15) They found rare variants located in the GCM2 C-terminal conserved inhibitory domain (CCID) in 7 of the 40 kindreds with FIHP. GCM2 variants can be found in various functional domains of the human GCM2 protein, but those with transcription-activating functions are usually located within the CCID region. (15, 18) GCM2 variants have been reported not only in FIHP but also in sporadic PHPT. (28, 29) The prevalence of GCM2 variants in sporadic PHPT ranges from approximately 1.5% to 26.9% depending on ethnicity, and is particularly high in the Ashkenazi Jewish population. (18, 28, 30) There are limited studies on the Asian population, and one study found that the prevalence of GCM2 mutation with trans-activating function in Chinese PHPT patients was 1.3%. (18) In our study, one patient out of 107 had the GCM2 variant (c.1162A>G [p.Lys388Glu]), which is located in the CCID region. This same variant was previously reported in a study of Chinese patients with sporadic PHPT. (18) They screened 232 patients diagnosed with PHPT and found two cases with the variant c.1162A>G (p.Lys388Glu) of GCM2. Cases with variant c.1162A>G (p.Lys388Glu) had carcinoma pathology. Considering its prevalence, location, and transcription activity, we speculated that this variant in our patient could be associated with the development of PHPT.

There were concerns that PHPT patients with GCM2 variant could have an aggressive clinical phenotype, a high rate of multiglandular disease, and a low rate of biochemical cure. (31) Our patient with GCM2 variant was diagnosed with PHPT during health check-up and did not have any clinical manifestations, including renal stones and low bone mass. The patient underwent right inferior parathyroidectomy, and the pathology revealed parathyroid adenoma. After the surgery, the patient achieved biochemical cure and was under routine follow-up without recurrence. However, the patient’s age at diagnosis was 27 years, indicating possible involvement of genetic components in disease development. On NGS testing, no pathogenic or likely pathogenic variant was found, and variant c.1162A>G (p.Lys388Glu) in GCM2 was reported as VUS.

APC gene mutations are a well-known pathogenic mutations of familial adenomatous polyposis (FAP). Notably, germline mutations in the APC gene were identified in a patient with sporadic MEN1, metastatic papillary thyroid cancer, and FAP. (32) It has been suggested that APC gene variant might be involved in the pathogenesis of tumors in the parathyroid and thyroid glands. APC gene variants have also been found in parathyroid cancers. (17) The aberrant WNT/β-catenin signaling in parathyroid cancer could be due to a loss of expression or alteration of the APC gene (33).

In this study, two missense variants c.6754C>G (p.Pro2252Ala) and c.8017A>G (p.Arg2673Gly) of the APC gene were identified and classified as VUSs in patients with benign sporadic PHPT. The patients were diagnosed with PHPT at relatively young ages of 33 and 45 years. Of interest, APC mutations were also observed in patients with parathyroid cancer and in those with FHH. Among the 13 parathyroid cancer patients in this study, two patients carried APC variants (c.890C>T [p.Thr297Ile] and c.5378C>G (p.Ala1793Gly]) and did not harbor any other pathogenic or likely pathogenic variants. The other patient with APC variant (c.7969G>A[p.Val2657Ile]) was diagnosed with FHH and concurrently had the AP2S1 variant, classified as VUS. These five patients with APC variants were relatively young at the time of diagnosis, and two were diagnosed with parathyroid cancer. It is possible that the APC variants in these patients are involved in the development of PHPT.

Three patients with VUSs concurrently carried pathogenic or likely pathogenic variants. One patient with a WT1 variant (c.1139G>A [p.Arg380Gln]), reported as VUS, had a pathogenic CDC73 mutation (c.376C>T [p.Arg126Ter]) and was diagnosed with parathyroid cancer. Pathogenic CDC73 mutation is suspected to cause parathyroid cancer, and the WT1 variant could be an incidental finding from NGS.

The second patient carried the likely pathogenic LRP5 mutation (c.731C>T [p.Thr244Met]) and ESR2 variant (c.1541C>T [p.Pro514Leu]), classified as VUS. LRP5 mutation was not only reported in osteoporosis (34), but has also been associated with parathyroid tumors. (21, 35) In this patient, the LRP5 mutation could have played a role in the development of PHPT and osteoporosis. In addition, based on a study reporting estrogen receptor involvement in parathyroid adenoma, (36) there is a possibility that ESR2 variant also played a role in the development of PHPT in this case.

Lastly, a patient with CASR variant (c.613C>T [p.Arg205Cys]), reported as VUS, also had likely pathogenic VHL mutation (c.24G>A [p.Trp8Ter]). Although VHL mutation was reported as a likely pathogenic variant, the phenotype of VHL mutation is unclear in this patient, which might be due to variable expression of VHL mutation. (37) In contrast, CASR variant is associated with PHPT in previous studies. (38–40) Therefore, we speculated that although it was reported as VUS, CASR variant was involved in the development of PHPT and VHL mutation did not present its phenotype in this patient.

Genetic testing is usually indicated in some patients with PHPT who are at high risk of carrying a mutation: those with familial PHPT or parathyroid cancer (41) However, other clinical indications for genetic testing are still unclear, and the role of genetic testing in PHPT is often overlooked. In this study, 13 out of 107 study subjects (12.1%) had pathogenic or likely pathogenic variants, and 76.9% of the subjects with positive genetic test results had at least one of these clinical characteristics: 1) age < 40 years, 2) family history of PHPT, 3) multiglandular PHPT, or 4) recurrent PHPT. These clinical characteristic are generally consistent with previous studies, (3) and this implies that patients with these clinical characteristics need to undergo genetic testing.

Interestingly, 38.5% of patients with positive genetic testing had recurrent PHPT, but none of them had persistent PHPT. This different prevalence of recurrent or persistent PHPT in patients with positive test genetic testing might be due to the different clinical or genetic characteristics between the two. In our study, the patients who showed persistent PHPT were mainly due to the residual tissues of parathyroid adenoma or hyperplasia after the surgery or ectopic tissue which was not identified at the first operation. In contrast, the patients with recurrent PHPT were more likely to have genetic alterations, such as MEN1 or parathyroid cancer, so that they had the recurred disease even after the complete resection of the initial parathyroid tumor. In clinical practice, recurrent or persistent PHPT has been considered from the same point of view. However, based on the results in this study, patients with recurrent PHPT are at higher risk of having genetic alterations, thereby, should be urged to undergo genetic study. However, since this study included only small number of the patients, further study with large number of patients with recurrent or persistent PHPT is required.

The age of the subjects at the time of diagnosis did not differ between the groups according to the genetic test results. This might be due to a selection bias. Patients with classical sporadic form of PHPT and aged above 50 years would be less likely to undergo genetic testing and thus were not included in this study. In contrast, young patients were easily suspected to have genetic abnormalities, underwent genetic testing, and were enrolled in this study retrospectively. Therefore, there might be selection bias and the difficulty in determining the age difference between the groups with and without positive test results. In addition, the number of subjects with positive genetic test results was relatively small, leading to statistically insignificant results.

There are several other limitations to this study. This study included heterogeneous groups of PHPT, familial PHPT, benign sporadic PHPT, and parathyroid cancer. However, the number of subjects with familial PHPT and parathyroid cancer may not be sufficient to detect novel gene mutations. Second, the NGS panel used in this study did not include all candidate genes related to parathyroid disease. Lastly, because two different sequencing panels were used among the study patients, it might have affected the results.

In this study, we analyzed the NGS results of patients with PHPT. We speculated that although some variants were reported as VUSs, they could be associated with the development of the disease. In particular, as well as patients with familial PHPT or parathyroid cancer, which are classical indications for NGS, patients with young age or recurrent disease should be urged to undergo genetic testing. Advances in genetics and the declining cost of genetic testing may lead to its wider utilization in the future, thus helping in the diagnosis and management of PHPT patients and their relatives. We believe that this study provides insights into the genetic background of PHPT and provides a better approach for genetic counseling. Further studies are warranted to investigate the genetic abnormalities in PHPT pathogenesis and the role of NGS in PHPT in clinical practice.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Institutional Review Board of Severance Hospital, Yonsei University Health System, Seoul, Korea. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Conceptualization: H-SP, NH, and YR. Methodology: H-SP, NH, DW, and YR. Formal analysis: H-SP and YR. Investigation: H-SP, YL, NH, and YR. Writing—original draft preparation: H-SP, and YL. Writing—review and editing: H-SP, YL, NH, DW, and YR. Supervision: YR. All authors have read and agreed to the published version of the manuscript.

This study was supported by Hanim Precision Medicine Center of Yonsei University Health System under Grant number (6-2021-0208).

The 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.

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