Genetics of hereditary forms of primary hyperparathyroidism

Primary hyperparathyroidism (PHPT), a relatively common disorder characterized by hypercalcemia with raised or inappropriately normal serum parathyroid hormone (PTH) concentrations, may occur as part of a hereditary syndromic disorder or as a non-syndromic disease. The associated syndromic disorders include multiple endocrine neoplasia types 1–5 (MEN1-5) and hyperparathyroidism with jaw tumor (HPT-JT) syndromes, and the non-syndromic forms include familial hypocalciuric hypercalcemia types 1–3 (FHH1-3), familial isolated hyperparathyroidism (FIHP), and neonatal severe hyperparathyroidism (NS-HPT). Such hereditary forms may occur in > 10% of patients with PHPT, and their recognition is important for implementation of gene-specific screening protocols and investigations for other associated tumors. Syndromic PHPT tends to be multifocal and multiglandular with most patients requiring parathyroidectomy with the aim of limiting end-organ damage associated with hypercalcemia, particularly osteoporosis, nephrolithiasis, and renal failure. Some patients with non-syndromic PHPT may have mutations of the MEN1 gene or the calcium-sensing receptor (CASR), whose loss of function mutations usually cause FHH1, a disorder associated with mild hypercalcemia and may follow a benign clinical course. Measurement of the urinary calcium-to-creatinine ratio clearance (UCCR) may help to distinguish patients with FHH from those with PHPT, as the majority of FHH patients have low urinary calcium excretion (UCCR < 0.01). Once genetic testing confirms a hereditary cause of PHPT, further genetic testing can be offered to the patients’ relatives and subsequent screening can be carried out in these affected family members, which prevents inappropriate testing in normal individuals.


Physiology of calcium homeostasis
Serum calcium is maintained within a narrow range (< 0.4 mmol/L), with extracellular calcium concentrations monitored by parathyroid gland chief cells.PTH secretion from the parathyroid glands is predominantly determined by extracellular calcium concentrations [Ca2 +] by the calcium-sensing receptor (CaSR; encoded by the CASR gene located on chromosome 3q13.33-q21.1;Figs. 1 and 2).In addition to the direct effect of extracellular [Ca2 +], PTH is also suppressed by circulating 1,25-dihydroxyvitamin D (calcitriol or active vitamin D) concentrations which act on the vitamin D receptor (VDR), and by fibroblast growth factor 23 (FGF23) concentrations via its action on the fibroblast growth factor receptor (FGFR), in association with α-Klotho.FGF23 is released by osteocytes in response to raised extracellular phosphate and predominantly acts by increasing renal phosphate excretion and by inhibiting the renal conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D (Fig. 2).The CaSR is a G-protein coupled  [13,14] d Germline MAX mutations and pheochromocytomas in association with other endocrine tumors have been reported in 11 cases (PHPT, pituitary adenoma, and PNETs) [15][16][17][18][19][20][21][22] e PHPT has been reported in four cases in patients with MEN5 f CDC73 mutations are reported to account for ~ 12% of patients with hereditary PHPT [23,24] * There are three classical types of MEN2 syndrome, as follows: MEN2A (now referred to as MEN2), MEN2B (now referred to as MEN3) which is characterized by the occurrence of aggressive MTC, and pheochromocytoma in association with a Marfinoid habitus, mucosal neuromas, medullated corneal nerve fibers, and intestinal ganglioneuromas; and familial MTC, in which MTC is the sole manifestation.MEN2 has also been reported to be associated with cutaneous lichen sclerosis and Hirschsprung's disease

Pathology of PHPT and hypercalcemic disorders
The current WHO 2022 classification of parathyroid tumors aims to pathologically distinguish parathyroid disease secondary to germline mutations, i.e., multiglandular multiple parathyroid adenomas as seen with syndromic forms of PHPT (e.g., MEN1 due to MEN1 mutations) or parathyroid carcinoma due to CDC73 mutations, from that of parathyroid hyperplasia, usually seen in patients with secondary hyperparathyroidism (e.g., chronic renal failure) [25].The syndromic forms of PHPT are due to LOF of tumor suppressor genes, e.g., MEN1, CDKN1B, and CDC73, with patients harboring germline CDC73 mutations having a Fig. 1 Examples of genetic changes associated with familial hypocalciuric hypercalcemia (FHH).In FHH types 1-3, inactivating mutations in the CaSR, GNA11, or AP2S1 lead to loss of function of signaling through the CaSR pathway and therefore require higher extracellular calcium concentrations (red arrow) to suppress PTH secretion.These germline genetic changes affect all parathyroid cells higher occurrence of parathyroid carcinomas [23,26] or increased oncogenic signaling (e.g., activating RET mutations; Table 1).Some inherited non-syndromic forms of PHPT may be associated with MEN1 [10,11], CASR, e.g., FHH1, and neonatal severe hyperparathyroidism (NS-HPT) [12,27], or CDC73 mutations [26,28,29].However, it is important to note that the majority of LOF mutations of the CaSR and its signaling pathway components, the G-protein alpha 11 subunit (Gα11, encoded by GNA11) and the adaptor-protein 2 sigma subunit (AP2σ encoded by AP2S1), result in FHH1, FHH2, and FHH3, respectively (Fig. 1).The impaired signaling via the CaSR pathway in these disorders results in a higher set-point for the CaSR, which leads to hypercalcemia in association with plasma PTH concentrations that are in the normal reference range (~ 80%) or elevated [30].In sporadic parathyroid adenomas, overexpression of CCDN1 has been observed in 20-40% of tumors [9] and loss of expression of the CaSR has been reported in up to 90% (36/40) of tumors [31].Additionally, promoter methylation of the CASR promoter 2 was found in 45% (18/40) of tumors with increased expression of the repressive histone mark trimethylation of lysine 9 on histone 3 (H3K9me3) [31].Biochemically, elevated serum [Ca2 +] and normal or elevated PTH concentrations may be indistinguishable between the different causes of hereditary PHPT and sporadic PHPT [3,27,32].

Multiple endocrine neoplasia 1
Multiple endocrine neoplasia 1 (MEN1) is an autosomal dominant hereditary multiple endocrine neoplasia syndrome due to a germline heterozygous LOF mutations in the MEN1 gene, located on chromosome 11q13.1,which encodes for the 610 amino acid tumor suppressor menin.Multifocal tumors develop in endocrine glands after a second hit to the remaining functional MEN1 allele, consistent with Knudson's two-hit hypothesis.MEN1 is characterized by tumors in the parathyroids, pituitary, and pancreas, although other tumors can be found, including adrenal cortical adenomas and bronchopulmonary and thymic tumors [3].The prevalence of MEN1 is reported to be between 1 and 3 per 100,000 [33].PHPT is the most common endocrine disorder, with ~ 75% penetrance by the age of 50 years and > 90% by 70 years in MEN1 patients [34,35].MEN1 accounts for the majority of people presenting with PHPT due to a hereditary cause, which is in part due to MEN1 being the most common hereditary disorder associated with PHPT and is highly penetrant.More than 1500 individual MEN1 mutations have been reported in patients with MEN1 syndrome, with the majority resulting in protein-truncating variants with no clear correlation between genotype and phenotype [10,36].
PHPT is characterized by multiple clonal parathyroid tumors that previously were classified histologically as parathyroid hyperplasia, a term which has subsequently changed with the latest WHO guidelines [25].Menin is a scaffold protein and plays an integral role in epigenetic regulation: for example, it is required for the formation of the active histone mark trimethylation of lysine 4 on histone 3 (H3K4me3).One study reported that four parathyroid adenomas associated with MEN1 syndrome showed no global change in H3K4me3 levels by immunohistochemistry when compared to two normal parathyroid tissue samples and seven sporadic parathyroid adenomas [37].In another study of parathyroid tissue, menin loss was reported to be associated with increased DNA methylation, a DNA mark associated with transcriptional repression [38] and 12 human MEN1-associated and one sporadic parathyroid adenoma with a L338P missense MEN1 mutation were found to have increased global DNA methylation when compared to twelve sporadic parathyroid adenomas with no MEN1 gene mutations and nine normal parathyroid tissue samples [38].Finally, menin loss in seven separate parathyroid adenomas from patients with MEN1 has been reported to be associated with a reduction in the expression of the VDR when compared to both sporadic adenomas (n = 12) and normal parathyroid tissue (n = 6) [37].

Mutation-negative MEN1
It is estimated that 10-30% of patients with a MEN1-like phenotype have no genetic mutation found in the MEN1 gene, and these patients present with endocrine neoplasms at a later age and have a similar life expectancy to that of the general population [39].CDKN1B mutations (MEN4) have been reported in ~ 1.5% of patients with mutation-negative MEN1, thereby being reclassified as having MEN4 syndrome.Germline mutations in other CDKIs have been found in 0.5-1% of patients, including CDKN1A (p21), CDKN2C (p18), and CDKN2B (p15) [40].Genetic analysis for MEN1 mutations usually involves sequencing the coding region of MEN1 (exons 2-10); however, mutations involving the promoter region (for example, a 596 bp deletion in the MEN1 5′UTR) have been reported in a MEN1 kindred with no MEN1 mutation in the coding region [41].The significance of this deletion was tested in vivo, which reported ~ 80% reduction in MEN1 mRNA and ~ 80% reduction in menin protein expression [41].Other causes of mutation-negative MEN1 syndrome may include the chance co-occurrence of two endocrine tumors without an underlying germline predisposition syndrome or a germline mutation in a gene not commonly screened for as part of a MEN1 panel (e.g., aryl hydrocarbon receptor-interacting protein (AIP OMIM: 605,555) mutations in familial isolated pituitary adenoma) with the co-occurrence of sporadic PHPT [42].

Multiple endocrine neoplasia type 4
Multiple endocrine neoplasia type 4 (MEN4) is characterized by germline mutations in CDKN1B, located on chromosome 12p13.1.CDKN1B transcription requires the active histone mark H3K4me3 which is maintained by a functioning menin.CDKN1B encodes for the 196 amino acid, p27 kip1 or p27, a nuclear protein which is involved in cell cycle regulation and inhibits cycle progression at G1. Tumors associated with MEN4 include PHPT (75%), pituitary adenomas (44%), pancreatic neuroendocrine tumors (PNETs), papillary thyroid cancer, and renal, thymic, and reproductive organ tumors [47,48].MEN4 (previously termed MENX) was initially discovered in a rat [49], which developed highly penetrant multiple neuroendocrine tumors within the first year of life, with the causative gene (CDKN1B) discovered a few years later [50].A recent case series and comprehensive literature review of MEN4 reported a total of 32 unique CDKN1B variants associated with MEN4 (with six located in the 5′UTR) from 22 studies [13].Since then, a further two unique CDKN1B variants have been reported in association with familial PHPT [14].The overall prevalence of PHPT in the entire cohort was ~ 42%, with 53.2% diagnosed with PTHP by the age of 60 years [13].

Multiple endocrine neoplasia type 5 and MAX mutations
MYC-associated protein X (MAX) is a 160 amino acid protein encoded by the MAX gene, located on chromosome 14q23.3.The MAX protein typically forms a heterodimer with the MYC family of proteins and is involved in cellular proliferation [51].Heterozygous LOF MAX mutations, which cause hereditary paraganglioma-pheochromocytoma syndrome, have also been reported with other endocrine and non-endocrine tumors [15,52].Multiple endocrine tumors have been associated with germline LOF MAX mutations, and these including pituitary adenomas and PNETs [15][16][17][18][19]52]; therefore, it has been suggested that germline LOF MAX mutations have been suggested may cause multiple endocrine neoplasia type 5 (MEN5) [16].There have been four reported cases of MAX mutations in association with PHPT [15][16][17]20].Given the rare number of case reports of PHPT in association with MAX mutations, further study is required to determine the role of MAX mutations in syndromic PHPT [53].

Hyperparathyroidism-jaw tumor syndrome
Cell division cycle 73 (CDC73; previously hyperparathyroidism type 2 (HRPT2)) is located on chromosome 1q31.2and encodes a 531 protein, parafibromin; it was initially discovered in 26 affected kindreds with hyperparathyroidismjaw tumor syndrome (HPT-JT) [54].HPT-JT is characterized by PHPT in up to 95% of patients and ossifying fibromas in the jaw in 25-50% [28,[54][55][56].HPT-JT is also associated with renal tumors including hamartomas, Wilm's tumors, and uterine tumors [55].Importantly, parathyroid carcinoma is over-represented in patients with CDC73 germline or somatic mutations, which suggests that parafibromin plays an important tumor suppressor role in the parathyroid gland [23,26].Parafibromin is a nuclear protein with both tumor-suppressive and oncogenic properties.Parafibromin can exert its antiproliferative effect by interacting with nuclear beta-catenin by polymerase associated factor 1 (PAF1) complex [57,58] and by decreasing the expression of cyclin D1 [59,60], for example, by H3K9 methylation at CCND1 and by suppression of the c-myc proto-oncogene [60,61].Loss of function of CDC73 is usually by proteintruncating variants seen in up to 80% of germline variants causing HPT-JT [28].In a cohort of 68 patients from 29 kindreds with HPT-JT, 85% presented with PHPT as their initial manifestation, with a median age of 26 years (interquartile range: 20-35 years) [23].Of the patients with PHPT, 65% had parathyroid adenomas and 31% had features of parathyroid carcinoma [23].PHPT is typically due to a single parathyroid adenoma, although multiple parathyroid tumors have also been reported [62,63].CDC73 mutations are also seen in FIHP [62].

Familial isolated hyperparathyroidism
Familial isolated hyperparathyroidism (FIHP) may be due to incomplete penetrance of mutations causing syndromic PHPTs, as the genes found in FIHP overlap, for example, MEN1, CDC73, or CASR [3,12,26,28,29,64].However, there is no genotype-phenotype correlation between patients with these LOF mutations and FIHP [6,53].Recently, activating mutations in GCM2 located on chromosome 6p24.2,which encodes the 506 amino acid transcription factor GCMb, have been reported in 18% of patients with FIHP [65], with specific variants enriched among different ethnic backgrounds [66].GCMb is important for parathyroid gland development, as evidenced by Gcm2 knockout mice developing hypoparathyroidism [67] and LOF GCM2 mutations causing familial isolated hypoparathyroidism [68].GCM2 activating mutations have been reported to be enriched in patients with both FIHP and sporadic PHPT; however, the penetrance appears to be low and further studies are needed [69,70].

Familial hypocalciuric hypercalcemia
Familial hypocalciuric hypercalcemia (FHH) is a relatively common disorder with an estimated prevalence of 74 per 100,000 [71].FHH is caused by inactivating mutations in either CASR (FHH1), GNA11 (FHH2) [72], or AP2S1 (FHH3) [73].FHH patients require a higher extracellular [Ca2 +] to activate the CaSR pathway, mobilize intracellular calcium, and activate mitogen-activated protein kinases (Fig. 1) [74,75].Therefore, patients with FHH tend to have a higher serum [Ca2 +] and normal or elevated PTH concentrations.CASR LOF mutations lead to a decreased ability of the kidney to excrete calcium relative to serum [Ca2 +], which results in relative hypocalciuria [76].FHH1 is the most common form, accounting for ~ 65% of cases and, depending on the location and amino acid change in the CASR gene, determines the degree of loss of function and the severity of hypercalcemia [12].There are > 230 different CASR variants that have been reported to be associated with FHH1.FHH2 is due to LOF mutations in GNA11 located on chromosome 19p13.3which encodes for Gα11.Gα11 binds to the intracytoplasmic tail of the CaSR and is responsible for signal transduction [77].Only four variants in GNA11 have been reported in FHH2 (T54M [78], namely, L135Q [72], I200del [72], and F220S) [79].Pathogenic variants in GNA11 causing FHH2 are rare and occur in < 1% of individuals undergoing genetic testing for FHH [80].FHH3 is due to LOF mutations in the AP2S1 gene found on chromosome 19q13.32and encodes for the protein AP2σ.FHH3 causes < 10% of cases of FHH [73].AP2σ is integral for CaSR endocytosis via clathrin-mediated pits and for receptor trafficking and CaSR signaling potentiation (Fig. 1) [77].The prevalence of FHH3 has been reported at ~ 7.8 per 100,000, and mutations causing FHH3 most commonly involve the R15 residue [81].Classical teaching describes FHH as a benign condition that needs to be distinguished from PHPT to prevent FHH patients from undergoing inappropriate parathyroidectomy [82].One of the main distinguishing features of FHH compared to PHPT is that of low urinary calcium excretion (UCCR of < 0.01) and is seen in ~ 80% of patients with FHH type 1 and in < 20% of patients with PHPT.Other clues indicating FHH include a personal or family history of recurrence of hypercalcemia post-parathyroidectomy.However, FHH is not always a benign condition and has been associated with renal calculi, osteoporosis, and pancreatitis [12].For patients with signs and/or symptoms suggestive of symptomatic hypercalcemia, case reports have shown efficacy with the use of cinacalcet [83].Cinacalcet is a calcimimetic that is able to stimulate the CaSR, leading to a decrease in serum [Ca2 +] [79,84].

Neonatal severe hyperparathyroidism
Neonatal severe hyperparathyroidism (NS-HPT) is caused by either homozygous or compound heterozygous LOF CASR mutations.NS-HPT may also occur in a child with a paternally inherited (or de novo) heterozygous CASR mutation, born to a normocalcemic mother.NS-HPT usually presents within the first 6 months of life with life-threatening hypercalcemia, skeletal demineralization, bony deformities, fractures, constipation, dehydration, and failure to thrive.Patients with NS-HPT usually require urgent parathyroidectomy [3,27].There have been 12 case reports of successful treatment of NS-HPT with cinacalcet (with genetically confirmed CASR mutations), and three cases reported a lack of response to cinacalcet.Four of these 12 patients had a heterozygous CASR mutation R185Q (one inherited and three de novo) [85][86][87]; seven with inherited homozygous mutations, including R69H, G613E, and Y789fs [88][89][90][91][92]; one with a compound heterozygous mutation, C582Y and P682L [93]; and a homozygous donor splice site mutation in intron 5 [94].All three cases reported with no response to cinacalcet were patients with homozygous CASR mutations at D99H, R690H, and R69H [95].

Treatment
Special care needs to be taken in patients with syndromic PHPT as the majority present with multifocal multiglandular disease at an earlier age than sporadic PHPT; therefore, operative type, risk of recurrence, risk of post-surgical hypoparathyroidism, and age of the patient must be taken into consideration.Additionally, given the higher incidence of parathyroid carcinoma in patients with CDC73 mutations, surveillance frequency and type of parathyroid operation will be different compared to other forms of syndromic PHPT [96].For sporadic PHPT, patients should be considered for parathyroidectomy when there is significant risk or presence of symptomatic PHPT, for example, nephrocalcinosis or nephrolithiasis, renal failure with a creatinine clearance < 60 mL/min, hypercalciuria defined as > 250 mg/ day in women and > 300 mg/day in men, minimal trauma fracture, bone mineral density by T-score ≤ − 2.5, or serum calcium level > 0.25 mmol/L above the upper limit of normal, or in patients who present < 50 years of age [96].For patients in whom parathyroidectomy is contraindicated, treatment with cinacalcet may be considered: its efficacy has been reported in individuals with FHH, NS-HPT, and MEN1 [85-88, 92, 94, 97-99].

Genetic testing for PHPT
At present, not all patients with PHPT are tested for germline mutations in genes associated with PHPT.There has been one large multicenter study that examined 1085 patients with MEN2A which found that only 10 cases presented initially with PHPT, and nine of these 10 patients were found to have synchronous MTC [100].This suggests that the pick-up rate for diagnosing pathogenic RET mutations causing MEN2A syndrome in patients presenting only with PHPT is low and that screening for RET mutations in this scenario may not be helpful.Patients with a clinical suspicion of a hereditary form of PHPT (e.g., occurring < 30 years old, multiglandular disease, parathyroid carcinoma, first-degree relative of a known mutation carrier, or other clinical features associated with a syndromic form of PHPT) [3,4,96] should undergo genetic testing as this will help guide PHTP management (e.g., parathyroidectomy) and determine if screening for other tumors is required (e.g., pituitary and pancreatic neuroendocrine screening in MEN1 syndrome), while it will help determine whether family members should also be tested.For patients with a clinical suspicion of a hereditary form of PHPT, genetic testing should be undertaken; however, it is unclear if targeted genes should be tested or a PHPT panel (e.g., MEN1, RET, CDKN1B, CDC73, CASR, GNA11, and AP2S1) [53]

Conclusion
PHPT is a relatively common disorder and is associated with a genetic cause in ~ 10% of cases.Of the syndromic forms of hereditary PHPT, the MEN1 syndrome is the most common.Importantly, FHH is not as rare as originally thought and to prevent patients from undergoing inappropriate parathyroidectomy, an increased uptake of genetic testing may help with clinical decision-making.However, it is still unclear if targeted genetic testing of specific genes or testing with a global PHPT panel is the most appropriate way forward.

Fig. 2
Fig.2Parathyroid hormone (PTH) synthesis and/or secretion can be decreased in the chief cell of the parathyroid gland by different mechanisms, which include the following: increased extracellular calcium concentrations by activation of the calcium-sensing receptor (CaSR), activation of the fibroblast growth factor (FGF)/αKlotho receptor complex by FGF23, and activation of the retinoid X receptor (RXR)/ vitamin D receptor complex by 1,25-dihydroxyvitamin D. In bone, PTH acts on the osteoblast to secrete receptor activator of nuclear factor kappa-B ligand (RANKL), which acts on the osteocytes to release calcium, thereby increasing extracellular calcium.FGF23 is released