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Objective: The aim of this study was to analyze variants of the gene glial cells missing-2 (GCM2), encoding a parathyroid cell-specific transcription factor, in familial hypoparathyroidism and in familial isolated hyperparathyroidism (FIHP) without and with parathyroid carcinoma., Design: We characterized 2 families with hypoparathyroidism and 19 with FIHP in which we examined the mechanism of action of GCM2 variants., Methods: Leukocyte DNA of hypoparathyroid individuals was Sanger sequenced for CASR, PTH, GNA11 and GCM2 mutations. DNA of hyperparathyroid individuals underwent MEN1, CDKN1B, CDC73, CASR, RET and GCM2 sequencing. The actions of identified GCM2 variants were evaluated by in vitro functional analyses., Results: A novel homozygous p.R67C GCM2 mutation which failed to stimulate transcriptional activity in a luciferase assay was identified in affected members of two hypoparathyroid families. Oligonucleotide pull-down assay and in silico structural modeling indicated that this mutant had lost the ability to bind the consensus GCM recognition sequence of DNA. Two novel (p.I383M and p.T386S) and one previously reported (p.Y394S) heterozygous GCM2 variants that lie within a C-terminal conserved inhibitory domain were identified in three affected individuals of the hyperparathyroid families. One family member, heterozygous for p.I138M, had parathyroid carcinoma (PC), and a heterozygous p.V382M variant was found in another patient affected by sporadic PC. These variants exerted significantly enhanced in vitrotranscriptional activity, including increased stimulation of the PTH promoter., Conclusions: We provide evidence that two novel GCM2 R67C inactivating mutations with an inability to bind DNA are causative of hypoparathyroidism. Additionally, we provide evidence that two novel GCM2 variants increased transactivation of the PTH promoter in vitro and are associated with FIHP. Furthermore, our studies suggest that activating GCM2 variants may contribute to facilitating more aggressive parathyroid disease.

Context: Paget disease of bone (PDB) is a metabolic bone disease whose genetic cause remains unknown in up to 50% of familial patients., Objective: Our aim was to investigate the underlying genetic defect in a large pedigree with a severe, early onset, autosomal dominant form of PDB across 3 generations., Methods: Whole exome sequencing was performed in affected and unaffected family members, and then mutation screening was replicated in a sample of PDB patients with early-onset, polyostotic PDB., Results: We identified a frameshift D107Rfs*3 mutation in PFN1 (encoding for profilin 1, a highly conserved regulator of actin-polymerization and cell motility) causing the truncation of the C-terminal part of the protein. The mutation was also detected in a 17-year-old asymptomatic family member who upon biochemical and radiological analyses was indeed found to be affected. Sequencing of the entire PFN1 coding region in unrelated PDB patients identified the same mutation in 1 patient. All mutation carriers had a reduced response to bisphosphonates, requiring multiple zoledronate infusions to control bone pain and achieve biochemical remission over a long term. In vitro osteoclastogenesis in peripheral blood mononuclear cells (PBMCs) from mutation carriers showed a higher number of osteoclasts with PDB-like features. A similar phenotype was observed upon PFN1 silencing in murine bone marrow-derived monocytes, suggesting that the frameshift PFN1 mutation confers a loss of function in profilin 1 activity that induces PDB-like features in the osteoclasts, likely due to enhanced cell motility and actin ring formation., Conclusions: Our findings indicate that PFN1 mutation causes an early onset, polyostotic PDB-like disorder., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Patients with primary aldosteronism (PA) have a high prevalence of osteoporosis (OP) and fractures (Fx). We evaluated the presence of PA in patients admitted to our metabolic bone disease outpatient clinic., Design: Study conducted on an in- and outpatient basis in a referral Italian endocrinology unit., Methods: A total of 2632 patients were evaluated. 2310 were excluded because they were taking drugs known to affect bone or mineralocorticoids metabolism or were diagnosed to have a secondary cause of osteoporosis. The remaining 322 subjects (304 females, 18 males) took part in the study. Bone mineral density (BMD) and thoracic and lumbar spine vertebral morphometry were performed by dual X-ray absorptiometry. All patients were screened for PA with aldosterone-to-renin ratio. In those who had positive results, confirmatory tests were performed., Results: Among 322 subjects, 213 were osteoporotics and 109 were not. PA was diagnosed in eleven out of 213 osteoporotic patients (5.2%) and one out of 109 non-osteoporotic subjects (0.9%, P  = 0.066). PA was observed in the 26.1% of patients with the concomitant presence of osteoporosis, hypertension and hypercalciuria. Compared with patients without PA, patients with PA had mean values of urinary calcium excretion, 4.8 ± 2.5 mmol/day vs 7.6 ± 3.2 mmol/day, P  < 0.001 and serum PTH levels, 5.4 pmol/L vs 7.3 pmol/L, P  < 0.01, significantly higher., Conclusions: PA should be considered among the causes of secondary OP., (© 2017 European Society of Endocrinology.)

Context: Familial isolated hypoparathyroidism (FIH) is a genetically heterogeneous disorder due to mutations of the calcium-sensing receptor (CASR), glial cells missing-2 (GCM2), guanine nucleotide binding protein α11 (GNA11), or parathyroid hormone (PTH) genes. Thus far, only four cases with homozygous and two cases with heterozygous mutations in the PTH gene have been reported., Objective: To clinically describe an FIH family and identify and characterize the causal gene mutation., Design: Genomic DNA of the family members was subjected to CASR, GCM2, GNA11, and PTH gene mutational analysis. Functional assays were performed on the variant identified., Participants: Six subjects of a three-generation FIH family with three affected individuals having severe hypocalcemia and inappropriately low serum PTH., Results: No mutations were detected in the CASR, GCM2, and GNA11 genes. A heterozygous variant that segregated with the disease was identified in PTH gene exon 2 (c.41T>A; p.M14K). This missense variant, in the hydrophobic core of the signal sequence, was predicted in silico to impair cleavage of preproPTH to proPTH. Functional assays in HEK293 cells demonstrated much greater retention intracellularly but impaired secretion into the medium of the M14K mutant relative to wild type. The addition of the pharmacological chaperone, 4-phenylbutyric acid, led to a reduction of cellular retention and increased accumulation in the cell medium of the M14K mutant., Conclusions: We report a heterozygous PTH mutation in an FIH family and demonstrate accumulation of the mutant intracellularly and its impaired secretion. An accurate genetic diagnosis in such hypoparathyroid patients is critical for appropriate treatment and genetic counseling., (Copyright © 2017 Endocrine Society)

Context: Glial cells missing-2 (GCM2) is key for parathyroid gland organogenesis. Its persistent expression in the adult parathyroid raises the possibility that overactive forms play a role in the evolution of parathyroid hyperactivity or tumorigenesis. A GCM2 c.844T → G; p.Y282D missense variant has been described within a transactivation inhibitory domain (amino acids 263-352)., Objective: The aims of the study were to 1) assess the frequency of Y282D in Italian primary hyperparathyroidism (PHPT) and control (C) populations, 2) test for association of 282D with PHPT and its phenotypic features, and 3) compare the transactivation potency of GCM2 282D relative to wild-type Y282., Subjects and Methods: Subjects included a large southern Italian cohort (310 PHPT and 433 C) and 2 replication cohorts from northern Italy. Association of 282D with PHPT was tested in all cohorts and with phenotypic features in the larger PHPT cohort. An in vitro GCM promoter-luciferase reporter assay was conducted in HEK293 cells., Results: 282D was significantly increased in the PHPT group, with a minor allele frequency of 0.066 compared with 0.029 in the C group (P = .0008), in the discovery cohort and was more prevalent in the replication cohorts. Combined analysis (510 PHPT and 665 C) yielded a likelihood ratio of 2.27 (95% confidence interval = 1.50-3.42; P < .0001). The 282D variant was not associated with serum calcium, phosphate, creatinine, or PTH levels or with bone mineral density, fractures, or renal stones in the PHPT group. The 282D variant had significantly greater transcriptional activity than the wild-type Y282 (17× basal vs 12× basal; P < 0.05)., Conclusion: The higher frequency of GCM2 282D in PHPT and enhanced transcriptional activity of this variant supports the notion that it could contribute causally to parathyroid tumorigenesis.

Background: Inactivating mutations of the calcium-sensing receptor (CaSR), of the G-protein subunit α11 (GNA11) and of the adaptor-related protein complex 2, sigma 1 subunit (AP2S1) genes are responsible for familial hypocalciuric hypercalcaemia (FHH). The aim of this study was to analyse prevalence and pathogenicity of CaSR, GNA11 and AP2S1 mutations in patients with an FHH phenotype and to compare them with a sample of patients with primary hyperparathyroidism (PHPT) in order to identify the most useful laboratory parameter for a differential diagnosis., Methods: Patients with an FHH phenotype were studied with polymerase chain reaction amplification and direct sequencing of the entire CaSR, GNA11 and AP2S1 coding sequences. Novel mutations were introduced in a Myc-tagged human wild-type (WT) CaSR cDNA-expressing vector, and functional assay was performed on human embryonic kidney cells evaluating expression and function of mutated proteins., Results: Among 16 FHH patients, none had an inactivating GNA11 or AP2S1 mutation while 3 (18.8%) carried a CaSR mutation and 10 (62.5%) at least one CaSR polymorphism. Within the latter group, 7 of 10 patients had more than one polymorphism (4.1 ± 2.1 per patient). Two novel CaSR mutations [c.2120A>T (E707V) and c.2320G>A (G774S)] were identified: the E707V mutation prevented CaSR expression (western blot), whereas the G774S mutation determined a reduced receptor sensitivity to calcium (IP3 assay). PHPT patients showed significantly (P < 0.001) higher serum calcium, parathyroid hormone, urinary calcium and calcium-creatinine clearance ratio (CCCR) and significantly lower serum phosphate than FHH ones., Conclusions: FHH should be clearly differentiated by PHPT to avoid unnecessary surgery: CCCR could be a useful screening tool while genetic analysis should include the two novel CaSR mutations herein described. The role of multiple polymorphisms deserves further investigation in patients with an FHH phenotype., (© The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Objective: To examine factors, in addition to bone mineral density (BMD), such as the common calcium-sensing receptor (CASR) gene polymorphisms, associated with vertebral fracture (VFx) risk in primary hyperparathyroidism (PHPT)., Design and Methods: A cross-sectional analysis of 266 Caucasian PHPT seen as outpatients. Serum calcium (sCa) phosphate metabolism parameters were measured. BMD was assessed by dual-energy X-ray absorptiometry (expressed as Z-score) at lumbar spine (Z-LS) and femoral neck, morphometric VFx by radiograph, and CASR A986S/R990G genotypes by PCR amplification and genomic DNA sequencing., Results: Fractured patients (n=100, 37.6%) had lower sCa (10.8±0.7 mg/dl) and Z-LS BMD (-1.0±1.44), higher age (61±10 years), and prevalence (51%) of ≥1 S alleles of the CASR A986S single-nucleotide polymorphism (SNP; AS/SS), than those not fractured (n=166, 11.2±1.0 mg/dl, -0.57±0.97, 58±13 years, and 38% AS/SS, respectively, P<0.05 for all comparisons). Logistic regression, with VFx as dependent variable, showed independent risks associated with increased age (OR 1.03, 95% CI 1.01-1.06, P=0.006), decreased sCa (OR 1.86, 95% CI 1.28-2.7, P=0.001), and Z-LS BMD (OR 1.4, 95% CI 1.12-1.7, P=0.002) and presence of AS/SS (OR 1.8, 95% CI 1.1-2.9, P=0.05). The presence of two out of three factors (age ≥58 years, sCa <10.8 and Z-LS BMD≤-1.0, and AS/SS genotype) gave an overall OR of 4.2 (95% CI 2.25-7.85, P<0.0001)., Conclusions: In PHPT, VFx is associated positively with age, negatively with sCa and spinal BMD, and presence of at least one copy of the CASR A986S SNP., (© 2014 European Society of Endocrinology.)

Background and Objective: Single nucleotide polymorphisms (SNPs) of the calcium-sensing receptor (CASR) gene at the regulatory region were associated with idiopathic calcium nephrolithiasis. To confirm their association with nephrolithiasis, we tested patients with primary hyperparathyroidism (PHPT)., Design: A genotype-phenotype association study., Methods: In all, 332 PHPT patients and 453 healthy controls were genotyped for the rs7652589 (G>A) and rs1501899 (G>A) SNPs sited in the noncoding regulatory region of the CASR gene. Allele, haplotype, and diplotype distribution were compared between PHPT patients and controls, and in stone forming and stone-free PHPT patients., Results: The allele frequency at rs7652589 and rs1501899 SNPs was similar in PHPT patients and controls. The A minor alleles at these two SNPs were more frequent in stone forming (n=157) than in stone-free (n=175) PHPT patients (rs7652589: 36.9 vs 27.1%, P=0.007; rs1501899: 37.1 vs 26.4%, P=0.003). Accordingly, homozygous or heterozygous PHPT patients for the AA haplotype (n=174, AA/AA or AA/GG diplotype) had an increased stone risk (odds ratio 1.83, 95% confidence interval 1.2-2.9, P=0.008). Furthermore, these PHPT patients had higher serum concentrations of ionized calcium and parathyroid hormone (1.50 ± 0.015 mmol/l and 183 ± 12.2 pg/ml) than patients with the GG/GG diplotype (n=145, 1.47 ± 0.011 mmol/l (P=0.04) and 150 ± 11.4 pg/ml (P=0.049)). Using a logistic regression model, the increase in stone risk in PHPT patients was predicted by AA/AA or AA/GG diplotype, the highest tertile of serum ionized calcium values and the lowest tertile of age., Conclusions: Polymorphisms located in the regulatory region of the CASR gene may increase susceptibility of the PHPT patients to kidney stone production.

Context: Inactivating mutations of the calcium-sensing receptor (CASR) are implicated in different hypercalcemic syndromes, including familial hypocalciuric hypercalcemia (FHH), primary hyperparathyroidism (PHPT), and familial isolated hyperparathyroidism (FIHP). However, molecular diagnostics applied to large nonselected hypercalcemic cohorts from a single center have not been reported., Objective: Our objective was to describe the prevalence, type, and potential pathogenicity of CASR mutations in a series of cases with FHH (n = 17), PHPT (n = 165), and FIHP (n = 3) and controls (n = 198) presenting at a single endocrine clinic., Subjects: All were prospectively evaluated at the "Casa Sollievo della Sofferenza" Hospital in southern Italy over a 3-yr period., Methods: CASR screening was conducted by denaturing HPLC. The variant CASRs were functionally characterized by transient transfection studies in kidney cells in vitro., Results: A single novel missense variant was identified in one PHPT case. However, in FHH probands, mutations were found in eight of 17 (47%). With a hypercalcemic family member, mutation detection rate in FHH rose to seven of eight (87%), whereas only one of nine sporadic cases was positive, and none of the three FIHP cases had detectable CASR mutations. Five missense variant CASRs, identified in control subjects, performed as wild type in functional assays, whereas the missense mutant CASRs identified in the FHH patients, and in the one PHPT case, exhibited significant impairment. A novel intronic mutation (IVS4-19a-->c) found in one FHH family, created an abnormally spliced product in an in vitro minigene assay., Conclusion: CASR testing, with functional analysis, provides critical confirmatory evidence in the differential diagnosis of hypercalcemic states.

Introduction: Three single-nucleotide polymorphisms in the calcium-sensing receptor gene (CASR) encoding the missense substitutions A986S, R990G, and Q1011E have been associated with normal variation in extracellular calcium homeostasis, both individually and in haplotype combination. The aim of this study was to examine haplotype associations in primary hyperparathyroidism (PHPT)., Patients and Methods: Patients with sporadic PHPT (n = 237) were recruited from endocrine clinics and healthy controls (n = 433) from a blood donor clinic, and levels of serum calcium, albumin, and PTH were measured. In PHPT patients, urinary calcium/creatinine clearances and bone mineral density at spine and femoral neck were measured and the presence of kidney stones and vertebral fractures identified. The CASR single-nucleotide polymorphisms were haplotyped by allele-specific sequencing., Results: Four haplotypes (ARQ, SRQ, AGQ, and ARE) of eight were observed, in keeping with significant linkage disequilibrium, but haplotype frequencies did not show significant Hardy-Weinberg disequilibrium. The SRQ haplotype was more common in PHPT (125 of 474 alleles) than in controls (170 of 866 alleles, P = 0.006) and showed a significant (P = 0.006) gene-dosage effect. There was no significant association between haplotype and bone mineral density or fractures, but association with kidney stones was significant (P = 0.0007). In the stone-forming subgroup, the SRQ haplotype was underrepresented and AGQ overrepresented. Patients bearing the AGQ haplotype had an odds ratio of 3.8 (95% confidence interval, 1.30-11.3) for presentation with renal stones compared with the rest., Conclusion: Our data indicate that the CASR SRQ haplotype is significantly associated with PHPT in our population. Within the PHPT patient population, the AGQ haplotype is significantly associated with kidney stones.

Context: Mutations of the HRPT2 gene have recently been implicated in the development of parathyroid carcinoma., Objective: The objective of this study was early diagnosis of parathyroid tumor in a family with germline HRPT2 mutation., Patients, Methods, and Results: In a 40-yr-old male previously treated for parathyroid atypical adenoma, we screened the 17 translated HRPT2 exons and their exon-intron boundaries and found a germline frameshift mutation in exon 7 (685delAGAG) predicting a premature stop codon at nucleotides 767-769. Nine family members (age, 33.9 +/- 19.8 yr, mean +/- SD) also carry the mutation, but eight have had normal serum calcium. Biochemical and ultrasonographic evaluation uncovered a 27-yr-old hypercalcemic carrier niece with an atypical parathyroid adenoma, and a 43-yr-old normocalcemic carrier sister was found by ultrasonography to have an extrathyroidal nodule, which proved to be parathyroid carcinoma. The index case, 12 yr after surgery, was normocalcemic, but ultrasonography revealed an extrathyroidal nodule in the contralateral hemithyroid tissue that proved to be atypical adenoma., Conclusions: Our report confirms that germline mutations of HRPT2 gene may be associated with multiple parathyroid neoplasms. Our experience suggests that longitudinal surveillance by serum biochemistry alone may not be 100% sensitive, and addition of routine neck ultrasonography is a readily accepted adjunct that may facilitate earlier disease detection in some families.

Blood ionized calcium (iCa) is a quantitative trait subject to genetic influence. iCa is maintained in a narrow range through the action of the calcium-sensing receptor (CASR) controlling PTH secretion and calcium excretion. A CASR single nucleotide polymorphism (SNP) prevalent in Caucasian populations (A986S) has shown significant association with iCa in a cohort of young women, but association with the neighboring SNPs, R990G and Q1011E, has not been examined. We studied 377 unrelated adults (184 men and 193 women) recruited as healthy adults from a blood donor clinic. The subjects were not taking any medications, nor did they have disorders of calcium metabolism. Relative frequencies for the CASR 986S, 990G, and 1011E minor alleles were 24%, 4%, and 3% respectively. At the A986S locus, subjects with the AA genotype had significantly lower iCa (P = 0.0001) than subjects with one or two S alleles (mean +/- se, 1.221 +/- 0.003 vs. 1.239 +/- 0.003 mmol/liter). For the R990G site, subjects with the RR genotype had higher iCa than those with one copy of the 990G allele (1.230 +/- 0.002 vs. 1.213 +/- 0.007 mmol/liter; P = 0.032). With respect to the 1011 locus, iCa was lower in QQ genotype subjects than in the QE group (1.227 +/- 0.002 vs. 1.255 +/- 0.008 mmol/liter; P = 0.002). After resolution of phase for the doubly heterozygous subjects, analysis was conducted on haplotypes across all three loci. As expected, subjects with SRQ and ARE haplotypes are relatively hypercalcemic, and those with AGQ are hypocalcemic, relative to subjects with the common ARQ haplotype. Multiple regression analysis with clinical covariates (age, sex and menopausal status, creatinine, and PTH) showed that 16.5% of the total variance in iCa may be explained, and the seven CASR haplotypes contribute significantly (P < 0.0001) and substantially (49.1% of the explained variance) to the model, with the following corrected iCa means: ARQ/AGQ, 1.21 +/- 0.01; ARQ/ARQ, 1.22 +/- 0.01; ARQ/SRQ, 1.24 +/- 0.01; SRQ/AGQ, 1.24 +/- 0.03; SRQ/SRQ, 1.25 +/- 0.01; ARQ/ARE, 1.25 +/- 0.01; and SRQ/ARE, 1.27 +/- 0.01. Our data confirm the association between iCa and the A986S locus and suggest that R990G and Q1011E are also predictive. Given the significant between-population variations in frequency of variant alleles in this CASR SNP cluster, tri-locus haplotyping may prove to be more informative in studies of association between variation in CASR and disease.