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The most common causes of hypercalcemia are primary hyperparathyroidism (PHPT) and malignancy. Parathyroid carcinoma (PC), causing a severe PHPT, is the rarest parathyroid tumor. A diagnosis of PC is challenging because the clinical profile overlaps with that of benign counterpart. Surgery is the mainstay treatment. CDC73 mutations have been detected in up to 80% of sporadic PCs. Ectopic production of parathyroid hormone (PTH) by malignant nonparathyroid tumors is a rare condition accounting for less than 1% of hypercalcemia of malignancy. PTH secretion can be considered an aberration in the tissue specificity of gene expression and may involve heterogeneous molecular mechanisms.

Context The pathogenesis of nephrolithiasis in primary hyperparathyroidism (PHPT) remains to be elucidated. The latest guidelines suggest parathyroidectomy in patients with asymptomatic PHPT with hypercalciuria (> 400 mg/d) and increased stone risk profile. Objective The objective of this work is to evaluate the association of urinary stone risk factors and nephrolithiasis in patients with asymptomatic sporadic PHPT and its clinical relevance. Design A total of 157 consecutive patients with sporadic asymptomatic PHPT were evaluated by measurement of serum and 24-hour urinary parameters and kidney ultrasound. Results Urinary parameters were tested in the univariate analysis as continuous and categorical variables. Only hypercalciuria and hypomagnesuria were significantly associated with nephrolithiasis in the univariate and multivariate analysis adjusted for age, sex, body mass index, estimated glomerular filtration rate, parathyroid hormone, 25-hydroxyvitamin D, serum calcium, and urine volume (odds ratio, OR 2.14 [1.10-4.56]; P = .04; OR 3.06 [1.26-7.43]; P = .013, respectively). Hypomagnesuria remained associated with nephrolithiasis in the multivariate analysis (OR 6.09 [1.57-23.5], P = .009) even when the analysis was limited to patients without concomitant hypercalciuria. The urinary calcium/magnesium (Ca/Mg) ratio was also associated with nephrolithiasis (univariate OR 1.62 [1.27-2.08]; P = .001 and multivariate analysis OR 1.74 [1.25-2.42], P = .001). Hypomagnesuria and urinary Ca/Mg ratio had a better, but rather low, positive predictive value compared with hypercalciuria. Conclusions Hypomagnesuria and urinary Ca/Mg ratio are each associated with silent nephrolithiasis and have potential clinical utility as risk factors, besides hypercalciuria, for kidney stones in asymptomatic PHPT patients. The other urinary indices that have been commonly thought to be associated with kidney stones in PHPT are not supported by our results.

ContextAtypical parathyroid adenomas (APAs) are neoplasms with uncertain malignant potential but lack unequivocal histological signs of malignancy.ObjectiveThis work aims to retrospectively evaluate the clinical and biochemical profiles of patients with APA, the outcome after parathyroidectomy (PTX), and the presence of CDC73 germline and somatic mutations.MethodsThis monocentric study was conducted on consecutive patients undergoing PTX for primary hyperparathyroidism (PHPT) between June 2000 and December 2020. Fifty-eight patients with a confirmed histopathological diagnosis of APA, and age- and sex-matched controls with parathyroid adenoma (PA) were also included.ResultsFifty-four patients had sporadic PHPT and 4 had familial isolated hyperparathyroidism (FIHP). Thirty-four patients (59%) had symptomatic disease. Serum calcium and parathyroid hormone (PTH) levels were significantly higher in symptomatic compared to asymptomatic patients (P = .048 and .008, respectively). FIHP patients were younger than their sporadic counterparts (30 ± 17 years vs 55 ± 13 years). APA patients had significantly higher serum calcium and PTH levels and lower 25-hydroxyvitamin D concentration, bone mineral density, and T score at one-third distal radius compared to those with PA. Four of 56 APA patients displayed a CDC73 germline mutation. No somatic CDC73 mutation was identified in 24 tumor specimens. The mean follow-up after surgery was 60 ± 56.4 months. All but 6 patients (90%), 5 with apparently sporadic PHPT and 1 with FIHP, were cured after surgery.ConclusionThe large majority of patients with APA, despite a moderate/severe phenotype, have a good prognosis. Germline CDC73 mutation–positive patients had a higher rate of persistent/recurrent disease. CDC73 gene alterations do not seem to have a relevant role in the tumorigenesis of sporadic APA.

Parathyroid carcinoma (PC) is one of the rarest and aggressive malignancies of the endocrine system. In some instances, the histological diagnosis remains uncertain unless there is evidence of gross local invasion or secondary spread. The identification of molecular markers could improve the diagnostic accuracy of these lesions. The expression of 740 genes involved in the tumor progression processes was assessed in 8 parathyroid adenomas (PAs), 17 non-metastatic and 10 metastatic PCs using NanoString technology. Clustering analysis and Ingenuity Pathway Analysis (IPA) were interrogated to compare the gene expression profiles among the three analyzed groups and to evaluate the potential role of differentially expressed genes, respectively. The 103 differentially expressed genes between metastatic PCs and PAs are able to discriminate perfectly the two groups from a molecular point of view. The molecular signatures identified in non-metastatic PCs vs PAs and in metastatic PCs vs non-metastatic PCs comparisons, although with some exceptions, seem to be histotype-specific IPA reveals that hepatic fibrosis/hepatic stellate cell activation and GP6 signaling pathway are involved in malignant behavior of parathyroid tumors, whereas the activation of the HOTAIR regulatory pathway are involved in the metastatization process. Our investigation identified differentially expressed genes in non-metastatic PCs mainly encoding ECM proteins and in metastatic PCs driving endothelial-to-mesenchymal transition or encoding mediators of angiogenesis. The identified genes might be promising molecular markers potentially useful in the clinical practice for the early diagnosis and prognosis of PC.

Purpose: Previously in 1987, a 21-year-old-male was diagnosed with primary hyperparathyroidism (PHPT) when a right inferior parathyroid adenoma was removed. PHPT recurred after three and six years and on both occasions was cured by resection of parathyroid adenomas. At 52-years-of-age, the patient developed a late-onset hypoparathyroidism (HP), even though postsurgical HP typically occurs as a transient or permanent form soon after neck surgery. The purpose of this work was to report the follow-up of the patient and to review prior cases of late-onset postsurgical HP.\ud \ud Methods: Prior cases of late-onset postsurgical HP were searched and reviewed focusing on clinical and biochemical features.\ud \ud Results: The patient’s asymptomatic hypocalcemia with total serum calcium at 8.2 mg/dL was initially documented in September 2018; PTH was inappropriately low at 15 ng/mL. In\ud February 2020, a mild hypocalcemia was confirmed with low-normal PTH at 15 ng/mL. Autoimmune and familial causes of HP were ruled out including the presence of stimulating autoantibodies against calcium-sensing receptor. Instead, a progressive damage or atrophy of the parathyroid gland(s) ensuing years after surgery is believed to have led to the patient’s hypocalcemia. All 19 previously reported cases of late-onset postsurgical HP occurred after thyroid surgery, with no examples of the condition being found following parathyroidectomy.\ud \ud Conclusions: The case highlights the rare occurrence of late-onset postsurgical HP in a patient who had had multiple parathyroidectomies for PHPT. Thus, monitoring serum calcium,\ud phosphate and PTH during follow-up of such patients is recommended.

The 4th International Workshop for the management of asymptomatic PHPT included, among the criteria for parathyroidectomy, the presence of hypercalciuria (dUCa> 400 mg/day) and increased biochemical stone risk profile. The aim of the present study was to evaluate the biochemical stone risk profile in 176 consecutive patients (143 females and 33 males) with asymptomatic PHPT. We recorded clinical and biochemical data, including 24 hours urinary measurements of the following parameters: volume and pH, creatinine, calcium, magnesium, sodium, potassium, ammonium, uric acid, oxalate, citrate, phosphate, inorganic sulphate and chloride and kidney ultrasound. In our cohort dUCa> 400mg/day showed a low sensitivity and positive predictive value (PPV) for nephrolithiasis with high specificity (46.2, 32.7, 73.0% respectively), while hypercalciuria by 4 mg/kg/bw (d-UCa>4mg/kg) had a high sensibility, with low PPV and specificity (79.5, 27.7, 40.1%) Daily hypomagnesuria (d-HypoMg), but not any other urinary parameter, was an independent predictor of nephrolithiasis in the univariate (OR 2.97 CI 1.27-7.09 P=0.014) and multivariate analyses adjusting for age, sex, BMI, and eGFR (OR 3.13 CI 1.17-8.42 P=0.02). d-HypoMg was relatively lower in the regression analysis with urinary calcium in patients with nephrolithiasis compared with those without. The mean ratio between (dUCa) and (dUMg) was higher in patients with nephrolithiasis compared with those without (4.6±2.0 vs 3.3±4.1; P In patients with hypercalciuria (>400 mg/24-hour) dUMg was positively correlated with dUCa in those without nephrolithiasis (r=0.50, β=0.2, P=0.002) but not in those with nephrolithiasis (r=0.05, β= 0.014; P=0.8). In patients without hypercalciuria we found that hypomagnesuria remained a predictor of nephrolithiasis using either 400 mg/die (P=0.002, OR 5.12 (1.84-14.24) or 4 mg/kg bw (P=0.014, OR 6.24 (1.45-26.8). Moreover, the OR for nephrolithiasis improved using the combination of d-HypoMg with d-UCa>4mg/kg (OR 8.12, CI 1.92-34.18, P=0.004), but not with dUCa> 400mg/day. The current urinary calcium threshold of >400 mg/24-hour has a low sensitivity in detecting nephrolithiasis; our data suggest that sensitivity, specificity and positive predictive value could be improved including dUMg, dUCa/dUMg ratio and the combination of d-HypoMg with d-UCa>4mg/kg in the stone risk evaluation.

Unfortunately, the 13th author name has been published incorrectly in the original publication.

The latest guidelines of the 4th International Workshop on Asymptomatic Primary Hyperparathyroidism (aPHPT) reintroduced hypercalciuria (i.e. urinary calcium > 400 mg/day) as criterion for surgery. However, the value of hypercalciuria as a predictor of nephrolithiasis and the correct cut-off values still need to be confirmed. To evaluate the prevalence of silent kidney stones in a large series of patients with aPHPT and the sensibility, specificity and predictive value of different cut-off values of hypercalciuria in identifying patients with nephrolithiasis. One hundred seventy-six consecutive patients with aPHPT were evaluated at our Institution by serum and urinary parameters and kidney ultrasound. Silent nephrolithiasis was found in 38 (21.6%) patients. In the univariate and multivariate model, hypercalciuria was a predictor of nephrolithiasis using the criterion of 400 mg/24 h [(OR 2.30, (1.11–4.82) P = 0.025], 4 mg/kg/bw [OR 2.65, (1.14–6.25) P = 0.023], gender criterion [OR 2.79, (1.15–6.79) P = 0.023] and the cut-off value derived from the ROC analysis [(> 231 mg/24 h) OR 5.02 (1.68–14.97) P = 0.004]. Despite these several predictive criteria, however, hypercalciuria had a low positive predictive value (PPV), ranging from 27.4 to 32.7%. Hypercalciuria is a predictor of nephrolithiasis, but its PPV is low.

Familial isolated hyperparathyroidism (FIHP) is a rare inherited disease accounting for 1% of all cases of primary hyperparathyroidism (PHPT). It is genetically heterogeneous being associated with mutations in different genes, including MEN1, CDC73, CASR, and recently GCM2. The aim of the study was to further investigate the molecular pathogenesis in Italian FIHP kindreds. We used whole exome sequencing (WES) in the probands of seven unrelated FIHP kindreds. We carried out a separate family-based exome analysis in a large family characterized by the co-occurrence of PHPT with multiple tumors apparently unrelated to the disease. Selected variants were also screened in 18 additional FIHP kindreds. The clinical, biochemical, and pathological characteristics of the families were also investigated. Three different variants in GCM2 gene were found in two families, but only one (p.Tyr394Ser), already been shown to be pathogenic in vitro, segregated with the disease. Six probands carried seven heterozygous missense mutations segregating with the disease in the FAT3, PARK2, HDAC4, ITPR2 and TBCE genes. A genetic variant in the APC gene co-segregating with PHPT (p.Val530Ala) was detected in a family whose affected relatives had additional tumors, including colonic polyposis. We confirm the role of GCM2 germline mutations in the pathogenesis of FIHP, although at a lower rate than in the previous WES study. Further studies are needed to establish the prevalence and the role in the predisposition to FIHP of the novel variants in additional genes.

Atypical parathyroid adenomas represent a group of intermediate form of parathyroid neoplasms of uncertain malignant potential which show some atypical histological features that represent a challenge for the differential diagnosis with parathyroid carcinomas. They may occur as sporadic or as a part of hereditary syndromes. The molecular signature of these neoplasms is still unknown and the germline CDC73 mutations appears to be the most common anomaly in this setting suggesting that these cases might represent variants of the hyperparathyroidism-jaw tumor syndrome. The identification of markers predicting the outcome is of great importance to guide an adequate postoperative monitoring and, the same time, relieve of the anxiety of relatively strict monitoring patients not at risk. This review will summarize the current knowledge of the clinical, biochemical, molecular and histological profile of atypical parathyroid adenomas.

Parathyroid carcinoma (PC) is a rare neoplasia difficult to diagnose preoperatively. It mainly occurs as a sporadic disease but also as part of familial PHPT. At variance with patients with the benign counterpart, the phenotype of these patients is characterized by a severe primary hyperparathyroidism (PHPT). The clinical features are mostly due to the effects of the excessive secretion of PTH by the functioning tumor and hypercalcemia rather than to the tumor burden. The prognosis is poor and unmanageable hypercalcemia accounts for death in the majority of cases. The best chance of cure is surgery, although persistent or recurrent disease occurs in about 50% of patients. Somatic loss-of-function mutations of CDC73 gene, encoding parafibromin, are the most frequent genetic alterations occurring in PCs. Mutations of the PRUNE2 gene, alterations of the PI3K/AKT/mTOR pathway and amplification of the CCND1 gene have been recently detected in PCs. Alteration of microRNA profile and methylation pattern have been identified in PCs. The recent studies have better defined the genomic landscape of PCs and represent major progress toward a full molecular characterization of this neoplasia and development of novel therapeutic options.

The aim of this study was to carry out genetic screening of the MEN1, CDKN1B and AIP genes, both by direct sequencing of the coding region and multiplex ligation-dependent probe amplification (MLPA) assay in the largest monocentric series of Italian patients with Multiple Endocrine Neoplasia type 1 syndrome (MEN1) and Familial Isolated Hyperparathyroidism (FIHP). The study also aimed to describe and compare the clinical features of MEN1 mutation-negative and mutation-positive patients during long-term follow-up and to correlate the specific types and locations of MEN1 gene mutations with onset and aggressiveness of the main MEN1 manifestations. A total of 69 index cases followed at the Endocrinology Unit in Pisa over a period of 19 years, including 54 MEN1 and 15 FIHP kindreds were enrolled. Seven index cases with MEN1 but MEN1 mutation-negative, followed at the University Hospital of Cagliari, were also investigated. FIHP were also tested for CDC73 and CaSR gene alterations. MEN1 germline mutations were identified in 90% of the index cases of familial MEN1 (F-MEN1) and in 23% of sporadic cases (S-MEN1). MEN1 and CDC73 mutations accounted for 13% and 7% of the FIHP cohort, respectively. A CDKN1B mutation was identified in one F-MEN1. Two AIP variants of unknown significance were detected in two MEN1-negative S-MEN1. A MEN1 positive test best predicted the onset of all three major MEN1-related manifestations or parathyroid and gastro-entero-pancreatic tumors during follow-up. A comparison between the clinical characteristics of F and S-MEN1 showed a higher prevalence of a single parathyroid disease and pituitary tumors in sporadic compared to familial MEN1 patients. No significant correlation was found between the type and location of MEN1 mutations and the clinical phenotype. Since all MEN1 mutation-positive sporadic patients had a phenotype resembling that of familial MEN1 (multiglandular parathyroid hyperplasia, a prevalence of gastro-entero-pancreatic tumors and/or the classic triad) we might hypothesize that a subset of the sporadic MEN1 mutation-negative patients could represent an incidental coexistence of sporadic primary hyperparathyroidism and pituitary tumors or a MEN1 phenocopy, in our cohort, as in most cases described in the literature.

Juvenile primary hyperparathyroidism is uncommon and more symptomatic than the adult counterpart. The aim of this prospective monocentric study, conducted in a tertiary referral center, was to evaluate the clinical, biochemical, and densitometric data, and the outcome of a series of patients with juvenile primary hyperparathyroidism. The study group included 154 patients with sporadic and familial juvenile primary hyperparathyroidism, aged ≤40 years. Relative frequency of sporadic and familial forms, comparison of the clinical and biochemical characteristics, rate of cure after parathyroidectomy and the outcome of patients not undergoing surgery were evaluated. Familial cases (n = 42) were younger, less frequently females, and had milder disease compared to sporadic cases (n = 112). No difference was observed in biochemical and densitometric parameters. Among patients undergoing parathyroidectomy (n = 116), familial cases had a higher rate of multigland disease and a higher persistence/relapse rate compared to sporadic cases (73 vs. 3.6% and 48.1 vs. 5.7%, respectively). Patients who did not undergo parathyroidectomy had stable clinical, biochemical, and densitometric parameters during follow-up (median 27 months). Using the cut-off age of 25 years, there was no difference in clinical, biochemical and densitometric parameters between younger and older patients, with the exception of parathyroid hormone and phosphate, which were significantly lower and higher, respectively, in patients

MEN1 is the main gene responsible for tumorigenesis of syndromic and sporadic primary hyperparathyroidism (PHPT). Germline mutations of the CDKN1B/p27Kip gene have been associated with multiple endocrine tumors in rats and humans. To evaluate the involvement of the CDKN1B gene and its relationship with MEN1 in sporadic PHPT, we carried out sequencing and loss of heterozygosity analyses of the CDKN1B gene in 147 sporadic parathyroid adenomas. p27 immunohistochemistry and genetic screening of the MEN1 gene were performed in 50 cases. Three germline CDKN1B variants (c.-80C>T, c.-29_-26delAGAG, c.397C>A) were identified in 3/147 patients. Reduction of CDKN1B gene transcription rate was demonstrated in vitro for the novel c.-80C>T and the c.-29_-26delAGAG variants. Loss of p27 expression was detected in the tumor carrying the c.-29_-26delAGAG variant. Two tumors carrying the CDKN1B variants also harbored a MEN1 mutation. Fifty-four percent of 50 CDKN1B mutation-negative tumors had a reduction of p27 nuclear staining. Somatic MEN1 mutations, identified in 15/50 samples, significantly segregated in tumors negative for nuclear and cytoplasmic p27 staining. The germline nature of the CDKN1B mutations suggests that they might predispose to PHPT. The lack of somatic CDKN1B mutations in our samples points to a rare involvement in parathyroid adenomas, despite the frequent loss of nuclear p27 expression. MEN1 biallelic inactivation seems to be directly related to down-regulation of p27 expression through the inhibition of CDKN1B gene transcription.

Inactivating mutations of the multiple endocrine neoplasia 1 (MEN1) gene cause MEN1 syndrome, characterized by primary hyperparathyroidism (pHPT), and parathyroid and gastro-entero-pancreatic pituitary tumors. At present, only 14 cases of malignant parathyroid tumor have been associated with the syndrome, with 6 cases carrying an inactivating mutation of the MEN1 gene. The present study presents the case of a 48-year-old female who presented with multigland pHPT and multiple pancreatic lesions. The patient underwent surgery several times for the excision of parathyroid hyperplasia, carcinoma and adenoma. The MEN1 gene was screened, revealing three variants (in cis) at the intron/exon 3 boundary (IVS2-3G>C, c.497A>T and c.499G>T) detected on the DNA of the proband, not shared by her relatives. RNA sequencing revealed that the IVS2-3C>G variant caused the skipping of the exon 3. Therefore, the present study reports on a novel rare association of MEN1 syndrome and parathyroid carcinoma. The reported splicing mutation was previously identified in subjects who always developed malignant lesions; thus, a possible genotype-phenotype association may be considered.

The molecular pathogenesis of primary hyperparathyroidism is still largely unknown. The aryl hydrocarbon receptor interacting protein (AIP) gene has a major role in the pathogenesis of familial isolated pituitary adenoma.We evaluated the involvement of the AIP gene in sporadic parathyroid adenomas.We performed direct sequencing and multiplex ligation-dependent probe amplification analyses of the AIP gene in a large series of sporadic parathyroid adenomas. Loss of heterozygosity (LOH) at the AIP locus was studied, and aryl hydrocarbon receptor interacting protein immunostaining was also performed. In addition, alterations in the MEN1 gene were studied.A somatic AIP mutation, substitution of arginine with glutamine at codon 304 (R304Q), was identified in 2 of 132 tumors. The mutation was germline in both cases despite the nonfamilial presentation. Heterozygous AIP large deletions were detected in 29 cases including 1 of the 2 mutated tumors, confirming a biallelic inactivation of the AIP gene. The AIP-mutated tumor with LOH showed decreased AIP immunostaining compared with normal parathyroid. LOH at the MEN1 locus, which often shared LOH at the AIP locus, was found in one third of tumors. Somatic MEN1 mutations were found in the 1 of the 2 AIP-mutated tumors and in 22 parathyroid adenomas. In addition, multiplex ligation-dependent probe amplification analysis revealed 1 large deletion of the MEN1 gene in 1 patient.The AIP gene is rarely involved in parathyroid adenomas, but the germline nature of the mutations suggests that it might predispose to primary hyperparathyroidism. MEN1 gene alterations occur in a substantial proportion of sporadic parathyroid adenomas.

Early onset of primary hyperparathyroidism (PHPT) and multiglandular involvement suggest a familial form in which germline mutation of a PHPT-related gene(s) and a somatic event at the same locus can be often demonstrated. We investigated the involvement of multiple endocrine neoplasia type 1 (MEN1) and HRPT2 genes in a 39-year-old man with recurrent PHPT. PHPT was firstly diagnosed at the age of 21 and the patient had two recurrences separated by extended periods of normocalcemia. This unusual history prompted us to investigate other family members and study the MEN1 and HRPT2 genes. An HRPT2 germline missense mutation in exon 3 (R91P) was found in the index case, which was associated with different HRPT2 somatic alterations in each of the three examined parathyroid tumors. These findings are consistent with Knudson’s ‘two hit’ concept of biallelic inactivation of classical tumor suppressor genes. Screening of 15 asymptomatic relatives was negative for the R91P germline mutation. All the three abnormal parathyroid specimens showed cystic features at histology and were negative for parafibromin immunostaining. In one specimen, diffuse parafibromin staining was evident in a rim of normal parathyroid tissue surrounding the adenomatous lesion. Our study shows that different somatic genetic events at the HRPT2 locus are responsible for the asynchronous occurrence of multiple adenomas in a patient carrying an HRPT2 germline mutation. The finding of diffuse parafibromin staining in a rim of normal parathyroid tissue, but not in the contiguous adenomatous lesion, reinforces the concept that loss of parafibromin expression is responsible for the development of parathyroid tumors in this setting.

We investigated the involvement of the HRPT2 gene by loss of heterozygosity analysis and direct sequencing in a kindred with hyperparathyroidism-jaw tumor syndrome (HPT-JT) and three kindreds with familial isolated primary hyperparathyroidism (FIHP). Seven patients with sporadic parathyroid cancers and 35 with parathyroid adenomas with no family history of primary hyperparathyroidism or HPT-JT were also studied. A germline heterozygous substitution G to A was found in the donor splice site of intron 1 in one of the three FIHP families. No mutations were identified in the HPT-JT kindred. A somatic HRPT2 mutation was found in four of seven patients with parathyroid cancers, two of which were unreported frameshift mutations (195insT and 195insA) in exon 2. Consistent with recent findings, two of seven patients with sporadic parathyroid cancer had germline mutations. Four adenomas showed loss of heterozygosity at HRPT2, whereas a somatic HRPT2 mutation was found in one. In conclusion, we provide additional evidence for a strong association between HRPT2 gene mutations and sporadic parathyroid cancer. The finding that two of the seven patients with sporadic parathyroid cancer carried an HRPT2 germline mutation suggests that they might have occult HPT-JT. Our results also confirm the need for testing HRPT2 gene in FIHP families.

The calcium-sensing receptor (CaR) polymorphism A986S has been found to be associated with higher serum calcium levels in normal subjects, suggesting that this amino acid change might decrease the inhibitory activity of the mutated receptor, render the parathyroid cells more prone to proliferate, and eventually increase the risk of developing primary hyperparathyroidism (PHPT). The aim of the present study was to investigate the frequency of this and other 2 known CaR polymorphisms (R990G and Q1011 E) in patients with PHPT and their effect on its phenotype. We studied 103 Italian patients with PHPT and 148 healthy Italian subjects and we compared the results in 50 pairs matched for sex, age and geographic provenience. A fragment of exon 7 of the CaR gene, containing the 3 polymorphic loci of interest (A986S, R990G, and Q1011E), was amplified by PCR and sequenced. Serum calcium and PTH levels, BMD and other biochemical and clinical parameters were evaluated. The frequency distribution of the A9865, R990G, and Q1011 E polymorphisms in the 103 PHPT patients was 39.8%, 5.8%, and 2.0%, respectively. There was no difference in the frequency of the 3 CaR polymorphisms in the 50 matched pairs of patients and controls. We found no significant difference in several clinical and biochemical parameters between PHPT patients carrying or not the 986S allele. Finally, no relationship was observed between the 986S genotype and total and ionized serum calcium in control subjects. The A986S CaR polymorphism is the most common in Italian PHPT patients and the allotype AS does not appear to play a relevant role in the pathogenesis of PHPT and its severity. The A986S polymorphism does not correlate with serum calcium levels in normal Italian subjects.

Loss of heterozygosity (LOH) in the MEN1 region on chromosome 11q13 and MEN1 gene mutations have been found in a subset of sporadic parathyroid tumors. The question of whether these genetic abnormalities in the parathyroid tumors might influence the clinical and biochemical characteristics of primary hyperparathyroidism (PHPT) remains to be elucidated. The aim of the present study was to correlate the presence of MEN1 gene alterations in PHPT tumors with the clinical phenotype. Using microsatellite analysis for LOH at 11q13 and DNA sequencing of the coding exons, the MEN1 gene was studied in 38 parathyroid tumors of patients with sporadic PHPT. Fourteen tumors showed LOH at 11q13, and mutations of MEN1 gene were detected in 7 cases. The clinical and biochemical characteristics of patients were unrelated to the presence or absence of LOH and/or MEN1 gene mutations. In conclusion, MEN1 gene alterations are rather common in sporadic PHPT and their presence does not correlate with the clinical manifestations of the disease.

Summary OBJECTIVES Familial hyperparathyroidism may occur as part of hereditary syndromes, including multiple endocrine neoplasia types 1 and 2 (MEN1 and MEN2A), hyperparathyroidism-jaw tumour (HPT-JT) syndrome and familial isolated hyperparathyroidism (FIHP). It is unclear whether the latter is a distinct genetic entity or a variant of MEN1 or HPT-JT, where, because of reduced penetrance, only primary hyperparathyroidism (PHPT) is present. In the present study, we describe two unrelated Italian kindreds with FIHP, in which the clinical, histopathological and genetic analyses of the MEN1 gene and HPRT2 locus at 1q21-32 suggest that both might be a variant of MEN1 and HPT-JT syndromes. PATIENTS AND DESIGN We studied 16 members, aged 14–50 years, of two unrelated kindreds with FIHP. Genomic DNA was isolated from peripheral blood leucocytes in all family members, and from parathyroid tissue in those who underwent parathyroidectomy. MEASUREMENTS Ionized calcium and PTH were measured in all family members. The nine coding exons and 16 splice junctions of the MEN1 gene from constitutional DNA were amplified by the polymerase chain reaction (PCR) and sequenced. Parathyroid glands were obtained from five subjects. Allelic deletions (loss of heterozygosity, LOH) of chromosomes 11q13 and 1q21-q32 were assessed using PYGM and D11S449, and D1S215, D1S222, D1S428, D1S412, D1S413 and D1S477, respectively. Forward primers were conjugated with 5′ fluorescent dye. PCR products were analysed using an ABI PRISM 310 sequencer. RESULTS Five members of family 1 and three of family 2 had PHPT. A heterozygous deletion of 1 bp of the MEN1 gene in exon 10 (1785delA) was found in affected members of family 1. No MEN1 gene mutation was found in any member of family 2. LOH at 11q13 was observed in family 1 tumours, but not in those from family 2. Studies at 1q21-32 showed LOH in two family 2 tumours, whereas a retained heterozygosity was found in the remaining member. No LOH at 1q21-32 was found in family 1 tumours. The pathology of family 1 showed chief cell hyperplasia with a diffuse-nodular pattern of growth. Cut surface showed no cystic structures. Typical parathyroid adenoma was diagnosed in one member of family 2 and atypical adenoma in the remaining two. These tumours showed cystic structures. CONCLUSIONS In conclusion, we describe two unrelated kindreds with FIHP which, on the basis of histopathological and genetic studies, could be labelled as variants of the MEN1 and HPT-JT syndromes, respectively. Therefore, an extensive analysis of the genes involved in these diseases should be performed in families with familial isolated hyperparathyroidism to identify the subset linked to the MEN1 gene or to the HPRT2 locus.

Inactivating germline mutations of the CDKN1B gene encoding the nuclear cyclin-dependent kinase inhibitor P27kip1 protein have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to characterize in vitro the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by primary hyperparathyroidism due to multiglandular parathyroid involvement and gastro–entero–pancreatic tumors. We carried out subcellular localization experiments by transfection with plasmid vectors expressing the WT or mutant CDKN1B cDNA into the eukaryotic human cervix adenocarcinoma (HeLa) and GH3 cell lines. Results from western blotting studies indicated that fusion proteins were expressed at equal levels. The mutated protein was shorter compared with the WT protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells, WT P27 localized in the nucleus, whereas the P27_S125X protein was retained in the cytoplasm, predicting the loss of tumor-suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, because both WT and mutant alleles were determined to be present by sequencing the somatic DNA. Immunohistochemistry revealed a complete loss of nuclear expression of P27 in a parathyroid adenoma, which had been removed by the second surgery in the patient. In conclusion, our results confirm the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4.

A reduced expression of calcium-sensing receptor (CaR) messenger ribonucleic acid and protein accompanied by abnormalities in parathyroid cell proliferation and PTH secretion are present in primary hyperparathyroidism. We studied the expression of CaR protein by immunohistochemistry in 36 sporadic parathyroid adenomas and investigated the relationship between CaR expression and several preoperative clinical parameters, including the set-point of Ca(2+)-regulated PTH secretion (measured in vivo). The adenomas were classified in 4 categories according to the intensity of immunohistochemical staining: 5 (14%) showed a CaR staining intensity similar to that of normal parathyroid ( ), 10 (27%) showed moderate staining (++), 16 (45%) showed weak staining (+), and 5 (14%) were negative (-). The intensity of CaR staining was not related to preoperative serum Ca(2+), PTH levels or adenoma volume. Twenty-nine patients underwent preoperatively the calcium infusion test to evaluate the PTH-Ca(2+) set-point. Individual values of PTH-Ca(2+) set-point ranged from 1.38-1.93 mmol/L and were significantly correlated with basal Ca(2+) levels (r = 0.96; P: = 0. 0001) and adenoma volume (r = 0.5; P: = 0.01). The mean PTH-Ca(2+) set-point values were significantly different in the 4 groups of patients classified according to immunohistochemical staining intensity of their adenoma (P: = 0.025; F = 3.78); the mean PTH-Ca(2+) set-point was significantly higher in the groups classified as negative than in those classified as weak or moderate. No correlation was observed between the PTH-Ca(2+) set-point and basal PTH levels or between the percent maximal PTH inhibition and adenoma volume and basal PTH or Ca(2+) levels. In summary, our data suggest that there is a relationship between apparent CaR protein expression and PTH-Ca(2+) set-point abnormality, suggesting that a reduced receptor content might have an important role in the pathogenesis of primary hyperparathyroidism.

Inactivating mutations of the CDC73 tumor suppressor gene have been reported in parathyroid carcinomas (PC), in association with the loss of nuclear expression of the encoded protein, parafibromin. The aim of this study was to further investigate the role of the CDC73 gene in PC and evaluate whether gene carrier status and/or the loss of parafibromin staining might have an effect on the outcome of the disease. We performed genetic and immunohistochemical studies in parathyroid tumor samples from 35 patients with sporadic PC. Nonsense or frameshift CDC73 mutations were detected in 13 samples suitable for DNA sequencing. Six of these mutations were germline. Loss of parafibromin expression was found in 17 samples. The presence of the CDC73 mutation as well as the loss of parafibromin predicted a high likelihood of subsequent recurrence and/or metastasis (92.3%, P=0.049 and 94.1%, P=0.0017 respectively), but only the latter was associated with a decreased overall 5- and 10-year survival rates (59%, P=0.107, and 23%, P=0.0026 respectively). The presence of both the CDC73 mutation and loss of parafibromin staining compared with their absence predicted a lower overall survival at 10- (18 vs 84%, P=0.016) but not at 5-year follow-up. In conclusion, loss of parafibromin staining, better than CDC73 mutation, predicts the clinical outcome and mortality rate. The added value of CDC73 mutational analysis is the possibility of identifying germline mutations, which will prompt the screening of other family members.

Background Diagnosis of multiple endocrine neoplasia type 1 (MEN1) is commonly based on clinical criteria, and confirmed by genetic testing. In patients without known MEN1-related germline mutations, the possibility of a casual association between two or more endocrine tumors cannot be excluded and subsequent management may be difficult to plan. We describe a very uncommon case of functioning glucagonoma associated with primary hyperparathyroidism (pHPT) in which genetic testing failed to detect germline mutations of MEN-1 and other known genes responsible for MEN1. Case presentation The patient, a 65-year old woman, had been suffering for more than 1 year from weakness, progressive weight loss, angular cheilitis, glossitis and, more recently, skin rashes on the perineum, perioral skin and groin folds. After multidisciplinary investigations, functioning glucagonoma and asymptomatic pHPT were diagnosed and, since family history was negative, sporadic MEN1 was suspected. However, genetic testing revealed neither MEN-1 nor other gene mutations responsible for rarer cases of MEN1 (CDKN1B/p27 and other cyclin-dependent kinase inhibitor genes CDKN1A/p15, CDKN2C/p18, CDKN2B/p21). The patient underwent distal splenopancreatectomy and at the 4-month follow-up she showed complete remission of symptoms. Six months later, a thyroid nodule, suspected to be a malignant neoplasia, and two hyperfunctioning parathyroid glands were detected respectively by ultrasound with fine needle aspiration cytology and 99mTc-sestamibi scan with SPECT acquisition. Total thyroidectomy was performed, whereas selective parathyroidectomy was preferred to a more extensive procedure because the diagnosis of MEN1 was not supported by genetic analysis and intraoperative intact parathyroid hormone had revealed “adenoma-like” kinetics after the second parathyroid resection. Thirty-nine and 25 months after respectively the first and the second operation, the patient is well and shows no signs or symptoms of recurrence. Conclusions Despite well-defined diagnostic criteria and guidelines, diagnosis of MEN1 can still be challenging. When diagnosis is doubtful, appropriate management may be difficult to establish.

Bone morbidity remains a major problem even after successful renal transplantation. We investigated the role of calcium-sensing receptor (CaSR) polymorphisms and 25-hydroxyvitamin D levels on the persistence of secondary hyperparathyroidism (SHPT) and their relationships with vertebral fractures (VFx) in 125 renal allograft recipients transplanted 44 ± 23 months before. All patients underwent evaluation of the main biochemical parameters of calcium metabolism as well as vertebral and femoral bone density. In 87 patients, CaSR polymorphisms (A986S, R990G, and Q1011E) also were assessed. X-ray images of the lateral spine were obtained in 102 subjects to perform vertebral morphometry. High parathyroid hormone (PTH) and 25-hydroxyvitamin D lower than 80 nmol/L were found in 54% and 97% of patients, respectively, with 40% of these showing vitamin D levels lower than 30 nmol/L. VFx were detected in 57% of the subjects. After multiple adjustments, 25-hydroxyvitamin D, age, and hemodialysis duration, but not CaSR polymorphisms, were found to be significant predictors of high PTH, whereas age and time since transplant were positively related with lower 25-hydroxyvitamin D values. PTH and time since transplant were significantly associated with VFx. Patients with two or more VFx showed serum PTH levels 50% higher than patients without fractures. We therefore conclude that persistent SHPT is a very common feature after renal transplantation and that, unlike CaSR polymorphisms, low 25-hydroxyvitamin D is involved in its pathogenesis. High PTH levels, in turn, are associated with an increased VFx risk, which confirms the need for strategies aimed at lowering serum PTH in this setting as well. © 2010 American Society for Bone and Mineral Research.

HRPT2 and parafibromin studies improved the diagnostic accuracy in two patients with primary hyperparathyroidism (PHPT) referred to us after surgery, in whom the clinical data were at variance with the pathological diagnosis of adenoma and carcinoma, respectively. Patients were referred to us after parathyroidectomy. Patient #1 had had a 1.5-cm tumor easily removed with a histological diagnosis of parathyroid carcinoma and normocalcemia for 2 years. Re-examination of the histology showed no cardinal signs of parathyroid cancer. Patient #2, with severe PHPT, had had the removal of a 3.5-cm tumor described histologically as adenoma. Ten years later PHPT recurred and persisted despite removal of two mildly enlarged parathyroid glands that were histologically normal. Re-review of the initial histology showed a trabecular pattern, fibrous bands, and atypical mitoses, suggesting an atypical adenoma. Because of the suspicion that case #1 could be an atypical adenoma and case #2 a carcinoma further molecular studies were performed. No HRPT2 and parafibromin abnormalities were identified in patient #1, strongly indicating a benign lesion. In patient #2, an HRPT2 germline mutation was found (E115X in exon 4) and associated with no parafibromin staining. These data, together with the clinical features, supported the suspicion of a parathyroid carcinoma that was confirmed by histological examination of further slides of the tumor, showing capsular and vascular invasion. A lung 1.5-cm nodule detected by computed tomography was excised. Histology showed a metastasis of parathyroid carcinoma. HRPT2 gene studies improved the diagnostic accuracy in 2 parathyroid tumors that are of uncertain type.

Summary Objective Familial isolated primary hyperparathyroidism (FIPH) can result from either incomplete expression of a syndromic form of familial primary hyperparathyroidism [multiple endocrine neoplasia type 1 (MEN 1), hyperparathyroidism–jaw tumour syndrome (HPT-JT) or familial hypocalciuric hypercalcaemia (FHH)] or still unrecognized causes. Design Genetic analyses of MEN1, HRPT2 and CASR genes in FIHP. Patients Seven well-characterized Italian kindreds with FIHP, with negative clinical features for MEN 1, HPT-JT and FHH. The mean age (± SD) at diagnosis was 45 ± 17 years (range 18–70 years) in the probands and 42 ± 18 years (range 15–69 years) in the other affected subjects. Measurements Direct sequencing of germline DNA of the MEN1, HRPT2 and CASR genes from probands. The region of interest was amplified in some family members. Results Germline MEN1 mutations were detected in three kindreds. Multiglandular involvement was found in all but one affected subject belonging to the three kindreds with MEN1 mutations. In these patients persistence/relapse of the disease was observed unless an extensive parathyroidectomy (excision of 31/2 glands) had been performed, with the exception of one patient, who is currently normocalcaemic 168 months after excision of two glands. No mutations of MEN1, HRPT2 and CASR genes were identified in the remaining four families. Conclusions MEN1 genotyping appears worthwhile in FIHP families, as the finding of mutation(s) may predict multiglandular involvement and therefore have practical surgical implications, and prompt further investigation in the family, with the possibility of identifying new cases and beginning a programme of periodic surveillance for emergence of tumours in all carriers.

Summary OBJECTIVES Some histological features may suggest the malignant nature of a parathyroid tumour. However, the diagnosis of parathyroid cancer can only be definitively established in the presence of local invasion or metastases. DESIGN We further investigated the role of the retinoblastoma gene (Rb1) and the breast cancer susceptibility gene (BRCA2) in the differential diagnosis between benign and malignant parathyroid tumours by evaluating loss of heterozygosity (LOH) at these loci and Rb protein (pRb) immunohistochemistry. PATIENTS AND MEASUREMENTS Fifty-three parathyroid adenomas from patients with sporadic primary hyperparathyroidism (PHPT) and 10 parathyroid cancer specimens were studied. Microsatellite polymorphisms at the Rb1 and BRCA2 loci were polymerase chain reaction (PCR) amplified from each patient’s paired tumour and leucocyte DNA samples, using oligonucleotide primers flanking the repeat sequence. Immunohistochemical staining of pRb was carried out using a monoclonal antibody. RESULTS All but one of the 53 tumour‐leucocyte pairs was informative for at least one of the three polymorphic markers of the Rb1 gene. Fifteen adenomas (28·8%) showed LOH. Regarding the BRCA2 gene, 46 tumour‐ leucocyte pairs were informative and LOH was present in eight (17·4%). All six carcinomas had LOH for at least one marker at the Rb1 locus. LOH for the BRCA2 microsatellite was found in three of the five informative primary tumour samples. Immunohistochemical analysis revealed that all adenomas were positive and the number of pRb-positive cells varied significantly among different samples. The mean percentage of stained cells was 15·7%. Eleven of the 30 (36·7%) adenomas showed sparse positive staining, 13 (43·3%) intermediate staining and six (20%) extensive staining. All parathyroid cancers were entirely negative for pRb immunostaining. CONCLUSIONS Inactivation of the Rb1 gene is a common event in parathyroid tumorigenesis. Retention of heterozygosity seems to exclude parathyroid malignancy, which is suggested by the combined finding of LOH and lack of protein expression.

OBJECTIVE: To report a new mutation of the multiple endocrine neoplasia type 1 (MEN1) gene in an Italian kindred. DESIGN: The study included the female proband, aged 50 years, affected by primary hyperparathyroidism, insulinoma and prolactinoma, and ten relatives. Blood samples were obtained for biochemical and genetic analyses. Clinical screening tests included serum glucose, ionized calcium, intact parathyroid hormone, GH, insulin and prolactin. The coding sequence, including nine coding exons and 16 splice sites, was amplified by PCR and directly sequenced. RESULTS: Two additional cases of primary hyperparathyroidism were identified among the paternal family members. The sequence analysis showed a heterozygous T to C transition at codon 444 in exon 9, resulting in a leucine to proline substitution (L444P) in the patient and in the two paternal family members with primary hyperparathyroidism. The L444P amino acid change was absent in 50 normal subjects. The mutation determined the loss of a BlnI restriction site of the wild-type sequence and the creation of a new restriction EcoRII site. The patient, but not her paternal affected relatives, also had a common heterozygous polymorphism (D418D) in exon 9. CONCLUSIONS: A new MEN1 mutation (L444P) in exon 9 has been identified; this substitution caused the loss of a BlnI restriction site and the creation of a new EcoRII site.