Objective: HRPT2 gene mutations are associated with parathyroid carcinomas, and absence of parafibromin immunoreactivity has been suggested as a diagnostic marker of malignancy. The aim of our study was to extend parafibromin studies in a series of benign and malignant parathyroid tumors and cross-validate the results of immunohistochemistry with those of HRPT2 analysis. Design and patients: We performed parafibromin and cyclin D1 immunostaining and HRPT2 gene analysis using loss of heterozygosity studies and sequencing analysis in parathyroid specimens from 11 patients with carcinoma (eleven primary tumors, one skin, and four lung metastases), 22 with sporadic adenomas, and 4 with atypical adenomas. Results: Ten out of eleven parathyroid cancers were negative for parafibromin staining and showed HRPT2 gene abnormalities. The remaining sample was negative for immunostaining and genetic analyses. All but one sporadic adenomas showed parafibromin immunoreactivity and no HRPT2 gene abnormalities. The sample with negative immunostaining carried an HRPT2 mutation. Two atypical adenomas were positive and two negative with parafibromin staining. No HRPT2 abnormalities were found in these samples. Cyclin D1 expression was heterogeneous and there was no relationship between expression/expression level of cyclin D1 and parafibromin expression. Conclusions: We have shown that negative parafibromin staining is almost invariably associated with HRPT2 mutations and confirm that loss of parafibromin staining strongly predicts parathyroid malignancy. In clinical practice, these tests could be particularly useful in the subset of parathyroid tumors with equivocal histological examination. However, their diagnostic value in this setting remains to be proven.European Journal of Endocrinology 156 547-554

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.

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.

Objective: Identification and characterization of calcium-sensing receptor (CASR) mutations in four unrelated Italian kindreds with familial hypocalciuric hypercalcemia. Design: Clinical evaluation and genetic analysis of CASR gene. Functional characterization of mutated CASRs. Methods: Direct sequencing of CASR gene in genomic DNA. Studies of CASR-mediated increases in cytosolic calcium concentration [Ca 2C ] i in CASR-transfected COS-7 cells in vitro. Results: Four unreported heterozygous CASR mutations were identified, including three missense (H595Y, P748H, and C765W) and one splice site (IVS2C1GOC) mutation. The H595Y, P748H, and C765W mutant receptors, although expressed at normal levels on the cell surface, showed a reduced response in [Ca 2C ] i relative to the wildtype (WT) CASR to increasing extracellular calcium concentrations. Cotransfection experiments showed that the H595Y and P748H mutants did not affect the apparent affinity of the WT CASR for calcium, suggesting that they do not exert a dominantnegative effect. On the other hand, the co-transfected C765W mutant decreased the maximum response of the WT CASR to calcium, suggesting that it may reduce the effective concentration of the normal CASR on the cell surface or impair its maximal signaling capacity. Conclusions: Four CASR mutations were identified. The reduced functional responses to extracellular calcium and normal expression of the mutant receptors suggest that conformational changes account for altered CASR activity. Moreover, a reduced complement of normal CASRs in these heterozygous patients, perhaps combined with a mutant receptor-induced decrease in maximal activity of the WT receptor, may contribute to defective calcium-sensing in vivo.