Clinical and molecular description of the first Italian cohort of 33 subjects with hypophosphatasia.

Introduction: Hypophosphatasia (HPP) is a rare genetic disease caused by inactivating variants of the ALPL gene. Few data are available on the clinical presentation in Italy and/or on Italian HPP surveys. Methods: There were 30 suspected HPP patients recruited from different Italian tertiary cares. Biological samples and related clinical, biochemical, and anamnestic data were collected and the ALPL gene sequenced. Search for large genomic deletions at the ALPL locus (1p36) was done. Phylogenetic conservation and modeling were applied to infer the effect of the variants on the protein structure. Results: There were 21 ALPL variants and one large genomic deletion found in 20 out of 30 patients. Unexpectedly, NGS-driven differential diagnosis allowed uncovering three hidden additional HPP cases, for a total of 33 HPP subjects. Eight out of 24 coding variants were novel and classified as "pathogenic", "likely pathogenic", and "variants of uncertain significance". Bioinformatic analysis confirmed that all the variants strongly destabilize the homodimer structure. There were 10 cases with low ALP and high VitB6 that resulted negative to genetic testing, whereas two positive cases have an unexpected normal ALP value. No association was evident with other biochemical/clinical parameters. Discussion: We present the survey of HPP Italian patients with the highest ALPL mutation rate so far reported and confirm the complexity of a prompt recognition of the syndrome, mostly for HPP in adults. Low ALP and high VitB6 values are mandatory for the genetic screening, this latter remaining the gold standard not only to confirm the clinical diagnosis but also to make differential diagnosis, to identify carriers, to avoid likely dangerous therapy in unrecognized cases.

From Survey Results to a Decision-Making Matrix for Strategic Planning in Healthcare: The Case of Clinical Pathways.

BACKGROUND: It is a well-known fact that the information obtained from a survey can be used in a healthcare organizational analysis; however, it is very difficult to compare the different results found in the literature to each other, even through the use of metanalysis, as the methodology is often not consistent. METHODS: Data from a survey analyzing the organizational and managerial responses adopted in pathology-specific clinical pathways (CPs) during the first two waves of the COVID-19 pandemic were used for constructing a decisional matrix, a tool called SPRIS system, consisting of four different sheets. The first sheet reports the results of the survey and, using a streetlight color system, identifies strengths and weaknesses; the second one, by assigning a priority score, establishes the priority of intervention on each of the strengths and weaknesses identified; the third sheet reports the subjective items of the questionnaire in order to identify threats and opportunities and their probability of happening; in the last sheet, a SWOT Analysis is used to calculate the performance index of the whole organization. RESULTS: The SPRIS system, applied to data concerning the adaptation of four CPs to the COVID-19 pandemic, showed that, whereas all the CPs had a good performance index, some concerns remained unsolved and need be addressed. CONCLUSIONS: The SPRIS system showed to be an easily constructed tool that is able to give an overview of the organization analyzed by the survey and to produce an index that can be used in a direct quality comparison between different services or organizations.

Impact of the COVID-19 Pandemic on Clinical Pathways for Non-SARS-CoV-2 Related Diseases in the Lazio Region, Italy.

Clinical pathways (CPs) are multidisciplinary clinical governance tools necessary for the care management of the patients, whose aim is to outline the best practicable path within a health organization related to an illness or to a complex clinical situation. The COVID-19 pandemic emergency has created the need for an organizational renewal of care pathways based on the principles of "primary health care" recommended by the WHO. In Italy, the Hospitals and Local Health Authorities (ASL) have tried to guarantee the continuity of non-deferrable treatments and the maximum safety of both patients and health professionals. This study analyzes the organizational and managerial responses adopted in pathology-specific care pathways to assess how CPs as diagnostic tools responded to the COVID-19 pandemic in the first two waves. Twenty-four referents of Operational Units (UU OO) from Hospitals (AO) and Local Health Authorities (ASL) of the Lazio Region (Central Italy) that apply four different CPs responded to a survey, which analyzes the managerial and organizational responses of CPs in regard to different contexts. Results show that the structural and organizational adjustments of the CPs have made it possible to maintain an adequate level of care for specific treatment processes, with some common critical aspects that require improvement actions. The adjustments found could be useful for dealing with new outbreaks and/or new epidemics in order to try to mitigate the potential negative impact, especially on the most vulnerable patient categories.

Italian Association of Clinical Endocrinologists (AME) and International Chapter of Clinical Endocrinology (ICCE). Position statement for clinical practice: prolactin-secreting tumors.

Prolactinomas are the most frequent pituitary adenomas. Prolactinoma may occur in different clinical settings and always require an individually tailored approach. This is the reason why a panel of Italian neuroendocrine experts was charged with the task to provide indications for the diagnostic and therapeutic approaches that can be easily applied in different contexts. The document provides 15 recommendations for diagnosis and 54 recommendations for treatment, issued according to the GRADE system. The level of agreement among panel members was formally evaluated by RAND-UCLA methodology. In the last century, prolactinomas represented the paradigm of pituitary tumors for which the development of highly effective drugs obtained the best results, allowing to avoid neurosurgery in most cases. The impressive improvement of neurosurgical endoscopic techniques allows a far better definition of the tumoral tissue during surgery and the remission of endocrine symptoms in many patients with pituitary tumors. Consequently, this refinement of neurosurgery is changing the therapeutic strategy in prolactinomas, allowing the definitive cure of some patients with permanent discontinuation of medical therapy.

Normocalcemic primary hyperparathyroidism: an update.

Normocalcemic primary hyperparathyroidism (NPHPT) is diagnosed in the setting of elevated PTH concentrations with consistently normal albumin-adjusted and ionized serum calcium levels, in absence of secondary causes for elevated PTH concentrations. In order to confirm persistence of the hyperparathyroid state, PTH levels should be elevated on at least two occasions over a 3 to 6 months period. The prevalence of NPHPT depends on the population studied. Data from different studies are often not comparable; indeed, different criteria have been used to exclude secondary hyperparathyroidism. Notwithstanding such limits, the prevalence of NPHPT in studies including ionized calcium dosage was between 0.5% and 0.7%. Available data suggest that patients with NPHPT are likely to have more skeletal, kidney and metabolic complications compared to healthy subjects, but almost all studies suffer from possible misclassification of patients due to lack of ionized calcium dosage. The management of NPHPT is controversial in part due to lack of solid data about the natural history. However, surgical treatment is currently performed more frequently than in the past, although studies do not show, so far, a clear benefit from intervention.

Treatment of Acromegalic Osteopathy in Real-life Clinical Practice: The BAAC (Bone Active Drugs in Acromegaly) Study.

BACKGROUND: Vertebral fractures (VFs) are a frequent complication of acromegaly, but no studies have been so far published on effectiveness of antiosteoporotic drugs in this clinical setting. OBJECTIVE: To evaluate whether in real-life clinical practice bone active drugs may reduce the risk of VFs in patients with active or controlled acromegaly. STUDY DESIGN: Retrospective, longitudinal study including 9 tertiary care endocrine units. PATIENTS AND METHODS: Two hundred and forty-eight patients with acromegaly (104 males; mean age 56.00 ±â€…13.60 years) were evaluated for prevalent and incident VFs by quantitative morphometric approach. Bone active agents were used in 52 patients (20.97%) and the median period of follow-up was 48 months (range 12-132). RESULTS: During the follow-up, 65 patients (26.21%) developed incident VFs in relationship with pre-existing VFs (odds ratio [OR] 3.75; P < .001), duration of active acromegaly (OR 1.01; P = .04), active acromegaly at the study entry (OR 2.48; P = .007), and treated hypoadrenalism (OR 2.50; P = .005). In the entire population, treatment with bone active drugs did not have a significant effect on incident VFs (P = .82). However, in a sensitive analysis restricted to patients with active acromegaly at study entry (111 cases), treatment with bone active drugs was associated with a lower risk of incident VFs (OR 0.11; P = .004), independently of prevalent VFs (OR 7.65; P < .001) and treated hypoadrenalism (OR 3.86; P = .007). CONCLUSIONS: Bone active drugs may prevent VFs in patients with active acromegaly.

Rare Somatic MEN1 Gene Pathogenic Variant in a Patient Affected by Atypical Parathyroid Adenoma.

OBJECTIVE: Atypical parathyroid adenoma is a rare neoplasm, showing atypical histological features intermediate between classic benign adenoma and the rarest parathyroid carcinoma, whose the clinical behaviour and outcome is not yet understood or predictable. Up to date only two cases of atypical adenoma were found associated to a MEN1 syndrome, and only one was proved to carry a pathogenic variant of the MEN1 gene. DESIGN: We report the clinical, histologic, and molecular findings of a 44-year-old woman, presenting with a histologically proved atypical parathyroid adenoma with an apparent aggressive behaviour. METHODS AND RESULTS: CDC73 gene was screened at germline and somatic levels with no results. Whole exome sequencing performed on DNA extracted from blood leukocytes and tumour tissue revealed a somatic MEN1 gene heterozygous variant, c.912+1G > A, of the splicing donor site of exon 6. On immunohistochemistry, downregulation of the menin protein expression in the neoplastic cells was also observed. CONCLUSIONS: We report the second case of a rare association of a somatic MEN1 gene mutation in a patient with atypical parathyroid adenoma. We suggest that MEN1 gene could be an underestimate genetic determinant of these rare histological entities, and we highlight the utility of a complete genetic screening protocol, by the use of next-generation sequencing technology in such undetermined clinical cases with no frank clinical presentation.

MEN1 gene mutation with parathyroid carcinoma: first report of a familial case.

BACKGROUND: The occurrence of parathyroid carcinoma in multiple endocrine neoplasia type I (MENI) is rare and the 15 cases of malignant parathyroid tumor reported so far have been associated with MENI in individuals and not with multiple members within a family. METHODS: We report on a 61-year-old male, operated for a 7.3 cm parathyroid carcinoma infiltrating the esophagus. In his brother, a 4.6 cm parathyroid carcinoma was diagnosed histologically, while in the daughter, neck ultrasonography revealed 2 extrathyroidal nodules, yet to be excised. RESULTS: Screening of the MEN1 gene identified a known germline heterozygous missense mutation (c.1252G>A; p.D418N) in exon 9, in all affected subjects. CONCLUSIONS: The occurrence of parathyroid carcinoma in more than one affected member of a single MEN1 family represents the first reported familial case. This suggests that additional constitutional genetic mutations may contribute to the variation in malignant potential and clinical behavior of parathyroid tumors in MEN1.

Primary aldosteronism as a cause of secondary osteoporosis.

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.

Novel association of MEN1 gene mutations with parathyroid carcinoma.

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.

Vitamin D status in primary hyperparathyroidism: effect of genetic background.

Primary hyperparathyroidism (PHPT) is associated with hypovitaminosis D as assessed by serum total 25-hydroxyvitamin D (TotalD) levels. The aim of this study is to evaluate whether this is also the case for the calculated bioavailable 25-hydroxyvitamin D (BioD) or free 25-hydroxyvitamin D (FreeD), and whether the vitamin D status is influenced by genetic background. We compared vitamin D status of 88 PHPT patients each with a matched healthy family member sharing genetic background, i.e., first-degree relative (FDR), or not, namely an in-law relative (ILR). We compared TotalD and vitamin D-binding protein (DBP), using the latter to calculate BioD and FreeD. We also genotyped two common DBP polymorphisms (rs7041 and rs4588) likely to affect the affinity for and levels of vitamin D metabolites. TotalD was lower (p < 0.001) in PHPT (12.3 ± 6.6 ng/mL) than either family member group (FDR: 19.4 ± 12.1 and ILR: 23.2 ± 14.1), whether adjusted for DBP or not. DBP levels were also significantly lower (p < 0.001) in PHPT (323 ± 73 mg/L) versus FDR (377 ± 98) or ILR (382 ± 101). The differences between PHPT and control groups for TotalD, BioD, and FreeD were maintained after adjustment for season, gender, and serum creatinine. 25-hydroxyvitamin D, evaluated as total, free, or bioavailable fractions, is decreased in PHPT. No difference was seen between first-degree relative and in-law controls, suggesting that neither genetic nor non-genetic background greatly influences the genesis of the hypovitaminosis D seen in PHPT.

Somatostatin analogues according to Ki67 index in neuroendocrine tumours: an observational retrospective-prospective analysis from real life.

Somatostatin analogues (SSAs) have shown limited and variable antiproliferative effects in neuroendocrine tumours (NETs). Whether tumour control by SSAs depends on grading based on the 2010 WHO NET classification is still unclear. The aim of this study is to evaluate the efficacy of long-acting SSAs in NETs according to Ki67 index. An observational Italian multicentre study was designed to collect data in patients with gastro-entero-pancreatic or thoracic NETs under SSA treatment. Both retrospective and prospective data were included and they were analysed in line with Ki67 index, immunohistochemically evaluated in tumour samples and graded according to WHO classification (G1 = Ki67 index 0-2%, G2 = Ki67 index 3-20%, G3 = Ki67 index > 20%). Among 601 patients with NET, 140 with a histologically confirmed gastro-entero-pancreatic or thoracic NET or NET with unknown primary were treated with lanreotide autogel or octreotide LAR. An objective tumour response was observed in 11%, stability in 58% and progression in 31%. Objective response and tumour stability were not significantly different between G1 and G2 NETs. Progression free survival was longer but not significantly different in G1 than G2 NETs (median: 89 vs 43 months, p = 0.15). The median PFS was significantly longer in NETs showing Ki67 < 5% than in those showing Ki67 ≥ 5% (89 vs 35 months, p = 0.005). SSA therapy shows significant antiproliferative effects in well differentiated low/intermediate-proliferating NETs, not only G1 but also in G2 type. A Ki67 index of 5% seems to work better than 3% to select the best candidates for SSA therapy.

Increased prevalence of the GCM2 polymorphism, Y282D, in primary hyperparathyroidism: analysis of three Italian cohorts.

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.

Factors associated with vertebral fracture risk in patients with primary hyperparathyroidism.

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.

Identification and functional characterization of three NoLS (nucleolar localisation signals) mutations of the CDC73 gene.

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.

Over-supplementation of vitamin D in two patients with primary hyperparathyroidism.

OBJECTIVE: To describe the biochemical effects of an over-supplementation of vitamin D3 in two patients with primary hyperparathyroidism (PHPT). DESIGN: Two patients (A and B) with PHPT took erroneously 2,400,000 U (300,000 U/day for 8 days) and 4,500,000 U (300,000 U/day for 15 days) of cholecalciferol, respectively. They were followed for 4 months and ionized calcium, creatinine, PTH, 25 hydroxy-vitamin D, 1,25(OH)2D and urinary calcium/creatinine levels were measured. Finally, the patients were operated on and a parathyroid adenoma was removed in both. RESULTS: One week after the last dose of vitamin D, serum ionized calcium (iCa) rose from 1.35 to 1.41 mMol/L (n.r. 1.14-1.31) for patient A, and from 1.43 to 1.62 for patient B, while fasting urinary Calcium/Creatinine (uCa/Cr) augmented from 0.31 to 0.50 mg/mg, and from 0.32 to 0.55, respectively. During the follow-up, the average levels of iCa were 1.37 ± 0.03 and 1.48 ± 0.07 mMol/L, while those of uCa/Cr were 0.29 ± 0.13 and 0.32 ± 0.13, both iCa and uCa/Cr levels returning to baseline values within 4 months. CONCLUSIONS: The unintentional over-supplementation of vitamin D in the two PHPT patients caused a moderate and temporary increase of hypercalcemia and hypercalciuria and was not associated with clinical signs of toxicity.

CDC73 mutations and parafibromin immunohistochemistry in parathyroid tumors: clinical correlations in a single-centre patient cohort.

OBJECTIVE: To determine if molecular and immunohistochemical (IHC) features of the HRPT2/CDC73 gene and its product, parafibromin, predict the natural history of parathyroid malignancy, particularly atypical adenoma, as seen in a single-centre patient cohort. METHODS: Matched tumor and non-tumor tissues were obtained from 46 patients with parathyroid carcinoma (CA) (n = 15), atypical adenoma (AA) (n = 14) and typical adenoma (TA) (n = 17), as defined by standardized histopathological criteria. Exons and exon-intron boundaries of the CDC73 gene were sequenced to identify germline or somatic mutations. IHC staining for parafibromin was performed and scored as positive if nuclear staining was at least partially IHC-positive. RESULTS: Mutations of CDC73 were observed in 9/15 (60 %) CA, 2/14 (14 %) AA, and 1/17 (6 %) TA tumors. A recurrent two basepair mutation in exon 7 -- c.679_680delAG -- accounted for half of all identified mutations. Absence of parafibromin nuclear staining was noted in 8/12 (67 %) CA, 2/13 (15 %) AA, and 3/17 (18 %) TA tumors. Median follow up times were 88 months for CA, 76 months for AA, and 104 months for TA patients. One patient, a member of a previously reported multiplex family with a germline CDC73 mutation was found to have a second adenoma after removal of an atypical adenoma. CONCLUSIONS: Molecular screening and IHC are both useful tools in the differential diagnosis of parathyroid tumors, but both have limited sensitivity and specificity. CDC73 mutations and negative immunostaining were common in atypical adenomas, but no local recurrence was observed in any case with successful surgical removal after follow-up periods of 27 to 210 months.

CASR gene activating mutations in two families with autosomal dominant hypocalcemia.

BACKGROUND: Autosomal dominant hypocalcemia (ADH) is an endocrine disorder caused by activating mutations of the calcium-sensing receptor (CASR) gene which plays a major role in maintaining calcium homeostasis. Biochemical features of ADH are hypocalcemia and hypercalciuria with inappropriately low levels of parathyroid hormone (PTH). We report on two four-generation families affected by ADH. AIM: To identify mutations of CASR gene in subjects affected by familial idiopathic hypoparathyroidism. To perform functional assays of identified CASR variants by transient transfection on HEK293 cells. RESULTS: We identified two CASR variants (Q681R and P221L): the Q681R variant was novel while the P221L had been previously published. Functional assays on the Q681R variant showed that it did not alter the whole expression nor the correct plasmamembrane localization, but enhanced the signaling function, increasing the sensitivity of the receptor as compared to the WT. CONCLUSIONS: We report two activating CASR mutations in two families affected by ADH and the functional assays performed on the novel variant Q681R. Our work enlarged the spectrum of mutations of the CASR and contributed to a better elucidation of the protein function.

Bone involvement in aldosteronism.

In rats with aldosteronism, a reduction of bone mineral density (BMD) and cortical bone strength has been reported. Our study was aimed to evaluate bone involvement in patients with primary aldosteronism (PA). A total of 188 consecutive subjects with adrenal incidentaloma, observed between November 2009 and October 2011, were screened for PA with aldosterone-to-renin ratio. After confirmatory tests, in those who screened positive, 11 patients were diagnosed as PA and 15 patients were not (nPA). A serum/urinary biochemical profile, parathyroid hormone (PTH), BMD measured at lumbar spine (LS) and total and femoral neck (TN and FN) by dual X-ray absorptiometry, and conventional spinal radiographs (T(4) -L(4) ) were obtained in all subjects. PA patients had a significantly higher 24-hour urinary calcium (6.28 ± 1.85 versus 4.28 ± 1.18 mmol/d; p < 0.01), and PTH (9.8 [5.8-14.6], median [range] versus 5.3 [2.5-10.8] pmol/L; p < 0.01) than nPA patients. BMD expressed as Z-value at LS (-1.18 ± 0.99 versus 0.22 ± 1.12), FN (-0.85 ± 0.73 versus 0.01 ± 0.82), and TN (-0.49 ± 0.61 versus 0.39 ± 0.93) was lower in PA than in nPA (p = 0.003, p = 0.011, and p = 0.012, respectively). The prevalence of osteoporosis was higher in PA than in nPA (8/11, 72.7% versus 3/15, 20.0%; Fisher's exact test: p = 0.015). Vertebral fractures tended to be more prevalent in PA than in nPA (5/11, 45.5% versus 2/15, 13.3%; Fisher's exact test: p = 0.095). Logistic regression analysis showed that osteoporosis and morphometric vertebral fractures were associated with PA (odds ratio [OR], 15.4; 95% confidence interval [CI] = 1.83-130, p = 0.012; and OR, 30.4; 95%CI, 1.07-862, p = 0.045, respectively) regardless of age, body mass index (BMI), and LS-BMD. In 9 of 11 PA patients, 6 months after beginning of treatment (surgery or spironolactone) there was a significant reduction of urinary calcium excretion (p < 0.01) and PTH (p < 0.01), whereas in 5 of 11 PA patients, 1 year after beginning of treatment, BMD was significantly increased at LS, p < 0.01). In conclusion, PA is associated with osteoporosis, vertebral fractures, and increased urinary calcium excretion.