Pseudohypoparathyroidism: Focus on Cerebral and Renal Calcifications.

CONTEXT: Pseudohypoparathyroidism (PHP) is a group of disorders characterized by hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone (PTH) levels as a result of end-organ resistance to PTH. OBJECTIVE: To describe a cohort of 26 patients with PHP followed in a single tertiary center. METHODS: Clinical, biochemical, radiological, and genetic analysis of the GNAS gene in 26 patients recruited since 2002. RESULTS: Ten patients harbored a GNAS mutation, 15 epigenetic abnormalities at the GNAS locus, and 1 did not show genetic or epigenetic abnormalities. According to clinical, biochemical, and genetic features, patients were classified as PHP1A, PHP1B, and pseudopseudohypoparathyroidism. Patients with PHP1A had an earlier diagnosis and more cases with family history, Albright hereditary osteodystrophy (AHO) features, hormonal resistance, and hypertension. Obesity was a common feature. No difference in biochemical values was present among PHP1A and PHP1B. Intracerebral calcification occurred in 72% of patients with no difference among PHP1A and PHP1B subgroups. No significant difference was observed between patients with and without intracerebral calcification for the time-weighted average values of total serum calcium, phosphate, calcium-phosphate product, and PTH fold increase. A borderline association between cerebral calcification and age at the time of diagnosis (P = .04) was found in the whole cohort of patients. No renal calcifications were found in the overall cohort. CONCLUSION: Patients with PHP1A more frequently have AHO features as well as hypertension than patients with PHP1B. Patients with PHP presented a high rate of intracerebral calcification with no significant difference between subgroups. No increased risk of renal calcifications was also found in the entire cohort.

Inactivating PTH/PTHrP signaling disorders (iPPSDs): evaluation of the new classification in a multicenter large series of 544 molecularly characterized patients.

OBJECTIVE: Pseudohypoparathyroidism and related disorders belong to a group of heterogeneous rare diseases that share an impaired signaling downstream of Gsα-protein-coupled receptors. Affected patients may present with various combination of symptoms including resistance to PTH and/or to other hormones, ectopic ossifications, brachydactyly type E, early onset obesity, short stature and cognitive difficulties. Several years ago we proposed a novel nomenclature under the term of inactivating PTH/PTHrP signaling disorders (iPPSD). It is now of utmost importance to validate these criteria and/or improve the basis of this new classification. DESIGN: Retrospective study of a large international series of 459 probands and 85 relatives molecularly characterized. METHODS: Information on major and minor criteria associated with iPPSD and genetic results were retrieved from patient files. We compared the presence of each criteria according to the iPPSD subtype, age and gender of the patients. RESULTS: More than 98% of the probands met the proposed criteria for iPPSD classification. Noteworthy, most patients (85%) presented a combination of symptoms rather than a single sign suggestive of iPPSD and the overlap among the different genetic forms of iPPSD was confirmed. The clinical and molecular characterization of relatives identified familial history as an additional important criterion predictive of the disease. CONCLUSIONS: The phenotypic analysis of this large cohort confirmed the utility of the major and minor criteria and their combination to diagnose iPPSD. This report shows the importance of having simple and easily recognizable signs to diagnose with confidence these rare disorders and supports a better management of patients.

A severe inactivating PTH/PTHrP signaling disorder type 2 in a patient carrying a novel large deletion of the GNAS gene: a case report and review of the literature.

PURPOSE: Pseudohypoparathyroidism (PHP), characterized by multihormone resistance and Albright's hereditary osteodystrophy (AHO), is caused by GNAS mutations. Whole or partial gene deletions are rare. All disorders due to inactivating mutations of the GNAS gene are now classified as "inactivating PTH/PTHrP signaling disorder type 2" (iPPSD2). This study reports a family harboring a large GNAS gene deletion in order to improve the knowledge of genotype-phenotype correlation of this disease. METHODS: An 18-year-old man with severe diffuse soft ossifications and multihormone resistance underwent to clinical, biochemical, radiological, and genetic studies. A review of the literature of other cases of iPPSD2 due to GNAS large deletions was performed focusing on clinical and biochemical features. RESULTS: The proband presented signs of hypocalcemia and marked AHO features. Laboratory tests revealed hypocalcemia, high levels of serum phosphate, PTH, TSH, and calcitonin despite therapy with calcium carbonate, calcitriol, and levothyroxine. Diffuse soft tissue ossifications and brain calcifications were shown by radiological exams. Family history was remarkable for hypocalcemia, neurocognitive impairment, and cerebral calcifications in his brother and AHO features in the maternal grandfather. The proband's mother showed short stature, whereas physical examination of the father was unremarkable. Genetic analysis of the GNAS gene revealed an unreported large deletion encompassing exons 1-7 in the proband, brother, and mother. By reviewing the literature, only six other cases were described. CONCLUSIONS: We report a kindred harboring a large GNAS deletion. A genotype-phenotype correlation was observed in term of severity of tissue ossifications in the siblings but not in the mother.

FGF23 and Fetuin-A Interaction and Mesenchymal Osteogenic Transformation.

Recently, we found a strict bone association between Fibroblast growth factor 23 (FGF23) and Fetuin-A, both involved in cardiovascular and mineral bone disorders. In this study, an uninvestigated bone marrow positivity for both was found. Though the role of exogenous FGF23 on mesenchymal cells (MSCs) was reported, no information is as yet available on the possible production of this hormone by MSCs. To further analyze these uninvestigated aspects, we studied human primary cells and mouse and human cell lines by means of immunostaining, qRT-PCR, enzyme linked immunosorbent assays, chromatin immunoprecipitation, transfection, and a streamlined approach for the FGF23⁻Fetuin-A interaction called Duolink proximity ligation assay. Mesenchymal cells produce but do not secrete FGF23 and its expression increases during osteo-differentiation. Fibroblast growth factor 23 is also involved in the regulation of Fetuin-A by binding directly to the Fetuin-A promoter and then activating its transcription. Both FGF23 overexpression and addition induced an upregulation of Fetuin-A in the absence of osteo-inducer factors. Fibroblast growth factor 23 and Fetuin-A promoter were increased by osteo-inducer factors with this effect being abolished after FGF23 silencing. In conclusion, both FGF23 and Fetuin-A are present and strictly linked to each other in MSCs with FGF23 driving Fetuin-A production. This mechanism suggests a role for these two proteins in the osteoblast differentiation.

Inactivating PTH/PTHrP Signaling Disorders.

Pseudohypoparathyroidism (PHP), pseudo-PHP, acrodysostosis, and progressive osseous heteroplasia are heterogeneous disorders characterized by physical findings, differently associated in each subtype, including short bones, short stature, a stocky build, ectopic ossifications (features associated with Albright's hereditary osteodystrophy), as well as laboratory abnormalities consistent with hormone resistance, such as hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone (PTH) and thyroid-stimulating hormone levels. All these disorders are caused by impairments in the cAMP-mediated signal transduction pathway and, in particular, in the PTH/PTHrP signaling pathway: the main subtypes of PHP and related disorders are caused by de novo or autosomal dominantly inherited inactivating genetic mutations, and/or epigenetic, sporadic, or genetic-based alterations within or upstream of GNAS, PRKAR1A, PDE4D, and PDE3A. Here we will review the impressive progress that has been made over the past 30 years on the pathophysiology of these diseases and will describe the recently proposed novel nomenclature and classification. The new term "inactivating PTH/PTHrP signaling disorder," iPPSD: (1) defines the common mechanism responsible for all diseases, (2) does not require a confirmed genetic defect, (3) avoids ambiguous terms like "pseudo," and (4) eliminates the clinical or molecular overlap between diseases.

Fibrocartilaginous mesenchymoma of bone: a single-institution experience with molecular investigations and a review of the literature.

AIMS: Fibrocartilaginous mesenchymoma is a rare intraosseous lesion, with a total of 26 cases described in the literature. This study describes the clinical, radiological and histological features of eight new cases of fibrocartilaginous mesenchymoma collected at a single institution between 1982 and 2016. The presence of GNAS and IDH1/2 mutations and MDM2 amplification was explored to evaluate possible links between fibrocartilaginous mesenchymoma, fibrous dysplasia, de-differentiated chondrosarcoma and low-grade osteosarcoma. METHODS AND RESULTS: Eight new cases of fibrocartilaginous mesenchymoma of bone identified in our archives, dating from 1982 to 2016, were reviewed. The diagnosis was not performed on the initial biopsy in any of these cases, due mainly to the absence of obvious cartilaginous differentiation. On imaging, the tumour contained cartilaginous calcifications and showed a very strong uptake of contrast medium after injection. Histologically, the tumour was characterized by spindle cell proliferation mimicking a low-grade spindle cell sarcoma, associated with epiphyseal growth-plate-like nodules of cartilage and bone production. Molecularly, no GNAS and IDH1/2 mutations or MDM2 amplification were found in the cases analysed. Only one case recurred 1 year following intralesional resection. None died of disease. CONCLUSIONS: This very rare bone tumour has a typical radiological and histological pattern and a favourable survival outcome after treatment. Local recurrences can be prevented with complete surgery. Fibrocartilaginous mesenchymoma does not seem to be related genetically to fibrous dysplasia, low-grade osteosarcoma and de-differentiated chondrosarcoma.

From pseudohypoparathyroidism to inactivating PTH/PTHrP signalling disorder (iPPSD), a novel classification proposed by the EuroPHP network.

OBJECTIVE: Disorders caused by impairments in the parathyroid hormone (PTH) signalling pathway are historically classified under the term pseudohypoparathyroidism (PHP), which encompasses rare, related and highly heterogeneous diseases with demonstrated (epi)genetic causes. The actual classification is based on the presence or absence of specific clinical and biochemical signs together with an in vivo response to exogenous PTH and the results of an in vitro assay to measure Gsa protein activity. However, this classification disregards other related diseases such as acrodysostosis (ACRDYS) or progressive osseous heteroplasia (POH), as well as recent findings of clinical and genetic/epigenetic background of the different subtypes. Therefore, the EuroPHP network decided to develop a new classification that encompasses all disorders with impairments in PTH and/or PTHrP cAMP-mediated pathway. DESIGN AND METHODS: Extensive review of the literature was performed. Several meetings were organised to discuss about a new, more effective and accurate way to describe disorders caused by abnormalities of the PTH/PTHrP signalling pathway. RESULTS AND CONCLUSIONS: After determining the major and minor criteria to be considered for the diagnosis of these disorders, we proposed to group them under the term 'inactivating PTH/PTHrP signalling disorder' (iPPSD). This terminology: (i) defines the common mechanism responsible for all diseases; (ii) does not require a confirmed genetic defect; (iii) avoids ambiguous terms like 'pseudo' and (iv) eliminates the clinical or molecular overlap between diseases. We believe that the use of this nomenclature and classification will facilitate the development of rationale and comprehensive international guidelines for the diagnosis and treatment of iPPSDs.

Quantitative analysis of methylation defects and correlation with clinical characteristics in patients with pseudohypoparathyroidism type I and GNAS epigenetic alterations.

CONTEXT: Pseudohypoparathyroidism type I (PHP-I) includes two main subtypes, PHP-Ia and -Ib. About 70% of PHP-Ia patients, who show Albright hereditary osteodystrophy (AHO) associated with resistance toward multiple hormones (PTH/TSH/GHRH/gonadotropins), carry heterozygous mutations in the α-subunit of the stimulatory G protein (Gsα) exons 1-13, encoded by the guanine nucleotide binding-protein α-stimulating activity polypeptide 1 (GNAS), whereas the majority of PHP-Ib patients, who classically display hormone resistance limited to PTH and TSH with no AHO sign, have methylation defects in the imprinted GNAS cluster. Recently methylation defects have been detected also in patients with PHP and different degrees of AHO, indicating a molecular overlap between the two forms. OBJECTIVES: The objectives of the study were to collect patients with the following characteristics: clinical PHP-I (with or without AHO), no mutation in Gsα coding sequence, but the presence of GNAS methylation alterations and to investigate the existence of correlations between the degree of the epigenetic defect and the severity of the disease. PATIENTS AND METHODS: We quantified GNAS methylation alterations by both PCR-pyrosequencing and methylation specific-multiplex ligation-dependent probe amplification assay in genomic DNA from 63 patients with PHP-I and correlated these findings with clinical parameters (age at diagnosis; calcium, phosphorus, PTH, TSH levels; presence or absence of each AHO sign). RESULTS: By both approaches, the degree of the imprinting defect did not correlate with the onset of the disease, the severity of endocrine resistances, or with the presence/absence of specific AHO signs. CONCLUSIONS: Similar molecular alterations may lead to a broad spectrum of diseases, from isolated PTH resistance to complete PHP-Ia, and the degree of methylation alterations does not reflect or anticipate the severity and the type of different PHP/AHO manifestations.

Autosomal dominant pseudohypoparathyroidism type Ib: a novel inherited deletion ablating STX16 causes loss of imprinting at the A/B DMR.

CONTEXT: Pseudohypoparathyroidism type Ib (PHP-Ib) is a rare imprinting disorder characterized by end-organ resistance to PTH and, frequently, to thyroid-stimulating hormone. PHP-Ib familial form, with an autosomal dominant pattern of transmission (autosomal dominant pseudohypoparathyroidism type Ib [AD-PHP-Ib]), is typically characterized by an isolated loss of methylation at the guanine nucleotide-binding protein α-stimulating activity polypeptide 1 A/B differentially methylated region (DMR), secondary to genetic deletions disrupting the upstream imprinting control region in the syntaxin-16 (STX16) locus. However, deletions described up to now failed to account some cases of patients with a methylation defect limited to the A/B DMR; thus, it is expected the existence of other still unknown rearrangements, undetectable with conventional molecular diagnostic methods. OBJECTIVE: We investigated a PHP-Ib patient with a methylation defect limited to the A/B DMR and no known STX16 deletions to find the underlying primary genetic defect. PATIENT AND METHODS: A PHP-Ib patient (hypocalcaemia, hyperphosphataemia, raised serum PTH levels, no vitamin D deficiency) and his unaffected relatives were investigated by methylation specific-multiplex ligand-dependent probe amplification to search for novel pathogenetic defects affecting the guanine nucleotide-binding protein α-stimulating activity polypeptide 1 and STX16 loci. RESULTS: We report the clinical, biochemical, and molecular analysis of an AD-PHP-Ib patient with a novel STX16 deletion overlapping with previously identified STX16 deletions but that, unlike these genetic defects associated with AD-PHP-Ib, goes unnoticed with commonly used first-level diagnostic techniques. CONCLUSIONS: Our work highlights the importance of performing accurate investigations in PHP-Ib patients with methylation defects to allow precise genetic counseling because, in case of deletions, the segregation ratio is about 50% and the disease phenotype is transmitted in an autosomal dominant fashion via the mother.

A new structural rearrangement associated to Wolfram syndrome in a child with a partial phenotype.

Wolfram syndrome (WS) is a rare autosomal recessive disorder characterized by diabetes insipidus (DI), insulin-dependent diabetes mellitus (DM), optic atrophy (OA) and deafness caused by mutations in WFS1 gene (4p16.1), which encodes an endoplasmic reticulum protein, called Wolframin. We describe the case of an infant who presented hypernatremia and severe hypoplasia of the left eyeball with alteration of visual evoked potentials. Persistent hypernatremia, iposmolar polyuria and high plasma osmolality suggested DI, confirmed by a normal urine concentration after vasopressin test. Treatment with vasopressin allowed a normalization of sodium levels and urine output. Brain magnetic resonance imaging showed absence of the neurohypophysis hyperintense signal, normal adenohypophysis and optic tracts hypoplasia. The concomitant presence of DI and OA, even in the absence of DM and deafness, prompted the suspicion of WS and complete genetic analysis was performed. Genomic DNA sequencing of WFS1 showed no inactivating mutations described to date, but suggested a structural mutation as markers genotyping revealed a segmental paternal heterodisomy involving the upstream regulatory region (promoter and 5'UTR). cDNA sequencing revealed the coexistence of the wild-type transcript and two splice variants; one variant, probably benign, is known in literature and the other one causes the loss of exon 2, containing the translation initiation site. Western blot confirmed a marked protein reduction. During the clinical follow-up child's condition remained stable and glucose metabolism is still in the standard. In conclusion, the phenotype associated with this structural rearrangement, which substantially reduces the synthesis of Wolframin, confirms a tissue-specific pattern of expression of WFS1, suggests the presence of a different protein dosage sensitivity in different tissues and could be causative of DI and OA in our patient. The "incomplete" phenotype here described, usually absent in typical WS cases, is explained by the residual Wolframin expression that would preserve other organs, i.e. pancreatic islets. A careful longitudinal clinical follow-up will assess any changes in the phenotypic penetrance in our patient.

Filamin-A is essential for dopamine d2 receptor expression and signaling in tumorous lactotrophs.

CONTEXT: Dopamine agonists (DA) are the first choice treatment of prolactinomas. However, a subset of patients is resistant to DA, due to undefined dopamine D2 receptor (D2R) alterations. Recently, D2R was found to associate with filamin-A (FLNA), a widely expressed cytoskeleton protein with scaffolding properties, in melanoma and neuronal cells. OBJECTIVE: The aim of the study was to investigate the role of FLNA in D2R expression and signaling in human tumorous lactotrophs and rat MMQ and GH3 cells. DESIGN: We analyzed FLNA expression in a series of prolactinomas by immunohistochemistry and Western blotting. We performed FLNA silencing or transfection experiments in cultured cells from DA-sensitive or -resistant prolactinomas and in MMQ and GH3 cells, followed by analysis of D2R expression and signaling. RESULTS: We demonstrated reduced FLNA and D2R expression in DA-resistant tumors. The crucial role of FLNA on D2R was demonstrated by experiments showing that: 1) FLNA silencing in DA-sensitive prolactinomas resulted in 60% reduction of D2R expression and abrogation of DA-induced inhibition of prolactin release and antiproliferative signals, these results being replicated in MMQ cells that endogenously express FLNA and D2R; and 2) FLNA overexpression in DA-resistant prolactinomas restored D2R expression and prolactin responsiveness to DA, whereas this manipulation was ineffective in GH3 cells that express FLNA but not D2R. No alteration in FLNA promoter methylation was detected, ruling out the occurrence of epigenetic FLNA silencing in DA-resistant prolactinomas. CONCLUSIONS: These data indicate that FLNA is crucial for D2R expression and signaling in lactotrophs, suggesting that the impaired response to DA may be related to the reduction of FLNA expression in DA-resistant prolactinomas.

Pseudohypoparathyroidism and GNAS epigenetic defects: clinical evaluation of albright hereditary osteodystrophy and molecular analysis in 40 patients.

CONTEXT: The two main subtypes of pseudohypoparathyroidism (PHP), PHP-Ia and -Ib, are caused by mutations in GNAS exons 1-13 and methylation defects in the imprinted GNAS cluster, respectively. PHP-Ia patients show Albright hereditary osteodystrophy (AHO) and resistance toward PTH and additional hormones, whereas PHP-Ib patients do not have AHO, and hormone resistance appears to be limited to PTH and TSH. Recently, methylation defects have been detected in few patients with PHP and mild AHO, indicating a molecular overlap between the two forms. OBJECTIVES: The aim of the study was to screen patients with clinically diagnosed PHP-Ia for methylation defects and to investigate the presence of correlations between the molecular findings and AHO severity. PATIENTS AND METHODS: We investigated differential methylation of GNAS regions and STX16 microdeletions in genomic DNA from 40 patients with sporadic AHO and multihormone resistance, with no mutations in Gsalpha-coding GNAS exons. RESULTS: Molecular analysis showed GNAS cluster imprinting defects in 24 of the 40 patients analyzed. No STX16 deletion was detected. The presence of imprinting defects was not associated with the severity of AHO or with specific AHO signs. CONCLUSIONS: We report the largest series of the literature of patients with clinical AHO and multihormone resistance and no mutation in the Gsalpha gene. Our findings of frequent GNAS imprinting defects further confirm the existence of an overlap between molecular and clinical features of PHP-Ia and PHP-Ib and highlight the necessity of a new clinical classification of the disease that takes into account the recent knowledge on the molecular basis underlying these defects.