Localization of familial benign hypercalcemia, Oklahoma variant (FBHOk), to chromosome 19q13.

Calcium homeostasis by the kidneys and parathyroids is mediated by the calcium-sensing receptor (CaSR), which is located on 3q21-q24 and belongs to family C of the superfamily of G-protein coupled receptors that includes those for metabotropic glutamate, certain pheromones, and gamma-amino butyric acid (GABA-B). Inactivating CaSR mutations result in familial benign hypercalcemia (FBH), or familial hypocalciuric hypercalcemia (FHH), whereas activating mutations result in hypocalcemic hypercalciuria. However, not all FBH patients have CaSR mutations, which, together with the mapping of another FBH locus to 19p13.3, suggests that additional CaSRs or second messengers may be involved. These may be identified by positional cloning, and we therefore performed a genomewide search, using chromosome-specific sets of microsatellite polymorphisms, in an Oklahoma family with an FBH variant (FBHOk), for which linkage to 3q and 19p had been excluded. Linkage was established between FBHOk and eight chromosome 19q13 loci, with the highest LOD score, 6.67 (recombination fraction.00), obtained with D19S606. Recombinants further mapped FBHOk to a <12-cM interval flanked by D19S908 and D19S866. The calmodulin III gene is located within this interval, and DNA sequence analysis of the coding region, the 5' UTR, and part of the promoter region in an individual affected with FBHOk did not detect any abnormalities, thereby indicating that this gene is unlikely to be implicated in the etiology of FBHOk. This mapping of FBHOk to chromosome 19q13 will facilitate the identification of another CaSR or a mediator of calcium homeostasis.     

Differentiating Familial Hypocalciuric Hypercalcemia From Primary Hyperparathyroidism

ObjectiveBecause the clinical features of familial hypocalciuric hypercalcemia (FHH) overlap significantly with those of primary hyperparathyroidism (PHPT), various means of differentiating between the two diseases have been suggested. Here we present a review of the clinical delineation of these two diseases.MethodsReview of the English language literature on FHH and PHPT.ResultsFHH is a rare genetic disorder generally resulting in asymptomatic hypercalcemia of minimal clinical consequence. It is easily misdiagnosed as PHPT because both entities can manifest as hypercalcemia with an inappropriately normal or elevated level of parathyroid hormone. The 2 disorders differ in renal processing of calcium, and a number of indices of renal calcium excretion have been proposed to differentiate the 2 entities. However, the two disorders have considerable overlaps in their ranges on these indices making differentiation a challenge. There are many mutations in the calcium-sensing receptor (CaSR) gene associated with FHH and it is becoming increasingly recognized that the CaSR has broad functional variability.ConclusionThe calcium:creatinine clearance ratio (CCCR) is the consensus biochemical test to differentiate between PHPT and FHH. However, this test is still limited by a considerable indeterminate range, and definitive diagnosis of FHH requires genetic testing. A combination of clinical suspicion, biochemical testing, and genetic analysis is required to differentiate PHPT from FHH and thus spare patients with FHH from nontherapeutic operative treatment. (Endocr Pract. 2013;19:697-702)     

A patient with type 2 diabetes mellitus associated with mutations in calcium sensing receptor gene and mitochondrial DNA

A 44-year-old female with familial hypocalciuric hypercalcemia (FHH) due to a homozygous missense mutation (Pro40Ala) in calcium sensing receptor (CaSR) gene has type 2 diabetes mellitus. The identical heterozygous mutation of CaSR gene was observed in consanguineous parents and all other family members examined except her two sisters. Many subjects with abnormal glucose tolerance were observed in this family, which is compatible with maternal inheritance. Mitochondrial function of complex I (NADH-coenzyme Q reductase) activity in cybrid cells between mitochondrial DNA (mtDNA)-deleted (rho(0)) HeLa cells and mtDNA from the proband was decreased by 35%. The proband has eight substitutions and among these 4833 A/G is a missense substitution in NADH dehydrogenase 2 gene and may probably be a major pathogenic mutation of impaired complex I activity. These results suggest that coexistence of nuclear gene and mtDNA mutations may have caused or modified the development of abnormal glucose tolerance in this family.     

Amino Alcohol- (NPS-2143) and Quinazolinone-Derived Calcilytics (ATF936 and AXT914) Differentially Mitigate Excessive Signalling of Calcium-Sensing Receptor Mutants Causing Bartter Syndrome Type 5 and Autosomal Dominant Hypocalcemia

Introduction

Activating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants.

Methods

All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca²⁺]_o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca²⁺]_o in the presence or absence of NPS-2143, ATF936 or AXT914.

Results

All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca²⁺]_o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants.

Conclusion

The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.     

Le récepteur du calcium : un rôle central dans le métabolisme calcique

The extracellular calcium-sensing receptor (CaSR) enables the parathyroid gland cells to sense the extracellular calcium concentration, to adapt the amount of parathyroid hormone secreted by those glands and, in turn, by its action on kidney and bone, to maintain steady the extracellular calcium concentration. The prominent role of CaSR is illustrated by the fact that CaSR mutations are responsible for disorders in extracellular calcium metabolism. Drugs that either activate or inactivate CaSR will open new therapeutic opportunities in several areas of mineral metabolism.     

Primary hyperparathyroidism and familial hypocalciuric hypercalcemia: relationships and clinical implications

ObjectiveTo discuss the unusual occurrence of both familial hypocalciuric hypercalcemia (FHH) and primary hyperparathyroidism in the same patient and to explore potential mechanisms of association and issues related to clinical management.MethodsWe discuss the diagnosis, compare the clinical presentations of FHH and primary hyperparathyroidism, review the literature regarding patients who have presented with both disorders, and discuss management considerations. We also describe 2 patients who have both FHH (confirmed by genetic testing for a mutation in the gene encoding the calcium-sensing receptor [CASR]) and primary hyperparathyroidism.ResultsThe occurrence of both FHH and primary hyperparathyroidism in the same patient has been reported in a few cases, including 2 patients described here, one of whom was documented to have a novel CASR mutation. Inthose with clinical sequelae of hyperparathyroidism, parathyroidectomy has led to reduction, but not normalization, of serum calcium levels.ConclusionsThe coexistence of FHH and primary hyperparathyroidism should be considered in patients with hypercalcemia, hypophosphatemia, frankly elevated parathyroid hormone levels, and low urinary calcium excretion. Genetic testing for inactivating CASR gene mutations can confirm the diagnosis of FHH. Although surgical intervention does not resolve hypercalcemia, it may be beneficial by reducing the degree of hypercalcemia, alleviating the symptoms, and preventing potential complications of hyperparathyroidism. (Endocr Pract. 2012;18:412-417)     

Mapping of the calcium-sensing receptor gene (CASR) to human Chromosome 3q13.3-21 by fluorescence in situ hybridization,and localization to rat Chromosome 11 and mouse Chromosome 16

The calcium-sensing receptor (CASR), a member of the G-protein coupled receptor family, is expressed in both parathyroid and kidney, and aids these organs in sensing extracellular calcium levels. Inactivating mutations in the CASR gene have been described in familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). Activating mutations in the CASR gene have been described in autosomal dominant hypoparathyroidism and familial hypocalcemia. The human CASR gene was mapped to Chromosome (Chr) 3q13.3-21 by fluorescence in situ hybridization (FISH). By somatic cell hybrid analysis, the gene was localized to human Chr 3 (hybridization to other chromosomes was not observed) and rat Chr 11. By interspecific backcross analysis, the Casr gene segregated with D16Mit4 on mouse Chr 16. These findings extend our knowledge of the synteny conservation of human Chr 3, rat Chr 11, and mouse Chr 16.     

A spectrum of mutations in the second gene for autosomal dominant polycystic kidney disease (PKD2).

Recently the second gene for autosomal dominant polycystic kidney disease (ADPKD), located on chromosome 4q21-q22, has been cloned and characterized. The gene encodes an integral membrane protein, polycystin-2, that shows amino acid similarity to the PKD1 gene product and to the family of voltage-activated calcium (and sodium) channels. We have systematically screened the gene for mutations by single-strand conformation-polymorphism analysis in 35 families with the second type of ADPKD and have identified 20 mutations. So far, most mutations found seem to be unique and occur throughout the gene, without any evidence of clustering. In addition to small deletions, insertions, and substitutions leading to premature translation stops, one amino acid substitution and five possible splice-site mutations have been found. These findings suggest that the first step toward cyst formation in PKD2 patients is the loss of one functional copy of polycystin-2.     

A case of hypocalciuric hypercalcemia without family history.

Familial hypocalciuric hypercalcemia (FHH) is usually characterized by asymptomatic hypercalcemia, mild hypermagnesemia, and low urinary calcium excretion, and is occasionally associated with pulmonary fibrosis. It is inherited as an autosomal-dominant, and no sporadic case of hypocalciuric hypercalcemia has been heretofore reported. This report describes a patient with hypocalciuric hypercalcemia completely compatible with FHH but with no family history, suggesting that the most likely diagnosis is "nonfamilial" hypocalciuric hypercalcemia. We propose that the urinary excretion of calcium be examined in all patients with hypercalcemia, hypophosphatemia, and increased PTH before neck surgery, even if patients have no family history of hypercalcemia.     

A novel <Emphasis Type="Italic">CASR</Emphasis> mutation in a Tunisian FHH/NSHPT family associated with a mental retardation

The calcium-sensing receptor (CASR), a plasma membrane G-protein coupled receptor, is expressed in parathyroid gland and kidney, and controls systemic calcium homeostasis. Inactivating CASR mutations have previously been identified in patients with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). The aim of the present study is to determine the underlying molecular defect of FHH/NSHPT disease in a consanguineous Tunisian family. Mutation screening was carried out using RFLP-PCR and direct sequencing. We found that the proband is homozygous for a novel 15 bp deletion in the exon 7 (c.1952_1966del) confirming the diagnosis of NSHPT. All the FHH members were found to be heterozygous for the novel detected mutation. The mutation, p.S651_L655del, leads to the deletion of 5 codons in the second trans-membrane domain of the CASR which is thought to be involved in the processes of ligand-induced signaling. This alteration was associated with the evidence of mental retardation in the FHH carriers and appears to be a novel inactivating mutation in the CASR gene. Our findings provide additional support for the implication of CASR gene in the FHH/NSHPT pathogenesis.     

Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release <Emphasis Type="Italic">in vivo</Emphasis>

Background  

The calcium sensing receptor (CaSR) regulates serum calcium by suppressing secretion of parathyroid hormone; it also regulates renal tubular calcium excretion. Inactivating mutations of CaSR raise serum calcium and reduce urine calcium excretion. Thyroid C-cells (which make calcitonin) express CaSR and may, therefore, be regulated by it. Since calcium stimulates release of calcitonin, the higher blood calcium caused by inactivation of CaSR should increase serum calcitonin, unless CaSR mutations alter the responsiveness of calcitonin to calcium.     

Excessive Signal Transduction of Gain-of-Function Variants of the Calcium-Sensing Receptor (CaSR) Are Associated with Increased ER to Cytosol Calcium Gradient

In humans, gain-of-function mutations of the calcium-sensing receptor (CASR) gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA) and reducing expression of Plasma Membrane Calcium-ATPase (PMCA). Wild-type CaSR (hCaSR-wt) and its gain-of-function (hCaSR-R990G; hCaSR-N124K) variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate) receptor inputs to cell function.     

Insertion of an Alu sequence in the Ca(2+)-sensing receptor gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.

Missense mutations in the calcium-sensing receptor (CaR) gene have previously been identified in patients with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). We studied family members of a Nova Scotian deme expressing both FHH and NSHPT and found, by PCR amplification of CaR gene exons, that FHH individuals were heterozygous and NSHPT individuals were homozygous for an abnormally large exon 7. This is due to an insertion at codon 877 of an Alu-repetitive element of the predicted-variant/human-specific-1 subfamily. It is in the opposite orientation to the CaR gene and contains an exceptionally long poly(A) tract. Stop signals are introduced in all reading frames within the Alu sequence, leading to a predicted shortened mutant CaR protein. The loss of the majority of the CaR carboxyl-terminal intracellular domain would dramatically impair its signal transduction capability. Identification of the specific mutation responsible for the FHH/NSHPT phenotype in this community will allow rapid testing of at-risk individuals.     

Identification of a novel inactivating R465Q mutation of the calcium-sensing receptor

In this study, we describe a 52-year-old woman, who was diagnosed with familial benign hypocalciuric hypercalcemia (FBHH), a condition characterized by hypercalcemia, low urinary calcium excretion, and normal parathyroid hormone PTH levels, resulting from inactivating mutations of the calcium-sensing receptor (CaSR). In order to identify and characterize the underlying mutation in the CASR gene, direct sequence analysis of CASR exons 2-7 was performed, and functional activity was examined by transient transfection of human embryonic kidney (HEK-293) cells with wild-type and mutant CaSRs, followed by intracellular calcium measurement using fluorometry, and Western blot analysis. Sequence analysis demonstrated, in addition to the already described A986S polymorphism, a novel heterozygous G--> A substitution in CASR exon 5 that causes an arginine to glutamine substitution at codon 465 (R465Q). Functional analysis showed a rightward shift of the dose-response curve with a significant increase of the EC50 from 5.4 mM of the CaSR carrying the A986S polymorphism alone to 11.3 mM of the CaSR carrying the R465Q mutation in the presence of the A986S polymorphism. Western blot analysis of membrane protein revealed an even higher expression level of the R465Q mutant protein compared to wild-type CaSR. In conclusion, we identified a novel heterozygous loss-of-function R465Q mutation of the CASR gene, which is characterized by a blunted response to calcium stimulation, thereby causing FBHH.     

The family of mammalian small heat shock proteins (HSPBs): implications in protein deposit diseases and motor neuropathies

A number of neurological and muscular disorders are characterized by the accumulation of aggregate-prone proteins and are referred to as protein deposit or protein conformation diseases. Besides some sporadic forms, most of them are genetically inherited in an autosomal dominant manner, although recessive forms also exist. Although genetically very heterogeneous, some of these diseases are the result of mutations in some members of the mammalian small heat shock protein family (sHSP/HSPB), which are key players of the protein quality control system and participate, together with other molecular chaperones and co-chaperones, in the maintenance of protein homeostasis. Thus, on one hand upregulation of specific members of the HSPB family can exert protective effects in protein deposit diseases, such as the polyglutamine diseases. On the other hand, mutations in the HSPBs lead to neurological and muscular disorders, which may be due to a loss-of-function in protein quality control and/or to a gain-of-toxic function, resulting from the aggregation-proneness of the mutants. In this review we summarize the current knowledge about some of the best characterized functions of the HSPBs (e.g. role in cytoskeleton stabilization, chaperone function, anti-aggregation and anti-apoptotic activities), also highlighting differences in the properties of the various HSPBs and how these may counteract protein aggregation diseases. We also describe the mutations in the various HSPBs associated with neurological and muscular disorders and we discuss how gain-of-toxic function mechanisms (e.g. due to the mutated HSPB protein instability and aggregation) and/or loss-of-function mechanisms can contribute to HSPB-associated pathologies. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

Familial benign hypercalcemia--from clinical description to molecular genetics.

Familial benign hypercalcemia (or familial hypocalciuric hypercalcemia), a syndrome of lifelong hypercalcemia inherited as an autosomal dominant trait, is distinct from the multiple endocrine neoplasia syndromes and other forms of inherited parathyroid disease. Familial benign hypercalcemia results from the inappropriate secretion of parathyroid hormone despite hypercalcemia, enhanced renal tubular reabsorption of calcium (independent of parathyroid hormone), and apparent tissue resistance to adverse effects of hypercalcemia. Heterozygosity for the familial hypercalcemia trait is benign, although homozygosity for the trait may lead to severe neonatal primary hyperparathyroidism. Genetic linkage studies show that most persons affected with familial hypercalcemia have a mutation on the long arm of chromosome 3 (3cen-q21), although one phenotypically indistinguishable family appears to have a mutation on the short arm of chromosome 19 (19p), and another family has neither 3q nor 19p mutations. One group has recently shown mutations in a putative parathyroid cell-surface calcium receptor that are plausible causes for the chromosome 3q variant of the familial hypercalcemia syndrome. Perhaps the other genes for this syndrome encode proteins representing hitherto-unknown regulators of systemic calcium metabolism independent of parathyroid cell calcium sensing or proteins involved in signal transduction from the calcium receptor.     

A gene mutated in nephronophthisis and retinitis pigmentosa encodes a novel protein,nephroretinin, conserved in evolution

Nephronophthisis (NPHP) comprises a group of autosomal recessive cystic kidney diseases, which constitute the most frequent genetic cause for end-stage renal failure in children and young adults. The most prominent histologic feature of NPHP consists of development of renal fibrosis, which, in chronic renal failure of any origin, represents the pathogenic event correlated most strongly to loss of renal function. Four gene loci for NPHP have been mapped to chromosomes 2q13 (NPHP1), 9q22 (NPHP2), 3q22 (NPHP3), and 1p36 (NPHP4). At all four loci, linkage has also been demonstrated in families with the association of NPHP and retinitis pigmentosa, known as “Senior-Løken syndrome” (SLS). Identification of the gene for NPHP type 1 had revealed nephrocystin as a novel docking protein, providing new insights into mechanisms of cell-cell and cell-matrix signaling. We here report identification of the gene (NPHP4) causing NPHP type 4, by use of high-resolution haplotype analysis and by demonstration of nine likely loss-of-function mutations in six affected families. NPHP4 encodes a novel protein, nephroretinin, that is conserved in evolution—for example, in the nematode Caenorhabditis elegans. In addition, we demonstrate two loss-of-function mutations of NPHP4 in patients from two families with SLS. Thus, we have identified a novel gene with critical roles in renal tissue architecture and ophthalmic function.     

Minireview: the intimate link between calcium sensing receptor trafficking and signaling: implications for disorders of calcium homeostasis

The calcium-sensing receptor (CaSR) regulates organismal Ca(2+) homeostasis. Dysregulation of CaSR expression or mutations in the CASR gene cause disorders of Ca(2+) homeostasis and contribute to the progression or severity of cancers and cardiovascular disease. This brief review highlights recent findings that define the CaSR life cycle, which controls the cellular abundance of CaSR and CaSR signaling. A novel mechanism, termed agonist-driven insertional signaling (ADIS), contributes to the unique hallmarks of CaSR signaling, including the high degree of cooperativity and the lack of functional desensitization. Agonist-mediated activation of plasma membrane-localized CaSR increases the rate of insertion of CaSR at the plasma membrane without altering the constitutive endocytosis rate, thereby acutely increasing the maximum signaling response. Prolonged CaSR signaling requires a large intracellular ADIS-mobilizable pool of CaSR, which is maintained by signaling-mediated increases in biosynthesis. This model provides a rational framework for characterizing the defects caused by CaSR mutations and the altered functional expression of wild-type CaSR in disease states. Mechanistic dissection of ADIS of CaSR should lead to optimized pharmacological approaches to normalize CaSR signaling in disorders of Ca(2+) homeostasis.     

Calcium-sensing receptor modulates cell adhesion and migration via integrins

The calcium-sensing receptor (CaSR) is a family C G protein-coupled receptor that is activated by elevated levels of extracellular divalent cations. The CaSR couples to members of the G(q) family of G proteins, and in the endocrine system this receptor is instrumental in regulating the release of parathyroid hormone from the parathyroid gland and calcitonin from thyroid cells. Here, we demonstrate that in medullary thyroid carcinoma cells, the CaSR promotes cellular adhesion and migration via coupling to members of the integrin family of extracellular matrix-binding proteins. Immunopurification and mass spectrometry, co-immunoprecipitation, and co-localization studies showed that the CaSR and β1-containing integrins are components of a macromolecular protein complex. In fibronectin-based cell adhesion and migration assays, the CaSR-positive allosteric modulator NPS R-568 induced a concentration-dependent increase in cell adhesion and migration; both of these effects were blocked by a specific CaSR-negative allosteric modulator. These effects were mediated by integrins because they were blocked by a peptide inhibitor of integrin binding to fibronectin and β1 knockdown experiments. An analysis of intracellular signaling pathways revealed a key role for CaSR-induced phospholipase C activation and the release of intracellular calcium. These results demonstrate for the first time that an ion-sensing G protein-coupled receptor functionally couples to the integrins and, in conjunction with intracellular calcium release, promotes cellular adhesion and migration in tumor cells. The significance of this interaction is further highlighted by studies implicating the CaSR in cancer metastasis, axonal growth, and stem cell attachment, functions that rely on integrin-mediated cell adhesion.     

Loss-of-function mutations in the keratin 5 gene lead to Dowling-Degos disease

Dowling-Degos disease (DDD) is an autosomal dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation of the flexures. We performed a genomewide linkage analysis of two German families and mapped DDD to chromosome 12q, with a total LOD score of 4.42 ( theta =0.0) for marker D12S368. This region includes the keratin gene cluster, which we screened for mutations. We identified loss-of-function mutations in the keratin 5 gene (KRT5) in all affected family members and in six unrelated patients with DDD. These represent the first identified mutations that lead to haploinsufficiency in a keratin gene. The identification of loss-of-function mutations, along with the results from additional functional studies, suggest a crucial role for keratins in the organization of cell adhesion, melanosome uptake, organelle transport, and nuclear anchorage.