Heterozygous inactivating CaSR mutations causing neonatal hyperparathyroidism: function, inheritance and phenotype.

BACKGROUND: Homozygous inactivating mutations of the calcium-sensing receptor (CaSR) lead to neonatal severe hyperparathyroidism (NSHPT), whereas heterozygous inactivating mutations result in familial hypocalciuric hypercalcemia (FHH). It is unknown why in some cases heterozygous CaSR mutations cause neonatal hyperparathyroidism (NHPT) clinically similar to NSHPT but with only moderately elevated serum calcium. METHODS: A literature survey was conducted to identify patients with heterozygous CaSR mutations and NHPT. The common NHPT CaSR mutants R185Q and R227L were compared with 15 mutants causing only FHH in the heterozygous state. We studied in vitro calcium signaling including the functional consequences of co-expression of mutant and wild-type (wt) CaSR, patients' phenotype, age of disease manifestation and mode of inheritance. RESULTS: All inactivating CaSR mutants impaired calcium signaling of wt-CaSR regardless of the patients' clinical phenotype. The absolute intracellular calcium signaling response to physiologic extracellular calcium concentrations in vitro showed a high correlation with patients' serum calcium concentrations in vivo, which is similar in NHPT and FHH patients with the same genotype. Pedigrees of FHH families revealed that paternal inheritance per se does not necessarily lead to NHPT but may only cause FHH. CONCLUSIONS: There is a significant correlation between in vitro functional impairment of the CaSR at physiologic calcium concentrations and the severity of alterations in calcium homeostasis in patients. Whether a particular genotype leads to NHPT or FHH appears to depend on additional predisposing genetic or environmental factors. An individual therapeutic approach appears to be warranted for NHPT patients.

Calcium-sensing receptor sequencing in 21 patients with idiopathic or familial parathyroid disorder: pitfalls and characterization of a novel I32 V loss-of-function mutation.

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor with a crucial role in calcium homeostasis. Mutations in the CaSR gene may lead to specific parathyroid disorders due to either gain-of-function (autosomal dominant hypercalciuric hypocalcemia; ADHH) or loss-of-function (familial hypocalciuric hypercalcemia; FHH). Our aim was to evaluate CaSR mutations as a cause of disease in selected patients. We identified and recruited patients with phenotypes suggestive of CaSR-related parathyroid disorders. DNA was extracted, and CaSR gene was sequenced. Live-ratiometric measurements of intracellular [Ca(2+)] and Western blot assays for evaluation of MAPK phosphorylation in response to changes in extracellular [Ca(2+)] were performed in transiently transfected HEK-293T cells to functionally characterize mutants. A total of 21 patients were evaluated, seven of them with idiopathic hypoparathyroidism (suspected ADHH) and 14 with hyperparathyroidism (suspected FHH). In the latter group two patients were found to harbor missense mutations: a novel heterozygous I32 V mutation in a female index case and a sporadic known R185Q mutation in a 1-year-old girl. In-vitro functional studies showed that I32 V is an inactivating mutation. In our study, most patients had normal CaSR sequencing. This suggests that phenotypic pitfalls may occur at time of patients' selection for CaSR sequencing. In one patient with strong positive pre-test probability based on both familial history and appropriate phenotype, a novel I32 V mutation leading to FHH was identified and characterized. In cases of familial parathyroid disorders, CaSR sequencing should be performed, but if negative, one should consider involvement of alternative genes or mechanisms.

Cinacalcet monotherapy in neonatal severe hyperparathyroidism: a case study and review.

CONTEXT: Neonatal severe hyperparathyroidism (NSHPT) is a severe form of familial hypocalciuric hypercalcemia characterized by severe hypercalcemia and skeletal demineralization. In most cases, NSHPT is due to biallelic loss-of-function mutations in the CASR gene encoding the calcium-sensing receptor (CaSR), but some patients have heterozygous mutations. Conventional treatment consists of iv saline, bisphosphonates, and parathyroidectomy. OBJECTIVE: The aim of this project was to characterize the molecular basis for NSHPT in an affected newborn and to describe the response to monotherapy with cinacalcet. METHODS: Clinical and biochemical features were monitored as cinacalcet therapy was initiated and maintained. Genomic DNA was obtained from the proband and parents. The CASR gene was amplified by PCR and sequenced directly. RESULTS: The patient was a full-term male who developed hypotonia and respiratory failure soon after birth. He was found to have multiple fractures and diffuse bone demineralization, with a marked elevation in serum ionized calcium (1.99 mmol/L) and elevated serum levels of intact PTH (1154 pg/mL); serum 25-hydroxyvitamin D was low, and fractional excretion of calcium was reduced. The serum calcium level was not reduced by iv saline infusion. Based on an extensive family history of autosomal dominant hypercalcemia, a diagnosis of NSHPT was made, and cinacalcet therapy was initiated with a robust and durable effect. Molecular studies revealed a heterozygous R185Q missense mutation in the CASR in the patient and his father, whereas normal sequences for the CASR gene were present in the patient's mother. CONCLUSIONS: We describe the first use of cinacalcet as monotherapy for severe hypercalcemia in a newborn with NSHPT. The rapid and durable response to cinacalcet suggests that a trial of calcimimetic therapy should be considered early in the course of NSHPT.

Molecular and clinical analysis of a neonatal severe hyperparathyroidism case caused by a stop mutation in the calcium-sensing receptor extracellular domain representing in effect a human 'knockout'.

OBJECTIVE: Loss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaR(R392X)), in effect a full CAR 'knockout'. CASE REPORT: The infant (daughter of distant cousins) presented with hypercalcaemia (5.5-6  mmol/l corrected calcium (2.15-2.65)) and elevated PTH concentrations (650-950  pmol/l (12-81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels. DESIGN AND METHODS: The R392X stop codon was inserted into human CAR and the resulting mutant (CaR(R392X)) expressed transiently in HEK-293 cells. RESULTS: CaR(R392X) expressed as a 54  kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaR(R392X)-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity. CONCLUSIONS: Intriguingly, the patient remained normocalcaemic throughout childhood (2.5 mM corrected calcium, 11 pg/ml PTH (10-71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.

Neonatal hyperparathyroidism with a heterozygous calcium-sensing receptor (CASR) R185Q mutation: clinical benefit from cinacalcet.

UNLABELLED: Neonatal hyperparathyroidism can be caused by a heterozygous inactivating mutation in the calcium-sensing receptor. Calcimimetics, allosteric activators of the calcium-sensing receptor, may provide an effective means of reducing PTH secretion in such patients. OBJECTIVE/PATIENT: The objective of the study was to identify the molecular defect and to monitor the postnatal course of a 1-wk-old infant with elevated blood ionized calcium, serum PTH, and alkaline phosphatase and low calcium excretion. The parents were normocalcemic. METHODS: CASR gene mutation analysis was performed on genomic DNA of the proband and her parents. The infant was treated initially with pamidronate and then cinacalcet. RESULTS: A heterozygous mutation (R185Q, CGA > CAA) in exon 4 of the CASR gene was identified in the proband. The CASR gene of both parents was normal. At 1 wk of age, iv fluids and furosemide were initiated, but hypercalcemia, hyperparathyroidism, and low calcium excretion persisted. At 2 wk of age, a single iv dose of pamidronate resulted in hypocalcemia and further increase in PTH levels, but hypercalcemia recurred within 1 wk. At 3 wk of age, a single oral dose of cinacalcet resulted in decreased PTH levels at 2 h; blood-ionized calcium reached a nadir at 10 h. Three days later daily cinacalcet was initiated, resulting in normalization of ionized calcium. The suppression of serum PTH and reduction in total serum calcium was maintained long term. CONCLUSIONS: In neonatal hyperparathyroidism secondary to presumed de novo heterozygous CASR mutation, treatment with cinacalcet decreases PTH secretion and serum calcium levels and mitigates the need for parathyroidectomy.

Naturally-occurring mutation in the calcium-sensing receptor reveals the significance of extracellular domain loop III region for class C G-protein-coupled receptor function.

CONTEXT: Inactivating mutations of the calcium-sensing receptor (CaSR) cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Most mutations are clustered in the N-terminal and Cys-rich regions of the extracellular domain (ECD) and seven-transmembrane domain. Disease-causing mutations are uncommon in the C terminus of ECD. OBJECTIVE: The aim of the study was to characterize the CaSR mutations causing neonatal severe hyperparathyroidism in a consanguineous family. METHODS: Parathyroid glands from the index patient were stained for CaSR protein. The CaSR gene was sequenced, mutations were recreated in CaSR cDNA, and HEK293 cells were transfected with the CaSR mutant expression vector. Cellular CaSR targeting was detected by immunoblotting and immunocytochemistry; CaSR activity was assayed by inositol phosphate accumulation, MAPK activation, and single-cell microfluorimetry. RESULTS: Immunocytochemistry showed reduced intracellular CaSR in patient parathyroids. An in-frame homozygous deletion/insertion mutation, c.1031 > 1034 (delACAAinsT), replaced His344-Asn345 with a single Leu in CaSR loop III. The mutant reduced cell surface expression of CaSR in transfected HEK293 cells. Inositol phosphate accumulation, MAPK activation, and single-cell microfluorimetry revealed blunted signaling responses of the mutant receptor to changes in extracellular Ca(2+) concentration. CONCLUSION: Deletion of His344-Asn345 in the ECD loop III region affects cell surface targeting of CaSR in transfected cells and in affected parathyroid glands. Absence of conserved Asn345 may interfere with CaSR folding or glycosylation, leading to poor protein targeting to the cell membrane. This loss-of-function mutant indicates that the ECD loop III is required for CaSR activity.

Ophthalmic features of hypoparathyroidism-retardation-dysmorphism.

Hypoparathyroidism-retardation-dysmorphism (HRD; Sanjad-Sakati Syndrome; Online Mendelian Inheritance in Man [OMIM] #241410) is a rare recessive syndrome predominantly seen on the Arabian Peninsula and characterized by congenital hypoparathyroidism, intrauterine growth retardation, mental retardation, seizures, and a typical facial dysmorphism (prominent forehead, deep-set eyes, and abnormal external ears).(1,2) To date, the same homozygous deletion in TBCE (155-166del) has been reported in all Saudi Arabian patients with HRD(1) as well as in all Saudi Arabian patients with Kenny-Caffey syndrome (OMIM #244460),(1) a syndrome with a phenotype that resembles that of HRD but is characterized by the presence of normal intelligence, late closure of the anterior fontanelle, macrocephaly, and postnatal (rather than prenatal) growth retardation.(1,3) Nanophthalmos and corneal opacity have been documented in Kenny-Caffey syndrome patients,(4) but ocular disease has not been well-described in HRD. We describe the ocular features of four Saudi Arabian HRD children referred to our institution for ocular complaints noted by their parents.

The role of the extracellular calcium-sensing receptor in health and disease.

The calcium-sensing receptor has a key role in calcium homeostasis, it is involved in the regulation of the serum calcium level within minutes via the secretion and action of parathyroid and the excretion of calcium in the kidney in a negative feedback manner. Mutations of the calcium sensing receptor gene leads to inactivating and activating mutations resulting in diseases with hypercalcaemia and hypocalcaemia. The loss of function mutations are associated with familial benign hypocalciuric hypercalcaemia (FHH), an autosomal dominant disease characterised by lifelong mild hypercalcaemia, low urinary calcium excretion, and inappropriate high parathyroid hormone levels, sometimes difficult to distinguish from mild asymptomatic primary hyperparathyroidism. Patients with FHH did not profit from parathyroidectomy, a calcium lowering therapy is not necessary. The gain of function mutations of the calcium-sensing receptor are associated with autosomal dominant hypocalcaemia (ADH), a disease characterised by a generally asymptomatic hypocalcaemia, inappropriately high urinary calcium excretion and normal PTH levels. A therapy to raise the serum calcium concentration has to be done carefully and is only indicated in symptomatic patients, because of enhancement of hypercalciuria with the risk of nephrocalcinosis and nephrolithiasis. Molecular genetic analysis of the calcium sensing receptor gene facilitates the sometimes difficult diagnosis. The development of compounds modulating the calcium sensing receptor function and thereby the section of PTH may become an important role in treatment of diseases of calcium metabolism.

A novel homozygous deletion in the calcium-sensing receptor ligand-binding domain associated with neonatal severe hyperparathyroidism.

Neonatal severe hyperparathyroidism (NSHPT) is a life-threatening disorder usually caused by homozygous mutations occurring in the calcium-sensing receptor (CaR) gene. We examined an infant hospitalised with NSHPT for mutations in the CaR gene using heterozygous sequence analysis and confirmed this result by a restriction enzyme assay. Clinical management of this case, which was beset by other complications, involved control of the hypercalcemia and the effects of hyperparathyroidism by a combination of treatments prior to parathyroidectomy performed at 10 months. Mutational analysis demonstrated a homozygous 5 base-pair deletion in the CaR gene located at the 5' end of exon 4 which would result in a severely truncated, non-functional receptor with only the first 164 amino acids of the CaR followed by 23 amino acids of aberrant sequence. This is the first report of an out-of-frame deletion in the extracellular domain of the CaR associated with clinical disease.

Rickets-like radiological and biochemical features of neonatal mucolipidosis II (I-cell disease): report of two cases.

In this paper, two cases with mucolipidosis type II (I-cell disease) (proven in one presenting newborn and presumed in an elder deceased brother) are presented. These infants showed severe skeletal changes with diffuse periosteal new bone formation in long bones and ribs, marked osteopenia, and resorption of scapula, clavicula, and mandible. There was also irregular demineralization of metaphyses of long tubular bones, as seen in rickets. The activities of serum alkaline phosphatase and parathyroid hormone were markedly elevated. Phosphorus was decreased. Serum 1,25-dihydroxyvitamin D was slightly elevated, but 25-hydroxyvitamin D and calcium were normal. Dysostosis multiplex resembling rickets and very high alkaline phosphatase activity were due to defective osteoblastic activity, but the mechanism of elevated parathyroid hormone was not clear. We conclude that early skeletal manifestation of mucolipidosis type II is not clearly identified and that differentiation from congenital rickets or congenital hyperparathyroidism could be difficult. It is speculated that hyperparathyroidism in these patients could be related to the calcium-sensing receptor malfunction in the parathyroid gland.

Transient congenital hypoparathyroidism and 22q11 deletion.

CATCH-22 syndrome represents a spectrum of abnormalities associated with microdeletions of chromosome 22q11. We report a patient with transient congenital hypoparathyroidism, with severe neonatal hypocalcemia and spontaneous resolution in infancy, tetralogy of Fallot and thymic hypoplasia. Genetic confirmation of chromosome 22q11 deletion was made. Newborns with congenital hypoparathyroidism need genetic analysis and examination for anomalies associated with CATCH-22 syndrome.

Neonatal severe hyperparathyroidism, secondary hyperparathyroidism, and familial hypocalciuric hypercalcemia: multiple different phenotypes associated with an inactivating Alu insertion mutation of the calcium-sensing receptor gene.

Neonatal severe hyperparathyroidism (NSHPT) is considered an autosomal-recessive disorder, attributable in many cases to homozygous inactivating mutations of the Ca++-sensing receptor (CASR) gene at 3q13.3-21. Most heterozygotes are clinically asymptomatic but manifest as familial (benign) hypocalciuric hypercalcemia (FHH) with a laboratory profile that is variably and sometimes only marginally different from normal. In 5 NSHPT cases from 3 Nova Scotian families, we found homoallelic homozygosity for an insertion mutation in exon 7 of CASR that includes an Alu repeat element with an exceptionally long polyA tract. Four of the 5 NSHPT infants were treated by parathyroidectomy more than a decade ago and are well now. A fifth went undiagnosed until adulthood and has profound musculoskeletal and neurobehavioral deficits. Among 36 identified FHH heterozygotes are 3 individuals with an unexpected degree of hypercalcemia and elevated circulating parathyroid hormone levels consistent with secondary hyperparathyroidism. Two are obligately heterozygous offspring of NSHPT mothers with surgical hypoparathyroidism and variable compliance with vitamin D therapy. The other is an adult with coexistent celiac disease in whom hyperparathyroidism, probably secondary to vitamin D deficiency, led to surgery. In counseling affected families, the heterozygous state should not be considered entirely benign, since FHH heterozygotes, particularly infants, may be prone to secondary hyperparathyroidism and symptomatic hypercalcemia. In such families, molecular diagnosis will allow for unambiguous identification of at-risk individuals.

Sensing of extracellular Ca2+ by parathyroid and kidney cells: cloning and characterization of an extracellular Ca(2+)-sensing receptor.

The ability of the parathyroid cell to sense minute fluctuations in the extracellular ionized calcium concentration (Ca2+ o) is essential for maintaining mineral ion homeostasis. However, the mechanism(s) through which the parathyroid cell and other cells recognize and respond to changes in Ca2+ o has remained unclear. We recently isolated a cDNA encoding a Ca2+ o-sensing receptor from bovine parathyroid using expression cloning in Xenopus laevis oocytes. The receptor shows pharmacologic properties that are almost identical to those of the receptor on the parathyroid cell and, like the latter, stimulates phospholipase C in a G-protein-dependent manner. The amino acid sequence of the cloned receptor deduced from this cDNA predicts a protein with a molecular mass of 121 kd, which has three principal structural domains. The first is a 613 amino acid, putatively extracellular amino terminus which has several regions rich in acidic amino acids that may potentially be involved in binding Ca2+ and other polycationic agonists. The second comprises seven membrane-spanning segments that are characteristic of the superfamily of G-protein-coupled receptors, and the third is a 222 amino acid cytoplasmic tail. Transcripts for this Ca2+ o-sensing receptor are present in the parathyroid as well as in the kidney, thyroid, and brain. We next investigated the hypercalcemic disorders, familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, as possible examples of inherited abnormalities in this Ca2+ o-sensing receptor, since both disorders show abnormal Ca2+ o-sensing and/or handling in the kidney and parathyroid.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.

We demonstrate that mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), two inherited conditions characterized by altered calcium homeostasis. The Ca(2+)-sensing receptor belongs to the superfamily of seven membrane-spanning G protein-coupled receptors. Three nonconservative missense mutations are reported: two occur in the extracellular N-terminal domain of the receptor; the third occurs in the final intracellular loop. One mutated receptor identified in FHH individuals was expressed in X. laevis oocytes. The expressed wild-type receptor elicited large inward currents in response to perfused polyvalent cations; a markedly attenuated response was observed with the mutated protein. We conclude that the mammalian Ca(2+)-sensing receptor "sets" the extracellular Ca2+ level and is defective in individuals with FHH and NSHPT.

[Severe neonatal primary hyperparathyroidism: presentation of a case and review of the literature]. / Hiperparatiroidismo primario neonatal severo: presentación de un caso y revisión de la literatura.

We present a newborn baby with a diagnosis of neonatal severe primary hyperparathyroidism, based on the concomitant presence of hypercalcemia, hypophosphatemia and elevated values of parathormone. A national and international review on the subject is made and the differential diagnosis of this rare entity is discussed.