Caroline Silve

Delineating a CA2+ binding pocket within the venus flytrap module of the human calcium sensing receptor

The Ca2+-sensing receptor (CaSR) belongs to the class III G-protein-coupled receptors (GPCRs), which include receptors for pheromones, amino acids, sweeteners, and the neurotransmitters glutamate and γ-aminobutyric acid (GABA). These receptors are characterized by a long extracellular amino-terminal domain called a Venus flytrap module (VFTM) containing the ligand binding pocket. To elucidate the molecular determinants implicated in Ca2+ recognition by the CaSR VFTM, we developed a homology model of the human CaSR VFTM from the x-ray structure of the metabotropic glutamate receptor type 1 (mGluR1), and a phylogenetic analysis of 14 class III GPCR VFTMs. We identified critical amino acids delineating a Ca2+ binding pocket predicted to be adjacent to, but distinct from, a cavity reminiscent of the binding site described for amino acids in mGluRs, GABA-B receptor, and GPRC6a. Most interestingly, these Ca2+-contacting residues are well conserved within class III GPCR VFTMs. Our model was validated by mutational and functional analysis, including the characterization of activating and inactivating mutations affecting a single amino acid, Glu-297, located within the proposed Ca2+ binding pocket of the CaSR and associated with autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia, respectively, genetic diseases characterized by perturbations in Ca2+ homeostasis. Altogether, these data define a Ca2+ binding pocket within the CaSR VFTM that may be conserved in several other class III GPCRs, thereby providing a molecular basis for extracellular Ca2+ sensing by these receptors.

NHERF1 mutations and responsiveness of renal parathyroid hormone.

Impaired renal phosphate reabsorption, as measured by dividing the tubular maximal reabsorption of phosphate by the glomerular filtration rate (TmP/GFR), increases the risks of nephrolithiasis and bone demineralization. Data from animal models suggest that sodium-hydrogen exchanger regulatory factor 1 (NHERF1) controls renal phosphate transport. We sequenced the NHERF1 gene in 158 patients, 94 of whom had either nephrolithiasis or bone demineralization. We identified three distinct mutations in seven patients with a low TmP/GFR value. No patients with normal TmP/GFR values had mutations. The mutants expressed in cultured renal cells increased the generation of cyclic AMP (cAMP) by parathyroid hormone (PTH) and inhibited phosphate transport. These NHERF1 mutations suggest a previously unrecognized cause of renal phosphate loss in humans.

Recurrent PRKAR1A Mutation in Acrodysostosis with Hormone Resistance

The skeletal dysplasia characteristic of acrodysostosis resembles the Albrights hereditary osteodystrophy seen in patients with pseudohypoparathyroidism type 1a, but defects in the α-stimulatory subunit of the G-protein (GNAS), the cause of pseudohypoparathyroidism type 1a, are not present in patients with acrodysostosis. We report a germ-line mutation in the gene encoding PRKAR1A, the cyclic AMP (cAMP)-dependent regulatory subunit of protein kinase A, in three unrelated patients with acrodysostosis and resistance to multiple hormones. The mutated subunit impairs the protein kinase A response to stimulation by cAMP; this explains our patients hormone resistance and the similarities of their skeletal abnormalities with those observed in patients with pseudohypoparathyroidism type 1a.

PTHR1 mutations associated with Ollier disease result in receptor loss of function

PTHR1-signaling pathway is critical for the regulation of endochondral ossification. Thus, abnormalities in genes belonging to this pathway could potentially participate in the pathogenesis of Ollier disease/Maffucci syndrome, two developmental disorders defined by the presence of multiple enchondromas. In agreement, a functionally deleterious mutation in PTHR1 (p.R150C) was identified in enchondromas from two of six unrelated patients with enchondromatosis. However, neither the p.R150C mutation (26 tumors) nor any other mutation in the PTHR1 gene (11 patients) could be identified in another study. To further define the role of PTHR1-signaling pathway in Ollier disease and Maffucci syndrome, we analyzed the coding sequences of four genes (PTHR1, IHH, PTHrP and GNAS1) in leucocyte and/or tumor DNA from 61 and 23 patients affected with Ollier disease or Maffucci syndrome, respectively. We identified three previously undescribed missense mutations in PTHR1 in patients with Ollier disease at the heterozygous state. Two mutations (p.G121E, p.A122T) were present only in enchondromas, and one (p.R255H) in both enchondroma and leukocyte DNA. Assessment of receptor function demonstrated that these three mutations impair PTHR1 function by reducing either the affinity of the receptor for PTH or the receptor expression at the cell surface. These mutations were not found in DNA from 222 controls. Including our data, PTHR1 functionally deleterious mutations have now been identified in five out 31 enchondromas from Ollier patients. These findings provide further support for the idea that heterozygous mutations in PTHR1 that impair receptor function participate in the pathogenesis of Ollier disease in some patients.

Dominant-Negative GCMB Mutations Cause an Autosomal Dominant Form of Hypoparathyroidism

CONTEXTnHypoparathyroidism (HP) is characterized by low PTH levels, hypocalcemia, and hyperphosphatemia. Heterozygous mutations in pre-pro-PTH or the calcium-sensing receptor (CaSR) cause some forms of autosomal dominant HP (AD-HP). Furthermore, homozygous mutations in glial cells missing B (GCMB) have been implicated in autosomal recessive HP (AR-HP). In most other HP patients, however, the molecular defect remains undefined.nnnOBJECTIVEnOur objectives were to determine the genetic defect in the affected members of two unrelated families with AD-HP and define the underlying disease mechanism.nnnSUBJECTSnSeveral family members affected by AD-HP were investigated. The proband in family A had low calcium detected on routine blood testing, whereas the proband in family B had symptomatic hypocalcemia.nnnMETHODSnMutational analysis of the genes encoding pre-pro-PTH, CaSR, and GCMB was performed using PCR-amplified genomic DNA of the probands and other available members of each family. The identified GCMB mutants were characterized by Western blot analysis and luciferase reporter assay using DF-1 fibroblasts.nnnRESULTSnTwo novel heterozygous mutations located in the last GCMB exon (c.1389delT and c.1399delC in families A and B, respectively) were identified that both lead to frame-shifts and replacement of the putative second transactivation domain within carboxyl-terminal region by unrelated amino acid sequence. The mutant GCMB proteins were well expressed, and both showed dose-dependent inhibition of the transactivation capacity of wild-type protein in luciferase reporter assays.nnnCONCLUSIONSnThe dominant-negative effect observed in vitro for both GCMB mutations provides a plausible explanation for the impaired PTH secretion observed in the two unrelated families with AD-HP.

Abnormal sulfate metabolism in vitamin D-deficient rats.

To explore the possibility that vitamin D status regulates sulfate homeostasis, plasma sulfate levels, renal sulfate excretion, and the expression of the renal Na-SO4 cotransporter were evaluated in vitamin D-deficient (D-D-) rats and in D-D- rats rendered normocalcemic by either vitamin D or calcium/lactose supplementation. D-D- rats had significantly lower plasma sulfate levels than control animals (0.93+/-0.01 and 1.15+/-0.05 mM, respectively, P < 0.05), and fractional sulfate renal excretion was approximately threefold higher comparing D-D- and control rats. A decrease in renal cortical brush border membrane Na-SO4 cotransport activity, associated with a parallel decrease in both renal Na-SO4 cotransport protein and mRNA content (78+/-3 and 73+/-3% decreases, respectively, compared with control values), was also observed in D-D- rats. Vitamin D supplementation resulted in a return to normal of plasma sulfate, fractional sulfate excretion, and both renal Na-SO4 cotransport mRNA and protein. In contrast, renal sulfate excretion and renal Na-SO4 cotransport activity, protein abundance, and mRNA remained decreased in vitamin D-depleted rats fed a diet supplemented with lactose and calcium, despite that these rats were normocalcemic, and had significantly lower levels of parathyroid hormone and 25(OH)- and 1,25(OH)2-vitamin D levels than the vitamin D-supplemented groups. These results demonstrate that vitamin D modulates renal Na-SO4 sulfate cotransport and sulfate homeostasis. The ability of vitamin D status to regulate Na-SO4 cotransport appears to be a direct effect, and is not mediated by the effects of vitamin D on plasma calcium or parathyroid hormone levels. Because sulfate is required for synthesis of essential matrix components, abnormal sulfate metabolism in vitamin D-deficient animals may contribute to producing some of the abnormalities observed in rickets and osteomalacia.

GNAS-Related Loss-of-Function Disorders and the Role of Imprinting

GNAS (guanine nucleotide-binding protein, α stimulating) is a complex imprinted locus coding, besides the α-stimulatory subunit of the G protein, the paternally (extra-large, antisense and A/B) and maternally (neuroendocrine secretory protein) transcripts. Heterozygous mutations in the coding sequence of GNAS produce dominant phenotypes (combination of resistances to hormones signaling through G-protein-coupled receptors, osteodystrophy and obesity) that depend on the parental origin of the mutated allele. Likewise, alterations in the methylation at promoters of GNAS transcripts, associated or not with deletions of imprinting control regions in the nearby STX16 gene or within GNAS, prompt resistance to parathormone when affecting the maternal allele. Therefore, imprinting of GNAS is the determining factor for the variability of the phenotype. Knowledge of the various phenotypes is necessary for genetic counseling as well as an appropriate therapeutic balance between regular follow-up, prevention of disease complications and iatrogeny.

Long-Term Results of Continuous Subcutaneous Recombinant PTH (1-34) Infusion in Children with Refractory Hypoparathyroidism

Hypoparathyroidism in children is most often due to mutations in genes involved in parathyroid development and calcium homeostasis signaling. Some rare cases result from autoimmune attack on the parathyroid glands as a part of the type 1 polyglandular failure syndrome (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy). The majority of cases of pediatric hypoparathyroidism are well controlled under conventional treatment with calcium and vitamin D analogs. However, this treatment may be difficult to manage, especially in two situations: 1) in the context of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and 2) activating mutations in the calcium-sensing receptor. We successfully treated three patients with hypoparathyroidism with continuous subcutaneous administration of rhPTH(1-34) (recombinant human PTH(1-34)), two of which were refractory to conventional therapy.

Sodium-phosphate cotransporters, nephrolithiasis and bone demineralization.

Purpose of reviewWe discuss how recent findings obtained in disorders of phosphate metabolism in humans and in animal models have provided insights into the pathogenesis of renal stone formation and bone demineralization. Recent findingsMice that are null for the sodium-phosphate cotransporter (NPT)2a gene (NPT2a−/− mice) exhibit hypophosphataemia, increased urinary phosphate excretion, hypercalciuria and nephrolithiasis, but no bone demineralization. Mice null for the sodium-hydrogen exchanger regulatory factor (NHERF)1 (NHERF1−/− mice) also exhibit hypophosphataemia and increased renal phosphate excretion with decreased renal NPT2a expression, but they present with a severe sex-dependent bone demineralization. Heterozygous loss-of-function mutations in the NPT2a gene in humans induce hypophosphataemia, increased urinary phosphate excretion, hypercalciuria, nephrolithiasis in males (to date) and bone demineralization of variable severity in both sexes. Patients and experimental animals with increased circulating levels of fibroblast growth factor 23 present with hypophosphataemia, increased urinary phosphate excretion, inappropriate calcitriol synthesis and rickets/osteomalacia, but no nephrolithiasis except when treated. Low-phosphate diet in spontaneously hypercalciuric rats and disruption of the 1-α-hydroxylase gene in NPT2a−/− mice prevent renal stone formation. SummaryIncreased urinary phosphate excretion is a risk factor for renal calcium stone formation when it is associated with hypercalciuria. As yet undefined interplay between NPT2a, NHERF1 and possibly other cotransporters or associated proteins in bone cells may account for the diversity of bone phenotypes observed in disorders of phosphate metabolism with impaired renal phosphate reabsorption. The pathogenesis of both renal stone and bone demineralization appear to be affected by species, sex and mutation type, among other factors.

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

OBJECTIVEnDisorders 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.nnnDESIGN AND METHODSnExtensive 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.nnnRESULTS AND CONCLUSIONSnAfter 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.