BackgroundMapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward.ResultsWe report a simple but effective tool, the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT; http://www.flyrnai.org/diopt), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae. As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately 'cast a wide net' or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist).ConclusionsDIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.

In contrast to the regulation of calcium homeostasis, which has been extensively studied over the past several decades, relatively little is known about the regulation of phosphate homeostasis. Fibroblast growth factor 23 (FGF23) is part of a previously unrecognized hormonal bone-parathyroid-kidney axis, which is modulated by PTH, 1,25(OH)2-vitamin D (1,25(OH)2D), dietary and serum phosphorus levels. Synthesis and secretion of FGF23 by osteocytes are positively regulated by 1,25(OH)2D and serum phosphorus and negatively regulated, through yet unknown mechanisms, by the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and by dentin matrix protein 1 (DMP1). In turn, FGF23 inhibits the synthesis of 1,25(OH)2D, and it may negatively regulate the secretion of parathyroid hormone (PTH) from the parathyroid glands. However, FGF23 synergizes with PTH to increase renal phosphate excretion by reducing expression of the renal sodium-phosphate cotransporters NaPi-IIa and NaPi-IIc in the proximal tubules. Most insights gained into the regulation of phosphate homeostasis by these factors are derived from human genetic disorders and genetically engineered mice, which are reviewed in this paper.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare disorder of autosomal recessive inheritance that was first described in a large consanguineous Bedouin kindred. HHRH is characterized by the presence of hypophosphatemia secondary to renal phosphate wasting, radiographic and/or histological evidence of rickets, limb deformities, muscle weakness, and bone pain. HHRH is distinct from other forms of hypophosphatemic rickets in that affected individuals present with hypercalciuria due to increased serum 1,25-dihydroxyvitamin D levels and increased intestinal calcium absorption. We performed a genomewide linkage scan combined with homozygosity mapping, using genomic DNA from a large consanguineous Bedouin kindred that included 10 patients who received the diagnosis of HHRH. The disease mapped to a 1.6-Mbp region on chromosome 9q34, which contains SLC34A3, the gene encoding the renal sodium-phosphate cotransporter NaP(i)-IIc. Nucleotide sequence analysis revealed a homozygous single-nucleotide deletion (c.228delC) in this candidate gene in all individuals affected by HHRH. This mutation is predicted to truncate the NaP(i)-IIc protein in the first membrane-spanning domain and thus likely results in a complete loss of function of this protein in individuals homozygous for c.228delC. In addition, compound heterozygous missense and deletion mutations were found in three additional unrelated HHRH kindreds, which supports the conclusion that this disease is caused by SLC34A3 mutations affecting both alleles. Individuals of the investigated kindreds who were heterozygous for a SLC34A3 mutation frequently showed hypercalciuria, often in association with mild hypophosphatemia and/or elevations in 1,25-dihydroxyvitamin D levels. We conclude that NaP(i)-IIc has a key role in the regulation of phosphate homeostasis.

Calcitonin (CT) and parathyroid hormone (PTH), whose receptors belong to the same family of G proteincoupled receptors, share no amino acid sequence homology and selectively activate either CT or PTH receptors. We now show, however, that reciprocal hybrid ligands (CT/PTH and PTH/CT), which do not activate the "wildtype" receptors, activate PTH/CT and CT/PTH receptor chimeras, respectively. Our findings indicate that PTH and CT share a similar architecture with at least two functional, receptor-specific domains. These domains are sufficiently independent to permit synthetic hybrid ligands to efficiently activate appropriate receptor chimeras. Therefore, both ligands follow, despite their very different primary sequences, a common pattern of ligand-receptor interaction.The isolation of cDNAs encoding the receptors for secretin (1), calcitonin (CT) 1 (2), and parathyroid hormone (PTH)/PTHrelated peptide (PTHrP) (3, 4) established a new family of G protein-coupled receptors (GPRs) (5, 6). Members of this family, including some invertebrate receptors (7), 2,3 share, in addition to their overall structure with seven membrane-spanning helices, approximately 50 strictly conserved amino acids, including 8 important cysteines, an almost invariant amino acid sequence of the seventh membrane-spanning domain, and a similar intron/exon organization (10 -16). These findings suggest that this family of GPRs may have evolved from a common ancestral precursor. The ligands which activate these GPRs are similar in length, but lack, with the exception of the first 13 residues in PTH and PTHrP, any amino acid sequence homology. For both PTH-(1-34) and CT-(1-32), the importance of the amino terminus for bioactivity has been recognized, whereas the carboxyl-terminal portion contributes predominantly to receptor binding (17)(18)(19)(20)(21)(22)(23). Based on the limited data available, we and others had previously proposed that the carboxyl-terminal portion of these ligands determines specificity for the aminoterminal, extracellular receptor domains, while the amino termini of most ligands functionally interact with the membraneembedded receptor region (23-29). We have now tested this hypothesis more directly by constructing reciprocal chimeric ligands composed of portions of PTH and CT, and reciprocal chimeric receptors composed of portions of the PTH/PTHrP and CT receptor. These chimeras were designed such that the carboxyl-terminal portion of the ligand would match the aminoterminal, extracellular domain of the receptor, while the amino-terminal portion of the hybrid ligand would correspond to the membrane-embedded domains of the receptor and connecting loops. Our functional analysis of these hybrid ligands and chimeric receptors strengthens the proposed model of ligand-receptor interaction. Moreover, our studies imply that the receptors and their ligands are composed of functionally independent domains. ) were synthesized by the MGH Biopolymer Facility using Fmoc (N-(9-fluorenyl)methoxycarbonyl) technology (30). All peptides were H...

Maintenance of physiologic phosphate balance is of crucial biological importance, as it is fundamental to cellular function, energy metabolism, and skeletal mineralization. Fibroblast growth factor-23 (FGF-23) is a master regulator of phosphate homeostasis, but the molecular mechanism of such regulation is not yet completely understood. Targeted disruption of the Fgf-23 gene in mice (Fgf-23−/−) elicits hyperphosphatemia, and an increase in renal sodium/phosphate co-transporter 2a (NaPi2a) protein abundance. To elucidate the pathophysiological role of augmented renal proximal tubular expression of NaPi2a in Fgf-23−/− mice and to examine serum phosphate–independent functions of Fgf23 in bone, we generated a new mouse line deficient in both Fgf-23 and NaPi2a genes, and determined the effect of genomic ablation of NaPi2a from Fgf-23−/− mice on phosphate homeostasis and skeletal mineralization. Fgf-23−/−/NaPi2a−/− double mutant mice are viable and exhibit normal physical activities when compared to Fgf-23−/− animals. Biochemical analyses show that ablation of NaPi2a from Fgf-23−/− mice reversed hyperphosphatemia to hypophosphatemia by 6 weeks of age. Surprisingly, despite the complete reversal of serum phosphate levels in Fgf-23−/−/NaPi2a−/−, their skeletal phenotype still resembles the one of Fgf23−/− animals. The results of this study provide the first genetic evidence of an in vivo pathologic role of NaPi2a in regulating abnormal phosphate homeostasis in Fgf-23−/− mice by deletion of both NaPi2a and Fgf-23 genes in the same animal. The persistence of the skeletal anomalies in double mutants suggests that Fgf-23 affects bone mineralization independently of systemic phosphate homeostasis. Finally, our data support (1) that regulation of phosphate homeostasis is a systemic effect of Fgf-23, while (2) skeletal mineralization and chondrocyte differentiation appear to be effects of Fgf-23 that are independent of phosphate homeostasis.

The Brtl mouse model for type IV osteogenesis imperfecta improves its whole bone strength and stiffness between 2 and 6 months of age. This adaptation is accomplished without a corresponding improvement in geometric resistance to bending, suggesting an improvement in matrix material properties. Introduction:The Brittle IV (Brtl) mouse was developed as a knock-in model for osteogenesis imperfecta (OI) type IV. A Gly349Cys substitution was introduced into one col1a1 allele, resulting in a phenotype representative of the disease. In this study, we investigate the effect of the Brtl mutation on whole bone architecture, strength, and composition across a range of age groups. Materials and Methods: One-, 2-, 6-, and 12-month-old Brtl and wildtype (WT) mice were analyzed. Femurs were assessed at the central diaphysis for cortical geometric parameters using CT and were subsequently mechanically tested to failure by four-point bending. Matrix material properties were predicted using CT data to normalize data from mechanical tests. Raman spectroscopy and DXA were used to assess matrix composition. Results: Our findings show a postpubertal adaptation in which Brtl femoral strength and stiffness increase through a mechanism independent of changes in whole bone geometry. These findings suggest an improvement in the material properties of the bone matrix itself, rather than improvements in whole bone geometry, as seen in previous mouse models of OI. Raman spectroscopic results suggest these findings may be caused by changes in mineral/matrix balance rather than improvements in mineral crystallinity. Conclusions: Our findings parallel the currently unexplained clinical observation of decreased fractures in human OI patients after puberty. The Brtl mouse remains an important tool for investigating therapeutic interventions for OI.

Compound heterozygous and homozygous (comp/hom) mutations in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium (Na + )-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate wasting resulting in hypophosphatemia, correspondingly elevated 1,25(OH) 2 vitamin D levels, hypercalciuria, and rickets/osteomalacia. Similar, albeit less severe, biochemical changes are observed in heterozygous (het) carriers and indistinguishable from those changes encountered in idiopathic hypercalciuria (IH). Here, we report a review of clinical and laboratory records of 133 individuals from 27 kindreds, including 5 previously unreported HHRH kindreds and two cases with IH, in which known and novel SLC34A3 mutations (c.1357delTTC [p.F453del]; c.G1369A [p.G457S]; c.367delC) were identified. Individuals with mutations affecting both SLC34A3 alleles had a significantly increased risk of kidney stone formation or medullary nephrocalcinosis, namely 46% compared with 6% observed in healthy family members carrying only the wild-type SLC34A3 allele (P=0.005) or 5.64% in the general population (P,0.001). Renal calcifications were also more frequent in het carriers (16%; P=0.003 compared with the general population) and were more likely to occur in comp/hom and het individuals with decreased serum phosphate (odds ratio [OR], 0.75, 95% confidence interval [95% CI], 0.59 to 0.96; P=0.02), decreased tubular reabsorption of phosphate (OR, 0.41; 95% CI, 0.23 to 0.72; P=0.002), and increased serum 1,25(OH) 2 vitamin D (OR, 1.22; 95% CI, 1.05 to 1.41; P=0.008). Additional studies are needed to determine whether these biochemical parameters are independent of genotype and can guide therapy to prevent nephrocalcinosis, nephrolithiasis, and potentially, CKD.

Inorganic phosphate (P) is essential for signal transduction and cell metabolism, and is also an essential structural component of the extracellular matrix of the skeleton. P is sensed in bacteria and yeast at the plasma membrane, which activates intracellular signal transduction to control the expression of P transporters and other genes that control intracellular P levels. In multicellular organisms, P homeostasis must be maintained in the organism and at the cellular level, requiring an endocrine and metabolic P-sensing mechanism, about which little is currently known. This Review will discuss the metabolic effects of P, which are mediated by P transporters, inositol pyrophosphates and SYG1-Pho81-XPR1 (SPX)-domain proteins to maintain cellular phosphate homeostasis in the musculoskeletal system. In addition, we will discuss how P is sensed by the human body to regulate the production of fibroblast growth factor 23 (FGF23), parathyroid hormone and calcitriol to maintain serum levels of P in a narrow range. New findings on the crosstalk between iron and P homeostasis in the regulation of FGF23 expression will also be outlined. Mutations in components of these metabolic and endocrine phosphate sensors result in genetic disorders of phosphate homeostasis, cardiomyopathy and familial basal ganglial calcifications, highlighting the importance of this newly emerging area of research.