CYP24A1 and SLC34A1 genetic defects associated with idiopathic infantile hypercalcemia: from genotype to phenotype.

Loss of function mutations in the CYP24A1 gene, involved in vitamin D catabolism and in calcium homeostasis, are known to be the genetic drivers of both idiopathic infantile hypercalcemia (IIH) and adult renal stone disease. Recently, also defects in the SLC34A1 gene, encoding for the renal sodium-phosphate transporter NaPi-IIa, were associated with the disease. IIH typically affects infants and pediatric patients with a syndrome characterized by severe hypercalcemia, hypercalciuria, suppressed parathyroid hormone level and nephrolithiasis. In SLC34A1 mutated carriers, hypophosphatemia is also a typical biochemical tract. IIH may also persist undiagnosed into adulthood, causing an increased risk of nephrocalcinosis and renal complication. To note, a clinical heterogeneity characterizes IIH manifestation, principally due to the controversial gene-dose effect and, to the strong influence of environmental factors. The present review is aimed to provide an overview of the current molecular findings on the IIH disorder, giving a comprehensive description of the association between genotype and biochemical and clinical phenotype of the affected patients. We also underline that patients may benefit from genetic testing into a targeted diagnostic and therapeutic workflow.

The histone methyltransferase EZH2 is a therapeutic target in small cell carcinoma of the ovary, hypercalcaemic type.

Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a rare but aggressive and untreatable malignancy affecting young women. We and others recently discovered that SMARCA4, a gene encoding the ATPase of the SWI/SNF chromatin-remodelling complex, is the only gene recurrently mutated in the majority of SCCOHT. The low somatic complexity of SCCOHT genomes and the prominent role of the SWI/SNF chromatin-remodelling complex in transcriptional control of genes suggest that SCCOHT cells may rely on epigenetic rewiring for oncogenic transformation. Herein, we report that approximately 80% (19/24) of SCCOHT tumour samples have strong expression of the histone methyltransferase EZH2 by immunohistochemistry, with the rest expressing variable amounts of EZH2. Re-expression of SMARCA4 suppressed the expression of EZH2 in SCCOHT cells. In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to EZH2 shRNAs and two selective EZH2 inhibitors, GSK126 and EPZ-6438. EZH2 inhibitors induced cell cycle arrest, apoptosis, and cell differentiation in SCCOHT cells, along with the induction of genes involved in cell cycle regulation, apoptosis, and neuron-like differentiation. EZH2 inhibitors suppressed tumour growth and improved the survival of mice bearing SCCOHT xenografts. Therefore, our data suggest that loss of SMARCA4 creates a dependency on the catalytic activity of EZH2 in SCCOHT cells and that pharmacological inhibition of EZH2 is a promising therapeutic strategy for treating this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Comprehensive genomic profiling reveals inactivating SMARCA4 mutations and low tumor mutational burden in small cell carcinoma of the ovary, hypercalcemic-type.

OBJECTIVE: Small cell carcinoma of the ovary, hypercalcemic-type (SCCOHT) is a rare, extremely aggressive neoplasm that usually occurs in young women and is characterized by deleterious germline or somatic SMARCA4 mutations. We performed comprehensive genomic profiling (CGP) to potentially identify additional clinically and pathophysiologically relevant genomic alterations in SCCOHT. METHODS: CGP assessment of all classes of coding alterations in up to 406 genes commonly altered in cancer and intronic regions for up to 31 genes commonly rearranged in cancer was performed on 18 SCCOHT cases (16 exhibiting classic morphology and 2 cases exhibiting exclusive a large cell variant morphology). In addition, a retrospective database search for clinically advanced ovarian tumors with genomic profiles similar to SCCOHT yielded 3 additional cases originally diagnosed as non-SCCOHT. RESULTS: CGP revealed inactivating SMARCA4 alterations and low tumor mutational burden (TMB) (<6mutations/Mb) in 94% (15/16) of SCCOHT with classic morphology. In contrast, both (2/2) cases exhibiting only large cell variant morphology were hypermutated (TMB scores of 90 and 360mut/Mb) and were wildtype for SMARCA4. In our retrospective search, an index ovarian cancer patient harboring inactivating SMARCA4 alterations, initially diagnosed as endometrioid carcinoma, was re-classified as SCCOHT and responded to an SCCOHT chemotherapy regimen. CONCLUSION: The vast majority of SCCOHT demonstrate genomic SMARCA4 loss with only rare co-occurring alterations. Our data support a role for CGP in the diagnosis and management of SCCOHT and of other lesions with overlapping histological and clinical features, since identifying the former by genomic profile suggests benefit from an appropriate regimen and treatment decisions, as illustrated by an index patient.

SHORT syndrome with partial lipodystrophy due to impaired phosphatidylinositol 3 kinase signaling.

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.

Cytochrome P450-mediated metabolism of vitamin D.

The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field.

Chronic hypercalcaemia from inactivating mutations of vitamin D 24-hydroxylase (CYP24A1): implications for mineral metabolism changes in chronic renal failure.

BACKGROUND: Loss-of-function mutations of vitamin D-24 hydroxylase have recently been recognized as a cause of hypercalcaemia and nephrocalcinosis/nephrolithiasis in infants and adults. True prevalence and natural history of this condition are still to be defined. METHODS: We describe two adult patients with homozygous mutations and six related heterozygous carriers. Mineral and hormonal data in these patients were compared with that in 27 patients with stage 2-3 chronic kidney disease and 39 healthy adult kidney donors. RESULTS: Probands had recurrent nephrolithiasis, chronic hypercalcaemia with depressed parathyroid hormone (PTH) and increased 1,25(OH)(2)D levels; carriers had nephrolithiasis (two of six), hypercalciuria (two of six) and high or normal-high 1,25(OH)(2)D (four of four). Corticosteroids did not reduce plasma and urine calcium levels, but ketoconazole did, indicating that 1,25(OH)(2)D production is not maximally depressed despite coexisting hypercalcaemia, high 1,25(OH)(2)D and depressed PTH, and that 1,25(OH)(2)D degradation through vitamin D-24 hydroxylase is a regulator of plasma 1,25(OH)(2)D levels. Both probands had vascular calcifications and high bone mineral content. One developed stage 3b renal failure: in this patient 1,25(OH)(2)D decreased within normal limits as glomerular filtration rate (GFR) fell and PTH rose to high-normal values, yet hypercalcaemia persisted and the ratio of 1,25(OH)(2)D to GFR remained higher than normal for any degree of GFR. CONCLUSIONS: This natural model indicates that vitamin D-24 hydroxylase is a key physiologic regulator of calcitriol and plasma calcium levels, and that balanced reduction of 1,25(OH)(2)D and GFR is instrumental for the maintenance of physiologic calcium levels and balance in chronic kidney diseases.

Expression of 25-hydroxyvitamin D3-1alpha-hydroxylase in subcutaneous fat necrosis.

BACKGROUND: The most serious complication of subcutaneous fat necrosis (SCFN), a rare condition of the newborn characterized by indurated purple nodules, is hypercalcaemia. However, the mechanism for this hypercalcaemia remains unclear. OBJECTIVES: To determine whether the hypercalcaemia associated with SCFN involves expression of the vitamin D-activating enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase) in affected tissue. METHODS: Skin biopsies from two male patients with SCFN and hypercalcaemia were taken. The histological specimens were assessed using a polyclonal antibody against 1alpha-hydroxylase. RESULTS: Histology in both cases showed strong expression of 1alpha-hydroxylase protein (brown staining) within the inflammatory infiltrate associated with SCFN. This was consistent with similar experiments in other granulomatous conditions. CONCLUSIONS: Hypercalcaemia in SCFN appears to be due to abundant levels of 1alpha-hydroxylase in immune infiltrates associated with tissue lesions. This is consistent with previous observations of extrarenal 1alpha-hydroxylase in skin from other granulomatous conditions such as sarcoidosis and slack skin disease.

The detrimental role of inducible nitric oxide synthase in the pulmonary edema caused by hypercalcemia in conscious rats and isolated lungs.

We aim to test the hypothesis that hypercalcemia produces pulmonary edema (PE) and to elucidate the mechanism. Experimentations were carried out in conscious rats and isolated perfused rat lungs. We evaluated PE by lung weight changes, protein concentration in bronchoalveolar lavage, dye leakage, and microvascular permeability. Plasma nitrate/nitrite, methyl guanidine (MG), proinflammatory cytokines, procalcitonin levels, and histopathological examinations were evaluated. Immunochemical staining and reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in the lungs. Hypercalcemia was produced in the conscious rat and isolated perfused lungs. Calcitonin and L-N(6) (1-iminoethyl)-lysine (L-Nil) were administered before hypercalcemia to observe their effects. Hypercalcemia caused severe PE in rats. Pathological and immunochemical examinations revealed hemorrhagic edema with iNOS activity in the alveolar macrophages and epithelial cells. RT-PCR showed an increase in iNOS mRNA expression. Hypercalcemia increased nitrate/nitrite, MG, proinflammatory cytokines and procalcitonin levels. Pretreatment with calcitonin or L-Nil prevented these changes. In conclusion, hypercalcemia caused PE in conscious rats and isolated perfused rat lungs. The increases in nitrate/nitrite, free radicals, proinflammatory cytokines, procalcitonin and iNOS activity suggest that hypercalcemia induces a sepsis-like syndrome. The effect of hypercalcemia on the lung may involve iNOS and NO.

Cinacalcet Rectifies Hypercalcemia in a Patient With Familial Hypocalciuric Hypercalcemia Type 2 (FHH2) Caused by a Germline Loss-of-Function Gα11 Mutation.

G-protein subunit α-11 (Gα11 ) couples the calcium-sensing receptor (CaSR) to phospholipase C (PLC)-mediated intracellular calcium (Ca2+i ) and mitogen-activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss-of-function Gα11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα11 germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild-type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα11 , which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC-mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα11 proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα11 protein to impair CaSR-mediated Ca2+i and extracellular signal-regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα11 cleft region also impaired signaling by PLC. The loss-of-function associated with the Ser220 Gα11 mutant was rectified by treatment of cells with cinacalcet, which is a CaSR-positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα11 mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα11 germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα11 hydrophobic cleft region for CaSR-mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss-of-function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

In vitro adenylate cyclase-stimulating activity predicts the occurrence of humoral hypercalcemia of malignancy in nude mice.

A number of factors have been proposed as potential mediators of the syndrome of humoral hypercalcemia of malignancy (HHM), but to date no firm cause-and-effect relationship has been established. We attempted to establish such a relationship by determining whether the presence or absence of adenylate cyclase-stimulating activity (ACSA) in the media of cultured tumor cells predicted the occurrence of the syndrome of HHM when these cell lines were grown in nude mice in vivo. Conditioned media from 35 human renal carcinoma cell lines were surveyed for ACSA in the PTH-sensitive rat osteosarcoma 17/2.8 cell assay. 12 lines were positive (mean, 13.7-fold stimulation, range, 3.0 to 44.0), and 23 lines were negative (mean, 1.2-fold stimulation, range, 0.9 to 1.5). We were successful in establishing five of the positive and six of the negative lines in three to five nude mice per line. Mice implanted with the positive lines uniformly became hypercalcemic (mean serum calcium, 15.8 mg/dl), whereas mice implanted with the negative lines uniformly remained normocalcemic (mean serum calcium, 9.5 mg/dl), in spite of comparable mean tumor size. Acid-urea tumor extracts from each of four hypercalcemic animals contained potent in vitro ACSA (mean, 15.9-fold stimulation), while 5/5 extracts from normocalcemic animals did not (mean, 1.4-fold stimulation). Our study demonstrates that in this model system in vitro ACSA is a reliable predictive marker for HHM in vivo. Whether the protein responsible for this activity is also the mediator of the bone resorption seen in HHM remains to be demonstrated.

Insights into the pathological mechanisms of p85α mutations using a yeast-based phosphatidylinositol 3-kinase model.

In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdIns(4,5)P2 into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, ß, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85-p110 interactions typical of cancer.

Loss of SMARCA4 Expression Is Both Sensitive and Specific for the Diagnosis of Small Cell Carcinoma of Ovary, Hypercalcemic Type.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare ovarian neoplasm that occurs in young women and has a poor prognosis. The histologic diagnosis of SCCOHT can be challenging due to its rarity and relatively nonspecific histologic features, which range from the classic, first-described small cell morphology to a pattern in which there are large cells with abundant eosinophilic cytoplasm. Many entities can be in the differential diagnosis and to date, immunohistochemical stains have shown no distinctive profile and have been of limited aid. SMARCA4 (also known as BRG1) mutations have recently been reported at high frequency in these tumors. SMARCA4 is an important component of the SWI/SNF complex that regulates gene expression through alteration of nucleosome conformation. Studies to date have suggested that immunohistochemical loss of expression of SMARCA4 is associated with the presence of a SMARCA4 mutation in most cases. In this study, the sensitivity and specificity of the immunohistochemical loss of SMARCA4 expression for the diagnosis of SCCOHT is examined in the context of the differential diagnosis with other primary or metastatic ovarian tumors. All but one of the SCCOHT showed loss of SMARCA4 expression (16/17; 94%), while of 279 other tumors tested, only two tumors (one clear cell carcinoma and one ovarian melanoma) showed loss of SMARCA4 expression. We conclude that SMARCA4 immunohistochemistry is highly sensitive and specific for a diagnosis of SCCOHT and is of clinical utility in the differential diagnosis of poorly differentiated ovarian tumors.

Serum angiotensin-converting enzyme activity. Its use in the evaluation and management of hypercalcemia associated with sarcoidosis.

Serum angiotensin-converting enzyme (SACE) was measured in 14 patients (eight women and six men) with sarcoidosis and hypercalcemia. Thirteen patients were treated with prednisone, and 12 achieved normal or nearly normal serum calcium values. Two patients had coexistent hyperparathyroidism. Seven of eight patients with serial SACE measurements exhibited parallel falls in SACE and serum calcium levels. Eleven patients were successfully treated with alternate-day prednisone regimens. The data suggest that serial SACE measurements are useful in the evaluation and management of sarcoidosis with hypercalcemia. In patients with sarcoidosis, the reduction of SACE levels during glucocorticoid treatment may be due to a suppression of granuloma formation. Concomitant falls in serum calcium level suggest an important role of the granuloma or its cellular precursors in vitamin D metabolism.

Leiomyoblastoma associated with intractable hypercalcemia and elevated 1,25-dihydroxycholecalciferol levels. Treatment by hepatic enzyme induction.

A 42-year-old woman, with a previously resected jejunal leiomyoblastoma, was first seen with liver metastases 31/2 years after the tumor resection. Intractable malignant hypercalcemia appeared eight months later, together with renal insufficiency. No osteolytic lesions were detected. Levels of parathyroid hormone, cyclic adenosine monophosphate, and 1,25-dihydroxycholecalciferol (1,25[OH]2D) were not useful in distinguishing between the hypercalcemia of malignancy and concurrent hyperparathyroidism. Despite renal insufficiency, hypercalcemia, and subtotal parathyroidectomy, the 1,25(OH)2D levels remained elevated, consistent with the speculation that a tumor product stimulated 1-alpha-hydroxylation of 25-hydroxycholecalciferol. Phenytoin and phenobarbital (enzyme induction therapy), in combination with phosphorus and glucocorticoids, appeared to be useful in controlling the hypercalcemia.

Quantitation of CYP24A1 enzymatic activity with a simple two-hybrid system.

CONTEXT: Mutations of the CYP24A1 gene encoding the 24-hydroxylase (24OHase) that inactivates metabolites of vitamin D can cause hypercalcemia in infants and adults; in vitro assays of 24OHase activity have been difficult. OBJECTIVE: We sought an alternative assay to characterize a CYP24A1 mutation in a young adult with bilateral nephrolithiasis and hypercalcemia associated with ingestion of excess vitamin D supplements and robust dairy intake for 5 years. METHODS: CYP24A1 exons were sequenced from leukocyte DNA. Wild-type and mutant CYP24A1 cDNAs were expressed in JEG-3 cells, and 24OHase activity was assayed by a two-hybrid system. RESULTS: The CYP24A1 missense mutation L409S was found on only one allele; no other mutation was found in exons or in at least 30 bp of each intron/exon junction. Based on assays of endogenous 24OHase activity and of activity from a transiently transfected CYP24A1 cDNA expression vector, JEG-3 cells were chosen over HepG2, Y1, MA10, and NCI-H295A cells for two-hybrid assays of 24OHase activity. The apparent Michaelis constant, Km(app), was 9.0 ± 2.0 nM for CYP24A1 and 8.6 ± 2.2 nM for its mutant; the apparent maximum velocity, Vmax(app), was 0.71 ± 0.055 d(-1) for the wild type and 0.22 ± 0.026 d(-1) for the mutant. As assessed by Vmax/Km, the L409S mutant has 32% of wild-type activity (P = .0012). CONCLUSIONS: The two-hybrid system in JEG-3 cells provides a simple, sensitive, quantitative assay of 24OHase activity. Heterozygous mutation of CYP24A1 may cause hypercalcemia in the setting of excessive vitamin D intake, but it is also possible that the patient had another, unidentified CYP24A1 mutation on the other allele.

Increased level of immunoreactive neuron-specific enolase in thyroid C cells from dogs and guinea pigs after chronic hypercalcemia.

Immunocytochemical localization of neuron-specific enolase (NSE) in thyroid C cells was investigated in various mammalian species. In bovine thyroid glands most of the C cells were weakly immunoreactive to anti-NSE antiserum. In other mammalian species, including dogs, guinea pigs, rabbits, cats, pigs, rats, hamsters, mice, and monkeys, some C cells or only a few C cells were weakly immunoreactive to the antiserum. It seems that normal C cells contain NSE in small amounts only or are devoid of NSE. After chronically induced hypercalcemia, C cells revealed hypertrophic and hyperplastic features. Whereas immunoreactive calcitonin was markedly decreased, marked increase of immunoreactive NSE was observed in C cells of both dogs and guinea pigs; almost all C cells were filled with reaction product for NSE. After administration of ethylenethiourea for a period of 3-8 months, C cells revealed a marked decrease of secretory granules and appearance of vesicular inclusions of various sizes, which were immunoreactive to the calcitonin antiserum, indicating a disturbance of calcitonin synthesis. No immunoreactivity for NSE was observed in C cells from dogs and guinea pigs so treated. In rabbits showing hypocalcemic tetany, calcitonin immunoreactivity was very intense and NSE immunoreactivity was faint to negative in the C cells. Thus, the level of NSE in C cells was clearly connected with the functional activity of the cells.

Synthetic and partially-purified adenylate cyclase-stimulating proteins from tumors associated with humoral hypercalcemia of malignancy inhibit phosphate transport in a PTH-responsive renal cell line.

Hypophosphatemia and hyperphosphaturia characteristically occur in patients with humoral hypercalcemia of malignancy (HHM). To determine if a tumor product causes these abnormalities in phosphate metabolism, rather than, for example, hypercalcemia, we investigated the effect of partially-purified adenylate cyclase-stimulating activity (ACSA) from human and animal HHM-associated tumors on sodium-dependent phosphate transport (Na PiT) in a PTH-responsive renal epithelial cell line. Thirty minute exposure to 7 X 10(-10) MbPTH (1-34) equivalents of ACSA from the human and animal tumors, reduced NaPiT by 20% and 14%, respectively. We also recently isolated an adenylate cyclase-stimulating protein (hACSP) from two human tumors associated with HHM and identified a cDNA clone for this protein which encodes a 141 amino-acid peptide. Based on the deduced amino-acid sequence, we synthesized tyr36 (1-36) hACSP. This synthetic peptide induced a 22% decrease in the initial rate of NaPiT by the epithelial monolayer. Its inhibitory activity was roughly equipotent to that of bPTH (1-34). We conclude that the ACSP derived from HHM-associated tumors decreases phosphate transport in renal epithelial cells. This peptide appears to play a key role in mediating the changes in phosphate metabolism in this syndrome.

Kinetics of erythrocyte plasma membrane (Ca2+, Mg2+)ATPase in familial benign hypercalcemia.

The pathogenesis of familial benign hypercalcemia (FBH) is unknown. Possible explanations for the disorder include a set-point error in parathyroid gland regulation and intrinsic renal hyperreabsorption of calcium. Thus, FBH may involve an alteration in cellular calcium transport, especially in renal and parathyroid cells. A primary mediator of cellular calcium transport is (Ca2+,Mg2+)ATPase. Therefore, we examined in detail the kinetics of (Ca2+,Mg2+)ATPase activity in erythrocyte plasma membranes from 11 patients with FBH from 7 families, 5 patients with untreated primary hyperparathyroidism, and equal numbers of age- and sex-matched normal subjects. (Ca2+,Mg2+)ATPase activity was measured in isolated membranes as a function of free calcium (0.05-300 mumol/L) in the presence or absence of calmodulin (600 nmol/L) and as a function of calmodulin (0-1800 nmol/L). We found no significant differences in calcium- or calmodulin-dependent (Ca2+,Mg2+)ATPase kinetics between patients with FBH or primary hyperparathyroidism and their age- and sex-matched normal subjects. None of the kinetic parameters was correlated with serum calcium or serum PTH values. We postulate that a mechanism other than a global defect in (Ca2+,Mg2+)ATPase activity is responsible for the hypercalcemia in patients with FBH.

The bone isoenzyme of alkaline phosphatase in hypercalcaemic cancer patients.

Alkaline phosphatase (AP) is the classic marker of bone formation, especially in cancer patients, but the interpretation of its measurement is complicated by the existence of various circulating isoenzymes, especially of liver origin. The introduction of a mass measurement of the bone isoenzyme of AP (BAP) by an immunoradiometric assay has markedly improved the sensitivity and the specificity of the determination. We measured BAP and other markers of bone turnover in 46 patients with tumour-induced hypercalcaemia (TIH), which is an interesting model for evaluating markers of bone formation because of the uncoupling between bone formation and bone resorption found by histomorphometric techniques. The extent of bone metastatic involvement was evaluated by planimetry on bone scintigraphy. Mean (+/- S.D.) BAP concentrations were slightly higher in patients with TIH than in healthy subjects, 15.5 +/- 8.5 versus 12.4 +/- 3.5 micrograms/L (P < 0.05). However, the scatter of the data in TIH patients was quite marked. Increased values (10/46 patients, 22%) occurred only in patients with bone metastases. Total AP, gamma GT and BGP levels, as well as markers of bone resorption, were not significantly different between patients with or without bone metastases. BAP levels were significantly correlated with AP (rs = 0.63; P < 0.01) but not with BGP levels nor with markers of bone resorption. BAP levels were also correlated with the extent of bone uptake at scintigraphy (rs = 0.54; P < 0.01), but this was not the case for total AP or BGP. In the 36 patients re-evaluated when normocalcemic after pamidronate therapy, BAP levels increased from 16.3 +/- 9.2 to 22.2 +/- 21.3 micrograms/L (P < 0.05) but there were no significant changes in AP or BGP concentrations. In summary, our data confirm the existence of an uncoupling in bone turnover in TIH and indicate that cancer hypercalcaemia is another pathological condition characterised by a discordance between BAP and BGP concentrations. BAP levels appear to be a better reflection of bone metastatic involvement than total AP or BGP and their short-term increase after pamidronate therapy could reflect the recently described effects of bisphosphonates on osteoblasts.