Francesco Franceschelli

Influence of Calcium-Sensing Receptor Gene on Urinary Calcium Excretion in Stone-Forming Patients

Calcium-sensing receptor (CaSR) is a plasma membrane protein that regulates tubular reabsorption of Ca. To establish its role in idiopathic hypercalciuria, the association of urinary Ca excretion with the polymorphisms of CASR gene has been studied in healthy subjects and in hypercalciuric and normocalciuric Ca stone formers. CASR exon 7 single nucleotide polymorphisms (SNP), G/T at codon 986, G/A at codon 990, and C/G at codon 1011, were evaluated by PCR amplification and direct sequencing in 97 normocalciuric stone formers, 134 hypercalciuric stone formers, and 101 normocalciuric healthy controls. Four haplotypes were defined on the basis of CASR gene SNP: haplotype 1 was characterized by the most frequent sequence; haplotypes 2, 3, or 4 by the presence of a single polymorphic variant at codon 986, 990, or 1011, respectively. The relative risk of hypercalciuria was calculated with multinomial logistic regression and was significantly increased only in individuals carrying haplotype 3 (Odds ratio, 13.0 [95% confidence interval, 1.7 to 99.4]). Accordingly, Ca excretion was higher in subjects bearing haplotype 3, whereas those bearing haplotype 2 showed a slight increase of plasma Ca concentration. Multiple regression analysis showed that haplotype 3 explained 4.1% of the total variance of Ca excretion and 12.6% of the variance explained by the variables considered in the study. In conclusion, CASR gene could be a component of the complex genetic background regulating Ca excretion. Arg990Gly polymorphism could facilitate activation of CaSR and increase Ca excretion and susceptibility to idiopathic hypercalciuria.

Membrane binding sites and non-genomic effects of estrogen in cultured human preosteoclastic cells

Besides functional estrogen receptors, the presence of signalling cell surface binding sites for 17beta-estradiol (17betaE2) has been reported in osteoblast- and osteoclast-like cells, suggesting that 17betaE2 may influence bone remodelling by a dual mechanism of action: to affect gene expression mediated by the nuclear activity of the steroid-receptor complex, and to initiate rapid responses triggered by a signal-generating receptor on the cell surface. Recently, we demonstrated that the human pre-osteoclastic cell line FLG 29.1 bears functional estrogen receptors. In this study we examined FLG 29.1 cells for the presence of cell surface binding sites for 17betaE2, and whether 17betaE2 could elicit cell signalling. Using a cell-impermeant and fluorescent estrogen conjugate, 17beta-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, we demonstrated the presence of specific plasma membrane binding sites for 17betaE2. Stimulation of FLG 29.1 cells with low (1 nM) and high (1 microM) doses of 17betaE2 induced a prompt and significant (P < 0.05) increase of cellular pH, as measured in single cells using an image analysis system. In addition, both cAMP and cGMP were significantly increased by 17betaE2 with a dose-dependent response. Finally, a rapid increase of intracellular calcium ion concentration [Ca2+] was also induced by 1 nM 17betaE2, as measured in single cells using an image analysis system. Our findings strongly suggest a non-genomic action of 17betaE2 on osteoclast precursors.

A Novel Recessive Mutation of Fibroblast Growth Factor-23 in Tumoral Calcinosis

BACKGROUND Tumoral calcinosis is a rare disease characterized by hyperphosphatemia due to hypophosphaturia and by ectopic calcifications. Phosphatonins are important hormones that regulate phosphorus homeostasis. Tumoral calcinosis is a rare congenital disorder in which the differential diagnosis from other syndromes associated with extraskeletal calcifications may be difficult. Mutations in the UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-3 (GALNT3) and fibroblast growth factor-23 (FGF23) genes have been described. Mutational analysis is important for the early recognition of the disorder, for prevention of its complications, and for family screening strategies. We examined two unrelated white patients affected by tumoral calcinosis. METHODS The first patient was a woman with a history of an ectopic calcification in the left shoulder. The second patient was a man with a history of an ectopic calcification in the right buttock. Routine biochemistry and FGF-23 assays were performed on serum samples. Genomic DNA was extracted from peripheral blood. The FGF23 and GALNT3 genes were analyzed by direct sequencing. RESULTS A new homozygous H41Q codon 41, C-->A transversion at position 123 (c.123C>A) in exon 1 of the FGF23 gene was evidenced in both patients. No mutation of the GALNT3 gene was detected in these patients. As determined by an ELISA assay, intact FGF-23 circulating protein was low in both patients. CONCLUSIONS This is the fourth mutation of the FGF23 gene described in subjects with tumoral calcinosis.

Thyroid Cancer: Current Molecular Perspectives

The thyroid cancer is a rare oncological entity, representing no more than 1% of all human malignant neoplasms. Recently, it has been demonstrated a sharp increase in incidence of differentiated thyroid carcinoma, equally occurring in both sexes. So far, multiple genetic alterations have been identified in differentiated thyroid carcinoma, leading to investigate the clinical utility of genetic studies. In particular, molecular genetic approaches searching for gene mutations in the material collected by fine needle ago-biopsy may have a particular utility in small nodules and in those specimens with an indeterminate cytology. The expansion of knowledge about genetic mutations occurring in different thyroid tumors has characterized recent years, allowing the identification of a correlation between specific mutations and phenotypic characteristics of thyroid cancers, essential for their prognosis. This review will briefly report on the histological features and the new entity represented by thyroid microcarcinoma and will focus on both environmental and genetic aspects associated with the occurrence of thyroid cancer.

Calcium bioavailability from a calcium-rich mineral water, with some observations on method.

Goals The study was designed to determine whether high-calcium mineral water is an efficient additional source of dietary calcium, optimizing a method for calcium determination never used for mineral waters. Background It is generally agreed that an adequate calcium intake is necessary for the acquisition of an ideal peak bone mass and for the maintenance of the bone mineral density in adults, in postmenopausal women, and in the elderly. Mineral waters are calorie free, and some, with high calcium levels, might be significant sources of calcium. Study The availability of the calcium contained in a high-calcium mineral water was measured in 27 healthy subjects. In 8 subjects the calcium availability of the water was compared with the calcium availability ingested with milk at the same calcium load. Milk and water were labeled extrinsically with 30 mg 44Ca. Fractional absorption from the oral dose was determined from plasma samples using ICP-MS technique. Results At an ingested calcium load of 3.18 mmol, percentage of absorption for water averaged 22.53 ± 2.53 (mean ± SD) for men, 22.57 ± 2.10 (mean ± SD) for premenopausal women and 21.62 ± 3.12 (mean ± SD) for postmenopausal women. Percentage absorption from milk was 23.15 ± 4.06 (mean ± SD). Discussion The calcium from the mineral water is thus highly bioavailable, at least as bioavailable as milk calcium, and ICP-MS appears to represent a reliable and reproducible method for calcium absorption from alimentary sources.

Fibroblast growth factor 23 (FGF23) gene polymorphism in children with Kawasaki syndrome (KS) and susceptibility to cardiac abnormalities

BackgroundFibroblast Growth Factor (FGF) 23 influences endothelial integrity and few reports have studied the association between FGF23 and Kawasaki syndrome (KS), a childhood vasculitis displaying a high risk of subsequent cardiac abnormalities (CaA).AimTo investigate the genetic variation in the FGF23 gene in a cohort of KS children and its association with serum FGF23 levels and eventual development of CaA, including both coronary artery dilatations and aneurysms.Patients and methods84 Italian KS children were recruited; 24/84 (28.6%) developed CaA. Each patient underwent evaluation of serum FGF23 levels and FGF23 genotype: the frequency of the c.212-37insC (rs3832879) polymorphism in intron 1 was examined and compared with sex, age at disease onset, fever duration, laboratory data, and occurrence of CaA. Univariate statistical analysis of categorical parameters was performed by the Pearson’s Chi-square test or Fisher’s exact test as appropriate. Parametric variables were assessed by Student’s t-test for unpaired data. Independent predictors of disease were studied by a logistic regression model.Results28/84 patients carried the FGF23 polymorphism (33.3%) and had higher serum FGF23 levels (p < 0.01). FGF23 polymorphism was significantly associated with CaA compared to wild type FGF23 children (respectively, p = 0.03 and p = 0.05). The comparison with demographical, clinical or laboratory data was not significant.ConclusionsThe prevalent segregation of the c.212-37insC polymorphism in children with CaA advocates a possible functional FGF23 role in the predisposition to higher serum levels of FGF23 and potential occurrence of any coronary artery abnormalities in KS.

Aromatase expression and activity in the human leukaemic cell line FLG 29.1

The recent observation that estrogen synthesis occurs in osteoblast-like cells has suggested the aromatase activity as a possible local modulator of bone remodeling in post-menopausal women. To provide further insights into the androstenedione conversion to estrogen in bone-derived cells, we examined the human leukaemic cell line FLG 29.1, which is induced to differentiate toward the osteoclastic phenotype by TPA and TGF-beta1. Southern blot of RT-PCR products with a 32P-labeled cDNA probe for the human aromatase demonstrated that FLG 29.1 cells express aromatase mRNA. The enzyme activity, determined by measuring [3H]H2O release from [3H]androstenedione, obeyed Michaelis-Menten kinetic with apparent Km and Vmax values ranging from 5 to 10 nM and from 200 to 400 fmol/mg protein/6 h. Gene expression, enzyme activity and protein immunoreactivity, evaluated by immunocytochemistry, were stimulated in a time-dependent fashion by 5% charcoal-stripped FCS and by either 1-100 nM TPA or 0.01-0.5 ng/ml TGF-beta1, with maximal responses after 2-3 h exposure. After 24 h incubation of FLG 29.1 cells in the absence of these stimuli the aromatase mRNA and the protein were barely detectable. These findings demonstrate that cells of the osteoclastic lineage synthesize estrogen in vitro and that local cytokines, such as TGF-beta1, are able to induce androstenedione conversion.

Comparison of immuno- and HPLC-assays for the measurement of urinary collagen cross-links.

Pyridinoline (Pyr) and deoxypyridinoline (D-Pyr) are two cross-links of collagen molecules, that are present in the extracellular matrix and released during its degradation. Pyr is present in bone and cartilage, but not in significant amounts in other connective tissues and D-Pyr appears to be specific for bone tissue. Therefore, the urinary excretion of Pyr and D-Pyr might be a sensitive marker of bone matrix degradation. For the determination of urinary Pyr and D-Pyr two methods are available: a chromatographic method (HPLC) by which it is possible to measure separately Pyr and D-Pyr, and a new immunoassay which measures total free and low molecular weight pyridinoline released in the urine. We compared the results obtained by HPLC analysis of 205 urinary samples from normal subjects and patients affected by various bone disorders with those obtained by the immunoassay. The overall correlation coefficient between the results obtained by the two methods was 0.34. When calculated in a range of pyridinoline concentrations from 0 to 30, 30 to 60, and over 60 pmol/μmol creatinine the correlation coefficient was respectively − 0.094, 0.38, and 0.12. The two methods yielded variable profiles in the detection of circadian rhythms and these differences did not segregate with normal or pathological conditions. We conclude that the immunoassay proposed for the determination of urinary collagen cross-links is not immediately applicable to clinical use. The improvement of the antibody specificity will probably contribute to replace the HPLC method with the immunoassay.

Human Preosteoblastic Cell Culture from a Patient with Severe Tumoral Calcinosis-Hyperphosphatemia Due to a New GALNT3 Gene Mutation: Study of In Vitro Mineralization

Human disorders of phosphate (Pi) handling and skeletal mineralization represent a group of rare bone diseases. One of these disease is tumoral calcinosis (TC). In this study, we present the case of a patient with TC with a new GALNT3 gene mutation. We also performed functional studies using an in vitro cellular model. Genomic DNA was extracted from peripheral blood collected from a teenage Caucasian girl affected by TC, and from her parents. A higher capability to form mineralization nodules in vitro was found in human preosteoblastic cells of mutant when compared to wild-type controls. We found a novel homozygous inactivating splice site mutation in intron I (c.516-2a>g). A higher capability to form mineralization nodules in vitro was found in the mutant cells in human preosteoblastic cells when compared to wild-type controls. Understanding the functional significance and molecular physiology of this novel mutation will help to define the role of FGF23 in the control of Pi homeostasis in normal and in pathological conditions.

OP0276 Fibroblast growth factor (FGF23) gene polymorphism in kawasaki disease: A risk of coronary damage

Background Kawasaki disease may be complicated by coronary artery disease (CAD): genetic predisposition might play a role in the susceptibility both to KD and CAD. FGF23, a novel phosphaturic factor that influences phosphate renal reabsorption acts through FGF-receptor1 on the vasculature and heart. A study of our group (submitted work) has detected high serum FGF23 levels in all patienta with KD mainly in those with coronary artery involvement. Objectives To investigate FGF23 gene looking for possible mutations or polymorphisms responsible of abnormal serum FGF23 levels and evaluate a potential predisposition to CAD. Methods Genomic DNA extracted from peripheral blood and the 3 FGF23 exons, including the intron-exon boundary regions, were PCR-amplified and analyzed on ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA). The obtained sequences were compared to the wild type reference sequence of the FGF23 gene published on Genbank Database (NT-009759).Intact FGF23 was measured using an ELISA method (Immunotopics Inc. San Clemente, CA, USA). One hundred and eighteen KD Caucasian pts, 75M, 43F, median age 32.5 months were studied. Twenty eight out of 118 developed CAD. All underwent 2D-echocardiogram at admission, 15 days, 2, 6, 12 months. Pts with CAD were more closely controlled. Seventy-six sex-age-matched healthy children were used as controls, after clinical and laboratory exclusion of rheumatic, endocrinological and chronic renal diseases. Ethical Committee’s approval and informed consents by relatives were obtained. Pearson’s chi-square (χ2) analysis was performed to evaluate the distribution of FGF23 gene polymorphisms in the population. Analysis of covariance (ANCOVA), followed by LSD protected least significant difference, was performed to evaluate the correlation between FGF23 polymorphisms and serum FGF23 values. Results were expressed as means ±SEM. Results DNA analysis has shown a new C insertion in the intronic region between -36 and -37 nucleotide (rs3832879:NM_020638.2:c.212-37_212-36insC). Subjects without FGF23 polymorphism were indicated as WT, homozygous as pattern1 and heterozygous as pattern 2. To verify the frequency of the rs3832879 variant and evaluate the presence of polymorphic changes, DNA analysis of 100 Caucasian voluntaries confirmed this change. The χ2 analysis showed that 36.5% of the total population carried out the polymorphic site, and 63.5% not. Only 13.56% of KD pts without CAD had the FGF23 polymorphism, while it was present in 85.19% of pts with CAD (χ2: 41.2; df=1, p=0.001). ANCOVA analysis showed a statistically significant correlation between the presence of polymorphism and serum FGF23 levels. Pts WT had lower levels of serum FGF23 than heterozygotes and homozygotes (54.4±23 SD vs 51,9±28 and 135,3±35). All pts with FGF23 polymorphysm had higher FGF23 serum levels than those without (120±40 vs 38.2±5). Conclusions From our preliminary data the segregation of FGF23 genotype with the CD advocates a possible functional role of this new polymorphism in the predisposition to CAD in pts with KD. Disclosure of Interest None Declared