Orientador: Lilia F. R. de Souza Li / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T01:59:33Z (GMT). No. of bitstreams: 1 Andrade_SimoneCaixetade_M.pdf: 1991149 bytes, checksum: 4c5b783ab7776886c12977300fbb6af7 (MD5) Previous issue date: 2006 / Resumo: O CASR pertence à família C dos receptores que se acoplam à proteína G e é ativado quando interage com o cálcio extracelular, sendo responsável pelo ajuste do ¿set point¿ do cálcio extracelular por meio da regulação da secreção de PTH e excreção de cálcio. Mutações no Receptor Sensor de Cálcio (CASR) estão associadas a FHH (Hipercalcemia Hipocalciúrica Familiar) e NSHTP (Hiperparatireoidismo Neonatal Grave) quando inativadoras do receptor e ADH (Hipoparatireoidismo Autossômico Dominante) quando ativadoras. O Hiperparatireoidismo Neonatal Grave (NSHPT) é uma doença rara caracterizada por calcemias elevadas, próximas às consideradas incompatíveis à vida, associada ao aumento da concentração de PTH, desmineralização óssea grave e sintomas neonatais como hipotonia e baixo ganho ponderal. Trata-se de uma doença familiar, com pais portadores de Hipercalcemia Hipocalciúrica Familiar (FHH), uma doença autossômica dominante, geralmente assintomática, com calcemias elevadas ou no limite superior da normalidade, associada a concentrações de PTH normais, porém não suprimidas e hipocalciúria. ADH, por sua vez, cursam com desregulação no ajuste da concentração de cálcio extracelular, onde baixas concentrações de cálcio ativam o receptor e inibem a secreção de PTH pelas paratireóides e aumentam a excreção de cálcio pelos rins. Indivíduos afetados apresentam hipocalcemia, PTH no limite inferior ou abaixo do normal, hiperfosfatemia e hipercalciúria. O objetivo desse trabalho foi estudar duas famílias portadoras de NSHPT e FHH, identificar novas mutações e analisar o grau de expressão dos receptores mutados. Identificamos três mutações pontuais, nas posições c.1913.G>T, c.2.T>G e c.2244.C>G. Na família S encontramos a mutação c.1913.G>T que resulta em mudança de aminoácido Arginina por Leucina na posição 638 e a mutação silenciosa c.2244.C>G que não altera o aminoácido Prolina da posição 748. Na família J encontramos a mutação c.2.T>G que resulta em mudança do primeiro aminoácido Metionina e em perda da seqüência Kozak (AXXATGG). Um programa de análise para a previsão de seqüências utilizadas para início da tradução protéica, indicou que, na presença da mutação, o ATG com maior probabilidade de ser utilizado como o novo sítio de início de tradução localiza-se no exon 3, na mesma matriz de leitura original. Para análise da expressão do receptor, com a mutação no códon inicial de transcrição do receptor (p.M1?), inserimos no cDNA do CASR um fragmento correspondente à região -226 a 66 do CASR, contendo cinco potenciais seqüências Kozak. Para o estudo da expressão dos receptores mutados da família S inserimos as mutações no cDNA do CASR através de mutagênese sítio dirigida. Analisamos a expressão dos receptores mutados através do Western Blot. O receptor mutado p.R638L apresentou uma expressão similar ao receptor nativo e foram visualizadas as formas monoméricas correspondentes às bandas de 140kDa (forma imatura, parcialmente glicosilada) e 160kDa (forma madura e glicosilada) e bandas superiores maiores que 220kDa. A mutação c.2244.C>G é silenciosa e apresentou expressão similar à do receptor nativo. Em contraste, a expressão do receptor mutado p.M1? através do Western blot estava consideravelmente reduzida. Nos experimentos de imunocitoquímica, observamos que o receptor nativo foi bem expresso na superfície celular tanto em células não permeabilizadas, quanto permeabilizadas. Padrão semelhante foi observado para o receptor mutado p.R638L, indicando maturação e processamento apropriado no retículo endoplasmático enquanto que o receptor com a mutação p.M1? não foi visualizado na superfície celular de células não permeabilizadas e só foi identificado no interior de células permeabilizadas, sugerindo que o receptor mutado era retido no retículo endoplasmático não conseguindo se expressar na membrana plasmática / Abstract: The CASR belongs to family C of the G protein coupled receptors and it is activated by the interaction with extracellular calcium, which is responsible for adjusting extracellular calcium set point adjusting PTH and calcium excretion. Calcium Sensing Receptor mutations are related to Familial Hypocalciuric Hypercalcemia (FHH) and Neonatal Severe Hyperparathyroidism (NSHPT) when inactivating and to Autosomal Dominant Hypocalcemia (ADH) when activating. Neonatal Severe Hyperparathyroidism (NSHPT) is a rare disease characterized by hypercalcemia, calcium levels close to those incompatible with life, markedly elevated PTH levels, severe bone demineralization and neonatal symptoms as hypotonia and poor weight gain. Familial Hypocalciuric Hypercalcemia is a familial disease with Autosomal dominant inheritance, in which parents are usually affected, generally asymptomatic, mild ¿ to ¿ moderate hypercalcemia and normal PTH levels (but not suppressed) and hypocalciuria. In ADH, affected individuals¿ present hypocalcemia, PTH at the lower limit or normal range, hyperphosphatemia and hypercalciuria. The objective of this work was study of two families (S and J) with Neonatal Severe Hyperparathyroidism and Familial Hypocalciuric Hypercalcemia, search for new mutations and analyze the expression pattern of mutated receptors. Three new missense mutations were found: c.1913.G>T, c.2.T>G and c.2244.C>G. The mutation c.1913.G>T was identified at family S. and resulted in Arginine to Leucine change at codon 638. The silent mutation c.2244.C>G didn¿t change the amino acid Proline at codon 748. A novel mutation in exon 2, T to G transition at nucleotide 2, changing Metionine to Arginine was identified at family M. The mutation disrupts the original Kozak sequence (AXXATGG), altering the protein start site. Computational analysis using a program that predicts start sites showed that the putative new translation start site was in the exon 3 in frame. A portion of the gene containing the mutation and five cryptic Kozak sequences (-226 to 66) was used to analyze the expression of the mutant receptor (p.M1?). To analyze the expression pattern of Family S, the mutated cDNAs was inserted in a vector, using site direct mutagenesis. Western blot was performed to analyze the expression analysis of the mutated receptors. The p.R638L receptor showed similar expression pattern compared with the wild type receptor, presenting the monomeric forms of 140 (immature, partial glycosylated) and 160kDa (mature, glycosylated) and other forms higher than 200kDa. The mutation c.2244.C>G showed similar expression pattern compared with the wild type receptor. In contrast, Western blot expression levels of the mutant receptor p.M1? was dramatically reduced. Immunocytochemistry experiments showed strong staining at the cell surface of nonpermeabilized and permeabilized HEK293 cells expressing the wild type receptor. The same pattern was observed for the mutant receptor p.R638L, suggesting correct maturation and trafficking. While the mutant receptor p.M1? was not expressed on the cell surface and the staining was only identified inside permeabilized cells, suggesting that the mutant receptor was trapped within the endoplasmatic reticulum and was not expressed at the plasmatic membrane / Mestrado / Mestre em Farmacologia

Familial primary hyperparathyroidism (HPT) is a rare disorder that is treated surgically and mostly occurs in association with tumor-susceptibility syndromes, like multiple endocrine neoplasia and the hyperparathyroidism-jaw tumor syndrome. Familial hypercalciuric hypercalcemia (FHH) is another cause of hereditary hypercalcemia that generally is considered to require no treatment and is genetically and pathophysiologically distinct from HPT. Inactivating mutations in the calcium receptor gene cause FHH, whereas the down-regulated expression of the CaR in HPT never has been coupled to CaR gene mutations. Family screening revealed a hitherto unknown familial condition with characteristics of both FHH and HPT. The hypercalcemia was mapped to a point mutation in the intracellular domain of the CaR gene that was coupled to relative calcium resistance of the PTH release by transient expression in HEK 294 cells. Unusually radical excision of parathyroid glands was required to normalise the hypercalcemia. The mildly enlarged parathyroid glands displayed hyperplasia with nodular components. Frequent allelic loss on especially 12q was found and contrasts to findings in HPT. Allelic loss was also seen in loci typical for primary HPT like 1p, 6q and 15q, but not 11q13. Quantitative mRNA analysis showed that the glands had mild increase in a proliferation index (PCNA/GAPDH mRNA ratio) and mild reduction in genes important to parathyroid cell function, like CaR, PTH, VDR and LRP2. A previously unrecognized variant of hypercalcemia is explored that could be one explanation for persistent hypercalcemia after apparently typical routine operations for HPT. It also raises the issue of possibilities to treat FHH with parathyroidectomy provided it is radical enough.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 176-216).

<p>Familial primary hyperparathyroidism (HPT) is a rare disorder that is treated surgically and mostly occurs in association with tumor-susceptibility syndromes, like multiple endocrine neoplasia and the hyperparathyroidism-jaw tumor syndrome. Familial hypercalciuric hypercalcemia (FHH) is another cause of hereditary hypercalcemia that generally is considered to require no treatment and is genetically and pathophysiologically distinct from HPT. Inactivating mutations in the calcium receptor gene cause FHH, whereas the down-regulated expression of the CaR in HPT never has been coupled to CaR gene mutations. </p><p>Family screening revealed a hitherto unknown familial condition with characteristics of both FHH and HPT. The hypercalcemia was mapped to a point mutation in the intracellular domain of the CaR gene that was coupled to relative calcium resistance of the PTH release by transient expression in HEK 294 cells. Unusually radical excision of parathyroid glands was required to normalise the hypercalcemia. The mildly enlarged parathyroid glands displayed hyperplasia with nodular components. Frequent allelic loss on especially 12q was found and contrasts to findings in HPT. Allelic loss was also seen in loci typical for primary HPT like 1p, 6q and 15q, but not 11q13. Quantitative mRNA analysis showed that the glands had mild increase in a proliferation index (PCNA/GAPDH mRNA ratio) and mild reduction in genes important to parathyroid cell function, like CaR, PTH, VDR and LRP2. </p><p>A previously unrecognized variant of hypercalcemia is explored that could be one explanation for persistent hypercalcemia after apparently typical routine operations for HPT. It also raises the issue of possibilities to treat FHH with parathyroidectomy provided it is radical enough.</p>

Familial primary hyperparathyroidism (HPT) is a rare disorder that is treated surgically and mostly occurs in association with tumor-susceptibility syndromes, like multiple endocrine neoplasia and the hyperparathyroidism-jaw tumor syndrome. Familial hypercalciuric hypercalcemia (FHH) is another cause of hereditary hypercalcemia that generally is considered to require no treatment and is genetically and pathophysiologically distinct from HPT. Inactivating mutations in the calcium receptor gene cause FHH, whereas the down-regulated expression of the CaR in HPT never has been coupled to CaR gene mutations. Family screening revealed a hitherto unknown familial condition with characteristics of both FHH and HPT. The hypercalcemia was mapped to a point mutation in the intracellular domain of the CaR gene that was coupled to relative calcium resistance of the PTH release by transient expression in HEK 294 cells. Unusually radical excision of parathyroid glands was required to normalise the hypercalcemia. The mildly enlarged parathyroid glands displayed hyperplasia with nodular components. Frequent allelic loss on especially 12q was found and contrasts to findings in HPT. Allelic loss was also seen in loci typical for primary HPT like 1p, 6q and 15q, but not 11q13. Quantitative mRNA analysis showed that the glands had mild increase in a proliferation index (PCNA/GAPDH mRNA ratio) and mild reduction in genes important to parathyroid cell function, like CaR, PTH, VDR and LRP2. A previously unrecognized variant of hypercalcemia is explored that could be one explanation for persistent hypercalcemia after apparently typical routine operations for HPT. It also raises the issue of possibilities to treat FHH with parathyroidectomy provided it is radical enough.

Introduction Severe hypercalcemia from chronic gout is a rare phenomenon seen after the advent of newer drugs for its treatment. The hypercalcemia is secondary to either granuloma formation around the tophi or chronic immobilization from severe gouty arthritis. We present a patient with chronic tophaceous gout presenting with severe hypercalcemia and acute kidney injury. Case presentation A 63-year-old male patient with a past medical history of hypertension and chronic gout presented to the office with chronic, severe left knee pain. Initial evaluation of the knee with X-rays revealed destruction of the knee joint with cystic changes, and subsequent MRI with contrast showed soft tissue mass in the suprapatellar pouch with intraosseous extension and involvement of medial and lateral collateral ligament involvement. After interdisciplinary evaluation between radiology, orthopedic surgery, and oncology, this was concerning for highly aggressive pigmented villonodular synovitis of the knee, and a decision was made for the patient to undergo complete knee replacement. Perioperative workup was significant for severe hypercalcemia with a total calcium level of 13.2 mg/dl with ionized calcium of 7.2 mg/dl. Further evaluation into the cause of hypercalcemia revealed a low normal intact parathyroid hormone (PTH) level with normal phosphorus, calcidiol, and calcitriol levels. Other etiologies of hypercalcemia such as multiple myeloma, malignancies, metastatic disease, autoimmune, granulomatous, and infectious processes are excluded with extensive workup. The hypercalcemia is treated with fluids, diuretics, and bisphosphonates, eventually normalizing the calcium levels. The patient underwent total left knee replacement, and the mass identified was sent for biopsy. Biopsy revealed a prominent granulomatous reaction to amorphous crystals containing birefringent crystals under polarised light. Uniquely during our evaluation, vitamin D metabolites, uric acid, and PTH levels were normal despite the biopsy findings. The patient's calcium continued to be normal (8.4 to 10.4 mg/dl) over six months after the surgery. Thus, the scenario is supportive of hypercalcemia secondary to granulomatous inflammation around the large tophi. Conclusion Although rare, the knee joint is a site of severe tophaceous gout, and deposition of uric acid crystals can invoke a granulomatous reaction presenting with severe hypercalcemia as in our patient. Unique to our case, the patient can have benign lab findings on evaluation of hypercalcemia. Only a few case reports are illustrated in the literature, making our case and patient presentation unique.

Background: Lithium is the most effective long-term treatment for bipolar disease. It has, however,been associated with hypercalcemia and hyperparathyroidism. The aim of the study is to research howlithium associated hyperparathyroidism(LHPT)affects bone mineral density. Method: A sub-analysis was performed on an ongoing randomized prospective study evaluating the operation results from parathyroidectomy versus watchful waiting in 22patients with LHPT. The patients were followed-up for 2 years and their blood samples, bone mineral density (BMD) and FRAX assessment were analysed. The data from LHPT patients was also compared to a separate group of patients with primary hyperparathyroidism (PHPT) corresponding in age.Results: In comparing LHPT patients with PHPT apparent differences in the biochemical profile were detected, including elevated values of ionized Ca in PHPT (p=0.001), lower excretion of 24h urinary calcium in LHPT (p=0.003) and significantly higher values of PTH excretion in PHPT. LHPT showed tendencies to having better BMD (p=0.176). At 2-year follow-up of 8 LHPT patients, biochemicalvalues improved, suggesting cure, including lower risks of skeletal fractures. Discussion: The biochemical features in LHPT are distinctive from PHPT. However, each case is unique, and thebiochemicalvariety issimilar to PHPT. Confounding factors include age, sex, renal function and stability of the bipolar condition. Conclusions:The present study illustratesthat LHPT differs biochemically from PHPT. In comparison to PHPT, LHPT patients tend to have reduced BMD and the present study could not confirm the previous postulation that lithium could be protective of the skeleton. In conclusion, casesof LHPT should be assessed individually, since the clinical course is diverse. In patients risking fracture, parathyroidectomy should be considered.

Background: Lithium is the most effective long-term treatment for bipolar disease. It has, however,been associated with hypercalcemia and hyperparathyroidism. The aim of the study is to research howlithium associated hyperparathyroidism(LHPT)affects bone mineral density. Method: A sub-analysis was performed on an ongoing randomized prospective study evaluating the operation results from parathyroidectomy versus watchful waiting in 22patients with LHPT. The patients were followed-up for 2 years and their blood samples, bone mineral density (BMD) and FRAX assessment were analysed. The data from LHPT patients was also compared to a separate group of patients with primary hyperparathyroidism (PHPT) corresponding in age.Results: In comparing LHPT patients with PHPT apparent differences in the biochemical profile were detected, including elevated values of ionized Ca in PHPT (p=0.001), lower excretion of 24h urinary calcium in LHPT (p=0.003) and significantly higher values of PTH excretion in PHPT. LHPT showed tendencies to having better BMD (p=0.176). At 2-year follow-up of 8 LHPT patients, biochemicalvalues improved, suggesting cure, including lower risks of skeletal fractures. Discussion: The biochemical features in LHPT are distinctive from PHPT. However, each case is unique, and thebiochemicalvariety issimilar to PHPT. Confounding factors include age, sex, renal function and stability of the bipolar condition. Conclusions:The present study illustratesthat LHPT differs biochemically from PHPT. In comparison to PHPT, LHPT patients tend to have reduced BMD and the present study could not confirm the previous postulation that lithium could be protective of the skeleton. In conclusion, casesof LHPT should be assessed individually, since the clinical course is diverse. In patients risking fracture, parathyroidectomy should be considered.

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