Analysis of CASR gene variant in a child with idiopathic epilepsy and autism / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a child featuring idiopathic epilepsy and autism.@*METHODS@#Peripheral blood samples of the child and his parents were collected with informed consent for the extraction of genome DNA. Whole exome sequencing was carried out for the family trio. Candidate variants were verified by Sanger sequencing and bioinformatic analysis.@*RESULTS@#The proband was found to harbor a heterozygous nonsense c.3025C>T (p.Arg1009Ter) variant in exon 7 of the CASR gene exon 7, which may produce a truncated protein. Based on the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be deleterious and classified as possibly pathogenic (PVS1+PM2).@*CONCLUSION@#The c.3025C>T (p.Arg1009Ter) variant of the CASR gene probably underlay the disease in this child.

Clinical and genetic analysis of a child with neonatal severe parathyroidism / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a child with neonatal severe hyperparathyroidism.@*METHODS@#Genomic DNA was extracted from peripheral blood samples from the patient and her parents. Whole exome sequencing was carried out to screen potential mutations. Suspected mutation was verified by Sanger sequencing.@*RESULTS@#The proband was found to carry compound heterozygous variants c.179G>A (p.Cys60Tyr) and c.1525G>A (p.Gly509Arg) of the CaSR gene. The c.179G>A variant was derived from her mother and was unreported previously. The c.1525G>A variant was derived from her father and known to be pathogenic.@*CONCLUSION@#The compound heterozygous variants of c.179G>A and c.1525G>A of the CaSR gene probably underlie the disease in the patient. The results of genetic testing has enabled diagnosis and genetic counseling for her family.

Un caso de hipercalcemia hipocalciúrica de causa genética en niño prepúber / Genetic hypercalcemic hypocalciuria in a prepubertal boy

Un paciente de 12 años consultó por vómitos recurrentes asociados con cefaleas, con varios episodios durante 7 meses, y retraso ponderal secundario a esa sintomatología. Había recibido previamente un tratamiento con antibióticos e inhibidores de la bomba de protones, por diagnóstico de gastritis a Helicobacter pylori, después de biopsia gástrica realizada durante una videoendoscopía digestiva alta. Se desconoce su historia familiar porque es hijo adoptivo. Al examen físico el paciente estaba adelgazado, sin tumoración a nivel de cuello; presentaba genitales prepuberales. Como el paciente continuó con vómitos cíclicos recurrentes, siguieron exámenes complementarios donde se constató en 2 oportunidades hipercalcemia (13,2-13,6 mg/dl), acompañada de hipofosfatemia (2,7 mg/dl). Con un diagnóstico presuntivo de hiperparatiroidismo primario se realizaron dosajes de laboratorio: calcemia total e iónica elevada (12,1 y 5,6mg/dl respectivamente), fosfatemia baja (2,8 mg/dl), fosfatasa alcalina sérica normal (151 mU/ml), PTH sérica normal (47,1 pg/ml), 25(OH)vitamina D sérica adecuada (22 ng/ml). La ecografía de glándulas tiroides y paratiroides mostró una imagen redondeada hipoecoica, avascular, de 4 mm axial por 4 mm cefalocaudal, por 3 mm ánteroposterior en topografía paratiroidea derecha, planteándose la posibilidad de hipertrofia paratiroidea versus adenopatía. Se realizó estudio de paratiroides por imágenes: centellograma con 99mTc-MIBI y PET-CT con 18F-colina, pero no se constató captación anormal. Se realizaron nuevos estudios de laboratorio: en orina de 24 horas el calcio era de 19 mg, el cociente calcio/creatinina urinaria 0,03 mg/mg, la reabsorción tubular de fósforo normal (82%) y el cociente de las tasas de depuración de calcio y creatinina muy bajo (0,00046). El CTX sérico era bajo. El diagnóstico clínico fue de hipercalcemia hipocalciúrica; ante la falta de antecedentes familiares, se realizó un estudio de posibles mutaciones puntuales en el gen del receptor de calcio (CaSR), hallándose la presencia en heterocigosis de la mutación p.Arg185Gln (p.R185Q) en la posición 554 (c.554G>A) del exón 4 del gene CaSR. Esto implica el cambio de una arginina por glutamina en el codón 185 de la proteína, y confirma el origen genético de la hipercalcemia hipocalciúrica en nuestro paciente. La edad ósea era de 12 años, y se indicó un tratamiento con testosterona i.m. a bajas dosis para acelerar el desarrollo puberal; luego de 4 aplicaciones mensuales su talla se ha incrementado en 4 cm y su peso en 3 kg. Una aplicación subcutánea de denosumab (60 mg) no controló la hipercalcemia. Continuó por un año con hipoorexia y un episodio de vómitos por semana, pero actualmente tiene buen apetito y excelente tolerancia digestiva. Se le ha prescripto cinacalcet oral (AU)

A 12-year-old patient who consulted for recurrent vomiting associated with headaches, with several episodes for 7 months, and low body weight. The patient had previously received treatment with antibiotics and proton pump inhibitors, due to gastritis with Helicobacter pylori, after gastric biopsy performed during videoendoscopy. His family history is unknown because he is an adopted son. At physical examination the patient was thin, without neck tumor; he had prepubertal genitalia. As he patient continued with recurrent vomiting, he was admitted for further evaluation. Laboratory studies revealed hypercalcemia (13.2-13.6 mg/dl), accompanied by hypophosphatemia (2.7 mg/dl). With a presumptive diagnosis of primary hyperparathyroidism, complementary determinations were performed: total and high total and ionized serum calcium (12.1 and 5.6 mg/dl, respectively), normal serum alkaline phosphatase (151 mU/ml), and PTH (47.1 pg/ml), and normal serum 25(OH) vitamin D (32 ng/ml). The ultrasonography of thyroid and parathyroid glands showed a rounded hypoechoic, avascular image, 4 mm in diameter in the lower right parathyroid topography. A parathyroid imaging studies were performed: scintigraphy with 99mTc-MIBI and PET-CT with 18F-choline, but no abnormal uptake was observed. New laboratory studies were carried out: in 24-hour urine the calcium was 19 mg, the urinary calcium/creatinine ratio was 0.03 mg/mg, the tubular reabsorption of phosphorus was normal (82%) and the ratio of clearances rates of calcium and creatinine very low (0.00046). Serum CTX was low. The clinical diagnosis was hypocalciuric hypercalcemia; in the absence of a family history, a study of possible point mutations in the calcium receptor gene (CaSR) was carried out; there was a heterozygous mutation: p.Arg185Gln (p.R185Q) at position 554 (c.554G)>A) of exon 4 of the CaSR gene. This involves the exchange of an arginine for glutamine at codon 185 of the protein, and confirms the genetic origin of the hypocalciuric hypercalcemia in our patient. Bone age was 12 years, and a treatment with testosterone i.m. at low doses to accelerate pubertal development was started; after 4 monthly applications height has increased by 4 cm and weight by 3 kg. Loss of appetite and a weekly episode of postprandial vomiting continued during one yeas, but now his appetite is normal and vomiting has subsided. A subcutaneous application of denosumab (60 mg) did not control hypercalcemia. He has been prescribed oral cinacalcet (AU)

Hipercalcemia hipocalciúrica familiar: Descripción de un caso / Familial hypocalciuric hypercalcemia: A case report

La presencia de hipercalcemia mantenida obliga a realizar pruebas complementarias para determinar su origen. Es benigna y, generalmente, no requiere tratamiento. La secuenciación del gen CaSR confirma el diagnóstico y evita tratamientos innecesarios. Se presenta a un niño de 12 años, asintomático, con hipercalcemia persistente entre 11,4 y 12,2 mg/dl. El padre y dos hermanos tenían hipercalcemia asintomática. El análisis de laboratorio mostró valores de magnesio, fósforo y vitamina D normales y de hormona paratiroidea llamativamente normal para el valor de la hipercalcemia. Indice de calcio/creatinina urinario: 0,11 mg/mg; y calciuria de 24 h: 1,8 mg/kg/día. Ecografía abdominal, paratiroides, radiografías de huesos largos y densitometría ósea, normales. El estudio genético mostró mutación en exón 6 (c.1651A>G) del gen CaSR (en heterocigosis), confirmada en el padre y los hermanos.

The finding of persistent hypercalcemia suggests doing other medical tests to find the cause. Familial hypocalciuric hypercalcemia is usually benign and it requires no treatment. It is important to do CASR gene sequencing to avoid unnecessary treatments. We report a 12-year-old child, asymptomatic, with calcemia between 11.4 and 12.2 mg/dl. His father and two brothers presented asymptomatic hypercalcemia. The blood test with magnesium, phosphorus, 25(OH)Vit D was normal, remarkable normal parathyroid hormone for the level of hypercalcemia. Urinary calcium/creatinine ratio was 0,11 mg/dl and 24-hour urinary calcium was 1,8 mg/kg per day. Abdominal and parathyroid ecography, long bone radiographs and densitometry were normal. Genetic study showed a mutation, c.1651A>G, in exon 6 of the calciumsensing receptor gene, confirmed in father and brothers, too.

Hipercalcemia hipocalciurica familiar en una paciente con mutación del receptor de calcio: forma atípica de presentación y tratamiento con cinacalcet / Familial hypocalciuric hypercalcemia in a patient with calcium-sensing receptor mutation: atypical clinical presentation and treatment with cinacalcet

El hiperparatiroidismo familiar y la hipercalcemia hipocalciúrica familiar (HHF) constituyen un subgrupo heterogéneo de trastornos con herencia mendeliana, que representan en conjunto el 5% de las causas de hipercalcemia PTH dependiente. La HHF se asocia con mutaciones del gen del receptor sensor de calcio (CaSR). Esta entidad se manifiesta, en la mayoría de los casos, con la presentación asintomática y familiar de hipercalcemia e hipocalciuria y valores elevados o normales de hormona paratiroidea (PTH). Los avances en la biología molecular han contribuido al diagnóstico, evaluación del fenotipo de cada entidad y elección del tratamiento. Se describe el caso de una paciente con hipercalcemia estudiada a partir de una tumoración de cuello asociada con una glándula paratiroides quística. Luego de un exhaustivo proceso diagnóstico se halló en el estudio genético una mutación inactivante en el gen CaSR. Teniendo en cuenta la presencia de la relación clearance calcio/clearance creatinina <0,01 y la falta de respuesta al tratamiento quirúrgico, se consideró la entidad de HHF con forma de presentación atípica. La paciente, sin tratamiento, presentaba un progresivo incremento de la calcemia luego de la cirugía de las glándulas paratiroides, que no se controló con el uso de bifosfonatos y evolucionó con episodios de mareos y desmayos frecuentes sin causa neurológica o cardiovascular detectada. Por lo tanto, se inició el tratamiento con cinacalcet, con el cual se obtuvo una buena respuesta terapéutica: descenso de la calcemia y mejoría de la sintomatología luego de un año de su comienzo. El cinacalcet es una herramienta terapéutica de importancia en estos raros casos de HHF. (AU)

Familial hyperparathyroidism including familial hypocalciuric hypercalcemia (FHH) is an heterogeneous subgroup of disorders with Mendelian inheritance, that account for 5% of PTH dependent hypercalcemia. FHH is associated with mutations of the calcium receptor (CaSR) gene. This entity is manifested by hypercalcemia with hypocalciuria and high or normal levels of parathyroid hormone (PTH) generally asymptomatic and with familial presentation. Advances in molecular biology have contributed to the diagnosis, evaluation of the phenotype of each entity and the choice of treatment. We describe a patient with hypercalcemia diagnosed following the finding of a neck tumor associated with cystic parathyroids. After an exhaustive diagnostic process, an inactivating mutation in the CaSR gene was found. Considering the presence of a ratio clearance calcium / clearance creatinine <0.01 and the lack of response to surgical treatment, HHF entity with atypical presentation was considered. The patient exhibited progressive increase in serum calcium following parathyroid surgery, which was not controlled with the use of bisphosphonates and evolved into episodes of frequent dizziness and fainting, without neurological or cardiovascular causes. Treatment with cinacalcet was initiated, with a good therapeutic response. The use of cinacalcet is a useful therapeutic tool in these rare cases of FHH. (AU)

Genetic and Clinical Characteristics of Korean Patients with Isolated Hypoparathyroidism: From the Korean Hypopara Registry Study

Isolated hypoparathyroidism (IH) shows heterogeneous phenotypes and can be caused by defects in a variety of genes. The goal of our study was to determine the clinical features and to analyze gene mutations in a large cohort of Korean patients with sporadic or familial IH. We recruited 23 patients. They showed a broad range of onset age and various values of biochemical data. Whole exome sequencing was performed on two affected cases and one unaffected individual in a family. All coding exons and exon-intron borders of GCMB, CASR, and prepro-PTH were sequenced using PCR-amplified DNA. In one family who underwent the whole exome sequencing analysis, approximately 300 single nucleotide changes emerged as candidates for genetic alteration. Among them, we identified a functional mutation in exon 2 of GCMB (C106R) in two affected cases. Besides, heterozygous gain-of-function mutations in the CASR gene were found in other subjects; D410E and P221L. We also found one single nucleotide polymorphism (SNP) in the prepro-PTH gene, five SNPs in the CASR gene, and four SNPs in the GCMB gene. The current study represents a variety of biochemical phenotypes in IH patients with the molecular genetic diagnosis of IH.

New mutation in the CASR gene in a family with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) / Nova mutação no gene CASR em uma família com hipercalcemia hipocalciúrica familiar (HHF) e hiperparatireoidismo neonatal grave (HPTNG)

A loss of calcium-sensing receptor (CASR) function due to inactivating mutations can cause familial hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). NSHPT represents the most severe expression of FHH and courses as a life-threatening condition. The aim of this study was to identify and characterize a CASR mutation in a female infant brought to the health service due to dehydration, apathy, lack of breast feeding and severe hypercalcemia. Molecular analysis was performed on genomic DNA of the index case and her parents. A novel homozygous mutation (p.E519X) in CASR was identified in the proband; both mother and father had the same mutation in heterozygous state, confirming their FHH condition. The mutation results in a truncated and inactive protein due to the lack of transmembrane and intracellular domains. The identification of this novel CASR gene mutation established the basis of hypercalcemia in this family and allowed a genetic counseling.

Mutações inativadoras no gene do sensor do cálcio (CASR) podem causar hipercalcemia hipocalciúrica familiar (HHF) ou hiperparatireoidismo neonatal grave (HPTNSG). A HPTNS representa a forma mais grave da HHF cursando com risco de vida. O objetivo deste estudo foi identificar e caracterizar uma mutação no gene CASR de uma criança do sexo feminino levada ao hospital em decorrência de desidratação, apatia, dificuldade para mamar e hipercalcemia grave. A análise molecular foi realizada a partir do DNA genômico do caso índice e de seus pais. Uma nova mutação em homozigose (p.E519X) foi identificada no caso índice; ambos, mãe e pai, apresentaram a mesma mutação em heterozigose, o que os caracteriza como portadores de HHF. Essa alteração resulta em uma proteína truncada e inativa devido à falta dos domínios transmembrana e intracelular. A identificação dessa nova mutação estabeleceu a causa da hipercalcemia na família e permitiu o aconselhamento genético.

Autosomal Dominant Hypocalcemia Caused by an Activating Mutation of the Calcium-Sensing Receptor Gene: The First Case Report in Korea

Hypoparathyroidism is an abnormality of calcium metabolism characterized by low serum levels of parathyroid hormone in spite of hypocalcemia. The causes of hypoparathyroidism are numerous. Activating mutations in the calcium-sensing receptor (CaSR) gene are well-known causes of familial isolated hypoparathyroidism, also known as autosomal dominant hypocalcemia (ADH). Here we describe members of a Korean family with a heterozygous Pro221Leu mutation causing ADH. This case is the first report in Korea.

The calcium-sensing receptor and related diseases / O receptor sensor de cálcio e doenças associadas

The calcium-sensing receptor (CASR) adjusts the extracellular calcium set point regulating PTH secretion and renal calcium excretion. The receptor is expressed in several tissues and is also involved in other cellular functions such as proliferation, differentiation and other hormonal secretion. High extracellular calcium levels activate the receptor resulting in modulation of several signaling pathways depending on the target tissues. Mutations in the CASR gene can result in gain or loss of receptor function. Gain of function mutations are associated to Autossomal dominant hypocalcemia and Bartter syndrome type V, while loss of function mutations are associated to Familial hypocalciuric hypercalcemia and Neonatal severe hyperparathyroidism. More than one hundred mutations were described in this gene. In addition to calcium, the receptor also interacts with several ions and polyamines. The CASR is a potential therapeutic target to treatment of diseases including hyperparathyroidism and osteoporosis, since its interaction with pharmacological compounds results in modulation of PTH secretion.

O receptor sensor de cálcio (CASR) ajusta o set point do cálcio extracelular através da regulação da secreção de PTH e da excreção renal de cálcio. O receptor é expresso em diversos tecidos e também está envolvido em outras funções celulares como proliferação, diferenciação e secreção de outros hormônios. Concentrações altas de cálcio extracelular ativam o receptor resultando em modulação de inúmeras vias de sinais intracelulares dependendo do tecido-alvo. Mutações no gene do CASR podem resultar em ganho ou perda de função do receptor. Mutações com ganho de função são associadas à Hipocalcemia autossômica dominante e à Síndrome de Bartter tipo V, enquanto que mutações com perda de função são associadas à Hipercalcemia hipocalciúrica familiar e ao Hiperparatireoidismo neonatal grave. Mais de cem mutações foram descritas neste gene. Além do cálcio, o receptor também interage com inúmeros íons e poliaminas. CASR é um alvo terapêutico potencial para tratamento de doenças incluindo hiperparatireoidismo e osteoporose, pois a sua interação com compostos farmacológicos resulta em modulação da secreção de PTH.

A Case Report of Familial Benign Hypocalciuric Hypercalcemia: A Mutation in the Calcium-Sensing Receptor Gene

Familial benign hypocalciuric hypercalcemia (FBHH) is an autosomal dominant trait with high penetrance, clinically manifestating a relatively benign, lifelong, persistent hypercalcemia and hypocalciuria without hypercalcemic related complications. The calcium-sensing receptor (CaSR) plays an important role in the regulation of PTH secretion and calcium metabolism. Here we present a family with FBHH of an autosomal dominant inheritance. A heterozygous mutation of E297K (GAG -> AAG, exon 4) of CaSR gene was found in 3 family members. To our knowledge, it is the first confirmed case of FBHH with CaSR gene mutation in Korea.

The extracellular calcium sensing receptor is expressed in mouse mesangial cells and modulates cell proliferation

The extracellular calcium sensing receptor (CaSR) belongs to the type III family of G-protein-coupled receptors, a family that comprises the metabotropic glutamate receptor and the putative vomeronasal organ receptors. The CaSR plays an important role for calcium homeostasis in parathyroid cells, kidney cells and other cells to directly 'sense' changes in the extracellular calcium ion concentration ((Ca2+)o). The mesangial cells are known to be involved in many pathologic sequences through the mediation of altered glomerular hemodynamics, cell proliferation, and matrix production. In this study, we examined the expression of the CaSR in the mouse mesangial cell lines (MMC, ATCC number CRL-1927). Reverse transcription- polymerase chain reaction (RT-PCR) was perform with CaSR-specific primers, and this was followed by nucleotide sequencing of the amplified product; this process identified the CaSR transcript in the MMCs. Moreover, CaSR protein was present in the MMCs as assessed by Western blot and immunocytochemical analysis using a polyclonal antibody specific for the CaSR. Functionally, (Ca2+)o induced the increment of the intracellular calcium concentration ((Ca2+)i) in a dose-dependent manner. This (Ca2+)i increment by (Ca2+)o was attenuated by the pretreatment with a phospholipase C inhibitor (U73122) and also by a pretreatment with a CaSR antagonist (NPS 2390). The similar results were also obtained in IP3 accumulation by (Ca2+)o. To investigate the physiological effect of the CaSR, the effect of the (Ca2+)o on cell proliferation was studied. The increased (Ca2+)o (up to 10 mM) produced a significant increase in the cell numbers. This mitogenic effect of (Ca2+)o was inhibited by the co-treatment with a CaSR antagonist. From these results, the (Ca2+)o-induced (Ca2+)i elevation in the MMC is coupled with the extracellular calcium sensing receptor. Furthermore, (Ca2+)o produces a mitogenic effect in MMCs.

Hipercalcemia hipocalciurica debida a una mutacion de novo del gen del receptor sensor del calcio / Hypocalciuric hypercalcemia due to de novo mutation of the calcium sensing receptor

El objetivo de este trabajo es presentar el inusual caso clínico de una paciente de 34 años que consultó para establecer diagnóstico de certeza y conducta terapéutica ante una hipercalcemia asintomática, detectada en un examen dioquímico de rutina. La elevación de la calcemia en ausencia de inhibición de la secreción de parathormona y coincidente con una relación clearance de calcio/clearance de creatina inferior de 0.01, hicieron sospechar el diagnóstico de hipercalcemia hipocalciúrica familiar. La falta de antecedentes familiares llevó a realizar un estudio molecular de la paciente y su grupo familiar. Los resultados de los estudios nos permitieron concluir que la paciente es portadora de una mutación de novo (inactivante) del gen del receptor sensor del calcio. Se incluyen los datos de estudio molecular y una breve revisión bibliográfica del tema.

Effect of oophorectomy on expression of calcium sensing receptor mRNA in rat duodenal mucosa.

Calcium sensing receptor (CaR) in duodenal mucosa may be involved in active calcium absorption. Estrogen deficiency results in decreased intestinal calcium absorption. Effects of bilateral oophorectomy (OVX) have been studied on calcium homeostasis, bone mineral density (BMD) and CaR mRNA levels in duodenal mucosa at 4 weeks in adult female Sprague Dawley rats and compared with those in sham-operated and control group. There was no significant change in serum corrected calcium, inorganic phosphorous, calcidiol and intact parathyroid hormone in all the three groups. OVX rats had a significant decline in serum estrogen (E2) levels and alkaline phosphatase. They also had a significant decrease in BMD (DXA) at lumbar spine in vivo, and proximal and distal tibia in vitro while there was no significant change in serum E2 and BMD parameters in sham-operated and control rats. Northern blot analysis revealed no significant change in the CaR mRNA expression in duodenal mucosa in all three groups. The results suggests that CaR mRNA expression in duodenal mucosa is not affected by physiological circulating concentrations of estradiol in rats.