RESUMO
OBJECTIVES: The calcium-sensing receptor (CASR) is critical for maintenance of blood calcium in a narrow physiologic range. Naturally occurring mutations in the calcium-sensing receptor gene (CASR) cause hypocalcaemia or hypercalcaemia, and molecular diagnosis of these mutations is clinically important. Knowledge of SNP frequency and haplotype structure is essential in understanding molecular test results. DESIGN AND METHODS: Genotyping and haplotype analysis of 26 CASR SNPs (and a tetranucleotide insertion/deletion polymorphism) in control cohorts of Caucasian, Asian and African-American origin (n=1136, 88 and 104 chromosomes, respectively). RESULTS: The three SNPs in exon 7 (A986S, R990G, Q1011E) are the only common exonic variants in our cohorts, and synonymous exonic SNPs are uncommon. Linkage disequilibrium analysis of the Caucasian cohort (Haploview) showed that the CASR locus is divided into three haplotype blocks, coincident with 5' regulatory, coding, and 3' regulatory domains. CONCLUSIONS: These analyses provide an important framework for appropriate interpretation of CASR mutation screening now offered by a number of laboratories for the diagnosis of calcium disorders. They will assist in the study of CASR polymorphisms as predictors of complex disease states.
Assuntos
Variação Genética , Mutação , Receptores de Detecção de Cálcio/genética , Negro ou Afro-Americano/genética , Alelos , Povo Asiático/genética , Sequência de Bases , Estudos de Coortes , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Hipercalcemia/diagnóstico , Hipercalcemia/genética , Hipocalcemia/diagnóstico , Hipocalcemia/genética , Desequilíbrio de Ligação , Modelos Genéticos , Polimorfismo de Nucleotídeo Único , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , População Branca/genéticaRESUMO
CONTEXT: Inactivating mutations of the calcium-sensing receptor (CASR) are implicated in different hypercalcemic syndromes, including familial hypocalciuric hypercalcemia (FHH), primary hyperparathyroidism (PHPT), and familial isolated hyperparathyroidism (FIHP). However, molecular diagnostics applied to large nonselected hypercalcemic cohorts from a single center have not been reported. OBJECTIVE: Our objective was to describe the prevalence, type, and potential pathogenicity of CASR mutations in a series of cases with FHH (n = 17), PHPT (n = 165), and FIHP (n = 3) and controls (n = 198) presenting at a single endocrine clinic. SUBJECTS: All were prospectively evaluated at the "Casa Sollievo della Sofferenza" Hospital in southern Italy over a 3-yr period. METHODS: CASR screening was conducted by denaturing HPLC. The variant CASRs were functionally characterized by transient transfection studies in kidney cells in vitro. RESULTS: A single novel missense variant was identified in one PHPT case. However, in FHH probands, mutations were found in eight of 17 (47%). With a hypercalcemic family member, mutation detection rate in FHH rose to seven of eight (87%), whereas only one of nine sporadic cases was positive, and none of the three FIHP cases had detectable CASR mutations. Five missense variant CASRs, identified in control subjects, performed as wild type in functional assays, whereas the missense mutant CASRs identified in the FHH patients, and in the one PHPT case, exhibited significant impairment. A novel intronic mutation (IVS4-19a-->c) found in one FHH family, created an abnormally spliced product in an in vitro minigene assay. CONCLUSION: CASR testing, with functional analysis, provides critical confirmatory evidence in the differential diagnosis of hypercalcemic states.
Assuntos
Hipercalcemia/genética , Receptores de Detecção de Cálcio/genética , Idoso , Cálcio/sangue , Cálcio/urina , Estudos de Coortes , Biologia Computacional , Creatinina/sangue , DNA/genética , Bases de Dados Genéticas , Feminino , Variação Genética , Células HeLa , Humanos , Hipercalcemia/tratamento farmacológico , Hiperparatireoidismo Primário/sangue , Hiperparatireoidismo Primário/genética , Hiperparatireoidismo Primário/patologia , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Glândulas Paratireoides/patologia , Hormônio Paratireóideo/sangue , Linhagem , Fosfatos/sangue , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The calcium-sensing receptor (CASR), a plasma membrane G-protein-coupled receptor, is expressed in parathyroid gland and kidney, and controls systemic calcium homeostasis. Inactivating CASR mutations are associated with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism, and activating mutations cause autosomal dominant hypocalcemia (ADH). CASR mutation identification plays an important role in the clinical management of mineral metabolism disorders. We describe here a high-throughput method using screening with denaturing high performance liquid chromatography (DHPLC) to initially interrogate 12 amplicons covering translated exons and exon/intron boundaries, followed by sequencing of any amplicon with a modified melting curve relative to wild type, and direct sequencing of a 13th amplicon encoding the COOH-terminal tail to distinguish causative mutations from three common missense single nucleotide polymorphisms. A blinded analysis of 32 positive controls representing mutations throughout the CASR sequence, as well as 22 negative controls, yielded a concordance rate of 100%. We report eight novel and five recurrent FHH mutations, along with six novel and two recurrent ADH mutations. Thus, DHPLC provides a rapid and effective means to screen for CASR mutations.