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Título: Molecular genetics of familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism
Autores: Janicic, Natasa.
Fecha: 1998
Publicador: McGill University - MCGILL
Fuente:
Tipo: Electronic Thesis or Dissertation
Tema: Biology, Molecular.
Chemistry, Biochemistry.
Health Sciences, Medicine and Surgery.
Descripción: The concentration of extracellular ionized calcium (Ca2+ o) is the primary physiological regulator of parathyroid hormone (PTH) secretion. The parathyroid gland has a special ability to sense extracellular the calcium concentration and the mechanism whereby this is achieved became clearer when a parathyroid calcium-sensing receptor (CASR) was cloned and characterized. Inactivating mutations in the CASR gene have been identified as a cause of familial hypocalcemia hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT).
The goal of this thesis was genetic analysis of several families affected with FHH and NSHPT. Initially, we studied two families from Nova Scotia expressing both FHH and NSHPT. In order to localize the mutant gene haplotype analysis was first carried out using genetic markers for chromosome region 3q11.2-24. The CASR gene sequence was screened for mutations by sequence analysis implemented on either cloned genomic DNA or direct sequencing of PCR product. By PCR amplification of CASR gene exons it was found that FHH individuals were heterozygous and NSHPT individuals were homozygous for an abnormally large exon 7 due to an insertion of an Alu-repetitive element at codon 876. Later, we studied a third family from the same region and found the same type of mutation. However, one individual from that family had one allele of an even larger size providing evidence for de novo expansion of an exonic Alu insertion mutation. This is the first report of an Alu sequence insertion in the CASR gene and one of the rare examples of this type of mutation being present in the protein coding region.
Using fluorescence in situ hybridization (FISH), human CASR gene was mapped to chromosome 3q13.3-21. By somatic cell hybrid analysis, the gene was localized to human chromosome 3 and rat chromosome 11. By interspecific backcross analysis, the Casr gene segregated with D16Mit4 on mouse chromosome 16. These findings extend our knowledge of the synteny conservation of human chromosome 3, rat chromosome 11 and mouse chromosome 16.
The structural and functional consequences of the Alu insertion in exon 7 of the CASR gene were investigated. Stop signals are introduced in all reading frames within the Alu sequence, leading to a predicted shortened mutant protein. The effect of Alu insertion on transcription/translation was studied by protein truncation test (PTT). The Alu sequence was less efficiently transcribed due to stalling of the polymerase at the poly(T) tract. In vitro translation of the Alu transcript resulted in three truncated protein products whose sizes are consistent with the presence of predicted stop codons in all three reading frames. Western blot analysis was used to compare the expression of the mutant and the wild type receptor cDNA transiently transfected in human embryonic kidney (HEK293) cells. The Alu mutant receptor had a molecular mass ∼30kD smaller and showed decreased cell surface expression compared to the wild-type receptor. Interestingly, a CASR with a C-terminal truncation at residue 877 exhibited increased cell surface expression.
This thesis contributes to a better understanding of molecular genetics of the CASR gene. Furthermore, structure-function studies of an Alu insertion mutation in the CASR protein coding region provides a novel insight into the effect of this mutation on the gene-expression processes.
Idioma: en