Polymorphism Study Of Calcium-Sensing Receptor Gene (Casr)In Calcium Nephrolithiasis Affected Families
By: Hafza Ammara (2013-VA-865) | Dr. Muhammad YasirZahoor.
Contributor(s): Dr. Asif Nadeem | Ms. Huma Mujahid.
Material type: BookPublisher: 2015Description: 69p.Subject(s): Molecular Biology and BiotechnologyDDC classification: 2426-T Dissertation note: Nephrolithiasis is a multi-factorial kidney stone disease resulting from the combined influence of epidemiological, biochemical and genetic risk factors. Calcium-sensing receptorprotien is plasma membrane G protein-coupled receptors that regulate secretion of parathyroid hormoneand calcium re-absorption by kidney tubular cells. This protienis able to sense small changes in circulating calcium concentration and, once activated, it inhibits parathyroid hormone secretion and renal tubule calcium re-absorption. The CaSR gene protein islocated on chromosome 3q13 is one of the candidate gene explaining individual predispositions to calcium nephrolithiasis. CaSR gene is a predecessor for nephrolithiasis due to its role in calcium re-absorption. CaSRgene has seven exons and several mutations have been reported globally related to calcium nephrolithiasis. Twenty families affected with calcium nephrolithiasis having at least two affected individuals have been enrolled for this study. Ten families have already been analyzed for exon 3 & 4 in the laboratory. DNA has been extracted through inorganic extraction method from the blood of newly enrolled families. Primers have been designed for exon 5, 6 and 7 through Primer3 software. These exons have been sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer/ABI) and have been read in an automated sequencer, ABI Prism model 3730 (Perkin Elmer). We also screend the coding exon of CLDN14 genewhich is a membrane protein that regulates paracellular passage of ions and small solutesat epithelial tight junction.The overexpression of claudin-14 in the thick ascending limb of loop of henleof the kidney generates a renal phenotype characteristic with hypomagnesemiaand hypercalciuria that leads to the development of calcium nephrolithiasis. All of the sequences have been evaluated by using Clustal-W programs, Chromas and Bioedit software for mutational analysis.Sequence analysis of CaSR gene revealed one novel splice mutationC>G at position 63722 at exon 5 in one affected family.This variation is found in the intronic region of the gene.We found one missense mutation Q536R at exon six in three different affected families. And one synonymous single nucleotide polymorphism(SNP) C>G found at exon 7at rs2036400 in six different affected families.These SNPs showsa significant association of CaSRgene with nephrolithiasis. It will help to determine the risk factor and role of CaSR gene in inheritance of calcium nephrolithiasis. And it will also be used for genetic screening and prenatal diagnosis.Item type | Current location | Collection | Call number | Status | Date due | Barcode | Item holds |
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Thesis | UVAS Library Thesis Section | Veterinary Science | 2426-T (Browse shelf) | Available | 2426-T |
Nephrolithiasis is a multi-factorial kidney stone disease resulting from the combined influence of epidemiological, biochemical and genetic risk factors. Calcium-sensing receptorprotien is plasma membrane G protein-coupled receptors that regulate secretion of parathyroid hormoneand calcium re-absorption by kidney tubular cells. This protienis able to sense small changes in circulating calcium concentration and, once activated, it inhibits parathyroid hormone secretion and renal tubule calcium re-absorption. The CaSR gene protein islocated on chromosome 3q13 is one of the candidate gene explaining individual predispositions to calcium nephrolithiasis. CaSR gene is a predecessor for nephrolithiasis due to its role in calcium re-absorption. CaSRgene has seven exons and several mutations have been reported globally related to calcium nephrolithiasis.
Twenty families affected with calcium nephrolithiasis having at least two affected individuals have been enrolled for this study. Ten families have already been analyzed for exon 3 & 4 in the laboratory. DNA has been extracted through inorganic extraction method from the blood of newly enrolled families. Primers have been designed for exon 5, 6 and 7 through Primer3 software. These exons have been sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer/ABI) and have been read in an automated sequencer, ABI Prism model 3730 (Perkin Elmer).
We also screend the coding exon of CLDN14 genewhich is a membrane protein that regulates paracellular passage of ions and small solutesat epithelial tight junction.The overexpression of claudin-14 in the thick ascending limb of loop of henleof the kidney generates a renal phenotype characteristic with hypomagnesemiaand hypercalciuria that leads to the development of calcium nephrolithiasis.
All of the sequences have been evaluated by using Clustal-W programs, Chromas and Bioedit software for mutational analysis.Sequence analysis of CaSR gene revealed one novel splice mutationC>G at position 63722 at exon 5 in one affected family.This variation is found in the intronic region of the gene.We found one missense mutation Q536R at exon six in three different affected families. And one synonymous single nucleotide polymorphism(SNP) C>G found at exon 7at rs2036400 in six different affected families.These SNPs showsa significant association of CaSRgene with nephrolithiasis. It will help to determine the risk factor and role of CaSR gene in inheritance of calcium nephrolithiasis. And it will also be used for genetic screening and prenatal diagnosis.
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