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Eliecer Coto - One of the best experts on this subject based on the ideXlab platform.
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a new slc12a3 founder mutation p val647met in gitelman s syndrome patients of roma ancestry
Nefrologia, 2017Co-Authors: Helena Gilpena, Juan Gómez, Fernando Santos, Eliecer Coto, Mar Espino, Jose Ma Cea Crespo, Giannis Chantzopoulos, Filadelfia Komianou, Belen Alonso, Sara IglesiasAbstract:Abstract Background Gitelman's syndrome (GS) is an autosomal recessive disorder caused by mutations in the SLC12A3 gene. GS is characterized by hypokalaemic metabolic alkalosis, hypomagnesemia and hypocalciuria. Most of the reported patients of Roma ancestry are homozygous for an SLC12A3 intron 9 frameshifting mutation (c.1180+1G>T). Some forms of Bartter's syndrome result from mutations in the CLNCKB gene and clinically overlap with GS. Objectives To characterize a second SLC12A3 mutation in Roma patients negative for the intron 9 variant. Methods SLC12A3 and CLNCKB genes were analyzed by next-generation sequencing in two Spanish and Greek gypsy patients who were negative for the intron 9 splicing mutation. Sanger sequencing was performed to confirm the putative mutations in patients and family members. Results We identified a missense variant (p.Val647Met, c.1939G>A) in both cases, and both were homozygous for Met. This mutation was also found in three additional patients; two homozygous and one heterozygous compound with the intron 9 splicing mutation. This new SLC12A3 mutation seems to be characteristic of gipsy GS patients and was linked to the same haplotype in all cases, supporting a founder origin. All the patients showed biochemical features characteristic of GS. Conclusion We report a second founder mutation among GS patients of Roma ethnic background. The direct screening of this mutation would facilitate the characterization of patients who are negative for the more common intron 9 +1G>T mutation.
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A labor- and cost-effective non-optical semiconductor (Ion Torrent) next-generation sequencing of the SLC12A3 and CLCNKA/B genes in Gitelman’s syndrome patients
Journal of Human Genetics, 2014Co-Authors: Beatriz Tavira, Juan Gómez, Fernando Santos, Helena Gil, Victoria Alvarez, Eliecer CotoAbstract:Gitelman’s syndrome (GS) is a rare recessive disorder caused by mutations in the renal salt-handling genes SLC12A3 and CLCNKB . Our aim was to develop a next-generation sequencing (NGS) procedure for these genes based on two-tubes multiplex amplification of DNA pools and semiconductor sequencing with the Ion Torrent Personal Genome Machine (PGM). We created one pool with DNA from 20 GS patients previously Sanger sequenced for the coding exons of SLC12A3 . A total of 13 mutations present in 11 of these patients were used as control variants to validate the NGS procedure. The full coding sequence of SLC12A3 , CLCNKB and CLCNKA was amplified in only two Ampliseq tubes and processed and sequenced with the PGM. Large SLC12A3 and CLCNKB deletions were ascertained through multiplex ligation-dependent probe amplification in some patients. With the exception of the SLC12A3 exon 9, all the amplicons were successfully read and 12 of the 13 control variants were detected. The analysis of CLCNKB showed four putative mutations in the GS pool that were further assigned to specific patients. Two patients were heterozygous compounds for a single-nucleotide mutation and a large deletion at SLC12A3 or CLCNKB . We reported a NGS procedure that would facilitate the rapid and cost-effective large-scale screening of the three renal salt-handling genes. In addition to characterize the mutational spectrum of GS patients, the described procedure would facilitate the rapid and cost-effective screening of these genes at a population scale.
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a labor and cost effective non optical semiconductor ion torrent next generation sequencing of the slc12a3 and clcnka b genes in gitelman s syndrome patients
Journal of Human Genetics, 2014Co-Authors: Beatriz Tavira, Juan Gómez, Fernando Santos, Helena Gil, Victoria Alvarez, Eliecer CotoAbstract:A labor- and cost-effective non-optical semiconductor ( Ion Torrent ) next-generation sequencing of the SLC12A 3 and CLCNKA/B genes in Gitelman’s syndrome patients
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a new mutation intron 9 1 g t in the slc12a3 gene is linked to gitelman syndrome in gypsies
Kidney International, 2004Co-Authors: Eliecer Coto, Nikola Jeck, Victoria Alvarez, Julian Rodriguez, Rosario Stone, Cesar Loris, Luis M Rodriguez, Michel Fischbach, Hannsjorg W Seyberth, Fernando SantosAbstract:A new mutation (intron 9 +1 G>T) in the SLC12A3 gene is linked to Gitelman syndrome in Gypsies. Background Gitel syndrome is an inherited tubular disorder characterized by metabolic alkalosis, hypokalemia, and hypomagnesemia of renal origin and hypocalciuria. The majority of patients with Gitelman syndrome carry inactivating mutations in the SLC12A3 gene encoding the sodium-chloride cotransporter located in the distal convoluted tubule. The purpose of this study was to investigate the underlying mutation in Gitelman syndrome patients of Gypsy race from different geographic origin. Methods Twenty Gypsy patients with clinical and biochemical features of Gitelman syndrome were investigated by mutational analysis. The patients belonged to 12 unrelated Gypsy families living in four different European countries. The parents and unaffected siblings of each patient, as well as the DNA of a population of 200 healthy control patients, were also analyzed. Results All patients were homozygous for the same splice site mutation, guanine to thymine in the first position of intron 9 of SLC12A3 gene. This mutation was not found in the control population. Parents were heterozygous for the mutation. Despite sharing a common mutation, the clinical manifestations of the syndrome in the patients varied from lack of symptoms in six children to severe growth retardation in four. Conclusion Demonstration of a novel point mutation within the SLC12A3 gene in our cohort of Gypsy families with Gitelman syndrome is highly suggestive of a founder effect. This finding will facilitate the identification of the genetic defect in further cases of Gitelman syndrome among the Gypsy population. Our study represents the largest series ever published of patients with Gitelman syndrome having the same underlying mutation, and supports the lack of correlation between genotype and clinical phenotype in this disease.
Stephen D. Mccormick - One of the best experts on this subject based on the ideXlab platform.
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functional characterization and osmoregulatory role of the na k 2cl cotransporter in the gill of sea lamprey petromyzon marinus a basal vertebrate
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2020Co-Authors: Ciaran A Shaughnessy, Stephen D. MccormickAbstract:The present study provides molecular and functional characterization of Na+-K+-2Cl− cotransporter (NKCC1/Slc12a2) in the gills of sea lamprey (Petromyzon marinus), the most basal extant vertebrate ...
Mark A Knepper - One of the best experts on this subject based on the ideXlab platform.
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targeted single cell rna seq identifies minority cell types of kidney distal nephron
Journal of The American Society of Nephrology, 2021Co-Authors: Lihe Chen, Chun-lin Chou, Mark A KnepperAbstract:BACKGROUND Proximal tubule cells dominate the kidney parenchyma numerically, although less abundant cell types of the distal nephron have disproportionate roles in water and electrolyte balance. METHODS Coupling of a FACS-based enrichment protocol with single-cell RNA-seq profiled the transcriptomes of 9099 cells from the thick ascending limb (CTAL)/distal convoluted tubule (DCT) region of the mouse nephron. RESULTS Unsupervised clustering revealed Slc12a3+/Pvalb+ and Slc12a3+/Pvalb- cells, identified as DCT1 and DCT2 cells, respectively. DCT1 cells appear to be heterogeneous, with orthogonally variable expression of Slc8a1, Calb1, and Ckb. An additional DCT1 subcluster showed marked enrichment of cell cycle-/cell proliferation-associated mRNAs (e.g., Mki67, Stmn1, and Top2a), which fit with the known plasticity of DCT cells. No DCT2-specific transcripts were found. DCT2 cells contrast with DCT1 cells by expression of epithelial sodium channel β- and γ-subunits and much stronger expression of transcripts associated with calcium transport (Trpv5, Calb1, S100g, and Slc8a1). Additionally, scRNA-seq identified three distinct CTAL (Slc12a1+) cell subtypes. One of these expressed Nos1 and Avpr1a, consistent with macula densa cells. The other two CTAL clusters were distinguished by Cldn10 and Ptger3 in one and Cldn16 and Foxq1 in the other. These two CTAL cell types were also distinguished by expression of alternative Iroquois homeobox transcription factors, with Irx1 and Irx2 in the Cldn10+ CTAL cells and Irx3 in the Cldn16+ CTAL cells. CONCLUSIONS Single-cell transcriptomics revealed unexpected diversity among the cells of the distal nephron in mouse. Web-based data resources are provided for the single-cell data.
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targeted single cell rna seq identifies minority cell types of kidney distal nephron that regulate blood pressure and calcium balance
bioRxiv, 2020Co-Authors: Lihe Chen, Chunglin Chou, Mark A KnepperAbstract:ABSTRACT A major objective in modern biology is generation of comprehensive atlases of various organs identifying all cell types and their expressed genes. In kidney, extensive data exists for proximal tubule and collecting duct cells, but not for non-abundant intermediate epithelial cell types. Here, we coupled a FACS-enrichment protocol with single-cell RNA-seq analysis to profile the transcriptomes of 9099 cells from the nephron region adjacent to the macula densa. Clusters containing Slc12a3+/Pvalb+ and Slc12a3+/Pvalb- cells were identified as DCT1 and DCT2 cells. The DCT1 cells appear to be heterogeneously associated with variable expression of Slc8a1, Calb1, and Ckb among other mRNAs. No DCT2-specific transcripts were found. The analysis also identified two distinct cell types in the Slc12a1+ portion of Henle’s loop as well as Nos1+/Avpr1a+ macula densa cells. Thus, we identify unexpected cell diversity in the intermediate region of the nephron and create a web-based data resource for these cells.
Fernando Santos - One of the best experts on this subject based on the ideXlab platform.
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a new slc12a3 founder mutation p val647met in gitelman s syndrome patients of roma ancestry
Nefrologia, 2017Co-Authors: Helena Gilpena, Juan Gómez, Fernando Santos, Eliecer Coto, Mar Espino, Jose Ma Cea Crespo, Giannis Chantzopoulos, Filadelfia Komianou, Belen Alonso, Sara IglesiasAbstract:Abstract Background Gitelman's syndrome (GS) is an autosomal recessive disorder caused by mutations in the SLC12A3 gene. GS is characterized by hypokalaemic metabolic alkalosis, hypomagnesemia and hypocalciuria. Most of the reported patients of Roma ancestry are homozygous for an SLC12A3 intron 9 frameshifting mutation (c.1180+1G>T). Some forms of Bartter's syndrome result from mutations in the CLNCKB gene and clinically overlap with GS. Objectives To characterize a second SLC12A3 mutation in Roma patients negative for the intron 9 variant. Methods SLC12A3 and CLNCKB genes were analyzed by next-generation sequencing in two Spanish and Greek gypsy patients who were negative for the intron 9 splicing mutation. Sanger sequencing was performed to confirm the putative mutations in patients and family members. Results We identified a missense variant (p.Val647Met, c.1939G>A) in both cases, and both were homozygous for Met. This mutation was also found in three additional patients; two homozygous and one heterozygous compound with the intron 9 splicing mutation. This new SLC12A3 mutation seems to be characteristic of gipsy GS patients and was linked to the same haplotype in all cases, supporting a founder origin. All the patients showed biochemical features characteristic of GS. Conclusion We report a second founder mutation among GS patients of Roma ethnic background. The direct screening of this mutation would facilitate the characterization of patients who are negative for the more common intron 9 +1G>T mutation.
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A labor- and cost-effective non-optical semiconductor (Ion Torrent) next-generation sequencing of the SLC12A3 and CLCNKA/B genes in Gitelman’s syndrome patients
Journal of Human Genetics, 2014Co-Authors: Beatriz Tavira, Juan Gómez, Fernando Santos, Helena Gil, Victoria Alvarez, Eliecer CotoAbstract:Gitelman’s syndrome (GS) is a rare recessive disorder caused by mutations in the renal salt-handling genes SLC12A3 and CLCNKB . Our aim was to develop a next-generation sequencing (NGS) procedure for these genes based on two-tubes multiplex amplification of DNA pools and semiconductor sequencing with the Ion Torrent Personal Genome Machine (PGM). We created one pool with DNA from 20 GS patients previously Sanger sequenced for the coding exons of SLC12A3 . A total of 13 mutations present in 11 of these patients were used as control variants to validate the NGS procedure. The full coding sequence of SLC12A3 , CLCNKB and CLCNKA was amplified in only two Ampliseq tubes and processed and sequenced with the PGM. Large SLC12A3 and CLCNKB deletions were ascertained through multiplex ligation-dependent probe amplification in some patients. With the exception of the SLC12A3 exon 9, all the amplicons were successfully read and 12 of the 13 control variants were detected. The analysis of CLCNKB showed four putative mutations in the GS pool that were further assigned to specific patients. Two patients were heterozygous compounds for a single-nucleotide mutation and a large deletion at SLC12A3 or CLCNKB . We reported a NGS procedure that would facilitate the rapid and cost-effective large-scale screening of the three renal salt-handling genes. In addition to characterize the mutational spectrum of GS patients, the described procedure would facilitate the rapid and cost-effective screening of these genes at a population scale.
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a labor and cost effective non optical semiconductor ion torrent next generation sequencing of the slc12a3 and clcnka b genes in gitelman s syndrome patients
Journal of Human Genetics, 2014Co-Authors: Beatriz Tavira, Juan Gómez, Fernando Santos, Helena Gil, Victoria Alvarez, Eliecer CotoAbstract:A labor- and cost-effective non-optical semiconductor ( Ion Torrent ) next-generation sequencing of the SLC12A 3 and CLCNKA/B genes in Gitelman’s syndrome patients
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a new mutation intron 9 1 g t in the slc12a3 gene is linked to gitelman syndrome in gypsies
Kidney International, 2004Co-Authors: Eliecer Coto, Nikola Jeck, Victoria Alvarez, Julian Rodriguez, Rosario Stone, Cesar Loris, Luis M Rodriguez, Michel Fischbach, Hannsjorg W Seyberth, Fernando SantosAbstract:A new mutation (intron 9 +1 G>T) in the SLC12A3 gene is linked to Gitelman syndrome in Gypsies. Background Gitel syndrome is an inherited tubular disorder characterized by metabolic alkalosis, hypokalemia, and hypomagnesemia of renal origin and hypocalciuria. The majority of patients with Gitelman syndrome carry inactivating mutations in the SLC12A3 gene encoding the sodium-chloride cotransporter located in the distal convoluted tubule. The purpose of this study was to investigate the underlying mutation in Gitelman syndrome patients of Gypsy race from different geographic origin. Methods Twenty Gypsy patients with clinical and biochemical features of Gitelman syndrome were investigated by mutational analysis. The patients belonged to 12 unrelated Gypsy families living in four different European countries. The parents and unaffected siblings of each patient, as well as the DNA of a population of 200 healthy control patients, were also analyzed. Results All patients were homozygous for the same splice site mutation, guanine to thymine in the first position of intron 9 of SLC12A3 gene. This mutation was not found in the control population. Parents were heterozygous for the mutation. Despite sharing a common mutation, the clinical manifestations of the syndrome in the patients varied from lack of symptoms in six children to severe growth retardation in four. Conclusion Demonstration of a novel point mutation within the SLC12A3 gene in our cohort of Gypsy families with Gitelman syndrome is highly suggestive of a founder effect. This finding will facilitate the identification of the genetic defect in further cases of Gitelman syndrome among the Gypsy population. Our study represents the largest series ever published of patients with Gitelman syndrome having the same underlying mutation, and supports the lack of correlation between genotype and clinical phenotype in this disease.
Victoria Alvarez - One of the best experts on this subject based on the ideXlab platform.
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A labor- and cost-effective non-optical semiconductor (Ion Torrent) next-generation sequencing of the SLC12A3 and CLCNKA/B genes in Gitelman’s syndrome patients
Journal of Human Genetics, 2014Co-Authors: Beatriz Tavira, Juan Gómez, Fernando Santos, Helena Gil, Victoria Alvarez, Eliecer CotoAbstract:Gitelman’s syndrome (GS) is a rare recessive disorder caused by mutations in the renal salt-handling genes SLC12A3 and CLCNKB . Our aim was to develop a next-generation sequencing (NGS) procedure for these genes based on two-tubes multiplex amplification of DNA pools and semiconductor sequencing with the Ion Torrent Personal Genome Machine (PGM). We created one pool with DNA from 20 GS patients previously Sanger sequenced for the coding exons of SLC12A3 . A total of 13 mutations present in 11 of these patients were used as control variants to validate the NGS procedure. The full coding sequence of SLC12A3 , CLCNKB and CLCNKA was amplified in only two Ampliseq tubes and processed and sequenced with the PGM. Large SLC12A3 and CLCNKB deletions were ascertained through multiplex ligation-dependent probe amplification in some patients. With the exception of the SLC12A3 exon 9, all the amplicons were successfully read and 12 of the 13 control variants were detected. The analysis of CLCNKB showed four putative mutations in the GS pool that were further assigned to specific patients. Two patients were heterozygous compounds for a single-nucleotide mutation and a large deletion at SLC12A3 or CLCNKB . We reported a NGS procedure that would facilitate the rapid and cost-effective large-scale screening of the three renal salt-handling genes. In addition to characterize the mutational spectrum of GS patients, the described procedure would facilitate the rapid and cost-effective screening of these genes at a population scale.
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a labor and cost effective non optical semiconductor ion torrent next generation sequencing of the slc12a3 and clcnka b genes in gitelman s syndrome patients
Journal of Human Genetics, 2014Co-Authors: Beatriz Tavira, Juan Gómez, Fernando Santos, Helena Gil, Victoria Alvarez, Eliecer CotoAbstract:A labor- and cost-effective non-optical semiconductor ( Ion Torrent ) next-generation sequencing of the SLC12A 3 and CLCNKA/B genes in Gitelman’s syndrome patients
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a new mutation intron 9 1 g t in the slc12a3 gene is linked to gitelman syndrome in gypsies
Kidney International, 2004Co-Authors: Eliecer Coto, Nikola Jeck, Victoria Alvarez, Julian Rodriguez, Rosario Stone, Cesar Loris, Luis M Rodriguez, Michel Fischbach, Hannsjorg W Seyberth, Fernando SantosAbstract:A new mutation (intron 9 +1 G>T) in the SLC12A3 gene is linked to Gitelman syndrome in Gypsies. Background Gitel syndrome is an inherited tubular disorder characterized by metabolic alkalosis, hypokalemia, and hypomagnesemia of renal origin and hypocalciuria. The majority of patients with Gitelman syndrome carry inactivating mutations in the SLC12A3 gene encoding the sodium-chloride cotransporter located in the distal convoluted tubule. The purpose of this study was to investigate the underlying mutation in Gitelman syndrome patients of Gypsy race from different geographic origin. Methods Twenty Gypsy patients with clinical and biochemical features of Gitelman syndrome were investigated by mutational analysis. The patients belonged to 12 unrelated Gypsy families living in four different European countries. The parents and unaffected siblings of each patient, as well as the DNA of a population of 200 healthy control patients, were also analyzed. Results All patients were homozygous for the same splice site mutation, guanine to thymine in the first position of intron 9 of SLC12A3 gene. This mutation was not found in the control population. Parents were heterozygous for the mutation. Despite sharing a common mutation, the clinical manifestations of the syndrome in the patients varied from lack of symptoms in six children to severe growth retardation in four. Conclusion Demonstration of a novel point mutation within the SLC12A3 gene in our cohort of Gypsy families with Gitelman syndrome is highly suggestive of a founder effect. This finding will facilitate the identification of the genetic defect in further cases of Gitelman syndrome among the Gypsy population. Our study represents the largest series ever published of patients with Gitelman syndrome having the same underlying mutation, and supports the lack of correlation between genotype and clinical phenotype in this disease.