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Manuel Palacin - One of the best experts on this subject based on the ideXlab platform.
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inducible slc7a7 knockout mouse model recapitulates lysinuric protein intolerance disease
International Journal of Molecular Sciences, 2019Co-Authors: Susanna Bodoy, Maria Pia Sperandeo, Meritxell Espinoguarch, Fernando Sotillo, Aida Ormazabal, Gianfranco Sebastio, Antonio Zorzano, Rafael Artuch, Manuel PalacinAbstract:Lysinuric protein intolerance (LPI) is a rare autosomal disease caused by defective cationic amino acid (CAA) transport due to mutations in SLC7A7, which encodes for the y+LAT1 transporter. LPI patients suffer from a wide variety of symptoms, which range from failure to thrive, hyperammonemia, and nephropathy to pulmonar alveolar proteinosis (PAP), a potentially life-threatening complication. Hyperammonemia is currently prevented by citrulline supplementation. However, the full impact of this treatment is not completely understood. In contrast, there is no defined therapy for the multiple reported complications of LPI, including PAP, for which bronchoalveolar lavages do not prevent progression of the disease. The lack of a viable LPI model prompted us to generate a tamoxifen-inducible Slc7a7 knockout mouse (Slc7a7−/−). The Slc7a7−/− model resembles the human LPI phenotype, including malabsorption and impaired reabsorption of CAA, hypoargininemia and hyperammonemia. Interestingly, the Slc7a7−/− mice also develops PAP and neurological impairment. We observed that citrulline treatment improves the metabolic derangement and survival. On the basis of our findings, the Slc7a7−/− model emerges as a promising tool to further study the complexity of LPI, including its immune-like complications, and to design evidence-based therapies to halt its progression.
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Digenic Inheritance in Cystinuria Mouse Model.
PloS one, 2015Co-Authors: Meritxell Espino, Manuel Palacin, Mariona Font-llitjós, Clara Vilches, Eduardo Salido, Esther Prat, Miguel López De Heredia, Virginia NunesAbstract:Cystinuria is an aminoaciduria caused by mutations in the genes that encode the two subunits of the amino acid transport system b0,+, responsible for the renal reabsorption of cystine and dibasic amino acids. The clinical symptoms of cystinuria relate to nephrolithiasis, due to the precipitation of cystine in urine. Mutations in SLC3A1, which codes for the heavy subunit rBAT, cause cystinuria type A, whereas mutations in SLC7A9, which encodes the light subunit b0,+AT, cause cystinuria type B. By crossing Slc3a1-/- with SLC7A9-/- mice we generated a type AB cystinuria mouse model to test digenic inheritance of cystinuria. The 9 genotypes obtained have been analyzed at early (2- and 5-months) and late stage (8-months) of the disease. Monitoring the lithiasic phenotype by X-ray, urine amino acid content analysis and protein expression studies have shown that double heterozygous mice (SLC7A9+/-Slc3a1+/-) present lower expression of system b0,+ and higher hyperexcretion of cystine than single heterozygotes (SLC7A9+/-Slc3a1+/+ and SLC7A9+/+Slc3a1+/-) and give rise to lithiasis in 4% of the mice, demonstrating that cystinuria has a digenic inheritance in this mouse model. Moreover in this study it has been demonstrated a genotype/phenotype correlation in type AB cystinuria mouse model providing new insights for further molecular and genetic studies of cystinuria patients.
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New insights into cystinuria: 40 new mutations, genotype–phenotype correlation, and digenic inheritance causing partial phenotype
Journal of medical genetics, 2005Co-Authors: Mariona Font-llitjós, Manuel Palacin, Luigi Bisceglia, Leopoldo Zelante, Ferran Rousaud, L. De Sanctis, Maite Jiménez-vidal, M. Di Perna, Virginia NunesAbstract:Objective: To clarify the genotype–phenotype correlation and elucidate the role of digenic inheritance in cystinuria. Methods: 164 probands from the International Cystinuria Consortium were screened for mutations in SLC3A1 (type A) and SLC7A9 (type B) and classified on the basis of urine excretion of cystine and dibasic amino acids by obligate heterozygotes into 37 type I (silent heterozygotes), 46 type non-I (hyperexcretor heterozygotes), 14 mixed, and 67 untyped probands. Results: Mutations were identified in 97% of the probands, representing 282 alleles (86.8%). Forty new mutations were identified: 24 in SLC3A1 and 16 in SLC7A9 . Type A heterozygotes showed phenotype I, but mutation DupE5-E9 showed phenotype non-I in some heterozygotes. Type B heterozygotes showed phenotype non-I, with the exception of 10 type B mutations which showed phenotype I in some heterozygotes. Thus most type I probands carried type A mutations and all type non-I probands carried type B mutations. Types B and A mutations contributed to mixed type, BB being the most representative genotype. Two mixed cystinuria families transmitted mutations in both genes: double compound heterozygotes (type AB) had greater aminoaciduria than single heterozygotes in their family. Conclusions: Digenic inheritance is an exception (two of 164 families), with a limited contribution to the aminoaciduria values (partial phenotype) in cystinuria. Further mutational analysis could focus on one of the two genes ( SLC3A1 preferentially for type I and SLC7A9 for type non-I probands), while for mixed probands analysis of both genes might be required, with priority given to SLC7A9 .
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SLC7A9-deficient mice develop cystinuria non-I and cystine urolithiasis
Human molecular genetics, 2003Co-Authors: Lídia Feliubadaló, Antonio Zorzano, Manuel Palacin, Ferran Rousaud, Maria L. Arbonés, Sandra Mañas, Josep Chillarón, Joana Visa, Margot Rodés, Virginia NunesAbstract:Cystinuria is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in urolithiasis of cystine. Cystinuria is caused by defects in the amino acid transport system b0,+ (i.e. the rBAT/b0,+AT heteromeric complex). Mutations in SLC3A1, encoding rBAT, cause cystinuria type A, characterized by a silent phenotype in heterozygotes (phenotype I). Mutations in SLC7A9, encoding b0,+AT, cause cystinuria type B, in which heterozygotes in most cases hyperexcrete cystine and dibasic amino acids (phenotype non-I). To facilitate in vivo investigation of b0,+AT in cystinuria, SLC7A9 knockout mice have been generated. Expression of b0,+AT protein is completely abolished in the kidney of SLC7A9-/- mice ('Stones'). In contrast, Stones expressed significant amounts of rBAT protein, which is covalently linked to unidentified light subunit(s). Stones mice present a dramatic hyperexcretion of cystine and dibasic amino acids, while SLC7A9+/- mice show moderate but significant hyperexcretion of these amino acids (phenotype non-I). Forty-two per cent of Stones mice develop cystine calculi in the urinary system. Calculi develop during the first month of life and grow throughout the life span of the animals. Histopathology in kidney reveals typical changes for urolithiasis (tubular and pelvic dilatation, tubular necrosis, tubular hyaline droplets and chronic interstitial nephritis). The fact that some Stones mice, generated in a mixed genetic background, develop cystine calculi from an early age, while others do not develop them in their first year of life, suggests the involvement of modifier genes in the lithiasis phenotype. Thus, Stones provide a valid model of cystinuria which can be used in the study of genetic, pharmacological and environmental factors involved in cystine urolithiasis.
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SLC7A9 mutations in all three cystinuria subtypes
Kidney International, 2002Co-Authors: Daniel Leclerc, Manuel Palacin, Marylise Boutros, Daniel SuhAbstract:SLC7A9 mutations in all three cystinuria subtypes. Background Cystinuria is an inherited disorder of cystine and dibasic amino acid transport in kidney. Subtypes are defined by the urinary cystine excretion patterns of the obligate heterozygous parents: Type I/N (fully recessive or silent); Type II/N (high excretor); Type III/N (moderate excretor). The first gene implicated in cystinuria ( SLC3A1 ) is associated with the Type I urinary phenotype. A second cystinuria gene ( SLC7A9 ) was recently isolated, and mutations of this gene were associated with dominant (non-Type I) cystinuria alleles. Here we report genotype-phenotype studies of SLC7A9 mutations in a cohort of well-characterized cystinuria probands and their family members. Methods Individual exons of the SLC7A9 gene were screened by single strand conformation polymorphism (SSCP) analysis and sequencing of abnormally migrating fragments. Results Seven mutations were identified. A singlebp insertion (799insA) was present in four patients: on Type III alleles in two patients and on Type II alleles in two patients. These results suggest that Type II and Type III may be caused by the same mutation and, therefore, other factors must influence urinary cystine excretion. A 4bp deletion in intron 12 (IVS12+4delAGTA) and a missense mutation (1245G→A, A354T) were identified on Type III alleles. A nonsense codon (1491G→T, E436X) and a possible splicing mutation (IVS9-17G→A) were seen in a Type I/III patient, but the mutations could not be assigned to particular alleles. Of additional interest were two missense mutations (316T→C, I44T and 967C→T, P261L) linked to Type I alleles. Conclusion Our results provide evidence that some SLC7A9 mutations may be associated with fully recessive (Type I) forms of cystinuria. We also demonstrate SLC7A9 mutations in dominant Types II and III cystinuria. The finding of SLC7A9 mutations in all three subtypes underscores the complex interactions between specific cystinuria genes and other factors influencing cystine excretion. A simpler phenotypic classification scheme (recessive and dominant) for cystinuria is warranted.
Thomas Eggermann - One of the best experts on this subject based on the ideXlab platform.
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No evidence for point mutations in the novel renal cystine transporter AGT1/SLC7A13 contributing to the etiology of cystinuria
BMC nephrology, 2018Co-Authors: Kathrin Olschok, Sven Lahme, Udo Vester, Ingo Kurth, Thomas EggermannAbstract:Cystinuria is caused by the defective renal reabsorption of cystine and dibasic amino acids, and results in cystine stone formation. So far, mutations in two genes have been identified as causative. The SLC3A1/rBAT gene encodes the heavy subunit of the heterodimeric rBAT-b0,+AT transporter, whereas the light chain is encoded by the SLC7A9/ b0,+AT gene. In nearly 85% of patients mutations in both genes are detectable, but a significant number of patients currently remains without a molecular diagnosis. Thus, the existence of a further cystinuria gene had been suggested, and the recently identified AGT1/SLC7A13 represents the long-postulated partner of rBAT and third cystinuria candidate gene. We screened a cohort of 17 cystinuria patients for SLC7A13 variants which were negative for SLC3A1 and SLC7A9 mutations. Despite strong evidences for an involvement of SLC7A13 mutations in cystinuria, we could not confirm a relevant role of SLC7A13 for the disease. With the exclusion of SLC7A13/AGT1 as the third cystinuria gene accounting for the SLC3A1 and SLC7A9 mutation negative cases, it becomes obvious that other genetic factors should be responsible for the cystinuria phenotype in nearly 15% of patients.
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eLS - Molecular Genetics of Cystinuria
eLS, 2012Co-Authors: Thomas EggermannAbstract:Among the kidney stone diseases, cystinuria (OMIM 220100) is unique as it is exclusively caused by genomic alterations. Cystinuria is associated with mutations in the SLC3A1 and the SLC7A9 genes encoding the subunits rBAT and b0,+AT of the renal b0,+ transporter. Mutations in SLC3A1 are usually inherited autosomal recessively, but SLC7A9 mutations result in a broad clinical variability. The detection rate for mutations is influenced by the ethnic origin of the patient and by the functional significance of the variant. As a result, mutations cannot be detected in 100% of alleles, but it reaches approximately 85%. In case of SLC3A1, large rearrangements in 2p21 cause cystinuria and severe hypotonia (hypotonia–cystinuria syndrome – HCS). Meanwhile, a large number of mutations in both genes have been reported, several of these variants were functionally analysed. Thereby, the aetiology of the disease as well as the physiological mechanisms of the renal trafficking of cystine could be deciphered. Key Concepts: Cystinuria is characterised by the defect transport of cystine, resulting in cystine stone formation. With SLC3A1 and SLC7A9, two disease causing genes have been identified. SLC3A1 and SLC7A9 encode the two subunits of the amino acid transporter b0,+. Mutations in SLC3A1 are predominantly associated with an autosomal recessive inheritance, whereas SLC7A9 mutations show an autosomal dominant inheritance with an incomplete penetrance. Homozygosity for large genomic deletions affecting both the SLC3A1 and the PREPL gene in 2p21 cause the hypotonia–cystinuria syndrome. Keywords: cystinuria; molecular Genetics; SLC3A1; SLC7A9; mutations; hypotonia-cystinuria syndrome
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Molecular Genetic Testing in Cystinuria
International Journal of Human Genetics, 2011Co-Authors: Thomas Eggermann, Sabrina Spengler, Julia Wirth, Sven LahmeAbstract:Cystinuria (OMIM 220100) is caused by the defective transport of cystine and the dibasic amino acids in the proximal renal tubule and in the epithelial cells of the gastrointestinal tract. We analysed a cohort of 26 unrelated cystinuria patients diagnosed on the basis of stone formation. Direct sequencing of all coding regions and exon-intron boundaries of the SLC3A1 and SLC7A9 genes allowed us to identify 26 different mutations in 23 out of the 26 patients, in total they accounted for 40 affected chromosomes. Three of the 26 are novel mutations, two in SLC3A1 and one in SLC7A9. Interestingly, two of our patients carried three mutations in SLC3A1 each, one patient was mixed heterozygous for SLC3A1 and SLC7A9 mutations. In summary, these findings expand the spectrum of SLC3A1 and SLC7A9 mutations and confirm the heterogeneity and complexity of cystinuria. If we assume an autosomal recessive inheritance of the disease, our detection rate was 88.5% and thereby relatively high in comparison to other studies. Nevertheless we have to consider that at least SLC7A9 mutations are often dominant, we therefore think that our effective detection rate is higher. Additionally, the broad pathophysiological consequences of SLC7A9 mutations make an individual prognosis and genetic counselling difficult.
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Functional characterization of SLC7A9 polymorphisms assumed to influence the cystinuria phenotype.
Clinical nephrology, 2006Co-Authors: Eva Brauers, Schmidt C, Klaus Zerres, Thomas EggermannAbstract:Cystinuria is a hereditary disorder of cystine and dibasic amino acid transport across the luminal membrane of renal tubules and intestine, resulting in recurrent nephrolithiasis. While mutations in the SLC3-A 1 gene cause type I cystinuria, patients with non-type I cystinuria mostly carry mutations in the SLC7A9 gene. However, there is evidence that further genetic factors cause and influence the cystinuria phenotype. We recently demonstrated that specific polymorphisms in Exons 3, 4, 5 and 6 of the SLC7A9 gene show a significant different allelic distribution between cystinuria patients and controls. To determine the role of the variants in these exons we transfected COS7 cells with expression constructs containing different haplotypes. Sequencing of cDNAs derived from the resulting SLC7A9 mRNAs did not reveal any differences in expression between the minigenes of the specific haplotypes. Our findings suggest that the variants in SLC7A9 do not influence splicing behavior. Thus, the reasons for the different allelic distribution between cystinuria patients and controls remain unclear. It is conceivable that this distribution is caused by the association of certain alleles with so far undetected mutations in the gene.
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Functional analysis of a new splice site mutation, c.605-3C > A, in the cystinuria gene SLC7A9 leading to exon skipping
Molecular Genetics and Metabolism, 2005Co-Authors: Schmidt C, Sven Lahme, Klaus Zerres, Thomas EggermannAbstract:Abstract Cystinuria is a hereditary disorder of cystine and dibasic amino acid transport across the luminal membrane of renal tubules and intestine, resulting in recurrent nephrolithiasis. While mutations in the SLC3A1 gene cause type I cystinuria, patients with non-type I cystinuria carry mutations in the SLC7A9 gene. Up to now, more than 80 mutations in SLC3A1 and 50 in SLC7A9 have been reported in the literature. While deletions, duplications, and truncating mutations can often unambiguously classified to be pathogenic, the functional relevance of base pair substitutions is often difficult to predict. To determine the functional relevance of a new splice site mutation in intron 5 of SLC7A9 , c.605-3C > A, we transfected COS7 cells with expression constructs containing the wild-type and mutant allele, respectively. cDNAs derived from the resulting SLC7A9 mRNAs were sequenced. By this approach we could demonstrate that the mutant allele c.605-3A causes exon skipping and therefore represents a splice site mutation. To the best of our knowledge, this is the first splice site mutation in a cystinuria gene with a proven functional consequence.
Rima Rozen - One of the best experts on this subject based on the ideXlab platform.
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SLC7A9 mutations in all three cystinuria subtypes.
Kidney international, 2002Co-Authors: Daniel Leclerc, Marylise Boutros, Daniel Suh, Manuel Palacı́n, James R. Ellis, Paul Goodyer, Rima RozenAbstract:Cystinuria is an inherited disorder of cystine and dibasic amino acid transport in kidney. Subtypes are defined by the urinary cystine excretion patterns of the obligate heterozygous parents: Type I/N (fully recessive or silent); Type II/N (high excretor); Type III/N (moderate excretor). The first gene implicated in cystinuria (SLC3A1) is associated with the Type I urinary phenotype. A second cystinuria gene (SLC7A9) was recently isolated, and mutations of this gene were associated with dominant (non-Type I) cystinuria alleles. Here we report genotype-phenotype studies of SLC7A9 mutations in a cohort of well-characterized cystinuria probands and their family members. Individual exons of the SLC7A9 gene were screened by single strand conformation polymorphism (SSCP) analysis and sequencing of abnormally migrating fragments. Seven mutations were identified. A single bp insertion (799insA) was present in four patients: on Type III alleles in two patients and on Type II alleles in two patients. These results suggest that Type II and Type III may be caused by the same mutation and, therefore, other factors must influence urinary cystine excretion. A 4bp deletion in intron 12 (IVS12+4delAGTA) and a missense mutation (1245G-->A, A354T) were identified on Type III alleles. A nonsense codon (1491G-->T, E436X) and a possible splicing mutation (IVS9-17G-->A) were seen in a Type I/III patient, but the mutations could not be assigned to particular alleles. Of additional interest were two missense mutations (316T-->C, I44T and 967C-->T, P261L) linked to Type I alleles. Our results provide evidence that some SLC7A9 mutations may be associated with fully recessive (Type I) forms of cystinuria. We also demonstrate SLC7A9 mutations in dominant Types II and III cystinuria. The finding of SLC7A9 mutations in all three subtypes underscores the complex interactions between specific cystinuria genes and other factors influencing cystine excretion. A simpler phenotypic classification scheme (recessive and dominant) for cystinuria is warranted.
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Is the SLC7A10 gene on chromosome 19 a candidate locus for cystinuria
Molecular genetics and metabolism, 2001Co-Authors: Daniel Leclerc, James R. Ellis, Paul Goodyer, Rima RozenAbstract:One of the genes (SLC7A9) that causes cystinuria, an inborn error of amino acid transport, is localized to 19q13. Close examination of human genomic DNA sequences has identified a similar gene (SLC7A10) that also maps to the 19q13.1 region and is highly expressed in kidney. The homologies between SLC7A9 and SLC7A10 are likely the result of gene duplication. SLC7A10 is known to encode a protein with a function similar to that of the SLC7A9 gene product. To determine if mutations in the SLC7A10 gene could also cause cystinuria, we characterized the primary genomic structure and sequenced the 11 exons and surrounding sequences from 10 unrelated patients with cystinuria. We identified one missense mutation which may account for cystinuria in one family. We also observed one intronic change, as well as one silent mutation, that were seen only in cystinuria patients. We therefore suggest that the SLC7A10 gene warrants further investigation as another candidate gene for cystinuria.
Virginia Nunes - One of the best experts on this subject based on the ideXlab platform.
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Digenic Inheritance in Cystinuria Mouse Model.
PloS one, 2015Co-Authors: Meritxell Espino, Manuel Palacin, Mariona Font-llitjós, Clara Vilches, Eduardo Salido, Esther Prat, Miguel López De Heredia, Virginia NunesAbstract:Cystinuria is an aminoaciduria caused by mutations in the genes that encode the two subunits of the amino acid transport system b0,+, responsible for the renal reabsorption of cystine and dibasic amino acids. The clinical symptoms of cystinuria relate to nephrolithiasis, due to the precipitation of cystine in urine. Mutations in SLC3A1, which codes for the heavy subunit rBAT, cause cystinuria type A, whereas mutations in SLC7A9, which encodes the light subunit b0,+AT, cause cystinuria type B. By crossing Slc3a1-/- with SLC7A9-/- mice we generated a type AB cystinuria mouse model to test digenic inheritance of cystinuria. The 9 genotypes obtained have been analyzed at early (2- and 5-months) and late stage (8-months) of the disease. Monitoring the lithiasic phenotype by X-ray, urine amino acid content analysis and protein expression studies have shown that double heterozygous mice (SLC7A9+/-Slc3a1+/-) present lower expression of system b0,+ and higher hyperexcretion of cystine than single heterozygotes (SLC7A9+/-Slc3a1+/+ and SLC7A9+/+Slc3a1+/-) and give rise to lithiasis in 4% of the mice, demonstrating that cystinuria has a digenic inheritance in this mouse model. Moreover in this study it has been demonstrated a genotype/phenotype correlation in type AB cystinuria mouse model providing new insights for further molecular and genetic studies of cystinuria patients.
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New insights into cystinuria: 40 new mutations, genotype–phenotype correlation, and digenic inheritance causing partial phenotype
Journal of medical genetics, 2005Co-Authors: Mariona Font-llitjós, Manuel Palacin, Luigi Bisceglia, Leopoldo Zelante, Ferran Rousaud, L. De Sanctis, Maite Jiménez-vidal, M. Di Perna, Virginia NunesAbstract:Objective: To clarify the genotype–phenotype correlation and elucidate the role of digenic inheritance in cystinuria. Methods: 164 probands from the International Cystinuria Consortium were screened for mutations in SLC3A1 (type A) and SLC7A9 (type B) and classified on the basis of urine excretion of cystine and dibasic amino acids by obligate heterozygotes into 37 type I (silent heterozygotes), 46 type non-I (hyperexcretor heterozygotes), 14 mixed, and 67 untyped probands. Results: Mutations were identified in 97% of the probands, representing 282 alleles (86.8%). Forty new mutations were identified: 24 in SLC3A1 and 16 in SLC7A9 . Type A heterozygotes showed phenotype I, but mutation DupE5-E9 showed phenotype non-I in some heterozygotes. Type B heterozygotes showed phenotype non-I, with the exception of 10 type B mutations which showed phenotype I in some heterozygotes. Thus most type I probands carried type A mutations and all type non-I probands carried type B mutations. Types B and A mutations contributed to mixed type, BB being the most representative genotype. Two mixed cystinuria families transmitted mutations in both genes: double compound heterozygotes (type AB) had greater aminoaciduria than single heterozygotes in their family. Conclusions: Digenic inheritance is an exception (two of 164 families), with a limited contribution to the aminoaciduria values (partial phenotype) in cystinuria. Further mutational analysis could focus on one of the two genes ( SLC3A1 preferentially for type I and SLC7A9 for type non-I probands), while for mixed probands analysis of both genes might be required, with priority given to SLC7A9 .
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SLC7A9-deficient mice develop cystinuria non-I and cystine urolithiasis
Human molecular genetics, 2003Co-Authors: Lídia Feliubadaló, Antonio Zorzano, Manuel Palacin, Ferran Rousaud, Maria L. Arbonés, Sandra Mañas, Josep Chillarón, Joana Visa, Margot Rodés, Virginia NunesAbstract:Cystinuria is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in urolithiasis of cystine. Cystinuria is caused by defects in the amino acid transport system b0,+ (i.e. the rBAT/b0,+AT heteromeric complex). Mutations in SLC3A1, encoding rBAT, cause cystinuria type A, characterized by a silent phenotype in heterozygotes (phenotype I). Mutations in SLC7A9, encoding b0,+AT, cause cystinuria type B, in which heterozygotes in most cases hyperexcrete cystine and dibasic amino acids (phenotype non-I). To facilitate in vivo investigation of b0,+AT in cystinuria, SLC7A9 knockout mice have been generated. Expression of b0,+AT protein is completely abolished in the kidney of SLC7A9-/- mice ('Stones'). In contrast, Stones expressed significant amounts of rBAT protein, which is covalently linked to unidentified light subunit(s). Stones mice present a dramatic hyperexcretion of cystine and dibasic amino acids, while SLC7A9+/- mice show moderate but significant hyperexcretion of these amino acids (phenotype non-I). Forty-two per cent of Stones mice develop cystine calculi in the urinary system. Calculi develop during the first month of life and grow throughout the life span of the animals. Histopathology in kidney reveals typical changes for urolithiasis (tubular and pelvic dilatation, tubular necrosis, tubular hyaline droplets and chronic interstitial nephritis). The fact that some Stones mice, generated in a mixed genetic background, develop cystine calculi from an early age, while others do not develop them in their first year of life, suggests the involvement of modifier genes in the lithiasis phenotype. Thus, Stones provide a valid model of cystinuria which can be used in the study of genetic, pharmacological and environmental factors involved in cystine urolithiasis.
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Functional analysis of mutations in SLC7A9, and genotype-phenotype correlation in non-Type I cystinuria.
Human molecular genetics, 2001Co-Authors: Mariona Font, Elon Pras, Virginia Nunes, Luigi Bisceglia, Lídia Feliubadaló, Xavier Estivill, Eliahu Golomb, Yitshak Kreiss, Adamo Pio D'adamo, Leopoldo ZelanteAbstract:Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.
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Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.
Nature genetics, 1999Co-Authors: Lídia Feliubadaló, Virginia Nunes, Ferran Rousaud, Mariona Font, Xavier Estivill, Eliahu Golomb, Jesús Purroy, Michael Centola, Ivona Aksentijevich, Yitshak KreissAbstract:Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12–13.1 (refs 3,4). We have identified a new transcript, encoding a protein (bo,+AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes bo,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.
Kay Thomas - One of the best experts on this subject based on the ideXlab platform.
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Non-contrast computed tomography characteristics in a large cohort of cystinuria patient
World Journal of Urology, 2020Co-Authors: Hannah Warren, Daniel Poon, Rohit Srinivasan, Kerushan Thomas, Giles Rottenberg, Matthew Bultitude, Kay ThomasAbstract:Purpose Cystine stones are widely considered hard and difficult to treat. Hounsfield Units (HU) are used in other stone types to estimate ‘hardness’ and treatments based on that finding. Our objective was to report mean HU of cystine stones in vivo in a large case series of cystinuria patients and assess for differences in genotype. Methods A prospective case series of cystinuria patients referred to a specialist centre was analysed. CT imaging was assessed by two independent radiologists to determine in vivo attenuation of cystine calculi. Mean HU was compared for both cystinuria genes (SLC3A1 and SLC7A9) using an independent t-test. Results 164 adult cystinuric patients were identified (55% male), median age 43 years (range 18–80). Median follow up was 31 months (IQR 10–62). Genetic data available for 153/164 (93%) demonstrated 97 SLC3A1 (63%) and 55 (36%) SLC7A9 mutations (39 homozygous, 16 heterozygous) and one heterozygous for both SLC3A1/SLC7A9. 107 patients had CT images available demonstrating calculi. Median HU across the cohort was 633 (5th to 95th centile 328–780). There was no difference in mean HU between SLC3A1 and SLC7A9 genotypes ( p = 0.68) or homo and heterozygous SLC7A9 ( p = 0.70). Mean HU correlated with stone size (Pearson correlation coefficient = 0.51, p
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Non-contrast computed tomography characteristics in a large cohort of cystinuria patients.
World journal of urology, 2020Co-Authors: Hannah Warren, Daniel Poon, Rohit Srinivasan, Kerushan Thomas, Giles Rottenberg, Matthew Bultitude, Kay ThomasAbstract:PURPOSE Cystine stones are widely considered hard and difficult to treat. Hounsfield Units (HU) are used in other stone types to estimate 'hardness' and treatments based on that finding. Our objective was to report mean HU of cystine stones in vivo in a large case series of cystinuria patients and assess for differences in genotype. METHODS A prospective case series of cystinuria patients referred to a specialist centre was analysed. CT imaging was assessed by two independent radiologists to determine in vivo attenuation of cystine calculi. Mean HU was compared for both cystinuria genes (SLC3A1 and SLC7A9) using an independent t-test. RESULTS 164 adult cystinuric patients were identified (55% male), median age 43 years (range 18-80). Median follow up was 31 months (IQR 10-62). Genetic data available for 153/164 (93%) demonstrated 97 SLC3A1 (63%) and 55 (36%) SLC7A9 mutations (39 homozygous, 16 heterozygous) and one heterozygous for both SLC3A1/SLC7A9. 107 patients had CT images available demonstrating calculi. Median HU across the cohort was 633 (5th to 95th centile 328-780). There was no difference in mean HU between SLC3A1 and SLC7A9 genotypes (p = 0.68) or homo and heterozygous SLC7A9 (p = 0.70). Mean HU correlated with stone size (Pearson correlation coefficient = 0.51, p 1000 are unlikely pure cystine, and in a known cystinuric would suggest conversion to another stone type.
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Associating mutations causing cystinuria with disease severity with the aim of providing precision medicine
BMC Genomics, 2017Co-Authors: Henry J. Martell, Kay Thomas, Kathie A. Wong, Juan F. Martin, Ziyan Kassam, Mark N. WassAbstract:Background Cystinuria is an inherited disease that results in the formation of cystine stones in the kidney, which can have serious health complications. Two genes (SLC7A9 and SLC3A1) that form an amino acid transporter are known to be responsible for the disease. Variants that cause the disease disrupt amino acid transport across the cell membrane, leading to the build-up of relatively insoluble cystine, resulting in formation of stones. Assessing the effects of each mutation is critical in order to provide tailored treatment options for patients. We used various computational methods to assess the effects of cystinuria associated mutations, utilising information on protein function, evolutionary conservation and natural population variation of the two genes. We also analysed the ability of some methods to predict the phenotypes of individuals with cystinuria, based on their genotypes, and compared this to clinical data. Results Using a literature search, we collated a set of 94 SLC3A1 and 58 SLC7A9 point mutations known to be associated with cystinuria. There are differences in sequence location, evolutionary conservation, allele frequency, and predicted effect on protein function between these mutations and other genetic variants of the same genes that occur in a large population. Structural analysis considered how these mutations might lead to cystinuria. For SLC7A9, many mutations swap hydrophobic amino acids for charged amino acids or vice versa, while others affect known functional sites. For SLC3A1, functional information is currently insufficient to make confident predictions but mutations often result in the loss of hydrogen bonds and largely appear to affect protein stability. Finally, we showed that computational predictions of mutation severity were significantly correlated with the disease phenotypes of patients from a clinical study, despite different methods disagreeing for some of their predictions. Conclusions The results of this study are promising and highlight the areas of research which must now be pursued to better understand how mutations in SLC3A1 and SLC7A9 cause cystinuria. The application of our approach to a larger data set is essential, but we have shown that computational methods could play an important role in designing more effective personalised treatment options for patients with cystinuria.
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Associating mutations causing cystinuria with disease severity with the aim of providing precision medicine
BMC Genomics, 2017Co-Authors: Henry Martell, Kay Thomas, Ziyan Kassam, Kathie Wong, Juan Martin, Mark N. WassAbstract:Cystinuria is an inherited disease that results in the formation of cystine stones in the kidney, which can have serious health complications. Two genes (SLC7A9 and SLC3A1) that form an amino acid transporter are known to be responsible for the disease. Variants that cause the disease disrupt amino acid transport across the cell membrane, leading to the build-up of relatively insoluble cystine, resulting in formation of stones. Assessing the effects of each mutation is critical in order to provide tailored treatment options for patients. We used various computational methods to assess the effects of cystinuria associated mutations, utilising information on protein function, evolutionary conservation and natural population variation of the two genes. We also analysed the ability of some methods to predict the phenotypes of individuals with cystinuria, based on their genotypes, and compared this to clinical data. Using a literature search, we collated a set of 94 SLC3A1 and 58 SLC7A9 point mutations known to be associated with cystinuria. There are differences in sequence location, evolutionary conservation, allele frequency, and predicted effect on protein function between these mutations and other genetic variants of the same genes that occur in a large population. Structural analysis considered how these mutations might lead to cystinuria. For SLC7A9, many mutations swap hydrophobic amino acids for charged amino acids or vice versa, while others affect known functional sites. For SLC3A1, functional information is currently insufficient to make confident predictions but mutations often result in the loss of hydrogen bonds and largely appear to affect protein stability. Finally, we showed that computational predictions of mutation severity were significantly correlated with the disease phenotypes of patients from a clinical study, despite different methods disagreeing for some of their predictions. The results of this study are promising and highlight the areas of research which must now be pursued to better understand how mutations in SLC3A1 and SLC7A9 cause cystinuria. The application of our approach to a larger data set is essential, but we have shown that computational methods could play an important role in designing more effective personalised treatment options for patients with cystinuria.
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The genetic diversity of cystinuria in a UK population of patients
BJU international, 2015Co-Authors: Kathie Wong, Matthew Bultitude, Mark N. Wass, R. Mein, Frances Flinter, Caroline Pardy, Kay ThomasAbstract:Objective To examine the genetic mutations in the first UK cohort of patients with cystinuria with preliminary genotype/phenotype correlation. Patients and Methods DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) were used to identify the mutations in 74 patients in a specialist cystinuria clinic in the UK. Patients with type A cystinuria were classified into two groups: Group M patients had at least one missense mutation and Group N patients had two alleles of all other types of mutations including frameshift, splice site, nonsense, deletions and duplications. The levels of urinary dibasic amino acids, age at presentation of disease, number of stone episodes and interventions were compared between patients in the two groups using the Mann–Whitney U-test. Results In all, 41 patients had type A cystinuria, including one patient with a variant of unknown significance and 23 patients had type B cystinuria, including six patients with variants of unknown significance. One patient had three sequence variants in SLC7A9; however, two are of unknown significance. Three patients had type AB cystinuria. Three had a single mutation in SLC7A9. No identified mutations were found in three patients in either gene. There were a total of 88 mutations in SLC3A1 and 55 mutations in SLC7A9. There were 23 pathogenic mutations identified in our UK cohort of patients not previously published. In patients with type A cystinuria, the presence of a missense mutation correlated to lower levels of urinary lysine (mean [se] 611.9 [22.65] vs 752.3 [46.39] millimoles per mole of creatinine [mm/MC]; P=0.02), arginine (194.8 [24.83] vs 397.7 [15.32] mm/MC; P