Cystinuria is an autosomal recessive disease caused by mutations in SLC3A1 ( rBAT) and SLC7A9 ( b
0,+
AT). Gene targeting of the catalytic subunit ( Slc7a9) in mice leads to excessive excretion of ...cystine, lysine, arginine, and ornithine. Here, we studied this non-type I cystinuria mouse model using gene expression analysis, Western blotting, clearance, and brush-border membrane vesicle (BBMV) uptake experiments to further characterize the renal and intestinal consequences of losing Slc7a9 function. The electrogenic and BBMV flux studies in the intestine suggested that arginine and ornithine are transported via other routes apart from system b
0,+
. No remarkable gene expression changes were observed in other amino acid transporters and the peptide transporters in the intestine and kidney. Furthermore, the glomerular filtration rate (GFR) was reduced by 30% in knockout animals compared with wild-type animals. The fractional excretion of arginine was increased as expected (∼100%), but fractional excretions of lysine (∼35%), ornithine (∼16%), and cystine (∼11%) were less affected. Loss of function of b
0,+
AT reduced transport of cystine and arginine in renal BBMVs and completely abolished the exchanger activity of dibasic amino acids with neutral amino acids. In conclusion, loss of Slc7a9 function decreases the GFR and increases the excretion of several amino acids to a lesser extent than expected with no clear regulation at the mRNA and protein level of alternative transporters and no increased renal epithelial uptake. These observations indicate that transporters located in distal segments of the kidney and/or metabolic pathways may partially compensate for Slc7a9 loss of function.
La cistinúria és una aminoacidúria hereditària autosòmica recessiva (tipus I, OMIM 220100) i dominant amb penetrança incompleta (tipus no I, OMIM 600918) caracteritzada per un defecte en el transport ...d'aminoàcids bàsics i cistina que afecta les cèl·lules epitelials del túbul renal i de l'intestí. Es manifesta per una hiperaminoacidúria de cistina i aminoàcid dibàsics. La cistina precipita formant càlculs renals que poden produir obstruccions, infeccions i insuficiència renal en alguns casos. S'han descrit tres fenotips: I, no-I i mixte. El 1994 es clonà el cDNA humà de SLC3A1 (que codifica per rBAT), es mapà al cromosoma 2p16, i el nostre grup trobà mutacions en pacients de cistinúria de tipus I. Aquestes mutacions causaven un defecte en el transport quan s'expresaven en oòcits.S'ha acotat el locus de cistinúria de tipus no I al cromosoma 19q13.1, en una zona compresa entre els marcadors C13 i D19S587 de 2,3 Mb. S'ha clonat el gen SLC7A9 que codifica per a la subunitat lleugera de rBAT, b0,+AT. S'ha realitzat un anàlisi exhaustiu de mutacions del gen SLC7A9 en pacients de cistinúria. Aquestes mutacions causen pèrdua de funció del sistema de transport b0,+.El 88% dels al·lels associats al fenotip no I presenten mutacions a SLC7A9 indicant que és el principal responsable del tipus no I. S'han identificat 52 i 24 mutacions noves a SLC7A9 i SLC3A1 en 164 famílies amb cistinúria de l'Internacional Cystinuria Consortium (ICC), que eleven el total de mutacions publicades a 66 i 105 a SLC7A9 i SLC3A1 respectivament.Les mutacions identificades per l'ICC expliquen el 90,5%, 87,6% i 89,3% de pacients amb cistinúria de tipus I, no I i mixt, respectivament. Les mutacions més freqüents de SLC3A1 i SLC7A9 són p.M467T (26,4% dels al·lels mutats) i p.G105R (27,4% dels al·lels mutats) respectivament.L'anàlisi del mRNA de vuit mutacions puntuals de SLC7A9, que no afecten zones consens de splicing, revela que tres d'elles (les freqüents p.R333W i c.614dupA i la rara c.586C>T) presenten alteracions en el splicing. Aquestes mutacions eleven el percentatge d'al·lels amb splicing erroni del 7% al 28%.Per estudis de correlació genotip-fenotip s'ha observat que majoritàriament les mutacions a SLC3A1 i SLC7A9 s'associen a fenotips I i no I respectivament, però aproximadament el 12% dels heterozigots de SLC7A9 presenten fenotip I, i només el 4% dels heterozigots de SLC3A1 (mutació dupE5-E9) presenten fenotip no I. Aquesta dissociació entre genotip i fenotip ha motivat la proposta d'una nova classificació de la cistinúria: tipus A, causat per dues mutacions a SLC3A1; tipus B: causat per dues mutacions a SLC7A9 ; i un possible tipus AB, causat per una mutació a cada un dels gens. Els nostres resultats demostren que l'herència digènica (AB) no dóna lloc a un desenvolupament complet de la malaltia (litiasi), encara que pot agreujar l'hiperexcreció d'aminoàcids.Aproximadament el 13% dels al·lels de pacients amb cistinúria de l'ICC no han estat explicats per mutacions a SLC3A1 o SLC7A9 Aquests al·lels podrien explicar-se per mutacions al promotor o introns, a polimorfismes o a mutacions a altres gens.S'han identificat dos polimorfismes de canvi d'aminoàcid (p.V142A i p.L223M) i tres a la regió promotora de SLC7A9 (c.1-313G>T i c.1-617G>T c.1-1314G>A) que estan associats a cistinúria, suggerint un possible paper d'aquests polimorfismes en la malaltia o l'existència de mutacions associades a aquests encara no identificades. D'altra banda, el polimorfisme IVS8+8C>A en el gen candidat SLC7A8 (LAT-2) sembla que està associat a cistinúria, suggerint un possible paper causatiu o modulador de LAT-2 en el fenotip cistinúric. S'ha descartat la implicació de SLC7A10 (asc-1) en la cistinúria. S'ha començat a generar un ratolí knockout per Slc7a8 (LAT-2) per conèixer el seu possible paper en el fenotip cistinúric i en els altres teixits on s'expressa.
Cystinuria is an autosomal recessive (type I, OMIM 220100) and dominant with low penetrance (type non-I, OMIM 600918) aminoaciduria due to a disorder of renal reabsorption of cystine and dibasic amino acids, which results in urolithiasis of cystine. Three cystinuria phenotypes have been described: I, non-I and mixed. Cystinuria is due to mutations in the heavy subunit rBAT (SLC3A1) and in the light subunit b0,+AT (SLC7A9) of the heteromeric amino acid transporter system b0,+. We narrowed down the locus for type non-I cystinuria on chromosome 19q13.1 to 2,3 Mb, between markers C13 and D19S587. Then cloned SLC7A9, the gene that encodes for the light subunit of rBAT, b0,+AT. We performed an exhaustive mutation analysis of SLC7A9 in cystinuria patients. These mutations caused loss of function of the system b0,+. We have identified 52 new mutations in SLC7A9 and 24 in SLC3A1 from 164 probands that increases the total number of mutations to 66 and 105 respectively. The mutated alleles reached an average of 86.8%. Mutations in SLC3A1 (type A) and in SLC7A9 (type B) accounted for 44.3% and 55.7% of the alleles identified. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. The mRNA analysis of eight point mutations in SLC7A9, which do not affect splicing consensus sequences, revealed that three of them showed aberrant splicing. These mutations increase the percentage of alleles with aberrant splicing from 7% to 28%.SLC3A1 heterozygotes showed phenotype I with the exception of some carriers of dupE5-E9, which showed phenotype non-I. SLC7A9 heterozygotes showed phenotype non-I, with the exception of eleven mutations. Therefore we proposed a new cystinuria classification based on genetic criteria: type A caused by two mutations in SLC3A1 (rBAT), type B caused by two mutations in SLC7A9 (bo,+AT) and, the possibility of type AB with one mutation on each of the above genes. Our results indicate that digenic inheritance contributes to the urine phenotype but none of the AB individuals presented cystine urolithiasis. There are still 13% of unexplained alleles. Our results suggest that these alleles may be due to amino acid change polymorphisms and to promoter variants in SLC7A9, rather than to mutations in a third gene.
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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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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