No abstract

Lysinuric protein intolerance (LPI) is a rare autosomal inherited disease caused by defective cationic aminoacid transport 4F2hc/y þ LAT-1 at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is a multisystemic disease with a variety of clinical symptoms such as hepatosplenomegaly, osteoporosis, hypotonia, developmental delay, pulmonary insufficiency or end-stage renal disease. The SLC7A7 gene, which encodes the y þ LAT-1 protein, is mutated in LPI patients. Mutation analysis of the promoter localized in intron 1 and all exons of the SLC7A7 gene was performed in 11 patients from 9 unrelated LPI families. Point mutation screening was performed by exon direct sequencing and a new multiplex ligation probe amplification (MLPA) assay was set up for large rearrangement analysis. Eleven SLC7A7-specific mutations were identified, seven of them were novel: p.L124P, p.C425R, p.R468X, p.Y274fsX21, c.625 þ 1G4C, DelE4-E11 and DelE6-E11. The novel large deletions originated by the recombination of Alu repeats at introns 3 and 5, respectively, with the same AluY sequence localized at the SLC7A7 3 0 region. The novel MLPA assay is robust and valuable for LPI molecular diagnosis. Our results suggest that genomic rearrangements of SLC7A7 play a more important role in LPI than has been reported, increasing the detection rate from 5.1 to 21.4%. Moreover, the 3 0 region AluY repeat could be a recombination hot spot as it is involved in 38% of all SLC7A7 rearranged chromosomes described so far.

Cystinuria is a hereditary disorder caused by a defect in the apical membrane transport system for cystine and dibasic amino acids in renal proximal tubules and intestine, resulting in recurrent urolithiasis. Mutations in SLC3A1 and SLC7A9 genes, that codify for rBAT/b 0,ϩ AT transporter subunits, cause type A and B cystinuria, respectively. In humans, cystinuria treatment is based on the prevention of calculi formation and its dissolution or breakage. Persistent calculi are treated with thiols [i.e., D-penicillamine (DP) and mercaptopropionylglycine (MPG)] for cystine solubilization. We have developed a new protocol with DP to validate our Slc7a9 knockout mouse model for the study of the therapeutic effect of drugs in the treatment of cystine lithiasis. We performed a 5-wk treatment of individually caged lithiasic mutant mice with a previously tested DP dose. To appraise the evolution of lithiasis throughout the treatment a noninvasive indirect method of calculi quantification was developed: calculi mass was quantified by densitometry of X-ray images from cystinuric mice before and after treatment. Urine was collected in metabolic cage experiments to quantify amino acids in DP-treated and nontreated, nonlithiasic mutant mice. We found significant differences between DP-treated and nontreated knockout mice in calculi size and in urinary cystine excretion. Histopathological analysis showed that globally nontreated mutant mice had more severe and diffuse urinary system damage than DP-treated mice. Our results validate the use of this mouse model for testing the efficacy of potential new drugs against cystinuria.

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.