Inducible Slc7a7 Knockout Mouse Model Recapitulates Lysinuric Protein Intolerance Disease.

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.

Gene expression profile of peripheral blood monocytes: a step towards the molecular diagnosis of celiac disease?

AIM: Celiac disease (CD) is a multifactorial autoimmune disease induced by ingestion of gluten in genetically predisposed individuals. Despite technological progress, the diagnosis of CD is still based on duodenal biopsy as it was 50 years ago. In this study we analysed the expression of CD-associated genes in small bowel biopsies of patients and controls in order to explore the multivariate pathway of the expression profile of CD patients. Then, using multivariant discriminant analysis, we evaluated whether the expression profiles of these genes in peripheral blood monocytes (PBMs) differed between patients and controls. PARTICIPANTS: Thirty-seven patients with active and 11 with treated CD, 40 healthy controls and 9 disease controls (Crohn's disease patients) were enrolled. RESULTS: Several genes were differentially expressed in CD patients versus controls, but the analysis of each single gene did not provided a comprehensive picture. A multivariate discriminant analysis showed that the expression of 5 genes in intestinal mucosa accounted for 93% of the difference between CD patients and controls. We then applied the same approach to PBMs, on a training set of 20 samples. The discriminant equation obtained was validated on a testing cohort of 10 additional cases and controls, and we obtained a correct classification of all CD cases and of 91% of the control samples. We applied this equation to treated CD patients and to disease controls and obtained a discrimination of 100%. CONCLUSIONS: The combined expression of 4 genes allows one to discriminate between CD patients and controls, and between CD patients on a gluten-free diet and disease controls. Our results contribute to the understanding of the complex interactions among CD-associated genes, and they may represent a starting point for the development of a molecular diagnosis of celiac disease.

Improving the estimation of celiac disease sibling risk by non-HLA genes.

Celiac Disease (CD) is a polygenic trait, and HLA genes explain less than half of the genetic variation. Through large GWAs more than 40 associated non-HLA genes were identified, but they give a small contribution to the heritability of the disease. The aim of this study is to improve the estimate of the CD risk in siblings, by adding to HLA a small set of non-HLA genes. One-hundred fifty-seven Italian families with a confirmed CD case and at least one other sib and both parents were recruited. Among 249 sibs, 29 developed CD in a 6 year follow-up period. All individuals were typed for HLA and 10 SNPs in non-HLA genes: CCR1/CCR3 (rs6441961), IL12A/SCHIP1 and IL12A (rs17810546 and rs9811792), TAGAP (rs1738074), RGS1 (rs2816316), LPP (rs1464510), OLIG3 (rs2327832), REL (rs842647), IL2/IL21 (rs6822844), SH2B3 (rs3184504). Three associated SNPs (in LPP, REL, and RGS1 genes) were identified through the Transmission Disequilibrium Test and a Bayesian approach was used to assign a score (BS) to each detected HLA+SNPs genotype combination. We then classified CD sibs as at low or at high risk if their BS was respectively < or ≥ median BS value within each HLA risk group. A larger number (72%) of CD sibs showed a BS ≥ the median value and had a more than two fold higher OR than CD sibs with a BS value < the median (O.R = 2.53, p = 0.047). Our HLA+SNPs genotype classification, showed both a higher predictive negative value (95% vs 91%) and diagnostic sensitivity (79% vs 45%) than the HLA only. In conclusion, the estimate of the CD risk by HLA+SNPs approach, even if not applicable to prevention, could be a precious tool to improve the prediction of the disease in a cohort of first degree relatives, particularly in the low HLA risk groups.

Potential celiac patients: a model of celiac disease pathogenesis.

BACKGROUND AND AIM: Potential celiacs have the 'celiac type' HLA, positive anti-transglutaminase antibodies but no damage at small intestinal mucosa. Only a minority of them develops mucosal lesion. More than 40 genes were associated to Celiac Disease (CD) but we still do not know how those pathways transform a genetically predisposed individual into an affected person. The aim of the study is to explore the genetic features of Potential CD individuals. METHODS: 127 'potential' CD patients entered the study because of positive anti-tissue transglutaminase and no mucosal lesions; about 30% of those followed for four years become frankly celiac. They were genotyped for 13 polymorphisms of 'candidate genes' and compared to controls and celiacs. Moreover, 60 biopsy specimens were used for expression studies. RESULTS: Potential CD bear a lighter HLA-related risk, compared to celiac (χ(2) = 48.42; p value = 1×10(-8)). They share most of the polymorphisms of the celiacs, but the frequency of c-REL* G allele was suggestive for a difference compared to celiac (χ(2) = 5.42; p value = 0.02). One marker of the KIAA1109/IL-2/IL-21 candidate region differentiated potentials from celiac (rs4374642: χ2 = 7.17, p value = 0.01). The expression of IL-21 was completely suppressed in potentials compared to celiacs (p value = 0.02) and to controls (p value = 0.02), in contrast IL-2, KIAA1109 and c-REL expression were over-expressed. CONCLUSIONS: Potential CD show genetic features slightly different from celiacs. Genetic and expression markers help to differentiate this condition. Potential CD is a precious biological model of the pathways leading to the small intestinal mucosal damage in genetically predisposed individuals.

Multiple common variants for celiac disease influencing immune gene expression.

We performed a second-generation genome-wide association study of 4,533 individuals with celiac disease (cases) and 10,750 control subjects. We genotyped 113 selected SNPs with P(GWAS) < 10(-4) and 18 SNPs from 14 known loci in a further 4,918 cases and 5,684 controls. Variants from 13 new regions reached genome-wide significance (P(combined) < 5 x 10(-8)); most contain genes with immune functions (BACH2, CCR4, CD80, CIITA-SOCS1-CLEC16A, ICOSLG and ZMIZ1), with ETS1, RUNX3, THEMIS and TNFRSF14 having key roles in thymic T-cell selection. There was evidence to suggest associations for a further 13 regions. In an expression quantitative trait meta-analysis of 1,469 whole blood samples, 20 of 38 (52.6%) tested loci had celiac risk variants correlated (P < 0.0028, FDR 5%) with cis gene expression.

Lysinuric protein intolerance: update and extended mutation analysis of the SLC7A7 gene.

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is caused by mutations in the SLC7A7 gene, which encodes the y(+)LAT-1 protein, the catalytic light chain subunit of a complex belonging to the heterodimeric amino acid transporter family. Coexpression of 4F2hc (the heavy chain subunit) and y(+)LAT-1 induces y(+)L activity (CAA transport). So far a total of 43 different mutations of the SLC7A7 gene, nine of which newly reported here, have been identified in a group of 130 patients belonging to at least 98 independent families. The mutations are distributed along the entire gene and include all different types of mutations. Five polymorphisms within the SLC7A7 coding region and two variants found in the 5'UTR have been identified. A genuine founder effect mutation has been demonstrated only in Finland, where LPI patients share the same homozygous mutation, c.895-2A>T. LPI patients show extreme variability in clinical presentation, and no genotype-phenotype correlations have been defined. This phenotypic variability and the lack of a specific clinical presentation have caused various misdiagnoses. At the biochemical level, the elucidation of SLC7A7 function will be necessary to understand precise disease mechanisms and develop more specific and effective therapies. In this review, we summarize the current knowledge of SLC7A7 mutations and their role in LPI pathogenesis.

Slc7a7 disruption causes fetal growth retardation by downregulating Igf1 in the mouse model of lysinuric protein intolerance.

The solute carrier family 7A member 7 gene (SLC7A7) encodes the light chain of the heterodimeric carrier responsible for cationic amino acid (CAA) transport across the basolateral membranes of epithelial cells in intestine and kidney. Mutations affecting SLC7A7 cause lysinuric protein intolerance (LPI), a multiorgan disorder with clinical symptoms that include visceromegaly, growth retardation, osteoporosis, hyperammonemia, and hyperdibasicaminoaciduria. Here, we describe the consequences of inactivating Slc7a7 in a mouse model of LPI. The Slc7a7 mutation was generated by high-throughput retroviral gene-trapping in embryonic stem cells. The Slc7a7(-/-) mouse displayed intrauterine growth restriction (IUGR), commonly leading to neonatal lethality. After heavy protein ingestion, the surviving adult animals presented metabolic derangement consistent with that observed in human LPI. IUGR was investigated by examining the expression of main factors controlling fetal growth. Insulin-like growth factor 1, the dominant fetal growth regulator in late gestation, was markedly downregulated as demonstrated by quantitative real-time RT-PCR, immunostaining and Western blot analysis in fetal liver. To further explore the pathophysiology of LPI, gene expression profiling analyses were carried out by DNA microarray technology in intestine and liver of adult Slc7a7(-/-) mice. Significant upregulation or downregulation (twofold or greater) was observed for 488 transcripts in intestine, and for 521 transcripts in the liver. The largest category of differentially expressed genes corresponds to those involved in transport according to Gene Ontology classification. This mouse model offers new insights into the pathophysiology of LPI and into mechanisms linking CAA metabolic pathways and growth control.

Lysinuric protein intolerance: identification and functional analysis of mutations of the SLC7A7 gene.

Lysinuric protein intolerance (LPI) is an inherited hyperdibasic aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is relatively common in Finland and a few clusters of patients are known in Italy and Japan. The SLC7A7 gene, mutated in LPI patients, encodes the y+LAT-1 protein which is the light subunit of a heterodimeric CAA transporter. We performed the mutation analysis in seven probands from five unrelated LPI families and identified five novel SLC7A7 mutations (p.M50K, p.T188I, p.R333M, p.Y457X, and c.499+?_629-?). By expression studies in X. laevis oocytes or patient's renal tubular cells, the functional analysis of altogether eight SLC7A7 mutations is here reported. Noteworthy, the p.R333M mutation, caused by a G to T transversion of the last nucleotide at 3' end of exon 7, disrupts a functional splicing motif generating misspliced transcripts. Three of the novel mutations were found in patients originating from Greece and Pakistan thus increasing the list of ethnic backgrounds where LPI mutant alleles are present. This reinforces the view that the rarity of LPI outside Finland might be ascribed to misdiagnosis of this disease.