Citrullinaemia (CTLN) is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase (ASS). Adult-onset type II citrullinaemia (CTLN2) is characterized by a liver-specific ASS deficiency with no abnormalities in hepatic ASS mRNA or the gene ASS (refs 1-17). CTLN2 patients (1/100,000 in Japan) suffer from a disturbance of consciousness and coma, and most die with cerebral edema within a few years of onset. CTLN2 differs from classical citrullinaemia (CTLN1, OMIM 215700) in that CTLN1 is neonatal or infantile in onset, with ASS enzyme defects (in all tissues) arising due to mutations in ASS on chromosome 9q34 (refs 18-21). We collected 118 CTLN2 families, and localized the CTLN2 locus to chromosome 7q21.3 by homozygosity mapping analysis of individuals from 18 consanguineous unions. Using positional cloning we identified a novel gene, SLC25A13, and found five different DNA sequence alterations that account for mutations in all consanguineous patients examined. SLC25A13 encodes a 3.4-kb transcript expressed most abundantly in liver. The protein encoded by SLC25A13, named citrin, is bipartite in structure, containing a mitochondrial carrier motif and four EF-hand domains, suggesting it is a calcium-dependent mitochondrial solute transporter with a role in urea cycle function.

The genetic architecture of complex traits underlying physiology and disease in most organisms remains elusive. We still know little about the number of genes that underlie these traits, the magnitude of their effects, or the extent to which they interact. Chromosome substitution strains (CSSs) enable statistically powerful studies based on testing engineered inbred strains that have single, unique, and nonoverlapping genetic differences, thereby providing measures of phenotypic effects that are attributable to individual chromosomes. Here, we report a study of phenotypic effects and gene interactions for 90 blood, bone, and metabolic traits in a mouse CSS panel and 54 traits in a rat CSS panel. Two key observations emerge about the genetic architecture of these traits. First, the traits tend to be highly polygenic: across the genome, many individual chromosome substitutions each had significant phenotypic effects and, within each of the chromosomes studied, multiple distinct loci were found. Second, strong epistasis was found among the individual chromosomes. Specifically, individual chromosome substitutions often conferred surprisingly large effects (often a substantial fraction of the entire phenotypic difference between the parental strains), with the result that the sum of these individual effects often dramatically exceeded the difference between the parental strains. We suggest that strong, pervasive epistasis may reflect the presence of several phenotypically-buffered physiological states. These results have implications for identification of complex trait genes, developmental and physiological studies of phenotypic variation, and opportunities to engineer phenotypic outcomes in complex biological systems. chromosome substitution ͉ genetic variation ͉ quantitative trait loci

The CCAAT/enhancer-binding protein ␤ (C/EBP␤) is required for adipocyte differentiation and maturation. We have studied the role of the transcription factor, C/EBP␤, in the development of diet-induced obesity. Mice with a deletion in the gene for C/EBP␤ (C/EBP␤ ؊/؊ ) and wild-type mice were fed a high-fat diet (60% fat) for 12 weeks. The C/EBP␤ ؊/؊ mice lost body fat, whereas the wild-type mice increased their total body fat on a high-fat diet. The C/EBP␤ ؊/؊ mice had lower levels of blood triglycerides, free fatty acids, cholesterol, and hepatic triglyceride accumulation compared with the wild-type mice, thus protecting them from diet-induced obesity and fatty liver on a high-fat diet. Deletion of C/EBP␤ gene resulted in greatly reducing hepatic lipogenic genes, acetyl CoA carboxylase, and fatty acid synthase and increasing the expression of ␤-oxidation genes in the brown adipose tissue. CO 2 production was significantly higher in the C/EBP␤ ؊/؊ mice as was the level of uncoupling protein (UCP)-1 and UCP-3 in the muscle. In conclusion, the transcription factor C/EBP␤ is an important regulator in controlling lipid metabolism and in the development of diet-induced obesity. Diabetes 56: 161-167, 2007

Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn ؊/؊ mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn ؊/؊ mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.

Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol.Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD ؉ ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.

Congenic strains continue to be a fundamental resource for dissecting the genetic basis of complex traits. Traditionally, genetic variants (QTLs) that account for phenotypic variation in a panel of congenic strains are sought first by comparing phenotypes for each strain to the host (reference) strain, and then by examining the results to identify a common chromosome segment that provides the best match between genotype and phenotype across the panel. However, this ‘‘common-segment’’ method has significant limitations, including the subjective nature of the genetic model and an inability to deal formally with strain phenotypes that do not fit the model. We propose an alternative that we call ‘‘sequential’’ analysis and that is based on a unique principle of QTL analysis where each strain, corresponding to a single genotype, is tested individually for QTL effects rather than testing the congenic panel collectively for common effects across heterogeneous backgrounds. A minimum spanning tree, based on principles of graph theory, is used to determine the optimal sequence of strain comparisons. For two traits in two panels of congenic strains in mice, we compared results for the sequential method with the common-segment method as well as with two standard methods of QTL analysis, namely, interval mapping and multiple linear regression. The general utility of the sequential method was demonstrated with analysis of five additional traits in congenic panels from mice and rats. Sequential analysis rigorously resolved phenotypic heterogeneity among strains in the congenic panels and found QTLs that other methods failed to detect.

Discovery of genes that confer resistance to diseases such as diet-induced obesity could have tremendous therapeutic impact. We previously demonstrated that the C57BL/6J-ChrA/J/NaJ panel of chromosome substitution strains (CSSs) is a unique model for studying resistance to diet-induced obesity. In the present study, three replicate CSS surveys showed remarkable consistency, with 13 A/J-derived chromosomes reproducibly conferring resistance to high-fat-diet-induced obesity. Twenty CSS intercrosses, one derived from each of the 19 autosomes and chromosome X, were used to determine the number and location of quantitative trait loci (QTLs) on individual chromosomes and localized six QTLs. However, analyses of mean body weight in intercross progeny versus C57BL/6J provided strong evidence that many QTLs discovered in the CSS surveys eluded detection in these CSS intercrosses. Studies of the temporal effects of these QTLs suggest that obesity resistance was dynamic, with QTLs acting at different ages or after different durations of diet exposure. Thus, these studies provide insight into the genetic architecture of complex traits such as resistance to diet-induced obesity in the C57BL/6J-ChrA/J/NaJ CSSs. Because some of the QTLs detected in the CSS intercrosses were not detected using a traditional C57BL/6J × A/J intercross, our results demonstrate that surveys of CSSs and congenic strains derived from them are useful complementary tools for analyzing complex traits.

Obesity is associated with increased susceptibility to dyslipidemia, insulin resistance, and hypertension, a combination of traits that comprise the traditional definition of the metabolic syndrome. Recent evidence suggests that obesity is also associated with the development of nonalcoholic fatty liver disease (NAFLD). Despite the high prevalence of obesity and its related conditions, their etiologies and pathophysiology remains unknown. Both genetic and environmental factors contribute to the development of obesity and NAFLD. Previous genetic analysis of high-fat, diet-induced obesity in C57BL/6J (B6) and A/J male mice using a panel of B6-ChrA/J/NaJ chromosome substitution strains (CSSs) demonstrated that 17 CSSs conferred resistance to high-fat, diet-induced obesity. One of these CSS strains, CSS-17, which is homosomic for A/J-derived chromosome 17, was analyzed further and found to be resistant to diet-induced steatosis. In the current study we generated seven congenic strains derived from CCS-17, fed them either a high-fat, simple-carbohydrate (HFSC) or low-fat, simple-carbohydrate (LFSC) diet for 16 weeks and then analyzed body weight and related traits. From this study we identified several quantitative trait loci (QTLs). On a HFSC diet, Obrq13 protects against diet-induced obesity, steatosis, and elevated fasting insulin and glucose levels. On the LFSC diet, Obrq13 confers lower hepatic triglycerides, suggesting that this QTL regulates liver triglycerides regardless of diet. Obrq15 protects against diet-induced obesity and steatosis on the HFSC diet, and Obrq14 confers increased final body weight and results in steatosis and insulin resistance on the HFSC diet. In addition, on the LFSC diet, Obrq 16 confers decreased hepatic triglycerides and Obrq17 confers lower plasma triglycerides on the LFSC diet. These congenic strains provide mouse models to identify genes and metabolic pathways that are involved in the development of NAFLD and aspects of diet-induced metabolic syndrome.