AGC2 (Citrin) Deficiency-From Recognition of the Disease till Construction of Therapeutic Procedures.

Can you imagine a disease in which intake of an excess amount of sugars or carbohydrates causes hyperammonemia? It is hard to imagine the intake causing hyperammonemia. AGC2 or citrin deficiency shows their symptoms following sugar/carbohydrates intake excess and this disease is now known as a pan-ethnic disease. AGC2 (aspartate glutamate carrier 2) or citrin is a mitochondrial transporter which transports aspartate (Asp) from mitochondria to cytosol in exchange with glutamate (Glu) and H+. Asp is originally supplied from mitochondria to cytosol where it is necessary for synthesis of proteins, nucleotides, and urea. In cytosol, Asp can be synthesized from oxaloacetate and Glu by cytosolic Asp aminotransferase, but oxaloacetate formation is limited by the amount of NAD+. This means an increase in NADH causes suppression of Asp formation in the cytosol. Metabolism of carbohydrates and other substances which produce cytosolic NADH such as alcohol and glycerol suppress oxaloacetate formation. It is forced under citrin deficiency since citrin is a member of malate/Asp shuttle. In this review, we will describe history of identification of the SLC25A13 gene as the causative gene for adult-onset type II citrullinemia (CTLN2), a type of citrin deficiency, pathophysiology of citrin deficiency together with animal models and possible treatments for citrin deficiency newly developing.

mRNA Therapy Improves Metabolic and Behavioral Abnormalities in a Murine Model of Citrin Deficiency.

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.

Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.

Chronic hepatitis without hepatic steatosis caused by citrin deficiency in a child.

Citrin deficiency manifests as both neonatal intrahepatic cholestasis (NICCD) during early infancy and adult-onset type II citrullinemia during adulthood. Hepatic steatosis is most frequently observed in patients with citrin deficiency. Thus, non-alcoholic fatty liver disease that is unrelated to being overweight is considered one of the clinical features of citrin deficiency in children and adults. However, it remains unknown whether citrin deficiency is a cause of chronic hepatitis in the absence of fatty changes to the liver that occur during childhood. We encountered an 8-year-old girl who showed no clinical features of NICCD during infancy and had persistently elevated transaminase levels for several years. Liver biopsy showed widening of the portal tracts with intense mononuclear cell infiltration and mild fibrosis but no fatty changes. However, she had peculiar dietary habits similar to those that have been observed in many patients with citrin deficiency. In addition, a slightly elevated plasma citrulline level and a high pancreatic secretory trypsin inhibitor level were detected by blood examination, and she was diagnosed with citrin deficiency. Analysis of the SLC25A13 gene revealed the presence of the compound heterozygous mutations 851del4 and IVS13 + 1G > A. Thus, citrin deficiency should be included in the differential diagnosis of chronic hepatitis in children, even in the absence of hepatic steatosis.

Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons.

Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.

Biochemical characteristics of neonatal cholestasis induced by citrin deficiency.

AIM: To explore differences in biochemical indices between neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and that with other etiologies. METHODS: Patients under 6 mo of age who were referred for investigation of conjugated hyperbilirubinaemia from June 2003 to December 2010 were eligible for this study. After excluding diseases affecting the extrahepatic biliary system, all patients were screened for the two most common SLC25A13 mutations; the coding exons of the entire SLC25A13 gene was sequenced and Western blotting of citrin protein performed in selected cases. Patients in whom homozygous or compound heterozygous SLC25A13 mutation and/or absence of normal citrin protein was detected were defined as having NICCD. Cases in which no specific etiological factor could be ascertained after a comprehensive conjugated hyperbilirubinaemia work-up were defined as idiopathic neonatal cholestasis (INC). Thirty-two NICCD patients, 250 INC patients, and 39 infants with cholangiography-confirmed biliary atresia (BA) were enrolled. Laboratory values at their first visit were abstracted from medical files and compared. RESULTS: Compared with BA and INC patients, the NICCD patients had significantly higher levels of total bile acid (TBA) [all measures are expressed as median (inter-quartile range): 178.0 (111.2-236.4) µmol/L in NICCD vs 112.0 (84.9-153.9) µmol/L in BA and 103.0 (70.9-135.3) µmol/L in INC, P = 0.0001]. The NICCD patients had significantly lower direct bilirubin [D-Bil 59.6 (43.1-90.9) µmol/L in NICCD vs 134.0 (115.9-151.2) µmol/L in BA and 87.3 (63.0-123.6) µmol/L in INC, P = 0.0001]; alanine aminotransferase [ALT 34.0 (23.0-55.0) U/L in NICCD vs 108.0 (62.0-199.0) U/L in BA and 84.5 (46.0-166.0) U/L in INC, P = 0.0001]; aspartate aminotransferase [AST 74.0 (53.5-150.0) U/L in NICCD vs 153.0 (115.0-239.0) U/L in BA and 130.5 (81.0-223.0) U/L in INC, P = 0.0006]; albumin [34.9 (30.7-38.2) g/L in NICCD vs 38.4 (36.3-42.2) g/L in BA and 39.9 (37.0-42.3) g/L in INC, P = 0.0001]; glucose [3.2 (2.0-4.4) mmol/L in NICCD vs 4.1 (3.4-5.1) mmol/L in BA and 4.0 (3.4-4.6) mmol/L in INC, P = 0.0014] and total cholesterol [TCH 3.33 (2.97-4.00) mmol/L in NICCD vs 4.57 (3.81-5.26) mmol/L in BA and 4.00 (3.24-4.74) mmol/L in INC, P = 0.0155] levels. The D-Bil to total bilirubin (T-Bil) ratio was significantly lower in NICCD patients [all measures are expressed as median (inter-quartile range): 0.54 (0.40-0.74)] than that in BA patients [0.77 (0.72-0.81), P = 0.001] and that in INC patients [0.74 (0.59-0.80), P = 0.0045]. A much higher AST/ALT ratio was found in NICCD patients [2.46 (1.95-3.63)] compared to BA patients [1.38 (0.94-1.97), P = 0.0001] and INC patients [1.48 (1.10-2.26), P = 0.0001]. NICCD patients had significantly higher TBA/D-Bil ratio [3.36 (1.98-4.43) vs 0.85 (0.72-1.09) in BA patients and 1.04 (0.92-1.14) in INC patients, P = 0.0001], and TBA/TCH ratio [60.7 (32.4-70.9) vs 24.7 (19.8-30.2) in BA patients and 24.2 (21.4-26.9) in INC patients, P = 0.0001] compared to the BA and INC groups. CONCLUSION: NICCD has significantly different biochemical indices from BA or INC. TBA excretion in NICCD appeared to be more severely disturbed than that of bilirubin and cholesterol.

Simple and rapid genetic testing for citrin deficiency by screening 11 prevalent mutations in SLC25A13.

Citrin deficiency is an autosomal recessive disorder caused by mutations in the SLC25A13 gene and has two disease outcomes: adult-onset type II citrullinemia and neonatal intrahepatic cholestasis caused by citrin deficiency. The clinical appearance of these diseases is variable, ranging from almost no symptoms to coma, brain edema, and severe liver failure. Genetic testing for SLC25A13 mutations is essential for the diagnosis of citrin deficiency because chemical diagnoses are prohibitively difficult. Eleven SLC25A13 mutations account for 95% of the mutant alleles in Japanese patients with citrin deficiency. Therefore, a simple test for these mutations is desirable. We established a 1-hour, closed-tube assay for the 11 SLC25A13 mutations using real-time PCR. Each mutation site was amplified by PCR followed by a melting-curve analysis with adjacent hybridization probes (HybProbe, Roche). The 11 prevalent mutations were detected in seven PCR reactions. Six reactions were used to detect a single mutation each, and one reaction was used to detect five mutations that are clustered in a 21-bp region in exon 17. To test the reliability, we used this method to genotype blind DNA samples from 50 patients with citrin deficiency. Our results were in complete agreement those obtained using previously established methods. Furthermore, the mutations could be detected without difficulty using dried blood samples collected on filter paper. Therefore, this assay could be used for newborn screening and for facilitating the genetic diagnosis of citrin deficiency, especially in East Asian populations.

The characteristics of food intake in patients with type II citrullinemia.

Some patients with citrin deficiency caused by SLC25A13 gene mutations develop adult-onset type II citrullinemia (CTLN2) with hepatic encephalopathy. A recent nutritional survey of 18 citrin-deficient subjects (age 1-33 y) confirmed a marked decrease in carbohydrate intake compared to an age-matched general Japanese population. However, a quantitative understanding of food intake in CTLN2 patients remains unclear, although qualitative dietary information has been reported. In order to elucidate the characteristics of daily nutrition of CTLN2 patients, the food intake of 5 male patients (age 39-52 y) was investigated in detail by the Food Frequency Questionnaire. In the present survey, the mean energy ratio of protein : fat : carbohydrate (PFC ratio) of the 5 patients was 19±3% : 44±5% : 37±4%, which was almost identical to previously reported data in younger citrin-deficient subjects (19±2% : 44±5% : 37±7%). Cereal intake was especially low in all CTLN2 patients at 309±33 g/d (56% of control), compared to that in an age-matched general Japanese population (553±197 g/d). Additionally, CTLN2 patients preferred high fat and protein foods. Commonly, fat intake declines with age in the general Japanese population, but this tendency was not observed in the 5 CTLN2 patients. The present results suggest that intakes of low-carbohydrate, high-protein and high-fat food was characteristic the 5 CTLN2 patients surveyed, as has been previously reported in younger citrin-deficient subjects, and that the PFC ratio may not be influenced by age or CTLN2-onset.

Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency.

The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency.

The mutation spectrum of the SLC25A13 gene in Chinese infants with intrahepatic cholestasis and aminoacidemia.

BACKGROUND: SLC25A13 gene mutations cause citrin deficiency, which leads to neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). Information on the mutation spectrum of SLC25A13 in the Chinese population is limited. The aim of this study was to explore the mutation spectrum of the SLC25A13 gene in Chinese infants with intrahepatic cholestasis and various forms of aminoacidemia. METHODS: Sequence analyses were performed on 39 infants with intrahepatic cholestasis and various forms of aminoacidemia. Novel mutations were subjected to homology and structural analyses. Western blots were performed when liver specimens available. RESULTS: Genetic testing revealed the presence of SLC25A13 gene mutations (9 heterozygotes, 6 homozygotes and 13 compound heterozygotes) in 28 infants. Subsequent Western blot analysis revealed 22 cases of citrin deficiency, accounting for 56.4% of the 39 patients. Twelve types of mutations, including nine known mutations and three novel mutations, were found. Of the 49 mutated alleles, known ones include 851del4 (26 alleles, 53.1%), 1638ins23 (6 alleles, 12.2%), IVSl6ins3kb (3 alleles, 6.1%), IVS6+5G>A (2 alleles, 4.1%), E601K (2 alleles, 4.1%) and IVS11+1G>A, R184X, R360X and R585H (1 allele each, 2.0%). The three novel mutations were a splice site change (IVS6+1G>A), a deletion mutation (1092_1095delT) and a missense mutation (L85P), each in one allele. CONCLUSIONS: The mutation spectrum of the SLC25A13 gene in a Chinese population of infants with intrahepatic cholestasis with various forms of aminoacidemia was found to be different from that of other population groups in East Asia. The SLC25A13 gene mutation is the most important cause of infantile intrahepatic cholestasis with various forms of aminoacidemia.

Citrin deficiency and current treatment concepts.

In this paper, we describe the historical aspects of citrin and citrin deficiency, characteristic food preference and food aversion of citrin-deficient subjects, and carbohydrate toxicity in relation to ureogenesis and issues of the conventional treatment procedures for hyperammonemia in citrin deficiency, leading to current treatment concepts for citrin deficiency. We also emphasize the importance of a citrin deficiency mouse model in elucidating the pathophysiology and developing novel therapeutics based on the pathophysiology, such as sodium pyruvate.

Histological findings in the livers of patients with neonatal intrahepatic cholestasis caused by citrin deficiency.

AIM: To characterize the histological features of the livers of patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), we studied specimens from 30 patients diagnosed with NICCD by genetically analyzing the SLC25A13 gene. METHODS: Liver biopsy specimens were subjected to hematoxylin-eosin, Azan, and Berlin-blue staining. RESULTS: Most specimens showed varying degrees of fibrosis. The degree of inflammation varied among the specimens, with half showing moderate or severe inflammatory changes. Fat deposition in hepatocytes was observed in almost all of the specimens, and severe fatty liver was noted in 20 (67%) of them. There was a mixture of two types of hepatocytes with macrovesicular or microvesicular fat droplets, and cholestasis was observed at a rate of 77%. Hemosiderin deposition, mostly mild and localized in periportal hepatocytes and macrophages in portal areas, was observed in 57% of the specimens. CONCLUSION: A combination of mixed macrovesicular and microvesicular fatty hepatocytes and the above-described findings, such as fatty liver, cholestasis, necroinflammatory reaction and iron deposition, are almost never observed in other liver diseases in infants and adults. We believe that NICCD is a disease with characteristic hepatopathological features.

[Therapeutic approaches for patients with adult-onset type II citrullinemia (CTLN2): effectiveness of treatment with low-carbohydrate diet and sodium pyruvate].

Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease characterized by highly elevated plasma levels of citrulline and ammonia due to the urea cycle dysfunction associated with citrin deficiency. Patients with CTLN2 show various neurological symptoms with hyperammonemia closely resembling those of hepatic encephalopathy. Since 1990, 26 CTLN2 patients (17 males and 9 females) have been admitted and treated at Shinshu University Hospital. Twelve of the 26 patients received living related partial liver transplantation (LRLT). After LRLT, neurological symptoms soon disappeared, and all patients returned to their previous social lives. Among the 14 patients that did not undergo LRLT, 6 died of intractable encephalopathy or the development of hepatic cancer, but 8 patients have had relatively good clinical courses (follow-up range 0.5-8 years) with oral intake of L-arginine and low-carbohydrate and relatively protein-rich diet. Six patients have been also given sodium pyruvate and the frequency of attacks of encephalopathy markedly decreased in 5 of 6 patients. Our observations indicated that liver transplantation is a very promising type of therapy but that other therapeutic approaches, including low-carbohydrate diet and pyruvate, are being established.

Low levels of citrin (SLC25A13) expression in adult mouse brain restricted to neuronal clusters.

The mitochondrial aspartate-glutamate carriers (AGC) aralar (SLC25A12) and citrin (SLC25A13) are components of the malate aspartate shuttle (MAS), a major intracellular pathway to transfer reducing equivalents from NADH to the mitochondrial matrix. Aralar is the main AGC isoform present in the adult brain, and it is expressed mainly in neurons. To search for the other AGC isoform, citrin, in brain glial cells, we used a citrin knockout mouse in which the lacZ gene was inserted into the citrin locus as reporter gene. In agreement with the low citrin levels known to be present in the adult mouse brain, beta-galactosidase expression was very low. Surprisingly, unlike the case with astroglial cultures that express citrin, no beta-galactosidase was found in brain glial cells. It was confined to neuronal cells within discrete neuronal clusters. Double-immunolabelling experiments showed that beta-galactosidase colocalized not with glial cell markers but with the pan-neuronal marker NeuN. The deep cerebellar nuclei and a few midbrain nuclei (reticular tegmental pontine nuclei; magnocellular red nuclei) were the regions where beta-galactosidase expression was highest, and it was up-regulated in fasted mice, as was also the case for liver beta-galactosidase. The results support the notion that glial cells have much lower AGC levels and MAS activity than neurons.

[Citrin deficiency is an important etiology for cholestatic liver disease in children].

OBJECTIVE: To explore the major etiological features of cholestatic liver disease (CLD) in children, and to investigate the molecular epidemiological distribution of SLC25A13 mutations in CLD. METHOD: A clinical cross-sectional investigation was performed on 63 CLD cases diagnosed from Oct. 2003 to Mar. 2009 in our department, including 36 males and 27 females. Their clinical data were collected, and etiology and prognosis were analyzed and summarized. Thirteen to 17 mutations in SLC25A13 gene were screened by means of procedures established previously by our group. Several SLC25AJ3 mutations were detected by direct sequencing of DNA fragments amplified by genomic DNA-PCR. RESULT: No specific etiologies were identified in 24 of the 63 cases. Among the 39 cases with identified etiologies, inherited metabolic diseases were on top of the list, including 6 kinds and 27 cases in total, i.e., neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD, 21 cases), transient galactosemia, tyrosinemia type I, galactose kinase deficiency, ornithine carbamoyl transferase deficiency and glycogen storage disease type I, followed by acquired causes (7 cases in total), such as total parenteral nutrition associated cholestasis (TPNAC), congenital syphilis and CMV hepatitis; and then biliary tract malformation (5 cases in total), including biliary atresia, Caroli's disease and gallbladder polyp, were the third. Ten of the 55 patients on follow-up have passed away, while the remaining 45 cases were improved or recovered clinically. SLC25A13 gene analysis were performed in 44 CLD subjects and 21 of them from 20 families (with 40 SLC25A13 alleles in total) were found to have mutations, and the seven mutations detected were 851-854del (23/40), IVS6 + 5G > A (6/40), IVS16ins3kb (3/40), 1638-1660dup (2/30), A541D (1/30), R319X (1/30) and G333D (1/30), respectively, and there were other 3 mutations (3/40) still needing identification in the remaining 3 alleles. CONCLUSION: The etiologies for CLD in some cases can not be identified. However, inherited metabolic diseases, including NICCD in particular, constitute common causative factors for CLD. Most of the CLD conditions can be improved, even recovered clinically, although some cases presented with poor prognosis. Seven mutations in SLC25A13 gene were detected, among which, 851-854del, IVS6 + 5G > A, IVS16ins3kb and 1638-1660dup were the leading four mutations, respectively.

[Failure to thrive and dyslipidemia caused by citrin deficiency: a novel clinical phenotype].

Two clinical phenotypes for citrin deficiency (CD) have been reported. One is adult-onset citrullinemia type II (CTLN2) and another is neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). A child with CD and who had failure to thrive (FTT) and dyslipidemia as main clinical manifestations is reported here. Both the weight-and length-for-age at 18 months dropped below the 3rd percentile in the corresponding WHO anthropometry percentile charts, while blood biochemical analysis revealed dramatically increased triglyceride and total cholesterol, together with reduced HDL-cholesterol. Inquiries revealed his aversion to rice and fondness for fish since the age of one year, a peculiar habit which could not be corrected. Since the age of two years, the peculiar diet became more obvious, and slightly increased citrulline and threonine levels were detected on blood amino acid analysis. At the age of two years and five months he was suspected to have CD. Since then, he has been fed in accordance with his own food preferences, and FTT improved gradually, with weight-for-age, in particular, recovering beyond the 3rd percentile at three years of age, and dyslipidemia was also ameliorated gradually. SLC25A13 gene analysis revealed a homozygote of 851del4, and CD was thus confirmed. Diet survey at four years and seven months revealed a fondness for high-protein and low-carbohydrate foods, such as seafood, meat, eggs and milk. This child presented with FTT and dyslipidemia as main clinical manifestations and this was a novel CD phenotype different from NICCD and CTLN2.

Citrin deficiency as a cause of chronic liver disorder mimicking non-alcoholic fatty liver disease.

BACKGROUND/AIMS: Citrin deficiency caused by SLC25A13 gene mutations develops into adult-onset type II citrullinemia (CTLN2) and may be accompanied with hepatic steatosis and steatohepatitis. As its clinical features remain unclear, we aimed to explore the characteristics of fatty liver disease associated with citrin deficiency. METHODS: The prevalence of hepatic steatosis in 19 CTLN2 patients was examined, and clinical features were compared with those of non-alcoholic fatty liver disease (NAFLD) patients without known SLC25A13 gene mutations. RESULTS: Seventeen (89%) CTLN2 patients had steatosis, and 4 (21%) had been diagnosed as having NAFLD before appearance of neuropsychological symptoms. One patient had steatohepatitis. Citrin deficiency-associated fatty livers showed a considerably lower prevalence of accompanying obesity and metabolic syndrome, higher prevalence of history of pancreatitis, and higher serum levels of pancreatic secretory trypsin inhibitor (PSTI) than fatty livers without the mutations. Receiver operating characteristic curve analyses revealed that a body mass index < 20kg/m(2) and serum PSTI>29ng/mL were associated with citrin deficiency. CONCLUSIONS: Patients presenting with non-alcoholic fatty liver unrelated to obesity and metabolic syndrome might have citrin deficiency, and serum PSTI may be a useful indicator for distinguishing this from conventional NAFLD.

Primary liver carcinoma exhibiting dual hepatocellular-biliary epithelial differentiations associated with citrin deficiency: a case report.

We report a 50-year-old male patient with primary liver carcinoma exhibiting dual hepatocellular and biliary epithelial differentiations associated with citrin deficiency (asymptomatic adult-onset type II citrullinemia, CTLN2). Although so far 14 CTLN2 patients with hepatocellular carcinoma have been reported, this report describes a unique case of liver carcinoma showing the features of both hepatocellular and cholangiocellular carcinoma. In addition to the clinical data of the 14 patients reported previously, the findings in our patient suggest that the citrin deficiency might be one of the key disorders causing hepatocellular carcinoma especially at younger ages and can also play an important role in hepatocarcinogenesis of the hepatic progenitor cells, which have the bipotential to differentiate into both hepatocytes and cholangiocytes.

Living donor liver transplantation for type II citrullinemia from a heterozygous donor.

Adult-onset type II citrullinemia (CTLN2) is a rare disorder of the urea cycle resulting in hyperammonemia, with a poor prognosis. Here we report a 48-year-old Japanese man who showed abnormal nocturnal behavior. Laboratory data indicated raised plasma concentrations of ammonia and citrulline, and a definitive diagnosis of CTLN2 was made by DNA analysis. Hyperammonemia was not improved by oral intake of branched-chain amino acids (BCAA), whereas venous infusion of BCAA was effective. Western blotting revealed heterozygotic expression of citrin protein in a liver biopsy specimen from the patient's brother. However, as symptomatic CTLN2 is very unusual in a heterozygotic carrier, we considered the brother suitable as a living-donor liver transplantation (LDLT) donor. The recipient's entire liver was removed, and replaced with the left liver graft. The plasma ammonia level remained low without infusion of BCAA after liver transplantation. From this case we conclude that venous infusion, rather than oral administration, of BCAA is useful for conservative treatment of CTLN2. However, liver transplantation is the only effective therapeutic option for CTLN2, and should be performed before irreversible encephalopathy occurs. Use of a graft from heterozygotic donors is permissible treatment for CTLN2.

Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency.

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.