Citrin Deficiency: Clinical and Nutritional Features.

SLC25A13 gene mutations are responsible for diseases related to citrin deficiency (CD), such as neonatal intrahepatic cholestasis caused by citrin deficiency and adult-onset type II citrullinemia (CTLN2). From childhood to adulthood, CD patients are apparently healthy due to metabolic compensation with peculiar dietary habits-disliking high-carbohydrate foods and liking fat and protein-rich foods. Carbohydrate overload and alcohol consumption may trigger the sudden onset of CTLN2, inducing hyperammonemia and consciousness disturbance. Well-compensated asymptomatic CD patients are sometimes diagnosed as having non-obese (lean) non-alcoholic fatty liver disease and steatohepatitis, which have the risk of developing into liver cirrhosis and hepatocellular carcinoma. CD-induced fatty liver demonstrates significant suppression of peroxisome proliferator-activated receptor α and its downstream enzymes/proteins involved in fatty acid transport and oxidation and triglyceride secretion as a very low-density lipoprotein. Nutritional therapy is an essential and important treatment of CD, and medium-chain triglycerides oil and sodium pyruvate are useful for preventing hyperammonemia. We need to avoid the use of glycerol for treating brain edema by hyperammonemia. This review summarizes the clinical and nutritional features of CD-associated fatty liver disease and promising nutritional interventions.

Hyperammonemia in a pregnant woman with citrullinemia type I: a case report and literature review.

BACKGROUND: Citrullinemia type I (CTLN1) is a rare urea cycle disorder (UCD) with few adult cases described so far. Diagnosis of late-onset CTLN1 is difficult, and delayed treatment may increase the risk of severe hyperammonemia. Pregnancy is an important risk factor for women with CTLN1. However, the clinical manifestations of CTLN1 in a pregnant woman may be mistaken for pregnancy side effects and ultimately delay a timely diagnosis. CASE PRESENTATION: A 34-year-old woman developed vomiting and disturbance of consciousness after 12 weeks of gestation. A blood test showed hyperammonemia (454 µg/dL) with normal liver function tests. She fell into a deep coma, and her serum ammonia level increased to 800 µg/dL. Continuous renal replacement therapy (CRRT) was administered as a diagnostic treatment for UCD and serum ammonia. This patient's case was complicated by co-infection; her dependents decided to withdraw life support and the patient died. She was diagnosed with CTLN1 by analyses of plasma amino acids, urinary orotic acid, and second-generation gene sequencing. DISCUSSION AND CONCLUSION: When a patient displays symptoms of emesis and disturbance of consciousness in early pregnancy, blood ammonia should be monitored, and UCD should be considered, particularly for patients with hyperammonemia in the absence of severe liver function abnormalities.

Gene Therapy in Combination with Nitrogen Scavenger Pretreatment Corrects Biochemical and Behavioral Abnormalities of Infant Citrullinemia Type 1 Mice.

Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.

Clinical manifestation and long-term outcome of citrin deficiency: Report from a nationwide study in Japan.

Citrin deficiency is an autosomal recessive disorder caused by mutations in the SLC25A13 gene. The disease can present with age-dependent clinical manifestations: neonatal intrahepatic cholestasis by citrin deficiency (NICCD), failure to thrive, and dyslipidemia by citrin deficiency (FTTDCD), and adult-onset type II citrullinemia (CTLN2). As a nationwide study to investigate the clinical manifestations, medical therapy, and long-term outcome in Japanese patients with citrin deficiency, we collected clinical data of 222 patients diagnosed and/or treated at various different institutions between January 2000 and December 2019. In the entire cohort, 218 patients were alive while 4 patients (1 FTTDCD and 3 CTLN2) had died. All patients <20 years were alive. Patients with citrin deficiency had an increased risk for low weight and length at birth, and CTLN2 patients had an increased risk for growth impairment during adolescence. Liver transplantation has been performed in only 4 patients (1 NICCD, 3 CTLN2) with a good response thereafter. This study reports the diagnosis and clinical course in a large cohort of patients with citrin deficiency and suggests that early intervention including a low carbohydrate diet and MCT supplementation can be associated with improved clinical course and long-term outcome.

Citrin deficiency ­ pathogenesis, clinical and biochemical manifestation, diagnostics, treatment / Deficyt cytrynu.

Citrin deficiency is an inherited metabolic disease caused by biallelic pathogenic variants in the SLC25A13 gene encoding the carrier protein called citrin. There are observed three characteristic clinical and biochemical age-dependent phenotypes: neonatal intrahepatic cholestasis caused by citrin deficiency, failure to thrive and dyslipidemia caused by citrin deficiency and adult-onset citrullinemia type 2. The paper presents the characteristics of the pathogenesis of citrin deficiency, clinical and biochemical delineation of individual phenotypes, differential diagnosis and treatment of citrin deficiency.

Therapeutic targeting of argininosuccinate synthase 1 (ASS1)-deficient pulmonary fibrosis.

Argininosuccinate synthase 1 (ASS1) serves as a critical enzyme in arginine biosynthesis; however, its role in interstitial lung diseases, particularly idiopathic pulmonary fibrosis (IPF), remains largely unknown. This study aims at characterization and targeting of ASS1 deficiency in pulmonary fibrosis. We find that ASS1 was significantly decreased and inversely correlated with fibrotic status. Transcriptional downregulation of ASS1 was noted in fibroblastic foci of primary lung fibroblasts isolated from IPF patients. Genetic manipulations of ASS1 studies confirm that ASS1 expression inhibited fibroblast cell proliferation, migration, and invasion. We further show that the hepatocyte growth factor receptor (Met) receptor was activated and acted upstream of the Src-STAT3 axis signaling in ASS1-knockdown fibroblasts. Interestingly, both arginine-free conditions and arginine deiminase treatment were demonstrated to kill fibrotic fibroblasts, attenuated bleomycin-induced pulmonary fibrosis in mice, as well as synergistically increased nintedanib efficacy. Our data suggest ASS1 deficiency as a druggable target and also provide a unique therapeutic strategy against pulmonary fibrosis.

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.

Successful treatment of adult-onset type II citrullinemia with a low-carbohydrate diet and L-arginine after DNA analysis produced a definitive diagnosis.

A 60-year-old male, who exhibited finger tremors, obnubilation, and hyperammonemia (409 µg/dL), was admitted to our hospital. Initially, we suspected that a portosystemic shunt had caused his hyperammonemia. However, his symptoms did not improve after balloon-occluded retrograde transvenous obliteration. He was subsequently found to have some peculiar eating habits, including a fondness for bean curd and peanuts, and an aversion to alcohol and sweets. Furthermore, marked citrullinemia (454.2 nmol/mL) was revealed, which led us to suspect adult-onset type II citrullinemia (CTLN2). DNA analysis of the patient and his mother, son, and daughter confirmed that he was homozygous for the c.852_855del mutation in the SLC25A13 gene, and his relatives were heterozygous for the c.852_855del mutation, which led to a definitive diagnosis. A low-carbohydrate diet and the administration of L-arginine ameliorated his symptoms. It is important to be aware that CTLN2 can occur in elderly patients. Thus, patients who exhibit symptoms of CTLN2 should be interviewed about their dietary habits and subjected to plasma amino acid analysis.In this report, we consider the metabolic disorders seen in citrin deficiency and the associated compensatory mechanisms in relation to the clinical features and treatment of CTLN2.

Clinical findings in five Turkish patients with citrin deficiency and identification of a novel mutation on SLC25A13.

Background Citrin deficiency (CD) is an autosomal recessive genetic disorder caused by a defect in the mitochondrial aspartate/glutamate antiporter, citrin. Three clinical manifestations have been described until today. Case presentation We reported 5 CD patients from two families. Four patients were male and one patient was female. Two of them have NICCD (neonatal intrahepatic cholestasis caused by citrin deficiency); three of them have CTLN2 (adult-onset type II citrullinemia). Both NICCD patients showed typical clinical and biochemical changes with a diagnosis confirmed by mutations in the SLC25A13 gene. We detected a previously unreported homozygous novel mutation c.478delC (L160Wfs*36 ) on the SLC25A13 gene. All of the CTLN2 patients were siblings. Proband was a 15-year-old mentally retarded and autistic male who had admitted to our emergency with disorientation. Laboratory data showed hyperammonemia and citrullinemia. Conclusions Two different profiles of age-related CD have been depicted with this article. It has been aimed to underline that the CD can be observed in different forms not only in neonatals or little infants but also in adolescents. This article is the first case series that covers both NICCD and CTLN2 cases together and that has been published in Turkey. Considering the fact that especially the majority of CTLN2 cases have been identified in Asian countries, our article has vital importance in terms of defining phenotypic features of the disease.

Citrullinemia Type 1: Behavioral Improvement with Late Liver Transplantation.

Citrullinemia Type 1 (also known as classic citrullinemia) is a rare autosomal recessive urea cycle disorder due to reduced activity of argininosuccinate synthetase 1; characterized by hyperammonemia leading to neurological damage. The authors report a case of an 8-y boy who was diagnosed with Citrullinemia Type 1 at birth which was anticipated prenatally due to family history. His diagnosis was confirmed as a homozygous mutation (Exon 15: c.1168G > A (p.G390R)) of ASS gene. Inspite of being on a protein-free diet and ammonia scavenging treatment; the patient developed recurrent episodes of encephalopathy and seizures; complicated with behavioral issues. The patient underwent living related liver-transplantation from his mother (heterozygous carrier of the same mutation). Peri-transplant management of ammonia and plasma amino acid levels is challenging and has been highlighted. It is important to consider liver transplantation as it corrects the genetic deficiency of ASS resulting in the reversal of neuro-behavioral changes, as was seen in index patient.

Hypomorphic citrullinaemia due to mutated ASS1 with episodic ataxia.

Children with citrullinaemia commonly present in the neonatal period with life-threatening hyperammonaemia and progressive encephalopathy. Less often, 'hypomorphic' or mild late-onset childhood or adult-onset forms may be seen with intermittent neurological symptoms or acute crisis in pregnancy. We describe an 11-year-old boy with late-onset citrullinaemia manifesting as brief episodes of ataxia triggered by minor febrile illnesses, significant citrullinaemia, mild hyperammonaemia, normal neurological examination and mild cerebellar atrophy. Targeted gene sequencing showed a homozygous, missense mutation c.815G>A (p.R272H) in exon 12 of ASS1 gene resulting in the amino acid substitution of histidine for arginine at codon 272. Our case highlights the importance of recognising urea cycle defects as a cause of intermittent neurological symptoms such as ataxia. Type-1 citrullinaemia may remain hypomorphic and needs a high index of suspicion.

Metabolically based liver damage pathophysiology in patients with urea cycle disorders - A new hypothesis.

The underlying pathophysiology of liver dysfunction in urea cycle disorders (UCDs) is still largely elusive. There is some evidence that the accumulation of urea cycle (UC) intermediates are toxic for hepatocyte mitochondria. It is possible that liver injury is directly caused by the toxicity of ammonia. The rarity of UCDs, the lack of checking of iron level in these patients, superficial knowledge of UC and an underestimation of the metabolic role of fumaric acid, are the main reasons that are responsible for the incomprehension of the mechanism of liver injury in patients suffering from UCDs. Owing to our routine clinical practice to screen for iron overload in severely ill neonates, with the focus on the newborns suffering from acute liver failure, we report a case of citrullinemia with neonatal liver failure and high blood parameters of iron overload. We hypothesize that the key is in the decreased-deficient fumaric acid production in the course of UC in UCDs that causes several sequentially intertwined metabolic disturbances with final result of liver iron overload. The presented hypothesis could be easily tested by examining the patients suffering from UCDs, for liver iron overload. This could be easily performed in countries with a high population and comprehensive national register for inborn errors of metabolism. CONCLUSION: Providing the hypothesis is correct, neonatal liver damage in patients having UCD can be prevented by the supplementation of pregnant women with fumaric or succinic acid, prepared in the form of iron supplementation pills. After birth, liver damage in patients having UCDs can be prevented by supplementation of these patients with zinc fumarate or zinc succinylate, as well.

Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency.

BACKGROUND: Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia. METHODS: HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells. RESULTS: Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme and mRNA. In fact, EVs from ASS1-knockdown HLSCs contained low amounts of ASS1 mRNA and protein, and were unable to restore urea production in hepatocytes differentiated from ASS1-HLSCs. CONCLUSIONS: Collectively, these results suggest that EVs derived from normal HLSCs may compensate the loss of ASS1 enzyme activity in hepatocytes differentiated from ASS1-HLSCs.

Mutations in the Human Argininosuccinate Synthetase (ASS1) Gene, Impact on Patients, Common Changes, and Structural Considerations.

Citrullinemia type 1 is an autosomal recessive urea cycle disorder caused by defects in the argininosuccinate synthetase (ASS) enzyme due to mutations in ASS1 gene. An impairment of ASS function can lead to a wide spectrum of phenotypes, from life-threatening neonatal hyperammonemia to a later onset with mild symptoms, and even some asymptomatic patients exhibiting an only biochemical phenotype. The disease is panethnic. In this update, we report 137 mutations (64 of which are novel), consisting of 89 missense mutations, 19 nonsense mutations, 17 mutations that affect splicing, and 12 deletions. The change p.Gly390Arg is by far the most common mutation and is widely spread throughout the world. Other frequent mutations (p.Arg157His, p.Trp179Arg, p.Val263Met, p.Arg304Trp, p.Gly324Ser, p.Gly362Val, and p.Arg363Trp), each found in at least 12 independent families, are mainly carried by patients from the Indian subcontinent, Turkey, Germany, and Japan. To better understand the disease, we collected clinical data of >360 patients, including all published information available. This information is related to the patients' genetic background, the conservation of the mutated residues and a structural rationalization of the effect of the most frequent mutations. In addition, we review ASS regulation, animal models, diagnostic strategies, newborn screening, and treatment options.

SLC25A13 cDNA cloning analysis using peripheral blood lymphocytes facilitates the identification of a large deletion mutation: Molecular diagnosis of an infant with neonatal intrahepatic cholestasis caused by citrin deficiency.

Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is an autosomal recessive disorder resulting from biallelic mutations of the SLC25A13 gene. Due to the lack of well­recognized clinical or biochemical diagnostic criteria, the definitive diagnosis of this disease relies on the genetic analysis of SLC25A13 at present. As novel large deletion/insertion mutations of the SLC25A13 gene are difficult to detect using routine DNA analytic approaches, the timely diagnosis of patients with these types of mutations remains a challenge. The present study aimed to examine SLC25A13 mutations in an infant with a suspected diagnosis of NICCD. DNA was extracted from blood samples, and SLC25A13 mutations were examined by screening for high­frequency mutations and Sanger sequencing. Reverse transcription-polymerase chain reaction and cDNA cloning analyses were then performed using peripheral blood lymphocytes (PBLs) to identify the obscure mutation. The results demonstrated that the infant was heterozygous for a paternally­inherited mutation, c.851_854del4, and a maternally­inherited large deletion, c.1019_1177+893del, which has not been reported previously. A positive diagnosis of NICCD was made, and the infant responded favorably to a galactose­free and medium­chain triglyceride­enriched formula. The present study confirmed the effectiveness of this formula in NICCD therapy, enriched the SLC25A13 mutational spectrum and supported the feasibility of cDNA cloning analysis using PBLs as a molecular tool for facilitating the identification of large SLC25A13 deletions.

Electroencephalography and transcranial Doppler ultrasonography in neonatal citrullinemia.

The authors present a case of citrullinemia with a genotype of argininosuccinate synthetase (ASS1), c.380 G>A (p.R127Q)/c.380 G>A (p.R127Q), in two alleles. A 3-day-old female infant presented with status epilepticus and coma. Laboratory data showed hyperammonemia and marked lactic acidosis in the blood and cerebrospinal fluid; electroencephalography showed severely suppressed cerebral activity and focal paroxysmal volleys of slow and sharp waves (< 1Hz) over the left hemisphere. Real-time transcranial Doppler ultrasonography showed a brain edema and high peaked systolic and low diastolic flows in basal, anterior, and middle cerebral arteries; however, immediately after a blood exchange transfusion, systolic flows were lower and diastolic flows were higher. The resistance indices were significantly different (means: 0.58 vs. 0.37; p=0.01). The patient was placed on diet therapy. After six blood exchange transfusions and peritoneal dialysis, her neurologic examination results and serum ammonia and lactate values were normal. The authors found that electroencephalography and transcranial Doppler ultrasonography were useful for the diagnosis and follow-up treatment of neonatal citrullinemia.

[A case of adult-onset type II citrullinemia (CTLN2) triggered by an overseas travel].

A 43-year-old male presented with abnormal behavior and consciousness disturbance on the day after traveling abroad and was admitted to our hospital. Laboratory tests showed hyperammonemia and hypercitrullinemia. The electro-encephalogram showed frontal dominant bilateral slow δ burst. He had a peculiar taste for nuts. But he didn't take nuts during the overseas travel for 3 days. The family history revealed that his younger brother died of a status epilepticus of unknown cause at the age of 29. These findings were compatible with hepatic encephalopathy due to adult-onset type II citrullinemia (CTLN2). Gene analysis provided a definite diagnosis of CTLN2. Diet and drug therapy have improved his condition. He is due to have liver transplantation which is the only established radical treatment for CTLN2 if his condition becomes worse. The present case shows that cessation of the habitual intake of nuts only for 3 days could lead to onset of CTLN2.

Liver transplantation versus conservative treatment for adult-onset type II citrullinemia: our experience and a review of the literature.

Adult-onset type II citrullinemia (CTLN2), an autosomal recessive disorder caused by a mutation in the SLC25A13 gene, is characterized by increased serum citrulline and ammonia levels. Patients with CTLN2 also display various neuropsychiatric symptoms. Many individuals with CTLN2 are fond of protein-rich and/or lipid-rich foods with an aversion to carbohydrate-rich foods. We herein report two cases of CTLN2 treated with living donor liver transplantation (LDLT) and provide a review of the pertinent literature. Case 1 was a 43-year-old man admitted to our hospital for repetitive episodes of consciousness disturbance. Case 2 was a 37-year-old man admitted to our hospital because of abnormal behavior associated with hyperammonemia. A definitive diagnosis of CTLN2 was accomplished by DNA analysis in both patients, who successfully underwent LDLT using liver segments from donor siblings with confirmed heterozygous gene expression. Case 2 also underwent conservative therapy with arginine and a high-fat, carbohydrate-restricted diet prior to LDLT. Postoperative recovery was uneventful and food was unrestricted in both patients. We also identified 77 cases of CTLN2 in the literature and reviewed them in terms of outcome of both liver transplantation and conservative therapy. The survival rate in patients treated by liver transplantation was 100%, whereas that in patients treated by conservative treatment showed improvement from 39.5% to 76.5% over the years. Liver transplantation is a practical treatment that fundamentally improves patient quality of life after transplantation. However, recent studies have suggested that arginine and sodium pyruvate administration combined with intensive nutritional support is also an effective therapy for CTLN2. Further development of conservative therapy may provide a safer, more affordable alternative to liver transplantation in the near future.