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Citrin deficiency is an autosomal recessive disorder caused by a defect of citrin resulting from mutations in the SLC25A13 gene. Intrahepatic cholestasis and various metabolic abnormalities, including hypoglycemia, galactosemia, citrullinemia, and hyperammonemia may be present in neonates or infants in the "neonatal intrahepatic cholestasis caused by citrin deficiency" (NICCD) form of the disease. Because at present, newborn screening (NBS) for citrin deficiency using citrulline levels in dried blood spots (DBS) can only detect some of the patients, we tried to develop a new evaluation system to more reliably detect newborns with citrin deficiency utilizing parameters already in place in present NBS methods. To achieve this goal, we re‐analyzed NBS profiles of amino acids and acylcarnitines in 96 NICCD patients, who were diagnosed through selective screening or positive family history. Hereby, we identified the combined evaluation of arginine (Arg), citrulline (Cit), isoleucine+leucine (Ile + Leu), tyrosine (Tyr), free carnitine (C0) / glutarylcarnitine (C5‐DC) ratio in DBS as potentially sensitive to diagnose citrin deficiency in pre‐symptomatic newborns. In particular, a scoring system using threshold levels for Arg (≥9 μmol/L), Cit (≥ 39 μmol/L), Ile + Leu (≥ 99 μmol/L), Tyr (≥ 96 μmol/L) and C0/C5‐DC ratio (≥327) was significantly effective to detect newborns who later developed NICCD, and could thus be implemented in existing NBS programs at no extra analytical costs whenever citrin deficiency is considered to become a novel target disease. [ABSTRACT FROM AUTHOR]

Citrin deficiency (CD) is a recessive, liver disease caused by sequence variants in the SLC25A13 gene encoding a mitochondrial aspartate–glutamate transporter. CD manifests as different age‐dependent phenotypes and affects crucial hepatic metabolic pathways including malate–aspartate‐shuttle, glycolysis, gluconeogenesis, de novo lipogenesis and the tricarboxylic acid and urea cycles. Although the exact pathophysiology of CD remains unclear, impaired use of glucose and fatty acids as energy sources due to NADH shuttle defects and PPARα downregulation, respectively, indicates evident energy deficit in CD hepatocytes. The present review summarizes current trends on available and potential treatments for CD. Baseline recommendation for CD patients is dietary management, often already present as a self‐selected food preference, that includes protein and fat‐rich food, and avoidance of excess carbohydrates. At present, liver transplantation remains the sole curative option for severe CD cases. Our extensive literature review indicated medium‐chain triglycerides (MCT) as the most widely used CD treatment in all age groups. MCT can effectively improve symptoms across disease phenotypes by rapidly supplying energy to the liver, restoring redox balance and inducing lipogenesis. In contrast, sodium pyruvate restored glycolysis and displayed initial preclinical promise, with however limited efficacy in adult CD patients. Ursodeoxycholic acid, nitrogen scavengers and L‐arginine treatments effectively address specific pathophysiological aspects such as cholestasis and hyperammonemia and are commonly administered in combination with other drugs. Finally, future possibilities including restoring redox balance, amino acid supplementation, enhancing bioenergetics, improving ureagenesis and mRNA/DNA‐based gene therapy are also discussed. [ABSTRACT FROM AUTHOR]

Objective: Citrin, the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), is structurally and mechanistically the most complex SLC25 family member, because it consists of three domains and forms a homo-dimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency. Methods: We have employed structural modeling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knockout of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation. Results: Using 33 pathogenic variants of citrin we clarify determinants of subcellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect. Conclusions: Our work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency.

Citrin deficiency is a congenital secondary urea cycle disorder lacking useful disease models for effective treatment development. In this study, human induced pluripotent stem cells (iPSCs) were generated from two patients with citrin deficiency and differentiated into hepatocyte-like cells (HLCs). Citrin-deficient HLCs produced albumin and liver-specific markers but completely lacked citrin protein and expressed argininosuccinate synthase only weakly. In addition, ammonia concentrations in a medium cultured with citrin-deficient HLCs were higher than with control HLCs. Sodium pyruvate administration significantly reduced ammonia concentrations in the medium of citrin-deficient HLCs and slightly reduced ammonia in HLCs differentiated from control iPSCs, though this change was not significant. Our results suggest that sodium pyruvate may be an efficient treatment for patients with citrin deficiency. Citrin-deficient iPSCs are a pathological liver model for congenital urea cycle disorders to clarify pathogenesis and develop novel therapies.

Abstract Deficiency of citrin, the liver-type aspartate-glutamate carrier, arises from biallelic mutations of the gene SLC25A13. Although citrin deficiency (CD) is associated with higher risk of hepatocellular carcinoma (HCC) in adult patients, this association remains inconclusive in pediatric cases. The patient in this paper had been diagnosed to have CD by SLC25A13 analysis at the age 10 months, and then in response to dietary therapy, her prolonged jaundice and marked hepatosplenomegaly resolved gradually. However, she was referred to the hospital once again due to recurrent abdominal distention for 2 weeks at her age 4 years and 9 months, when prominently enlarged liver and spleen were palpated, along with a strikingly elevated serum alpha-fetoprotein (AFP) level of 27605 ng/mL as well as a large mass in the right liver lobe and a suspected tumor thrombus within the portal vein on enhanced computed tomography. After 4 rounds of adjuvant chemotherapy, right hepatic lobectomy and portal venous embolectomy were performed at her age 5 years and 3 months, and metastatic hepatoblastoma was confirmed by histopathological analysis. Afterwards, the patient underwent 5 additional cycles of chemotherapy and her condition remained stable for 7 months after surgery. Unfortunately, hepatoblastoma recurred in the left lobe at the age 5 years and 10 months, which progressed rapidly into liver failure, and led to death at the age 6 years and 1 month. As far as we know, this is the the first case of hepatoblastoma in a patient with CD, raising the possibility of an association between these two conditions.

Deficiency of citrin, the liver-type aspartate-glutamate carrier, arises from biallelic mutations of the gene SLC25A13. Although citrin deficiency (CD) is associated with higher risk of hepatocellular carcinoma (HCC) in adult patients, this association remains inconclusive in pediatric cases. The patient in this paper had been diagnosed to have CD by SLC25A13 analysis at the age 10 months, and then in response to dietary therapy, her prolonged jaundice and marked hepatosplenomegaly resolved gradually. However, she was referred to the hospital once again due to recurrent abdominal distention for 2 weeks at her age 4 years and 9 months, when prominently enlarged liver and spleen were palpated, along with a strikingly elevated serum alpha-fetoprotein (AFP) level of 27605 ng/mL as well as a large mass in the right liver lobe and a suspected tumor thrombus within the portal vein on enhanced computed tomography. After 4 rounds of adjuvant chemotherapy, right hepatic lobectomy and portal venous embolectomy were performed at her age 5 years and 3 months, and metastatic hepatoblastoma was confirmed by histopathological analysis. Afterwards, the patient underwent 5 additional cycles of chemotherapy and her condition remained stable for 7 months after surgery. Unfortunately, hepatoblastoma recurred in the left lobe at the age 5 years and 10 months, which progressed rapidly into liver failure, and led to death at the age 6 years and 1 month. As far as we know, this is the the first case of hepatoblastoma in a patient with CD, raising the possibility of an association between these two conditions. [ABSTRACT FROM AUTHOR]

To find biochemical and molecular markers can assist in identifying serious liver damage of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) patients. 138 patients under 13 days to 1.1 year old diagnosed of NICCD in our center from 2004 to 2020. Base on the abnormal liver laboratory tests, we divided 138 patients into three groups: acute liver failure (ALF), liver dysfunction, and non-liver dysfunction groups, then compared their clinical, biochemical and, molecular data. 96 % of 138 patients had high levels of citrulline and high ratio of threonine to serine, which is the distinctive feature of plasma amino acid profile for NICCD. A total of 18.1 % of 138 patients had evidence of ALF who presented the most severity hepatic damage, 51.5 % had liver dysfunction, and the remaining 30.4 % presented mild clinical symptoms (non-liver dysfunction). In ALF group, the levels of citrulline, tyrosine, TBIL, ALP, and γ-GT was significantly elevated, and the level of ALB and Fisher ratio was pronounced low. Homozygous mutations of 1,638_1660dup, IVS6+5G.A, or IVS16ins3kb in SLC25A13 gene were only found in ALF and liver dysfunction groups. Supportive treatment including medium-chain triglyceride supplemented diet and fresh frozen plasma could be life-saving and might reverse ALF. High level of citrulline, tyrosine, TBIL, ALP, γ-GT, and ammonia, low level of albumin, and low Fisher ratio were predictors to suggest severe liver damage in NICCD patients who may go on to develop fatal metabolic disorder. Early identification and proper therapy is particularly important for these patients. [ABSTRACT FROM AUTHOR]

Citrin deficiency (CD) is an autosomal recessive disease caused by mutations of the SLC25A13 gene, plasma bile acid profiles detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) could be an efficient approach for early diagnosis of intrahepatic cholestasis. The aim of this study was to investigate the genetic testing and clinical characteristics of a series of patients with CD, and to analyse plasma bile acid profiles in CD patients. We retrospectively analysed data from 14 patients (12 males and 2 females, age 1–18 months, mean 3.6 months) with CD between 2015 and 2021, including demographics, biochemical parameters, genetic test results, treatment, and clinical outcomes. In addition, 30 cases (15 males and 15 females, age 1–20 months, mean 3.8 months) with idiopathic cholestasis (IC) served as a control group. Plasma 15 bile acid profiles were compared between the CD and IC groups. Eight different mutations of the SLC25A13 gene were detected in the 14 patients diagnosed with CD, of which three novel variants of the SLC25A13 gene were investigated, the c.1043C>T (p.P348L) in exon11, the c.1216dupG (p.A406 Gfs*13) in exon12 and the c.135G>C (p.L45F) in exon3. More than half of the patients with CD had prolonged neonatal jaundice, which was associated with significantly higher alpha-fetoprotein (AFP) levels, hyperlactatemia and hypoglycemia. The majority of patients were ultimately self-limited. Only one patient developed liver failure and died at the age of 1 year due to abnormal coagulation function. In addition, the levels of glycochenodeoxycholic acid (GCDCA), taurocholate (TCA), and taurochenodeoxycholic acid (TCDCA) were significantly increased in the CD group compared with those in the IC group. Three novel variants of the SLC25A13 gene were identified for the first time, providing a reliable molecular reference and expanding the SLC25A13 gene spectrum in patients with CD. Plasma bile acid profiles could be a potential biomarker for non-invasive early diagnosis of patients with intrahepatic cholestasis caused by CD. [ABSTRACT FROM AUTHOR]

The deficiency of CITRIN, the liver mitochondrial aspartate–glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(−/−) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (−/−) mice expressing liver specific transgenic aralar had a small (~ 4–6 nmoles x mg prot−1 x min−1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(−/−) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(−/−) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficiency.

Abstract Background Dietary management is the most important and effective treatment for citrin deficiency, as well as a decisive factor in the clinical outcome of patients. However, the dietary management ability of caregivers of children with citrin deficiency is generally poor, especially in East Asia where carbohydrate-based diets are predominant. The aim of this study was to identify the difficulties that caregivers encounter in the process of home-based dietary management, and the reasons responsible for these challenges. Results A total of 26 caregivers of children with citrin deficiency were recruited, including 24 mothers, one father, and one grandmother. Grounded theory was employed to identify three themes (covering 12 sub-themes) related to the dilemma of dietary management: dietary management that is difficult to implement; conflicts with traditional concepts; and the notion that children are only a part of family life. The first theme describes the objective difficulties that caregivers encounter in the process of dietary management; the second theme describes the underlying reasons responsible for the non-adherent behavior of caregivers; the third theme further reveals the self-compromise by caregivers in the face of multiple difficulties. Conclusions This study reflects the adverse effects of multi-dimensional contradictions on the adherence of caregivers to dietary management. These findings reveal that the dietary management of citrin deficiency is not only a rational process, rather it is deeply embedded in family, social, and dietary traditions.

Citrin, the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), is structurally and mechanistically the most complex SLC25 family member, because it consists of three domains and forms a homo-dimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency. We have employed structural modeling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knockout of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation. Using 33 pathogenic variants of citrin we clarify determinants of subcellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect. Our work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency. [Display omitted] • We study 33 citrin missense mutations causing citrin deficiency. • We identify elements crucial for transport within the carrier domain of citrin. • The transport activity of citrin is not calcium-regulated, as previously thought. • Pathogenic mutations in the N-terminal domain cause a mitochondrial import defect. • Citrin domains contribute to distinct pathogenic mechanisms in citrin deficiency. [ABSTRACT FROM AUTHOR]

BackgroundNeonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a common clinical phenotype of citrin deficiency in infants. Its phenotype is atypical, so genetic testing is quite necessary for the diagnosis.Case presentationWe report 4 patients with jaundice and low body weight. Furthermore, the biochemical examination of all showed abnormal liver function and metabolic changes. DNA samples of the patients were extracted and subjected to genetic screening. All candidate pathogenic variants were validated by Sanger sequencing, and CNVs were ascertained by qPCR. The genetic screening revealed 6 variants in 4 patients, and all patients carried compound heterozygous variants of SLC25A13. Importantly, 3 variants were newly discovered: a nonsense mutation in exon17 (c.1803C > G), a frameshift mutation in exon 11(c.1141delG) and a deletion of the whole exon11. Thus, four NICCD patients were clearly caused by variants of SLC25A13. Biochemical indicators of all patients gradually returned to normal after dietary adjustment.ConclusionsOur study clarified the genetic etiology of the four infants, expanded the variant spectrum of SLC25A13, and provided a basis for genetic counseling of the family. Early diagnosis and intervention should be given to patients with NICCD.

Citrin belongs to the SLC25 transport protein family found mostly in inner mitochondrial membranes. The family prototype, the ADP-ATP carrier, delivers ATP made inside mitochondria to the cellular cytoplasm and returns ADP to the mitochondrion for resynthesis of ATP. In pre-genomic 1981, I noticed that the protein sequence of the bovine ADP-ATP carrier consists of three related sequences, each containing two transmembrane α-helices traveling in opposite senses. Colleagues and I demonstrated that two other mitochondrial carriers had similar features. From emergent genomic sequences, it became apparent that they represented a large family of transport proteins with the same characteristic threefold repeats. The human genome encodes 53 members, but the functions of many were unknown. So, colleagues and I determined how to make these proteins in Escherichia coli and introduce them into liposomes to allow exploration of their transport functions. The 27 human family members to have been thus identified include citrin and the closely related protein aralar. Both exchange aspartate from the mitochondrial matrix for cytosolic glutamate plus a proton. Citrin is expressed predominantly in liver and non-excitable tissues, whereas aralar is the dominant form in the brain. Each has a membrane extrinsic N-terminal Ca 2+ -binding domain, a transport domain, and a C-terminal amphipathic α-helix. Human mutations in citrin impair the urea cycle, malate-aspartate shuttle, gluconeogenesis, amino acid breakdown, and energy metabolism leading to citrin deficiency. Currently, the complex etiology of this condition is poorly understood and new knowledge would help to improve diagnosis, therapies, and finding a cure. My aims are to seek a basic understanding of the etiology of citrin deficiency and to use that knowledge in improving diagnostic procedures and in developing new treatments and a cure., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The mitochondrial malate aspartate shuttle system (MAS) maintains the cytosolic NAD+/NADH redox balance, thereby sustaining cytosolic redox-dependent pathways, such as glycolysis and serine biosynthesis. Human disease has been associated with defects in four MAS-proteins (encoded by MDH1, MDH2, GOT2, SLC25A12) sharing a neurological/epileptic phenotype, as well as citrin deficiency (SLC25A13) with a complex hepatopathic-neuropsychiatric phenotype. Ketogenic diets (KD) are high-fat/low-carbohydrate diets, which decrease glycolysis thus bypassing the mentioned defects. The same holds for mitochondrial pyruvate carrier (MPC) 1 deficiency, which also presents neurological deficits. We here describe 40 (18 previously unreported) subjects with MAS-/MPC1-defects (32 neurological phenotypes, eight citrin deficiency), describe and discuss their phenotypes and genotypes (presenting 12 novel variants), and the efficacy of KD. Of 13 MAS/MPC1-individuals with a neurological phenotype treated with KD, 11 experienced benefits—mainly a striking effect against seizures. Two individuals with citrin deficiency deceased before the correct diagnosis was established, presumably due to high-carbohydrate treatment. Six citrin-deficient individuals received a carbohydrate-restricted/fat-enriched diet and showed normalisation of laboratory values/hepatopathy as well as age-adequate thriving. We conclude that patients with MAS-/MPC1-defects are amenable to dietary intervention and that early (genetic) diagnosis is key for initiation of proper treatment and can even be lifesaving. [ABSTRACT FROM AUTHOR]

Rare diseases, also known as orphan diseases, are a group of diseases that affect a relatively small number of people. Low incidence rates, atypical symptoms, imperfect diagnosis criteria and few treatment options impose medical, psychological and financial stress on the local healthcare system. The spectrum of liver diseases in China has changed in the past decades due to successful control of once highly prevalent viral hepatitis B and C. Furthermore, the increased awareness and improved availability of specific laboratory tests have also facilitated the diagnosis of rare diseases such as autoimmune, cholestatic and genetic liver diseases. Finally, considering the huge population, the total number of many rare liver diseases in China is not as rare as once deemed. In this mini-review article, we will outline the current clinical and epidemiological profiles of some rare liver diseases that are no longer rare in China. [ABSTRACT FROM AUTHOR]

Citrin deficiency is an autosomal recessive disorder caused by variants of the SLC25A13 gene. Although newborn screening (NBS) provides an opportunity for its early diagnosis and treatment, citrin deficiency detection rates remain lower than those estimated. Before 2018, NBS for citrin deficiency was based on citrulline levels alone. In June 2018, a second-tier molecular test was implemented to detect 11 common variants of the SLC25A13 gene and improve the NBS detection rates. This study compares the incidence rates and costs before and after the second-tier implementation. Prior to 2018, five subjects were diagnosed via NBS, and 12 of 555,449 newborns screened were missed. In comparison, 11 subjects were diagnosed out of 198,071 newborns screened after 2018, and there were no false-negatives. The citrin deficiency detection rate increased from 1/32,673 to 1/18,006 after the second-tier test was implemented, with only a minimal increase in the total cost. The number of false-positive in our cohort was tolerable. Subjects with citrin deficiency may present with borderline elevated citrulline levels; these can remain slightly elevated or increase considerably on retest. Four patients (80%) detected prior to second-tier testing and six patients (55%) detected after it was implemented were identified based on the citrulline levels alone. However, at the time of second blood sampling, the normal citrulline level of five subjects did not exclude a citrin deficiency diagnosis. Our study shows that it is vital and cost-effective to employ second-tier molecular testing to improve the detection of citrin deficiency by NBS. • This is the first report of a successful real-world, nationwide, two-tier newborn screening program for citrin deficiency. • Second-tier molecular testing in newborn screening is a cost-effective method to improve citrin deficiency detection rate. • NGS is recommended for those with only one common variant of SLC25A13 or those with persistently elevated citrulline levels. [ABSTRACT FROM AUTHOR]

Rare diseases, also known as orphan diseases, are a group of diseases that affect a relatively small number of people. Low incidence rates, atypical symptoms, imperfect diagnosis criteria and few treatment options impose medical, psychological and financial stress on the local healthcare system. The spectrum of liver diseases in China has changed in the past decades due to successful control of once highly prevalent viral hepatitis B and C. Furthermore, the increased awareness and improved availability of specific laboratory tests have also facilitated the diagnosis of rare diseases such as autoimmune, cholestatic and genetic liver diseases. Finally, considering the huge population, the total number of many rare liver diseases in China is not as rare as once deemed. In this mini‐review article, we will outline the current clinical and epidemiological profiles of some rare liver diseases that are no longer rare in China. [ABSTRACT FROM AUTHOR]

Background: Dietary management is the most important and effective treatment for citrin deficiency, as well as a decisive factor in the clinical outcome of patients. However, the dietary management ability of caregivers of children with citrin deficiency is generally poor, especially in East Asia where carbohydrate-based diets are predominant. The aim of this study was to identify the difficulties that caregivers encounter in the process of home-based dietary management, and the reasons responsible for these challenges.Results: A total of 26 caregivers of children with citrin deficiency were recruited, including 24 mothers, one father, and one grandmother. Grounded theory was employed to identify three themes (covering 12 sub-themes) related to the dilemma of dietary management: dietary management that is difficult to implement; conflicts with traditional concepts; and the notion that children are only a part of family life. The first theme describes the objective difficulties that caregivers encounter in the process of dietary management; the second theme describes the underlying reasons responsible for the non-adherent behavior of caregivers; the third theme further reveals the self-compromise by caregivers in the face of multiple difficulties.Conclusions: This study reflects the adverse effects of multi-dimensional contradictions on the adherence of caregivers to dietary management. These findings reveal that the dietary management of citrin deficiency is not only a rational process, rather it is deeply embedded in family, social, and dietary traditions. [ABSTRACT FROM AUTHOR]

Abstract Background Neonatal intrahepatic cholestasis caused by citrin deficiency (CD) is a rare inborn error of metabolism due to variants in the SLC25A13 gene encoding the calcium-binding protein citrin. Citrin is an aspartate-glutamate carrier located within the inner mitochondrial membrane. Case presentation We report on two siblings of Romanian-Vietnamese ancestry with citrin deficiency. Patient 1 is a female who presented at age 8 weeks with cholestasis, elevated lactate levels and recurrent severe hypoglycemia. Diagnosis was made by whole exome sequencing and revealed compound heterozygosity for the frameshift variant c.852_855del, p.Met285Profs*2 and a novel deletion c.(69 + 1_70–1)_(212 + 1_231–1)del in SLC25A13. The girl responded well to dietary treatment with a lactose-free, MCT-enriched formula. Her younger brother (Patient 2) was born 1 year later and also found to be carrying the same gene variants. Dietary treatment from birth was able to completely prevent clinical manifestation until his current age of 4.5 months. Conclusions As CD is a well-treatable disorder it should be ruled out early in the differential diagnosis of neonatal cholestasis. Due to the combination of hepatopathy, lactic acidosis and recurrent hypoglycemia the clinical presentation of CD may resemble hepatic mitochondrial depletion syndrome.

Abstract Background Citrin deficiency (CD) is a recessive metabolic disease caused by biallelic pathogenic variants in SLC25A13. Although previous studies have reported ketosis in CD, it was observed at the time of euglycemia or mild hypoglycemia. Blood ketone levels concomitant with symptomatic or severe hypoglycemia in CD have not been a topic of focus despite its importance in identifying the etiology of hypoglycemia and assessing the ability of fatty acid utilization. Herein, we describe a patient with CD who had repeated episodes of hypoglycemia with insufficient ketosis. Case presentation A 1-year-old boy with repetitive hypoglycemia was referred to us to investigate its etiology. The fasting load for 13 h led to hypoketotic hypoglycemia, indicating the possibility of partial β-oxidation dysfunction. A genetic test led to the diagnosis of CD. The hypoglycemic episodes disappeared after switching to a medium-chain triglyceride-containing formula. Conclusions This case report suggests that symptomatic or severe hypoglycemia in patients with CD could be associated with relatively low levels of ketone bodies, implying that β-oxidation in these patients might possibly be partially disrupted. When encountering a patient with hypoglycemia, clinicians should check blood ketone levels and bear in mind the possibility of CD because excessive intravenous administration of glucose can cause decompensated symptoms in patients with CD as opposed to other disorders presenting with hypoketotic hypoglycemia, such as fatty acid oxidation disorders. Further studies in a large-scale cohort are warranted to confirm our speculation.

Citrin deficiency belongs to a group of urea cycle disorders that can be identified during newborn screening by measuring citrulline, phenylalanine, methionine, and galactose levels. Early diagnosis of citrin deficiency is beneficial as disease-specific interventions such as permission of food preference and/or supplementation of medium-chain triglyceride can prevent metabolic decompensation. However, there are currently no laboratory tests for the diagnosis of citrin deficiency in routine clinical practice. Our retrospective study investigated the diagnostic characteristics of citrin deficiency during infancy at secondary newborn screening. The present study included 10 patients with citrin deficiency and 35 controls without the condition. The positive likelihood ratios for serum levels of blood urea nitrogen (BUN) levels were 6.8 at the first visit, 3.2 at age ≤ 60 days, and 17.5 at age ≤ 100 days. The serum BUN/creatinine ratio also showed a high positive likelihood ratio (3.9 at first visit, 16.0 at age ≤ 60 days, and 24.5 at age ≤ 100 days). Therefore, the serum BUN or BUN/creatinine ratio may help to identify patients with citrin deficiency during newborn screening. Further studies are required to confirm its diagnostic accuracy in a larger cohort and elucidate the underlying mechanisms involved.

Background: Citrin deficiency is a rare metabolic disorder prevalent in East and Southeast Asia that affects liver or neurological function throughout various life stages. While early diagnosis and dietary management can improve prognosis for infant onset disease, data on long-term neurocognitive outcomes is scarce., Purpose: This study aimed to clarify whether transient metabolic disturbances during early childhood have a lasting effect on the neurocognitive function of individuals with citrin deficiency., Methods: Thirty patients diagnosed with citrin deficiency prior to 1 year of age underwent neuropsychological assessments including attention-deficit/hyperactivity disorder (ADHD) and intelligence quotient (IQ). We compared the peak laboratory values during infancy between children who were versus were not later diagnosed with ADHD., Results: Neurocognitive assessments of 30 individuals with citrin deficiency aged 3-25 years revealed that full-scale IQ scores were normally distributed. Of this cohort, 47% (14 of 30) were diagnosed with ADHD: six, six, and two with the combined, inattentive, and hyperactive-impulsive types, respectively. This prevalence was higher than that in the general population (1.7-16%). Moreover, a one-unit increase in ammonia levels before 1 year of age was associated with a 1.023-fold increase in the likelihood of future hyperactivity-impulsivity symptoms (p = 0.038; 95% confidence interval, 1.001-1.046). Despite these findings, this long-term follow-up of individuals with citrin deficiency indicated that it had minimal impact on neurocognitive function, allowing for a generally normal life., Conclusion: Patients with a history of cholestasis caused by citrin deficiency during infancy have a greater incidence of ADHD than the general population, suggesting that metabolic disturbances during early childhood in individuals with citrin deficiency may have a long-term negative impact on their neurocognitive function.

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Hepatocellular carcinoma (HCC) is a longstanding issue in clinical practice and metabolic research. New clues in better understanding the pathogenesis of HCC might relate to the metabolic context in patients with citrin (aspartate-glutamate carrier 1) deficiency (CD). Because citrin-deficient liver (CDL) is subject to HCC, it represents a unique metabolic model to highlight the mechanisms of HCC promotion, offering different angles of study than the classical metabolic syndrome/obesity/non-alcoholic fatty liver disease (NAFLD)/HCC study axis. In turn, the metabolic features of HCC could shed light on the pathogenesis of CDL. Among these, HCC-induced re-activation of aralar-1 (aspartate-glutamate carrier 2), physiologically not expressed in the adult liver, might take place in CDL, so gene redundancy for mitochondrial aspartate-glutamate carriers would be exploited by the CDL. This proposed (aralar-1 re-activation) and known (citrate/malate cycle) adaptive mechanisms may substitute for the impaired function in CD and are consistent with the clinical remission stage of CD and CD improvement by medium-chain triglycerides (MCT). However, these metabolic adaptive benefits could also promote HCC development. In CD, as a result of PPARα down-regulation, liver mitochondrial fatty acid-derived acetyl-CoA would, like glucose-derived acetyl-CoA, be used for lipid anabolism and fuel nuclear acetylation events which might trigger aralar-1 re-activation as seen in non-CD HCC. A brief account of these metabolic events which might lead to aralar-1 re-activation in CDL is here given. Consistency of this account for CDL events further relies on the protective roles of PPARα and inhibition of mitochondrial and plasma membrane citrate transporters in non-CD HCC. • SLC25A13 (citrin) and SLC25A12 (aralar-1) are the two aspartate-glutamate carriers. • Patients with citrin deficiency (CD) are treated with medium-chain triglycerides (MCT). • CD is a cause of hepatocellular carcinoma (HCC) and aralar-1 is re-activated in non-CD HCC. • This re-activation might contribute to CD remission stage and disease improvement by MCT. • Whether MCT promote nuclear acetylations, aralar-1 re-activation, and HCC in CD remains unclear. [ABSTRACT FROM AUTHOR]

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Citrin deficiency caused by SLC25A13 genetic mutations is an autosomal recessive disease, and four prevalent mutations including c.851_854del, c.1638_1660dup, IVS6+5G>A, and IVS16ins3kb make up >80% of total pathogenic mutations within the Chinese population. However, suitable assays for detection of these mutations have not yet been developed for use in routine clinical practice. In the current study, a real-time PCR-based multicolor melting curve analysis (MMCA) was developed to detect the four prevalent mutations in one closed-tube reaction. The analytical and clinical performances were evaluated using artificial templates and clinical samples. All four mutations in the test samples were accurately genotyped via their labeling fluorophores and Tm values, and the standard deviations of Tm values were indicated to be

Objective: To determine the prevalence of citrin protein deficiency in neonates in Shijiazhuang, and further analyze the mutations of pathogenic genes. Methods: A total of 160 061 newborns in Shijiazhuang from January 2014 to December 2019 were detected for blood amino acid levels by tandem mass spectrometry (MS/ MS), and the genetic testing were performed for suspected infants. Results: Among the 160 061 newborns, 20 cases were screened as positive cases, including 1 case confirmed the diagnosis with SLC25A13 gene testing; and 1 case that was false negative in newborn screening. The latter child patient was admitted to the hospital due to yellowish skin aggravated in less than 1 month. The blood tandem mass spectrometry showed that citrulline and methionine were elevated. The results of urine organic acids showed that 4 -hydroxyphenyllactic acid and 4 - hydroxyphenylpyruvic acid were both significantly increased. The prevalence of citrin protein deficiency in Shijiazhuang is 2/160 061. The gene testing showed that one case was a compound heterozygous mutation of SLC25A13 gene. The mutation sites were c.1021+1G>A and c.851_854delTATG. There was no report of c.1021+ 1G>A in the human gene mutation database. Conclusions: The application of tandem mass spectrometry in neonatal disease screening can detect the citrin protein deficiency as early as possible. Some newborn patients may be false negative screening; the gene testing is helpful for the diagnosis of citrin protein deficiency. The prevalence of citrin deficiency in Shijiazhuang area is 2/160 061. In this paper, we reported a novel mutation site of SLC25A13 gene. [ABSTRACT FROM AUTHOR]

Urea cycle disorders (UCDs) are a group of rare metabolic conditions characterized by hyperammonemia and a broad spectrum of phenotypic severity. They are caused by the congenital deficiency in the eight biomolecules involved in urea cycle. In the present study, five cases of UCD were recruited and submitted to a series of clinical, biochemical, and genetic analysis with a combination of high throughput techniques. Moreover, in silico analysis was conducted on the identified missense genetic variants. Various clinical and biochemical indications (including profiles of amino acids and urinary orotic acids) of UCD were manifested by the five probands. Sequence analysis revealed nine diagnostic variants, including three novel ones, which caused Argininosuccinic aciduria (ASA) in one case, Carbamoyl phosphate synthetase 1deficiency (CPS1D) in two cases, Ornithine transcarbamylase deficiency (OTCD) in one case, and Citrin deficiency in 1case. Results of in silico biophysical analysis strongly suggested the pathogenicity of each the five missense variants and provided insight into their intramolecular impacts. In conclusion, this study expanded the genetic variation spectrum of UCD, gave solid evidence for counselling to the affected families, and should facilitate the functional study on the proteins in urea cycle. [ABSTRACT FROM AUTHOR]

Kiyoshi Hayasaka,1,2 Chikahiko Numakura1 1Department of Pediatrics, Yamagata University School of Medicine, Yamagata, Japan; 2Department of Pediatrics, Miyukikai Hospital, Kaminoyama, Japan Abstract: Citrin deficiency is a recessively inherited metabolic disorder with age-dependent clinical manifestations. It causes neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). Patients with NICCD present with intrahepatic cholestasis in the neonatal period and usually respond to the treatment with medium-chain triglyceride (MCT) supplement and lactose-restricted formula. In adulthood, CTLN2 develops in

Citrin deficiency is a hereditary disorder caused by SLC25A13 mutations and manifests as neonatal intrahepatic cholestasis (NICCD), failure to thrive and dyslipidemia (FTTDCD), and adult‐onset type II citrullinemia (CTLN2). Citrin is a component of the malate‐aspartate nicotinamide adenine dinucleotide hydrogen (NADH) shuttle, an essential shuttle for hepatic glycolysis. Hepatic glycolysis and the coupled lipogenesis are impaired in citrin deficiency. Hepatic lipogenesis plays a significant role in fat supply during growth spurt periods: the fetal period, infancy, and puberty. Growth impairment in these periods is characteristic of citrin deficiency. Hepatocytes with citrin deficiency cannot use glucose and fatty acids as energy sources due to defects in the NADH shuttle and downregulation of peroxisome proliferator‐activated receptor α (PPARα), respectively. An energy deficit in hepatocytes is considered a fundamental pathogenesis of citrin deficiency. Medium‐chain triglyceride (MCT) supplementation with a lactose‐restricted formula and MCT supplementation under a low‐carbohydrate diet are recommended for NICCD and CTLN2, respectively. MCT supplementation therapy can provide energy to hepatocytes, promote lipogenesis, correct the cytosolic NAD+/NADH ratio via the malate‐citrate shuttle and improve ammonia detoxification, and it is a reasonable therapy for citrin deficiency. It is very important to administer MCT at a dose equivalent to the liver's energy requirements in divided doses with meals. MCT supplementation therapy is certainly promising for promoting growth spurts during infancy and adolescence and for preventing CTLN2 onset. Intravenous administration of solutions containing fructose is contraindicated, and persistent hyperglycemia should be avoided due to glucose intoxication for patients receiving hyperalimentation or with complicating diabetes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sodium taurocholate cotransporting polypeptide (NTCP), a carrier protein encoded by the gene SLC10A1, is expressed in the basolateral membrane of the hepatocyte to uptake bile acids from plasma. As a new inborn error of bile acid metabolism, NTCP deficiency remains far from being well understood in terms of the clinical and molecular features. Citrin deficiency is a well-known autosomal recessive disease arising from SLC25A13 mutations, and in neonates or infants, this condition presents as transient intrahepatic cholestasis which usually resolves before 1 year of age. All the three patients in this paper exhibited cholestatic jaundice and elevated total bile acids in their early infancy, which were attributed to citrin deficiency by SLC25A13 genetic analysis. In response to feeding with lactose-free and medium-chain triglycerides-enrich formula, their clinical and laboratory presentations disappeared gradually while the hypercholanemia persisted, even beyond 1 year of age. On subsequent SLC10A1 analysis, they were all homozygous for the well-known pathogenic variant c.800C > T (p.Ser267Phe), and NTCP deficiency was thus definitely diagnosed. The findings in this paper indicated that NTCP deficiency could be covered up by citrin deficiency during early infancy; however, in citrin-deficient patients with intractable hypercholanemia following resolved cholestatic jaundice, NTCP deficiency should be taken into consideration.

Citrin deficiency initially presents as neonatal intrahepatic cholestasis (NICCD) and often resolves within first year of infancy. Failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD) has been recently proposed as a novel post-NICCD phenotype and its clinical features are still being established. Herein, we encountered a 2-year-old girl who was hospitalized for intermittent fever lasting 10 days. Besides pneumonia, we observed an NICCD-like phenotype with the presence of liver dysfunction, dyslipidemia, aminoacidemia, organic academia, and extremely high levels of alpha-fetoprotein (AFP). Genetic testing confirmed the diagnosis of citrin deficiency and, liver histology revealed she had already developed cirrhosis. Although, improvement of biochemical parameters and liver histology were observed after treatment that included dietary restrictions and symptomatic treatments, AFP levels remained elevated (>400 ng/ml) during a 3-year follow-up period. Moreover, liver magnetic resonance imaging (MRI) examination performed on the patient at age 5 revealed the development of multiple liver nodules with diffusion restriction on diffusion-weighted imaging (DWI). These observations highly indicate the possibility of hepatocellular carcinoma (HCC). Thus, this case reveals that an NICCD-like phenotype complicated with cirrhosis can exist during FTTDCD stage without any prior signs. It also emphasizes the necessity of monitoring AFP levels during follow-up for citrin deficiency patients with persistently high AFP level after treatment as FTTDCD may progress to HCC. Individualized treatment strategy for patients with FTTDCD also need to be explored.

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. [ABSTRACT FROM AUTHOR]

Sodium taurocholate cotransporting polypeptide (NTCP), a carrier protein encoded by the gene SLC10A1 , is expressed in the basolateral membrane of the hepatocyte to uptake bile acids from plasma. As a new inborn error of bile acid metabolism, NTCP deficiency remains far from being well understood in terms of the clinical and molecular features. Citrin deficiency is a well-known autosomal recessive disease arising from SLC25A13 mutations, and in neonates or infants, this condition presents as transient intrahepatic cholestasis which usually resolves before 1 year of age. All the three patients in this paper exhibited cholestatic jaundice and elevated total bile acids in their early infancy, which were attributed to citrin deficiency by SLC25A13 genetic analysis. In response to feeding with lactose-free and medium-chain triglycerides-enrich formula, their clinical and laboratory presentations disappeared gradually while the hypercholanemia persisted, even beyond 1 year of age. On subsequent SLC10A1 analysis, they were all homozygous for the well-known pathogenic variant c.800C > T (p.Ser267Phe), and NTCP deficiency was thus definitely diagnosed. The findings in this paper indicated that NTCP deficiency could be covered up by citrin deficiency during early infancy; however, in citrin-deficient patients with intractable hypercholanemia following resolved cholestatic jaundice, NTCP deficiency should be taken into consideration. [ABSTRACT FROM AUTHOR]

Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD), failure to thrive and dyslipidemia (FTTDCD), and adult‐onset type II citrullinemia (CTLN2). Owing to a defect in the NADH‐shuttle, citrin deficiency impairs hepatic glycolysis and de novo lipogenesis leading to hepatic energy deficit. To investigate the physiological role of citrin, we studied the growth of 111 NICCD‐affected subjects (51 males and 60 females) and 12 NICCD‐unaffected subjects (five males and seven females), including the body weight, height, and genotype. We constructed growth charts using the lambda‐mu‐sigma (LMS) method. The NICCD‐affected subjects showed statistically significant growth impairment, including low birth weight and length, low body weight until 6 to 9 months of age, low height until 11 to 13 years of age, and low body weight in 7 to 12‐year‐old males and 8‐year‐old females. NICCD‐unaffected subjects showed similar growth impairment, including low birth weight and height, and growth impairment during adolescence. In the third trimester, de novo lipogenesis is required for deposition of body fat and myelination of the developing central nervous system, and its impairment likely causes low birth weight and length. The growth rate is the highest during the first 6 months of life and slows down after 6 months of age, which is probably associated with the onset and recovery of NICCD. Adolescence is the second catch‐up growth period, and the proportion and distribution of body fat change depending on age and sex. Characteristic growth impairment in citrin deficiency suggests a significant role of citrin in the catch‐up growth via lipogenesis. [ABSTRACT FROM AUTHOR]

Abstract The human SLC25A13 gene encodes the liver type aspartate/glutamate carrier isoform 2 (AGC2, commonly named as citrin), which plays a key role in the main NADH-shuttle of human hepatocyte. Biallelic SLC25A13 mutations result in Citrin deficiency (CD). In order to identify the important regulatory region of SLC25A13 gene and elucidate the way how potential promoter mutations affect the citrin expression, we performed promoter deletion analysis and established the reporter constructs of luciferase gene-carrying SLC25A13 promoter containing several mutations located in putative transcription factor-binding sites. The luciferase activities of all promoter constructs were measured using a Dual-Luciferase Reporter Assay System. Bioinformatic analysis showed that the promoter of SLC25A13 gene lacks TATA box and obviously typical initiator element, but contains a CCAAT box and two GC box. Promoter deletion analysis confirmed the region from −221 to −1 upstream ATG was essential for SLC25A13 to maintain the promoter activity. We utilized dual-luciferase reporter system as function analytical model to tentatively assess the effect of artificially constructed promoter mutations on citrin expression, and our analysis revealed that mutated putative CCAAT box and GC box could significantly affect the citrin expression. Our study confirmed the important SLC25A13 promoter regions that influenced citrin expression in HL7702 cells, and constructed a function analytical model. This work may be useful to further identify the pathogenic mutations leading to CD in the promoter region. Highlights • Human SLC25A13 gene promoter is GC-rich and TATA-less with a dispersed transcription pattern. • A GC box and a CCAAT box were predicted in the region upstream of the translational start site. • The region from −221 to −1 upstream of translation initiation codon was essential for SLC25A13 transcription. • A function analytical model of SLC25A13 gene promoter was established. [ABSTRACT FROM AUTHOR]

Here, we report SLC25A13 c.1610_1612delinsAT mutation from India in a 13-year old boy who presented with recurrent episodes of delirium and hyperammonemia. This is the second case with this mutation; the first case was of Pakistani origin. The boy responded to diet modification, sodium benzoate and arginine supplementation. Furthermore, we have aimed to establish genotype-phenotype correlation of 79 cases of citrin deficiency (46 males and 33 females) reported in 24 studies from all over the world. Inverse association was observed between age of onset and jaundice ( r  = −0.73). Late age of onset was associated with delirium ( r  = 0.61), aggressive behaviour ( r  = 0.67), altered sensorium ( r  = 0.67) and tremors ( r  = 0.65). The most common mutations associated with citrin deficiency were c.851_854del4, IVS16ins3kb, 1638-1660dup with a frequency of 42.41%, 16.46% and 6.33%, respectively. The c.851_854del4 mutation showed positive association with alpha feto protein ( r  = 0.40), ammonia ( r  = 0.50) and tyrosine ( r  = 0.40) while showing inverse association with threonine ( r  = −0.55). The IVS16ins3kb mutation was associated with high total ( r  = 0.65) and conjugated bilirubin ( r  = 0.54) along with high aspartate transaminase ( r  = 0.49) while citrulline levels are lower ( r  = −0.36). To conclude, all cases of intrahepatic cholestasis and neuropsychiatric abnormalities should be evaluated for citrin deficiency. However, the ethnic group-specific mutation frequencies should be considered in implementing screening. [ABSTRACT FROM AUTHOR]

Citrin is a liver-specific mitochondrial aspartate−glutamate carrier encoded by SLC25A13. Citrin deficiency caused by SLC25A13 mutation results in carbohydrate toxicity, citrullinemia type II, and fatty liver diseases, the mechanisms of some of which remain unknown. Citrin shows a functional homolog in yeast aspartate-glutamate carrier (Agc1p) and agc1Δ yeasts are used as a model organism of citrin deficiency. Here, we found that agc1Δ yeasts decreased fat utilization, impaired NADH balance in peroxisomes, and decreased chronological lifespan. The activation of GPD1-mediated NAD+ regeneration in peroxisomes by GPD1 over-expression or activation of the malate−oxaloacetate NADH peroxisomal shuttle, by increasing flux in this NADH shuttle and over-expression of MDH3, resulted in lifespan extension of agc1Δ yeasts. In addition, over-expression of PEX34 restored longevity of agc1Δ yeasts as well as wild-type cells. The effect of PEX34-mediated longevity required the presence of the GPD1-mediated NADH peroxisomal shuttle, which was independent of the presence of the peroxisomal malate−oxaloacetate NADH shuttle and PEX34-induced peroxisome proliferation. These data confirm that impaired NAD+ regeneration in peroxisomes is a key defect in the yeast model of citrin deficiency, and enhancing peroxisome function or inducing NAD+ regeneration in peroxisomes is suggested for further study in patients’ hepatocytes.

Background: Citrin, encoded by SLC25A13, is a component of the malate-aspartate shuttle, which is the main NADH-transporting system in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD), which usually resolves within the first year of life. However, small numbers of adults with citrin deficiency develop hyperammonemic encephalopathy, adult-onset type II citrullinemia (CTLN2), which leads to death due to cerebral edema. Liver transplantation is the only definitive therapy for patients with CTLN2. We previously reported that a lactose (galactose)-restricted and medium-chain triglyceride (MCT)-supplemented formula is notably effective for patients with NICCD. Citrin deficiency may impair the glycolysis in hepatocytes because of an increase in the cytosolic NADH/NAD+ ratio, leading to an energy shortage. MCT administration can provide energy to hepatocytes and was expected to have a good effect on CTLN2. Methods: An MCT supplementation therapy under a low-carbohydrate formula was administered to five patients with CTLN2. Four of the patients had episodes of hyperammonemic encephalopathy, and one patient had postprandial hyperammonemia with no symptoms. Results: One of the patients displaying hyperammonemic encephalopathy completely recovered with all normal laboratory findings. Others notably improved in terms of clinical and or laboratory findings with no hyperammonemic symptoms; however, the patients displayed persistent mild citrullinemia and occasionally had postprandial mild hyperammonemia most likely due to an irreversible change in the liver. Conclusions: An MCT supplement can provide energy to hepatocytes and promote hepatic lipogenesis, leading to a reduction in the cytosolic NADH/NAD+ ratio. MCT supplementation under a low-carbohydrate formula could be a promising therapy for CTLN2 and should also be used to prevent CTLN2 to avoid irreversible liver damage.

Citrin deficiency is an autosomal recessive metabolic disorder, which is caused by pathogenic mutations in the SLC25A13 gene on chromosome 7q21.3, as the causative gene that encodes the liver type aspartate/glutamate carrier isoform 2 (AGC2). One of the main clinical presentations is neonatal intrahepatic cholestatic hepatitis caused by citrin deficiency. We report a Turkish child presented with prolonged neonatal jaundice associated with elevated plasma citrulline and galactosuria. NICCD was suspected at this point and mutation study of SLC25A13 showed that she was homozygous for the missense NM_014251.2:c.1354G>A (NP_055066.1:p. Val452Ile) (dbSNP: rs143877538) mutation. Dramatic response was observed to the dietary treatment with medium-chain triglycerides containing formula, ursodeoxycholic acid and fat-soluble vitamin supplementation. The minor allele frequency of this variant was given as nearly as 0.01 in the South Asian population; it seems like a disease causing variant. This is the first report of this variant in the Turkish and European population. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Citrin deficiency causes adult-onset type II citrullinemia (CTLN-2), which later manifests as severe liver steatosis and life-threatening encephalopathy. Long-standing energy deficit of the liver and brain may predispose ones to CTLN-2. Here, we compared the energy-driving tricarboxylic acid (TCA) cycle and fatty acid β-oxidation cycle between 22 citrin-deficient children (age, 3–13 years) with normal liver functions and 37 healthy controls (age, 5–13 years). TCA cycle analysis showed that basal plasma citrate and α-ketoglutarate levels were significantly higher in the affected than the control group (p < 0.01). Conversely, basal plasma fumarate and malate levels were significantly lower than those for the control (p < 0.001). The plasma level of 3-OH-butyrate derived from fatty acid β-oxidation was significantly higher in the affected group (p < 0.01). Ten patients underwent sodium pyruvate therapy. However, this therapy did not correct or attenuate such deviations in both cycles. Sodium pyruvate therapy significantly increased fasting insulin secretion (p < 0.01); the fasting sugar level remained unchanged. Our results suggest that citrin-deficient children show considerable deviations of TCA cycle metabolite profiles that are resistant to sodium pyruvate treatment. Thus, long-standing and considerable TCA cycle dysfunction might be a pivotal metabolic background of CTLN-2 development. [ABSTRACT FROM AUTHOR]

Objective: To investigate the clinical phenotype and gene variation conditions in neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), so as to provide a basis for genetic counseling and clinical diagnosis and treatment of the family. Methods: 11 cases of neonatal intrahepatic cholestasis who visited the Children's Hospital Affiliated to Zhengzhou University between February 2019 and March 2021 were selected as the study subjects. High-throughput sequencing technology was used to detect the gene variation condition in 11 neonatal patients and 100 normal control neonates. The suspicious loci and family members were verified by Sanger sequencing and QPCR technology. Results: All 11 children with NICCD had different degrees of jaundice and liver damage symptoms, combined with coagulation dysfunction and anemia ( n = 7), cardiac malformation ( n = 2), elevated myocardial enzymes ( n = 4), hyperlipidemia ( n = 1), hyperkalemia ( n = 1), persistent diarrhea ( n = 3), developmental delay ( n = 1). A total of 10 different types of SLC25A13 gene mutations were detected in 11 cases, including three frameshift mutations, two splicing changes, two missense mutations, one intron insertion, one nonsense mutation, and one heterozygous deletion. After reviewing literature and databases, c.1878delG(p.I627Sfs*73) and exon11 deletion were novel mutations that had not been reported at home or abroad. Conclusion: The clinical features of NICCD are non-specific, and genetic testing aids in the early and accurate diagnosis of the disease, providing an important basis for clinical treatment and genetic counseling for family members. In addition, the detection of novel mutation sites has enriched the SLC25A13 gene variation spectrum.

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 well-recognized 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 high-frequency 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 paternally-inherited mutation, c.851_854del4, and a maternally-inherited 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 galactose-free and medium-chain triglyceride-enriched 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. [ABSTRACT FROM AUTHOR]

Citrin deficiency, an inherited defect of the liver-type mitochondrial aspartate/glutamate carrier isoform (citrin), may cause impairment of glycolysis because of an increase in the cytosolic NADH/NAD+ ratio. We report a Japanese boy whose main complaint was recurrent hypoglycemic episodes. He was suspected as having citrin deficiency because of his peculiar preference for protein- and fat-rich food. His young sister also had a similar food preference. Both siblings were diagnosed with citrin deficiency by genetic analysis. The brother and sister underwent an oral glucose tolerance test (OGTT) at 10 and 7 yr of age, respectively. Blood glucose, ammonia, lactic acid, pyruvic acid, and insulin levels were monitored before starting the test, and then every 30 min. During this test, they maintained blood glucose levels until 180 min. At 210 min, they experienced vomiting, feeling ill, and decreased blood glucose levels (2.9 and 2.8 mmol/l in the brother and sister, respectively). The sister and brother recovered uneventfully by intravenous glucose injection. In a second OGTT, 4 months after medium-chain triglyceride (MCT) oil supplementation, they had no major symptoms and normal glucose levels were maintained, even after 240 min. Additionally, after MCT oil therapy, their food preference slightly changed as they started eating more carbohydrates. Our OGTT data suggest excess carbohydrate intake has adverse consequences in patients with citrin deficiency, including hypoglycemia after a few hours. MCT oil therapy may be effective in preventing such hypoglycemia and improving metabolic derangement, even during the so-called apparently healthy period. [ABSTRACT FROM AUTHOR]