Unveiling genetic variants: Tetra-primer ARMS-PCR diagnosis and structural insights into BLAD, BC, and DUMPS in Pakistani cattle herds.

BACKGROUND: Bovine leukocyte adhesion deficiency (BLAD), bovine citrullinemia (BC), and deficiency of Uridine monophosphate synthetase (DUMPS) are the common autosomal recessive disorders affecting the global dairy industry. BLAD leads to poor wound healing and recurrent infections. In BC, ammonia builds up leading to neurological disorders and death. DUMPS results in developmental abnormalities. METHODOLOGY: In this study, tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) based diagnostic tests were optimized for BLAD, BC, and DUMPS. A total of 250 animals (58 indigenous and 192 Holstein Friesian (HF)) were screened from all across Pakistan. In addition to validation of ARMS-PCR results through Sanger sequencing, the protein modeling provided structural insights of the disease-associated reported SNPs. Pathway analysis illustrated gene functions under normal and mutated conditions. Furthermore, haplotype and phylogenetic analysis of ASS1 (Argininosuccinate synthetase) gene were performed on study samples and NCBI retrieved sequences. RESULTS: The study's focus was to screen the herds for prevalence of carriers of genetic disorders, as they are the main source of disease dissemination. One animal was found carrier for BC, whereas no carriers were found for BLAD and DUMPS. The protein models corroborated the reported amino acid change in BLAD, and protein truncation in both BC and DUMPS proteins. SNPs found in NCBI retrieved sequences were either silent or missense and had no effect on protein structure. DNA network presented graphical illustration of haplotype interactions and phylogenetic analysis conferred evolutionary landscape of ASS1 gene. The combination of these approaches produced an in-depth genetic picture of BC in Pakistani cattle. CONCLUSION: The development of diagnostic tests and identification of the heterozygous BC sample underscores the significance of constant monitoring to avoid the unwanted dissemination of mutant alleles among Pakistani cattle, thereby promoting the general well-being and sustainability of the dairy sector.

Using preimplantation genetic testing for monogenic disease for preventing citrullinemia type 1 transmission.

Aim: The aim of this study is to investigate if Preimplantation Genetic Testing (PGT) can effectively identify unreported variants according to American College of Medical Genetics and Genomics (ACMG)to prevent citrullinemia type 1 affection. Design: This study involves a detailed case analysis of a family with history of citrullinemia type 1, focusing on the use of PGT for monogenic diseases (PGT-M). The genetic variants were identified using ACMG guidelines, and PGT was employed to prevent the inheritance of these variants. The study included haplotype analysis and Sanger sequencing to confirm the results. Results: The study identified previously unreported variations in the ASS1 gene causing citrullinemia type 1. PGT successfully prevented the transmission of these variants, resulting in the birth of a healthy fetus. However, challenges such as allele dropout (ADO) and gene recombination were encountered during haplotype analysis, which could potentially defeat the diagnosis. The study demonstrated that combining haplotype analysis with Sanger sequencing can enhance the accuracy of PGT. Conclusion: Preimplantation Genetic Testing (PGT) targeting likely pathogenic and pathogenic variants in the ASS1 gene, as rated by ACMG, allows the birth of healthy infants free from citrullinemia type 1. Additionally, the establishment of single haplotypes and Sanger sequencing can reduce the misdiagnosis rate caused by allele dropout (ADO) and genetic recombination.

Citrin deficiency-The East-side story.

Citrin deficiency (CD) is a complex metabolic condition due to defects in SLC25A13 encoding citrin, an aspartate/glutamate carrier located in the mitochondrial inner membrane. The condition was first described in Japan and other East Asian countries in patients who were thought to suffer from classical citrullinemia type 1, and was therefore classified as a urea cycle disorder. With an improved understanding of its molecular basis, it became apparent that a defect of citrin is primarily affecting the malate-aspartate shuttle with however multiple secondary effects on many central metabolic pathways including glycolysis, gluconeogenesis, de novo lipogenesis and ureagenesis. In the meantime, it became also clear that CD must be considered as a global disease with patients identified in many parts of the world and affected by SLC25A13 genotypes different from those known in East Asian populations. The present short review summarizes the (hi)story of this complex metabolic condition and tries to explain the relevance of including CD as a differential diagnosis in neonates and infants with cholestasis and in (not only adult) patients with hyperammonemia of unknown origin with subsequent impact on the emergency management.

Pseudodendritic keratitis in citrullinemia; a report of an unusual and novel ocular finding in this metabolic disorder.

Purpose: To report a 15 year old girl with citrullinemia type 1 and 2 accompanied by neurologic signs and symptoms and a novel ocular complaint in cornea like tyrosinemia type 2. Observations: A 15 year old female was admitted with decreased consciousness and neurologic signs and symptoms. Citrulinemia was discovered through metabolic testing. Later genetic studies revealed mutations in both ASS1 and SLC25A13 genes. Two years after the first presentation, the patient was re-admitted with complaints of bilateral photophobia and tearing. Biomicroscopic examination revealed bilateral corneal haziness with pseudodendritic lesions like tyrosinemia type 2 that were subsided with protein restriction and the use of urea cycle disease (UCD) formula. Conclusions and importance: Citrullinemia is the inherited autosomal recessive disorder of urea cycle that leads to ammonia and accumulation of other toxic substances in the blood. Two types of Citrullinemia have been defined. Citrullinemia type 1, caused by deficiency or reduction in argininosuccinate synthetase enzyme activity due to damaging mutation in ASS1 gene. Citrullinemia type 2 as another subtype is caused by the absence or dysfunction of the mitochondrial membrane carrier protein (SLC25A13), also called CITRIN. Pseudodendritic keratitis is a rare condition that may be seen with tyrosinemia type 2. The association of this ocular complaint with citrullinemia has not been described previously. Awareness of this phenomenon may improve the diagnosis and management of citrullinemia patients.

Harnessing Next-Generation Sequencing as a Timely and Accurate Second-Tier Screening Test for Newborn Screening of Inborn Errors of Metabolism.

In this study, we evaluated the implementation of a second-tier genetic screening test using an amplicon-based next-generation sequencing (NGS) panel in our laboratory during the period of 1 September 2021 to 31 August 2022 for the newborn screening (NBS) of six conditions for inborn errors of metabolism: citrullinemia type II (MIM #605814), systemic primary carnitine deficiency (MIM #212140), glutaric acidemia type I (MIM #231670), beta-ketothiolase deficiency (#203750), holocarboxylase synthetase deficiency (MIM #253270) and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (MIM # 246450). The custom-designed NGS panel can detect sequence variants in the relevant genes and also specifically screen for the presence of the hotspot variant IVS16ins3kb of SLC25A13 by the copy number variant calling algorithm. Genetic second-tier tests were performed for 1.8% of a total of 22,883 NBS samples. The false positive rate for these six conditions after the NGS second-tier test was only 0.017%, and two cases of citrullinemia type II would have been missed as false negatives if only biochemical first-tier testing was performed. The confirmed true positive cases were citrullinemia type II (n = 2) and systemic primary carnitine deficiency (n = 1). The false positives were later confirmed to be carrier of citrullinemia type II (n = 2), carrier of glutaric acidemia type I (n = 1) and carrier of systemic primary carnitine deficiency (n = 1). There were no false negatives reported. The incorporation of a second-tier genetic screening test by NGS greatly enhanced our program's performance with 5-working days turn-around time maintained as before. In addition, early genetic information is available at the time of recall to facilitate better clinical management and genetic counseling.

Update on the diagnosis and management of neonatal intrahepatic cholestasis caused by citrin deficiency: Expert review on behalf of the Asian Pan-Pacific Society for Pediatric Gastroenterology, Hepatology, and Nutrition.

Citrin deficiency is an autosomal recessive metabolic liver disease caused by mutations in the SLC25A13 gene. The disease typically presents with cholestasis, elevated liver enzymes, hyperammonemia, hypercitrullinemia, and fatty liver in young infants, resulting in a phenotype known as "neonatal intrahepatic cholestasis caused by citrin deficiency" (NICCD). The diagnosis relies on clinical manifestation, biochemical evidence of hypercitrullinemia, and identifying mutations in the SLC25A13 gene. Several common mutations have been found in patients of East Asian background. The mainstay treatment is nutritional therapy in early infancy utilizing a lactose-free and medium-chain triglyceride formula. This approach leads to the majority of patients recovering liver function by 1 year of age. Some patients may remain asymptomatic or undiagnosed, but a small proportion of cases can progress to cirrhosis and liver failure, necessitating liver transplantation. Recently, advancements in newborn screening methods have improved the age of diagnosis. Early diagnosis and timely management improve patient outcomes. Further studies are needed to elucidate the long-term follow-up of NICCD patients into adolescence and adulthood.

Adult-onset Type II Citrullinemia Developed under Dietary Restrictions during Imprisonment.

A 29-year-old man presented with liver damage, and a liver biopsy was performed, but the cause was unclear. Thereafter, he was referred to our hospital. We found that he had been unable to consume carbohydrates in his diet and preferred fried chicken since childhood. In addition, he had shown disturbance of consciousness and abnormal behavior while he had been in prison, where dietary intake had been restricted. A plasma amino acid analysis revealed hypercitrullinemia. Therefore, we suspected adult-onset type II citrullinemia (CTLN2). Genetic testing showed pathologic variations in the SLC25A13 gene, which allowed us to make a definite diagnosis of CTLN2.

Severity-adjusted evaluation of liver transplantation on health outcomes in urea cycle disorders.

PURPOSE: Liver transplantation (LTx) is performed in individuals with urea cycle disorders when medical management (MM) insufficiently prevents the occurrence of hyperammonemic events. However, there is a paucity of systematic analyses on the effects of LTx on health-related outcome parameters compared to individuals with comparable severity who are medically managed. METHODS: We investigated the effects of LTx and MM on validated health-related outcome parameters, including the metabolic disease course, linear growth, and neurocognitive outcomes. Individuals were stratified into "severe" and "attenuated" categories based on the genotype-specific and validated in vitro enzyme activity. RESULTS: LTx enabled metabolic stability by prevention of further hyperammonemic events after transplantation and was associated with a more favorable growth outcome compared with individuals remaining under MM. However, neurocognitive outcome in individuals with LTx did not differ from the medically managed counterparts as reflected by the frequency of motor abnormality and cognitive standard deviation score at last observation. CONCLUSION: Whereas LTx enabled metabolic stability without further need of protein restriction or nitrogen-scavenging therapy and was associated with a more favorable growth outcome, LTx-as currently performed-was not associated with improved neurocognitive outcomes compared with long-term MM in the investigated urea cycle disorders.

ASS1 deficiency is associated with impaired neuronal differentiation in zebrafish larvae.

Citrullinemia type 1 (CTLN1) is a rare autosomal recessive urea cycle disorder caused by deficiency of the cytosolic enzyme argininosuccinate synthetase 1 (ASS1) due to pathogenic variants in the ASS1 gene located on chromosome 9q34.11. Even though hyperammenomia is considered the major pathomechanistic factor for neurological impairment and cognitive dysfunction, a relevant subset of individuals presents with a neurodegenerative course in the absence of hyperammonemic decompensations. Here we show, that ASS1 deficiency induced by antisense-mediated knockdown of the zebrafish ASS1 homologue is associated with defective neuronal differentiation ultimately causing neuronal cell loss and consecutively decreased brain size in zebrafish larvae in vivo. Whereas ASS1-deficient zebrafish larvae are characterized by markedly elevated concentrations of citrulline - the biochemical hallmark of CTLN1, accumulation of L-citrulline, hyperammonemia or therewith associated secondary metabolic alterations did not account for the observed phenotype. Intriguingly, coinjection of the human ASS1 mRNA not only normalized citrulline concentration but also reversed the morphological cerebral phenotype and restored brain size, confirming conserved functional properties of ASS1 across species. The results of the present study imply a novel, potentially non-enzymatic (moonlighting) function of the ASS1 protein in neurodevelopment.

The management and clinical outcomes of pregnancies in women with urea cycle disorders: A review of the literature and results of an international survey.

An increasing number of women with urea cycle disorders (UCDs) are reaching child-bearing age and becoming pregnant. Improved diagnostics and increased awareness of inherited metabolic diseases has also led to more previously undetected women being diagnosed with a UCD during or shortly after pregnancy. Pregnancy increases the risk of acute metabolic decompensation with hyperammonemia-which can occur in any trimester, and/or the postpartum period, and may lead to encephalopathy, psychosis, coma, and even death, if not diagnosed promptly and treated appropriately. There are also (theoretical) concerns that a maternal UCD, or its treatment, may cause potential risks for the unborn child. Currently evidence on management and outcome of pregnancies in UCDs is limited to case reports and there are no clear guidelines. In order to inform management and investigate outcomes of pregnancies in women with a UCD, we performed a retrospective review of published cases and analyzed data collected from an international online survey. We conclude that, although risk during the intra- and postpartum period exists, multidisciplinary management by an experienced team and a prospective plan usually result in successful pregnancy, labor, delivery, and postpartum period. No deaths were reported in mothers managed accordingly. With the exception of male neonates with Ornithine Transcarbamylase deficiency, the clinical outcome of children born to mothers with UCDs appears positive, although follow-up is limited. The outcome for women presenting with a first acute metabolic decompensation during pregnancy or postpartum is less favorable. Deaths were associated with diagnostic delay/late management of hyperammonemia in previously undiagnosed women.

Case report: Diagnosis of ADCY5-related dyskinesia explaining the entire phenotype in a patient with atypical citrullinemia type I.

In this case study, we report the case of a 13-year-old girl with citrullinemia type 1 (MIM #215700), an autosomal recessive inherited disorder of the urea cycle, which was confirmed by the identification of a homozygous pathogenic variant in the argininosuccinate synthetase 1 (ASS1) gene. However, the patient presented abnormal hyperkinetic movements with global developmental delay and clinical signs that were not fully consistent with those of citrullinemia type 1 or with those of her siblings with isolated citrullinemia type 1. Exome sequencing showed the presence of a de novo heterozygous pathogenic variant in the adenylate cyclase type 5 (ADCY5) gene. The variant confirmed the overlap with the so-called ADCY5-related dyskinesia with orofacial involvement, which is autosomal dominant (MIM #606703), a disorder disrupting the enzymatic conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). In addition to the citrullinemia-related low-protein diet and arginine supplementation, the identification of this second disease led to the introduction of a treatment with caffeine, which considerably improved the dyskinesia neurological picture. In conclusion, this case highlights the importance of clinical-biological confrontation for the interpretation of genetic variants, as one hereditary metabolic disease may hide another with therapeutic consequences. Summary: This article reports the misleading superposition of two inherited metabolic diseases, showing the importance of clinical-biological confrontation in the interpretation of genetic variants.

Adult Onset Episodic Encephalopathy Due to Citrin Deficiency-A Case Report.

Hyperammonemia is a rare cause of adult episodic encephalopathy. Citrin deficiency resulting in citrullinemia type 2 (CTLN2) can lead to recurrent delirium in adults. Here we report a case of adult onset episodic encephalopathy due to citrin deficiency. A 40 years old male presented with one-year history of episodic encephalopathy triggered by high protein and fat diet. He also had chronic pancreatitis and subacute intestinal obstruction which is a novel manifestation of CTLN2. Evaluation showed elevated blood liver enzymes, ammonia, and citrulline. MRI brain showed frontal hyperintensities and bulky basal ganglia which have not been reported. Diagnosis was confirmed by next-generation sequencing which showed a novel variant c. 1591G > A in exon15 of SLC25A13. Hyperammonemic syndromes should be considered in differential diagnosis of episodic encephalopathy in adults. This report shows novel features of subacute intestinal obstruction and MRI findings in CTLN2 expanding spectrum of manifestation.

Pathogenesis and Management of Citrin Deficiency.

Citrin deficiency (CD) is a hereditary disorder caused by SLC25A13 mutations that manifests as neonatal intrahepatic cholestasis caused by CD (NICCD), failure to thrive and dyslipidemia caused by CD (FTTDCD), and adult-onset type 2 citrullinemia (CTLN2). Citrin, an aspartate-glutamate carrier primarily expressed in the liver, is a component of the malate-aspartate shuttle, which is essential for glycolysis. Citrin-deficient hepatocytes have primary defects in glycolysis and de novo lipogenesis and exhibit secondarily downregulated PPARα, leading to impaired ß-oxidation. They are unable to utilize glucose and free fatty acids as energy sources, resulting in energy deficiencies. Medium-chain triglyceride (MCT) supplements are effective for treating CD by providing energy to hepatocytes, increasing lipogenesis, and activating the malate-citrate shuttle. However, patients with CD often exhibit growth impairment and irreversible brain and/or liver damage. To improve the quality of life and prevent irreversible damage, MCT supplementation with a diet containing minimal carbohydrates is recommended promptly after the diagnosis.

Citrullinemia type II accompanied by mental derangement combined with multidrug resistance 3 decrease, case report.

Background: Here we report a rare case of citrullinemia type II (CTLN2) accompanied by mental derangement with a deficiency of multidrug resistance 3 (MDR3) in the liver. Case presentation: The clinical data of a 17-year-old girl were collected. Liver puncture was performed, and hepatic expression of MDR3 was determined by immunohistochemistry. Serum amino acids of the patient and her parents wwere determined by a chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC-MS). Genetic mutations of ABCB4 and SLC25A13 were screened by whole-exome sequencing. Immunohistochemical analysis showed a remarkably lower expression of MDR3. Mutation in ABCB4 gene was not found and whole-exome sequencing revealed the SLC25A13 mutation 852-855 del. Elevated serum levels of citrulline, homocitrulline, and homoarginine in the patient and her mother were found. Conclusions: We reported a rare case of CTLN2 combined with MDR3 deficiency, without mutation of ABCB4. The link between MDR3 down-expression and CTLN2 warrants further investigation. Meanwhile, clinicians need to further rule out the possibility of CTLN2 if MDR3 decreases in adolescent patients with mental disorders and abnormal liver function.

Citrullinemia and What Else?

INTRODUCTION: Citrullinemia type I (CTLN1) is a rare autosomal recessive metabolic disorder. Symptoms typically include vomiting, lethargy, seizures and coma. In neonatal presentation, death occurs in days if untreated. Survivors may evolve with neurocognitive dysfunction. RESULTS/CASE REPORT: Two 10 years old, non-identical, twin sisters (S1; S2) with CTLN1 were born after a 36W gestation: S1 by eutocic delivery and S2 by cesarean section with nuchal cord (Apgar score 5/10). On day four, S2 presented hyperammonemia with coma. S1 had no complications. Diagnosis followed that of S2. Neurocognitive development was monitored at 3 months - 4 years of age with Griffiths Scales: global development quotient kept within the average, but S2 had a deficit in language and eye and hand coordination. At 5 years, the neurocognitive abilities were evaluated using Wechsler Preschool and Primary Scale of Intelligence - Revised (WPPSI-R). S2 revealed difficulties in verbal area (vocabulary, comprehension and memorizing sentences), with a lower average verbal intelligence quotient (IQ). S1 had high average IQ. Due to learning difficulties, S2 was reassessment at 8 years old with Wechsler Intelligence Scale for Children - Third edition (WISC-III): full-scale IQ -"extremely low". CONCLUSION: These non-identical twin sisters share the same citrullinemia type 1 causing variants in the ASS1 gene. Nevertheless, their clinical presentation and neurocognitive evolution are diverse. Other factors, like the different genetic background and perinatal issues such as the type of delivery and its circumstances and the neonatal coma episode of S2 may explain the dissimilar evolution. Maximum ammonium levels (and its duration) are critical for the patients' neurodevelopment: 131 in S1 and 546 umol/l in S2.

Lethality rescue and long-term amelioration of a citrullinemia type I mouse model by neonatal gene-targeting combined to SaCRISPR-Cas9.

Citrullinemia type I is a rare autosomal-recessive disorder caused by deficiency of argininosuccinate synthetase (ASS1). The clinical presentation includes the acute neonatal form, characterized by ammonia and citrulline accumulation in blood, which may lead to encephalopathy, coma, and death, and the milder late-onset form. Current treatments are unsatisfactory, and the only curative treatment is liver transplantation. We permanently modified the hepatocyte genome in lethal citrullinemia mice (Ass1fold/fold) by inserting the ASS1 cDNA into the albumin locus through the delivery of two AAV8 vectors carrying the donor DNA and the CRISPR-Cas9 platform. The neonatal treatment completely rescued mortality ensuring survival up to 5 months of age, with plasma citrulline levels significantly decreased, while plasma ammonia levels remained unchanged. In contrast, neonatal treatment with a liver-directed non-integrative AAV8-AAT-hASS1 vector failed to improve disease parameters. To model late-onset citrullinemia, we dosed postnatal day (P) 30 juvenile animals using the integrative approach, resulting in lifespan improvement and a minor reduction in disease markers. Conversely, treatment with the non-integrative vector completely rescued mortality, reducing plasma ammonia and citrulline to wild-type values. In summary, the integrative approach in neonates is effective, although further improvements are required to fully correct the phenotype. Non-integrative gene therapy application to juvenile mice ensures a stable and very efficient therapeutic effect.

Type 1 citrullinemia patient with Brugada pattern undergoing general anesthesia for dental extractions: A case report.

Key Clinical Message: The perioperative control of ammonia, reduction of stress, and administration of drugs tolerated in type 1 citrullinemia and Brugada pattern allowed the successful and uneventful management of general anesthesia in the study patient. Abstract: The aim of this study was to report the targeted perioperative management of general anesthesia (GA) adopted for dental extractions in a rare patient with type 1 citrullinemia and Brugada pattern. A male, Caucasian, adult type 1 citrullinemia patient needed dental extractions under GA. The medical history showed neurodevelopmental impairment, growth retardation, epilepsy, and a Type 2 Brugada electrocardiographic pattern in the second precordial lead. The authors focused the anesthesiologic protocol on the prevention of hyperammonemia and fatal arrhythmias. Changes in diet and 10% glucose solution administration prevented protein catabolism due to the fasting period (ammonia was 44 µmol/L preoperatively and 46 µmol/L postoperatively; glycemia was 120 g/dL preoperatively and 153 g/dL postoperatively). The patient received a continuous electrocardiogram, noninvasive blood pressure, pulse oximeter, entropy monitoring, train-of-four monitoring, and external biphasic defibrillator pads. Midazolam, remifentanil, and dexamethasone were administered for pre-anesthesia; thiopental and rocuronium for induction; remifentanil and desflurane for maintenance; sugammadex for decurarization. After the intraligamentary injection of lidocaine 2% with epinephrine 1:100,000 for local anesthesia, the patient developed a transient Type 1 Brugada pattern that lasted a few minutes. The whole procedure lasted 30 min. The patient's discharge to ward occurred 3 h after the end of GA. The perioperative management of ammonia, reduction of stress, and administration of drugs tolerated in Type 1 citrullinemia and Brugada pattern allowed the successful and uneventful administration of GA in the study patient.

Functional identification of two novel variants and a hypomorphic variant in ASS1 from patients with Citrullinemia type I.

Background: Citrullinemia type I (CTLN1) is a rare autosomal recessive inborn error of the urea cycle caused by mutations in the gene encoding the arginosuccinate synthetase (ASS1) enzyme. Classic CTLN1 often manifests with acute hyperammonemia and neurological symptoms. Molecular genetic testing is critical for patient diagnosis. Methods: Three unrelated families with clinically suspected CTLN1 were included in this study. Potential pathogenic variants were identified using whole exome sequencing (WES) and validated using Sanger sequencing. Western blotting, quantitative PCR, immunofluorescent staining, and ELISA were used to assess functional changes in candidate ASS1 variants. Results: Five variants were identified, two of which were novel, and one has been reported, but its pathogenicity was not validated. The novel variant c.649-651del (p.P217del) and the 5'UTR variant (c.-4C>T) resulted in a decrease in ASS1 expression at both the protein and transcription levels. The other novel variant, c.1048C>T (p.Q350*), showed a marked decrease in expression at the protein level, with the formation of truncated proteins but an increased transcription. Both c.649_651del (p.P217del) and c.1048C>T (p.Q350*) showed a highly significant reduction in enzyme activity, while c.-4C>T had no effect. Conclusion: We identified two novel variants and a hypomorphic non-coding variant in ASS1 and validated the pathogenicity using functional studies. Our findings contribute to expanding the spectrum of ASS1 variants and understanding the genotype-phenotype relationships of CTLN1.

Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver.

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.

Analysis of ASS1 gene in ten unrelated middle eastern families with citrullinemia type 1 identifies rare and novel variants.

BACKGROUND: Citrullinemia type 1 (CTLN1) is a rare autosomal recessive disease caused by argininosuccinate synthetase (ASS) deficiency. Manifestations vary from the acute neonatal or "classic" form to a milder, late-onset, or "unconventional" form. To date, more than 93 variants in the ASS1 gene located on chromosome 9q43.11 (OMIM #215700) are reportedly responsible for CTLN1. Their incidence and distribution vary according to geographic origins and ethnicity, and a correlation, although not clearly delineated, has been established between the genotype and the phenotype of the disease. Though, in the Middle East, national descriptions of CTLN1 are still lacking. METHODS: A total of ten unrelated Middle Eastern families, five Lebanese, two Syrians, and three Iraqis with citrullinemia index cases, were included in this study. Upon informed consent, DNA was extracted from the whole blood of the index patients as well as their parents and siblings. Genetic analysis was carried out by Sanger sequencing of the ASS1 gene. RESULTS: Seven different variants were identified. Two novel variants, c.286C>A (p.(Pro96Thr), RNA not analyzed) in exon 5 and deletion c.685_688+6del(p.(Lys229Glyfs*4), RNA not analyzed) in exon 10, were found in one Lebanese and one Syrian family, respectively, and were correlated with early-onset and severe clinical presentation. Five other known variants: c.535T>C (p.(Trp179Arg), RNA not analyzed) in exon 8, c.787G>A (p.(Val263Met), RNA not analyzed) in exon 12, c.847G>A (p.(Glu283Lys), RNA not analyzed) in exon 13, c.910C>T (p.(Arg304Trp), RNA not analyzed) in exon 13, and c.1168G>A (p.(Gly390Arg), RNA not analyzed) in exon 15, were found in Lebanese, Syrian, and Iraqi families, and were associated with diverse clinical presentations. CONCLUSION: Two novel variants and five known variants were found in a total of ten unrelated Middle Eastern families.