[Newborn screening, clinical features and genetic analysis for Citrin deficiency in Henan province].

OBJECTIVE: To explore the prevalence, clinical features, genetic characteristics and prognosis of Citrin deficiency in Henan province of China. METHODS: A total of 986 565 neonates screened by tandem mass spectrometry at the Third Affiliated Hospital of Zhengzhou University from January 2013 to December 2021 were retrospectively analyzed. Analysis of SLC25A13 gene variants and parental verification were carried out for neonates suspected for Citrin deficiency by next-generation sequencing. The clinical, biochemical and genetic characteristics of Citrin deficiency patients were integrated to guide the diet treatment and follow up the growth and development. Paired-t test was used to compare the amino acid levels in the peripheral blood samples before and after the treatment. RESULTS: Nine cases of Citrin deficiency were diagnosed among the 986 565 neonates. Specific elevation of citrulline was observed in all of the 9 cases. Six variants were detected by genetic sequencing, among which c.852_855delTATG, c.615+5G>A, c.550C>T and IVS16ins3kb were known pathogenic variants, whilst c.1111_1112delAT and c.837T>A were unreported previously. The detection rate for c. 852_855delTATG was the highest (61.6%, 11/18), followed by IVS16ins3kb (16.7%, 3/18). The clinical symptoms of all patients were relieved after the treatment, and the blood amino acid profile and biochemical parameters were significantly improved by gradually falling within the normal range. By June 2022, all patients had shown a good prognosis. CONCLUSION: The prevalence of Citrin deficiency among neonates from Henan Province by tandem mass spectrometry is 1/109 618, and the carrier rate for the pathogenic variants of the SLC25A13 gene was 1/166. The c.852_855delTATG may be a hot spot variant among the patients. Discovery of the novel variants has enriched the mutational spectrum of the SLC25A13 gene. Above results have provided a basis for the early diagnosis, treatment, prognosis and genetic counseling for the affected families.

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.

Clinical and genetic analysis of 26 Chinese patients with neonatal intrahepatic cholestasis due to citrin deficiency.

BACKGROUND: Neonatal intrahepatic cholestasis due to citrin deficiency (NICCD) is an autosomal recessive disorder caused by SLC25A13 genetic mutations. We retrospectively analyzed 26 Chinese infants with NICCD (years 2014-2022) in Quanzhou City. METHODS: The plasma citrulline (CIT) concentration analyzed by tandem mass spectrometry (MS/MS), biochemical parameters and molecular analysis results are presented. RESULTS: Twelve genotypes were discovered. The relationship between the CIT concentration and genotype is uncertain. In total, 8 mutations were detected, with 4 variations, c.851_854delGTAT, c.615 + 5G > A, c.1638_1660dup and IVS16ins3kb, constituting the high-frequency mutations. Specifically, we demonstrated 2 patients with NICCD combined with another inborn errors of metabolism (IEM). Patient No. 22 possessed compound heterozygous mutations of c.615 + 5G > A and c.790G > A in the SLC25A13 gene accompanied by compound heterozygous variations of c.C259T and c.A155G in the PTS gene. Additionally, Patient No. 26 carried c.51C > G and c.760C > T in the SLC22A5 gene as well as c.615 + 5G > A and IVS16ins3kb in the SLC25A13 gene. CONCLUSIONS: We report a case of the simultaneous occurrence of primary carnitine deficiency (PCD) and NICCD.

Citrin deficiency due to SLC25A13 exon deletion in a Chinese infant: A case report.

INTRODUCTION: Citrin is a calcium-bound aspartate-glutamate carrier protein encoded by the gene SLC25A13, mutations of which can cause citrin deficiency, an autosomal recessive disorder. The manifestations of citrin deficiency include neonatal intrahepatic choledeposits caused by citrin deficiency (NICCD: OMIM#605814), intermediate growth disorders and dyslipidemia caused by citrin deficiency, and citrullinemia type II (OMIM#603471) in adults. NICCD is a classical metabolic disorder that causes cholestasis in newborns. PATIENT CONCERN AND CLINICAL FINDINGS: Here, we present the case of a 2-month-old male patient treated in our hospital on March 20, 2023, due to "postnatal skin xanthochromia and transaminases higher than normal values". Since birth, the child's skin had yellowed all over the body, and his condition did not improve after multiple medical treatments. DIAGNOSIS/INTERVENTION/OUTCOMES: The child underwent full exome gene testing at the age of 2 months and 13 days, and the results indicated heterozygous deletion of exon 3 of the SLC25A13 gene, while genetic testing of the parents revealed no gene mutations. The variant was preliminarily judged as being pathogenic according to the ACMG guidelines, and the patient was diagnosed with "citrin deficiency". Skin yellowing eventually subsided, and liver function returned to normal without special treatment. CONCLUSIONS: Here, we report a rare case of citrin deficiency caused by a heterozygous deletion of the SLC25A13 gene. This case increases the clinical phenotypic profile of NICCD, suggesting that clinicians must be vigilant regarding such genetic metabolic diseases in the clinic for early diagnosis and treatment. NICCD should always be considered in the differential diagnosis of neonatal cholestasis.

Role of genetic introgression in introducing mutant alleles in Bos indicus cattle and prevalence of lethal genetic disorders in Bos taurus × Bos indicus and Bos indicus cattle in India.

Fertility is an important trait associated with reproductive performance and animal welfare concern. Lethal alleles affect fertility through early embryonic death, abortions, and stillbirth depending on the genetic expression of the allele. Holstein Friesian and Jersey are two major Bos taurus breeds used widely for increasing milk yield along with purebreds of Bos indicus breeds like Gir, Kankrej, Sahiwal, and Tharparkar. In the present study, prevalence of lethal mutants in crossbred Holstein Friesian (CBHF, n = 2435), crossbred Jersey (CBJY, n = 2874), Gir (n = 3288), Kankrej (n = 593), Sahiwal (n = 965), and Tharparkar (n = 18) were studied. Heterozygous carrier animals were identified for bovine leukocyte adhesion deficiency (BLAD), Citrullinemia, complex vertebral malformation (CVM), Brachyspina, Holstein Haplotype 1 (HH1), Holstein Haplotype 3 (HH3),Holstein Haplotype 4 (HH4) and Jersey Haplotype 1 (JH1). Breed purity analysis confirmed inheritance of Bos taurus genes contributing to the presence of lethal mutant alleles like BLAD, Citrullinemia, HH1, and JH1 in apparently phenotypic Bos indicus animals. Screening and elimination of heterozygous carrier bulls/cows is essential to control fertility loss associated with lethal alleles.

[Analysis of clinical and genetic variation in neonatal intrahepatic cholestasis caused by citrin deficiency].

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.

Features of liver injury in 138 Chinese patients with NICCD.

OBJECTIVES: To find biochemical and molecular markers can assist in identifying serious liver damage of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) patients. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Clinical and ASS1 gene variant analysis of three Chinese pedigrees affected with Citrullinemia type I].

OBJECTIVE: To analyze the clinical and genetic characteristics of three Chinese pedigrees affected with Citrullinemia type I (CTLN1). METHODS: Three children diagnosed at the Children's Hospital Affiliated to Shandong University from 2017 to 2020 were selected as the study subjects. Genomic DNA was extracted from peripheral blood samples of the probands and their parents. Next generation sequencing (NGS) was carried out to detect pathological variants of the probands. Sanger sequencing was used for validating the candidate variant among the pedigrees. RESULTS: The probands have respectively carried compound heterozygous variants of c.207_209delGGA and c.1168G>A, c.349G>A and c.364-1G>A, c.470G>A and c.970G>A of the ASS1 gene, which were respectively inherited from their parents. CONCLUSION: The newly discovered c.207_209delGGA and c.364-1G>A variants have enriched the mutational spectrum of the ASS1 gene. And the mutation spectrum of Chinese CTLN1 patients is heterogeneous.

Genetic and clinical features of patients with intrahepatic cholestasis caused by citrin deficiency.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A novel SLC25A13 gene splice site variant causes Citrin deficiency in an infant.

Citrin deficiency is an autosomal recessive disorder associated with SLC25A13 gene pathogenic variants, with more than a hundred known at present. It manifests in neonates as failure to thrive and acute liver insufficiency. We herein describe a case of a 4-week-old infant who presented with insufficient weight gain and liver failure accompanied by hyperammonemia. She was diagnosed with Citrin deficiency after a thorough biochemical and molecular analysis including amino acid profile, DNA sequencing of genes of interest and RNA splice site evaluation, to reveal a yet unknown damaging variant of the SLC25A13 gene.

The prognosis of citrin deficiency differs between early-identified newborn and later-onset symptomatic infants.

BACKGROUND: The prognosis for patients with citrin deficiency is not always benign. This study examined the differences between patients identified early by newborn screening and patients identified later with cholestasis/hepatitis. MATERIALS AND METHODS: This retrospective study included 42 patients with genetically confirmed SLC25A13 mutations who were born between May 1996 and August 2019. Fifteen patients were identified during newborn screening (NBS group) and 27 patients were identified through the onset of cholestasis/hepatitis in infancy (clinical group). RESULTS: Overall, 90% of the patients presented with cholestasis, among whom 86% (31/36) recovered at a median age of 174 days. Compared with patients in the clinical group, patients in the NBS group were significantly younger at diagnosis and at cholestasis-free achievement; they also had significantly lower levels of peak direct bilirubin and liver enzymes. At the median follow-up age of 11.8 years, 21% of the patients had dyslipidemia, whereas 36% of the patients had failure to thrive. The overall mortality rate was 2.4%. Variant c.851_854del was the most frequent, constituting 44% of the mutant alleles. CONCLUSION: Patients identified early by NBS had a better prognosis, demonstrating the importance of a timely diagnosis of NICCD and the need for careful follow-up. IMPACT: Some cases of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) are not benign. Compared with patients identified later based on the presence of cholestasis/hepatitis, patients identified early by newborn screening have less severe cholestasis and are cholestasis-free at a significantly younger age. A timely diagnosis is needed, along with follow-up examinations that assess metabolic profile and body weight, to improve the long-term prognosis of NICCD patients.

Nicotinamide riboside rescues dysregulated glycolysis and fatty acid ß-oxidation in a human hepatic cell model of citrin deficiency.

Citrin deficiency (CD) is an inborn error of metabolism caused by loss-of-function of the mitochondrial aspartate/glutamate transporter, CITRIN, which is involved in both the urea cycle and malate-aspartate shuttle. Patients with CD develop hepatosteatosis and hyperammonemia but there is no effective therapy for CD. Currently, there are no animal models that faithfully recapitulate the human CD phenotype. Accordingly, we generated a CITRIN knockout HepG2 cell line using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 genome editing technology to study metabolic and cell signaling defects in CD. CITRIN KO cells showed increased ammonia accumulation, higher cytosolic ratio of reduced versus oxidized form of nicotinamide adenine dinucleotide (NAD) and reduced glycolysis. Surprisingly, these cells showed impaired fatty acid metabolism and mitochondrial activity. CITRIN KO cells also displayed increased cholesterol and bile acid metabolism resembling those observed in CD patients. Remarkably, normalizing cytosolic NADH:NAD+ ratio by nicotinamide riboside increased glycolysis and fatty acid oxidation but had no effect on the hyperammonemia suggesting the urea cycle defect was independent of the aspartate/malate shuttle defect of CD. The correction of glycolysis and fatty acid metabolism defects in CITRIN KO cells by reducing cytoplasmic NADH:NAD+ levels suggests this may be a novel strategy to treat some of the metabolic defects of CD and other mitochondrial diseases.

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.

The mutation spectrum of SLC25A13 gene in citrin deficiency: identification of novel mutations in Vietnamese pediatric cohort with neonatal intrahepatic cholestasis.

BACKGROUND: Citrin deficiency (CD), a disorder caused by mutations in the SLC25A13 gene, may result in neonatal intrahepatic cholestasis. This study was purposely to explore the mutation spectrum of SLC25A13 gene in Vietnamese CD patients. METHODS: The 292 unrelated CD patients were first screened for four high-frequency mutations by PCR/PCR-RFLP. Then, Sanger sequencing was performed directly for heterozygous or undetected patients. Novel mutations identified would need to be confirmed by their parents. RESULTS: 12 pathogenic SLC25A13 mutations were identified in all probands, including three deletions c.851_854del (p.R284Rfs*3), c.70-63_133del (p.Y24_72Ifs*10), and c.[1956C>A;1962del] (p.[N652K;F654Lfs*45]), two splice-site mutations (IVS6+5G>A and IVS11+1G>A), one nonsense mutations c.1399C>T (p.R467*), one duplication mutation c.1638_1660dup (p.A554fs*570), one insertion IVSl6ins3kb (p.A584fs*585), and four missense mutation c.2T>C (p.M1T), c.1231G>A (p.V411M), c.1763G>A (p.R588Q), and c.135G>C (p.L45F). Among them, c.851_854del (mut I) was the most identified mutant allele (91.78%) with a total of 247 homozygous and 42 heterozygous genotypes of carriers. Interestingly, two novel mutations were identified: c.70-63_133del (p.Y24_72Ifs*10) and c.[1956C>A;1962del] (p.[N652K;F654Lfs*45]). CONCLUSION: The SLC25A13 mutation spectrum related to intrahepatic cholestasis infants in Vietnam revealed a quite similar pattern to Asian countries' reports. This finding supports the use of targeted SLC25A13 mutation for CD screening in Vietnam and contributed to the SLC25A13 mutation spectra worldwide. It also helps emphasize the role of DNA analysis in treatment, genetic counseling, and prenatal diagnosis.

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

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

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

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

Improved diagnosis of citrin deficiency by newborn screening using a molecular second-tier test.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Pathogenic variants of the mitochondrial aspartate/glutamate carrier causing citrin deficiency.

Citrin deficiency is a pan-ethnic and highly prevalent mitochondrial disease with three different stages: neonatal intrahepatic cholestasis (NICCD), a relatively mild adaptation stage, and type II citrullinemia in adulthood (CTLN2). The cause is the absence or dysfunction of the calcium-regulated mitochondrial aspartate/glutamate carrier 2 (AGC2/SLC25A13), also called citrin, which imports glutamate into the mitochondrial matrix and exports aspartate to the cytosol. In citrin deficiency, these missing transport steps lead to impairment of the malate-aspartate shuttle, gluconeogenesis, amino acid homeostasis, and the urea cycle. In this review, we describe the geological spread and occurrence of citrin deficiency, the metabolic consequences and use our current knowledge of the structure to predict the impact of the known pathogenic mutations on the calcium-regulatory and transport mechanism of citrin.

Asymptomatic ASS1 carriers with high blood citrulline levels.

INTRODUCTION: Citrullinemia Type 1 (CTLN1) is an autosomal recessive disorder caused by variants in the ASS1 gene. This study intends to clarify the etiology of false positives in newborn screening for citrullinemia. METHOD: Newborns who had elevated dried-blood spot citrulline levels were enrolled, and medical records were reviewed retrospectively. Common ASS1 variants were screened using high-resolution melting analysis. RESULT: Between 2011 and 2021, 130 newborns received confirmatory testing for citrullinemia, 4 were found to be patients for CTLN1; 11 were patients with citrin deficiency; and 49 newborns were confirmed to be carrying one pathogenic ASS1 variant. The incidence of CTLN1 was 1 in 188,380 (95% confidence interval: 1 in 73,258 to 1 in 484,416). All ASS1 variants studied in this cohort were located in exons 11 to 15, which encode the tetrameric interface regions of the ASS1 protein. Among 10 ASS1 carriers with elevated citrulline levels and complete sequence data, four (40%) revealed additional non-benign ASS1 variants; in contrast, only 2 of the 26 controls (7.7%), with normal citrulline levels, had additional ASS1 variants. CONCLUSION: Heterozygote ASS1 variants may lead to a mild elevation of blood citrulline levels: about 2-6 times the population mean. Molecular testing and family studies remain critical for precise diagnosis, genetic counseling, and management.