Neonatal cholestasis is a prevalent disorder in pediatrics, primarily caused by biliary obstruction, genetic metabolic disorders, and infections [1,2]. Its complex etiology poses significant challenges for early diagnosis and may lead to adverse outcomes. Citrin deficiency (CD) is a common cause of infantile cholestasis among inborn errors of metabolism. SLC25A13 is the pathogenic gene of CD, which encodes the citrin protein. Citrin, also known as aspartate/glutamate carrier 2 (AGC2), is a mitochondrial inner membrane protein predominantly expressed in the liver that plays a vital role in energy metabolism. Additionally, it is a crucial component of the malate-aspartate nicotinamide adenine dinucleotide (NADH) shuttle system. Its deficiency disrupts the urea cycle, hepatic glycolysis, gluconeogenesis, and de novo lipogenesis, leading to a series of metabolic disturbances [3–5]. CD presents with three age-dependent clinical phenotypes: neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), post-NICCD (including failure to thrive and dyslipidemia caused by citrin deficiency, FTTDCD), and adolescent and adult citrin deficiency (AACD) [6–9].

NICCD mainly presents a series of clinical symptoms, such as cholestasis and elevated liver enzymes, and is often accompanied by various metabolic abnormalities, including citrullinemia, hypoglycemia, galactosemia and hyperammonemia [7]. The primary treatment involves early initiation of a lactose-free, medium-chain triglyceride (MCT)-enriched formula, combined with supportive hepatoprotective therapy. Most patients achieve significant symptomatic improvement within their first year of life. However, NICCD carries a risk of adverse long-term outcomes. These include post-infancy growth retardation, progression to cirrhosis requiring transplantation, and mortality due to severe complications [10].

As there are no universally accepted diagnostic criteria for NICCD, genetic testing is essential for confirmation. The accurate application of molecular diagnostic techniques is therefore critical for enabling timely diagnosis and effective intervention [11]. The SLC25A13 gene, located at chromosome 7q21.3 and responsible for citrin deficiency, consists of 18 exons and 17 introns, encoding a 675-amino acid protein [7]. More than 140 pathogenetic/likely pathogenetic variants have been reported [6]. Population-based studies indicate that the carrier frequency of pathogenic SLC25A13 variants in China is 1/45, and the predicted disease prevalence is approximately 88,000 affected individuals [12,13]. However, only a few hundred patients have been reported so far, which means the majority of patients have been either undiagnosed or misdiagnosed. Insufficient awareness of the disease remains the primary barrier to diagnosis. Therefore, this study aims to clarify the clinical features and genetic characteristics of NICCD, as well as prognosis, to enrich the present data of citrin deficiency in China and provide support for further improving the diagnosis and treatment of this disease.

The present study delineates the clinical and genetic characteristics of 60 NICCD patients, among whom 50 were followed up, with the longest follow-up duration reaching 12 years. NICCD patients present with a wide range of clinical manifestations. Three major hotspot mutations in SLC25A13 were identified in NICCD patients from Zhejiang, China: c.852_855delTATG, IVS16ins3kb, and c.615+5G>A. Following treatment with lactose-free and MCT-enriched formula, patients achieved rapid clinical resolution. Three patients progressed to FTTDCD. All patients remained clinically stable during follow‑up, suggesting that early intervention contributes to a favorable prognosis.

It is reported that chubby face features, along with AST/ALT >2, direct bilirubin(DB)/total bilirubin(TB)<0.67, and a standard deviation score for α-fetoprotein of 4 or greater, might serve as useful clinical indicators for diagnosing NICCD early in infancy [14]. In this study, 36.7 % of patients had a chubby face, 90.0 % had AST/ALT >2 and 98.3 % had DB/TB<0.67, which is consistent with the previously reported diagnostic indicators and further confirms the value of these clinical and biochemical features in the early identification of NICCD. Consistently, 96.3 % of the patients in the present study exhibited elevated alkaline phosphatase (ALP), and 85.2 % of the patients presented with an ALP/ALT ratio > 10. Previous studies have demonstrated that elevated ALP and an ALP/ALT ratio > 10 can help distinguish NICCD from non-NICCD patients [15,16]. These findings further support the role of ALP and the ALP/ALT ratio as reliable auxiliary diagnostic markers for NICCD.

NICCD is characterized by abnormal plasma amino acid profiles, particularly a significant increase in citrulline, making it a primary biomarker for NBS. However, citrulline levels may not be elevated immediately after birth, resulting in the missed detection of several NICCD patients during NBS [17,18]. In the present study, only 5.2 % of patients had elevated citrulline on NBS, compared with 96.7 % at clinical presentation. The authors also observed increased methionine (83.6 %), arginine (78.8 %), tyrosine (11.8 %), and long-chain fatty acids (78.4 %), likely due to compensatory amino acid metabolism and impaired hepatic long-chain acylcarnitine metabolism secondary to hepatocyte energy insufficiency. These findings further confirm the metabolic characteristics of NICCD and the limitations of NBS, supporting the use of multi-indicator detection or adjustment of cut-off values for early diagnosis.

Citrin deficiency represents a genetic metabolic disorder with a global distribution, affecting individuals of various ethnic backgrounds. It has been reported in Asia, North America, and Europe, with a higher prevalence among patients in East Asia. To date, at least 140 pathogenic or likely pathogenic SLC25A13 variants have been described [6]. c.852_855delTATG is the predominant variant in Thailand and Vietnam NICCD patients [6,16]. c.852_855delTATG, IVS16ins3kb, and c.1638_1660dup are the most common SLC25A13 gene mutation loci in the Indian population [19]. The main high-frequency variations in Japan are c.1177+1G>A, c.852_855delTATG, c.1311+1G>A, which account for 60 % of the total mutations [20]. The most common variations in Korea are IVS16ins3kb, c.852_855delTATG and c.1177+1G>A [21,22]. In UK patients, c.1763G>A is the major pathogenic SLC25A13 gene mutation [23]. Song et al. identified c.852_855delTATG, IVS16ins3kb, c.1638_1660dup, and c.615+5G>A as the four most prevalent variants in China [12,13]. However, the number of patients in Zhejiang Province is limited. In the present study, the authors analyzed patients originally from Zhejiang province and found that c.852_855delTATG, IVS16ins3kb, and c.615+5G>A were the three most common mutation types. NICCD is inherited in an autosomal recessive manner, and it is generally caused by biallelic mutations in the SLC25A13 gene. However, only a single heterozygous mutation was detected in four children in this study. Supported by the patients’ distinct clinical manifestations, specific alterations in blood metabolites and treatment responses, the diagnosis of NICCD was definitively verified. This may be attributed to factors such as the presence of large-fragment insertions or deletions in the homologous allele, as well as copy number variations, which cannot be detected by routine Sanger/NGS sequencing and panel sequencing.

The primary management for NICCD involves dietary intervention. Discontinuing breastfeeding and standard infant formula, and replacing them with a lactose-free, MCT-enriched formula, can lead to significant symptomatic improvement [24]. A large-scale Japanese follow-up study of 222 patients demonstrated that early dietary intervention improves both clinical symptoms and long-term prognosis [20]. A study by Abuduxikuer et al. of 61 patients diagnosed with NICCD found that poor prognosis was linked to delayed diagnosis and delayed treatment with lactose-free and/or medium-chain triglyceride-enriched (LF/MCT) formula [25]. In the present study, all patients were given dietary therapy after diagnosis or suspected diagnosis. After complementary foods were introduced, dietitians developed a specific protein-fat-carbohydrate ratio recipe for each patient, with 15–25 % protein, 40–50 % fat and 30–40 % carbohydrate recommended [24].

Of the 50 patients followed up, 94 % of patients achieved normal bilirubin levels within 6 months after discharge. Before dietary treatment, 26 patients (52 %) had growth retardation (HAZ and/or WAZ/BAZ < −2), while only 5 patients (10 %) remained growth-retarded at the most recent follow-up. These findings indicate the efficacy of dietary treatment. In addition, 68 % of patients showed a preference for foods rich in protein and lipid and/or an aversion to carbohydrate-rich foods. This special dietary habit may potentially represent a compensatory mechanism for the associated metabolic dysfunction [26].

Eleven patients still experienced episodes of hypoglycemia. Four patients had no hypoglycemic episodes after one year of age following the addition of MCT oil or intensified feeding, confirming the therapeutic effect of MCT in providing energy and improving hepatic metabolism [10]. Four patients experienced one hypoglycemic episode during morning fasting or illness, while one patient had two episodes. Two patients exhibited recurrent episodes of early-morning fasting hypoglycemia. Both failed to adhere to the dietary treatment and attend regular follow-up; one refused MCT oil supplementation, while the other used MCT oil irregularly. These factors may represent the key triggers for hypoglycemia recurrence in NICCD. After timely identification and treatment, no serious complications occurred. This highlights the importance of accurately identifying the clinical manifestations of NICCD and underscores the essential role of disease-related education for patients and their families.

The dyslipidemia associated with citrin deficiency is primarily attributed to two complementary mechanisms: the downregulation of the peroxisome proliferator-activated receptor alpha (PPARα) signaling pathway, which impairs fatty acid oxidation, and the activation of the glycerol-3-phosphate-carbohydrate responsive element-binding protein (G3P-ChREBP) pathway, which promotes hepatic de novo lipogenesis [27,28]. In this study, eleven patients presented with dyslipidemia, including nine with elevated total cholesterol, one with elevated triglycerides, and one with concurrent elevations in both total cholesterol and triglycerides. This may be related to poor dietary compliance, as excessive carbohydrate intake can exacerbate metabolic disturbances in citrin deficiency.

In the present study, three patients progressed to FTTDCD. All patients remained in stable condition without severe complications during the follow-up period. Most patients with NICCD have a generally good prognosis, but a small number of cases may develop severe complications, including liver failure, cirrhosis, hepatocellular carcinoma, necessitating liver transplantation, or even death [20,25,29]. Though symptoms resolve in some patients by adulthood, there remains a risk of progression to fatal CTLN2 decades later [20,27]. Therefore, long-term follow-up is essential for managing the prognosis of patients with NICCD, as it facilitates the early detection of abnormal changes and prevents disease progression.

The major limitation of this study is its retrospective design. Reliance on historical medical records and follow-up data inherently introduces the risk of information bias. Specifically, certain clinical data (including detailed dietary records and long-term treatment compliance) may have been incomplete or inaccurately documented, which could potentially confound the analysis of factors associated with disease progression and treatment outcomes. Furthermore, the patients were predominantly from Zhejiang Province, which may not represent the overall disease spectrum of citrin deficiency in China. Additionally, supplementary assays were not performed to detect potential secondary pathogenic variants in the homologous allele among these four patients.

In conclusion, the present study presents a retrospective analysis of the clinical, biochemical, genetic and follow-up data of patients with NICCD in China. Patients with NICCD exhibit a broad spectrum of clinical manifestations. c.852_855delTATG, IVS16ins3kb and c.615+5G>A are the mutation hotspots of the SLC25A13 gene in NICCD patients in Zhejiang, China. Early intervention with a specialized diet is crucial for effective management and a favorable prognosis. Education of pediatric patients and their families is of great importance.

None.

The datasets are not publicly available due to ethical restrictions and institutional privacy policies. The data contains sensitive information that could compromise the anonymity of the participants. Requests to access the data should be directed to the corresponding author.

Lingli Chen: Conceptualization, Data curation, Formal analysis, Writing – review & editing. Jingan Lou: Project administration, Writing – review & editing. Youyou Luo: Project administration, Writing – review & editing. Youhong Fang: Resources, Writing – review & editing. Mingfang Sun: Data curation, Writing – review & editing. Jindan Yu: Conceptualization, Resources, Formal analysis, Writing – review & editing.