Citrin deficiency (CD) is an autosomal recessive disease caused by SLC25A13 gene mutation. It includes three age-dependent clinical phenotypes: neonatal intrahepatic cholestasis caused by citrin deficiency(NICCD), adult-onset type II citrullinemia (CTLN2) and failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD) between NICCD and CTLN2 stages (1). NICCD is one of the most common genetic metabolic diseases causing infantile cholestatic hepatopathy, which is associated with persistent jaundice, abnormal coagulation function, hepatosplenomegaly, chubby face, growth retardation, metabolic abnormalities, etc (2)., but these features are not pathognomonic. So genetic analysis is regarded as a reliable method for the definite diagnosis of NICCD. With the development of genetic testing, we recognize that although NICCD is a pan-ethnic disease, it is still prevalent in East Asia. Meanwhile, China is a high-incidence area, the mutation carrier rate of SLC25A13 gene is as high as 1/65. The type of variation of SLC25A13 is also different in different regions. In China, c.852_855del4, c.1638_1660dup, IVS6 + 5G > A, and IVS16ins3kb are the four most common mutations of SLC25A13.In this study, we performed genetic screening on 4 patients with jaundice and every patient carried compound heterozygous mutations with a common variant. Unexpectedly, we also detected 3 novel variants of SLC25A13, including a nonsense mutation, a frameshift mutation, and a whole exon deletion. These findings enriched the variant spectrum of NICCD and guided clinical diagnosis and genetic counseling.

SLC25A13 is the pathogenic gene of NICCD, which encodes the citrin protein. Citrin plays significant roles in the urea cycle, malic acid-aspartate shuttle, and gluconeogenesis (3). As a result, variants of SLC25A13 can lead to metabolic abnormalities such as citrullinemia, hyperammonemia, hypoglycemia, and hyperlipidemia. Our study results were similar to those of the previous studies (4), some patients with NICCD showed low birth weight and low birth height according to sex and gestational age. At the same time, the patients with NICCD can also be accompanied by elevated liver enzymes, hyperbilirubinemia, significantly elevated alpha-fetoprotein, hypoalbuminemia, prolonged prothrombin time and fatty liver. Predictably, four patients in our study also had these characteristics. The sign of hepatic steatosis is common in patients with NICCD. While both the ultrasonography and liver biopsy of patient 3 did not indicate evidence of hepatic steatosis. Therefore, consistent with Miyamoto's report (5), NICCD cannot be ruled out even if hepatic steatosis is not seen in patients with cholestasis. None of the above changes are specific. A variety of neonatal intrahepatic cholestasis (such as cytomegalovirus hepatitis, Alagille syndrome, etc.) can manifest as jaundice, elevated liver enzyme, hepatosplenomegaly, and growth retardation. Meanwhile, other inherited metabolic diseases(such as galactosemia (6), hepatocerebral mitochondrial DNA depletion syndrome (7–9), arginylsuccinuria (10), etc.) can also have symptoms similar to NICCD. It can be seen that it is a big challenge to distinguish NICCD from other diseases. Therefore, we should actively conduct genetic testing for patients with unexplained infantile cholestasis to avoid misdiagnosis and missed diagnosis.

So far, more than 300 variants of SLC25A13 have been reported worldwide, including 292 single nucleotide variations, 28 deletion mutations, 8 duplation mutations, 4 microsatellite mutations, 3 insertion mutations, and 1 insertional deletion variant. In our study, four patients all harbored compound heterozygous variants of SLC25A13, and a total of 6 variant types were detected, among which three have not been reported: c.1803C > G, c.1141delG and exon 11 del. The common mutation c.1803C > G identified in patient1 and 2, which is located on exon 17 and is predicted to cause the early appearance of amino acid termination code (tyrosine > termination) in protein synthesis, and according to ACMG guidelines, pathogenicity analysis is PVS1 + PM2 + PP4, it is classified as likely pathogenic, so far as we know, which has not been reported previously. While in our study, it was found in two patients and two family members. After a detailed medical history we found no consanguinity between the two families; however, they all came from Hubei Province, China, which spans the Yangtze River and belongs to the central city in China. The previous large-scale studies on NICCD were generally in the south and north of China, with few central cities. As we all know, there is a significant difference in the mutation carrier rate of SLC25A13 gene between North and South populations along the boundary of 30° North latitude in China. The results of our study suggested that the carrier rates of c.1803C > G may be frequent in the central cities of China, and there are differences in the distribution of the mutation spectrum in different geographical areas. As for the relationship between genotype and phenotype, although some studies declared that there were likely to be phenotype–genotype correlations in the specific genotypes (11), in our study, no obvious genotype-phenotype relationship seems to be found. For example, heterozygous genotypes including c.1803C > G were found in patient 1 and 2, but their birth weight, AST, Fib, PA and prothrombin time have a marked difference. And patient 1 and 3 carried the heterozygous variants including IVS16ins3kb, but they didn't present the same clinical manifestations. So we do not hold that there is a significant genotype-phenotype relationship between patients with NICCD. The number of cases in our study was small, so further studies with more samples may be required. c.1141delG is a novel code-shifted mutation that has not been reported in relevant clinical cases and our reference population gene database. Moreover, it is predicted that it may lead to the early occurrence of amino acid termination ciphers in protein synthesis. According to the ACMG guidelines, the pathogenicity analysis is PVS1 + PM2 + PP4, so it is likely pathogenic. Exon deletion is a rare variant type among SLC25A13 variants, which are mainly caused by large fragment deletion, including exon deletion, such as c.329–154_468 + 2352del2646bp (deletion of exon 5) (12), c.70–862_c.212 + 3527 del4532bp (deletion of exon 3) (13), Ex16 + 74_IVS17-32del516 (deletion of exon 17) (14), c.329–1687 -c.468 + 3865del5692bp (deletion of exon 5) (15), c.1019_1177 + 893del (deletion of exon 11) (16), c.1312–2860_1452 + 988del (deletion of exon 14) (17), c.1312–4144_1452 + 3373del (deletion of exon 14) (17) and c.3251 _c.15 + 18443del21709bp (deletion of exon 1) (18). Partial abnormal splicing of partially intron regions leads to exome deletions in mRNA, such as IVS4ins6 kb (skipping of exon 5) (19), IVS11+1 G > A (skipping of exon 11) (20), IVS15 + 1G > T (skipping of exon 15) (21) and IVS13+1G > A (skipping of exon 13) (22). In this study, after high-throughput sequencing analysis, patient 4 was found to carry a common variant c.852_855del and be accompanied by copy number variation. In order to clarify the definite genetic etiology of the patient, qPCR verification was performed. Eventually, another variant (exon 11 del) was found in the patient. So, this patient was confirmed as NICCD caused by compound heterozygous variants of SLC25A13. The other three variants identified in this study: c.1663_1664insGAGATTACAGGTGGCTGCCCGGG (12, 23), IVS16ins3kb (15, 24–27), and c.852_855del (23, 24, 28–31) have been reported in multiple literatures and are three of the most common variants of SLC25A13 in China.

Conventional genetic screening, such as whole exon sequencing, can only detect point mutations or small deletions. However, it was found in a study by Tokuhara (32) that about 15% of the complex heterozygotes or homozygotes of the SLC25A13 gene could not be identified by the routine methods. Fortunately, the emergence of multiplex ligation-dependent probe amplification (MLPA) (20), Southern blots restriction fragment analysis (33), custom fluorescence in situ hybridization (FISH) (34), and qPCR (21) can help us better detect this part of the variants. Therefore, additional testing is recommended for the patients who are clinically considered for NICCD but have only one heterozygous mutation detected by conventional genetic screening.

After the dietary adjustment, the liver function such as ALT, GGT, TB, DB, TBA, ALB, AFP returned to normal at the last review (Table 3). The patients were all underweight at the time of their first visit to our hospital, although the follow-up time of our study was short (the maximum is approximately seven years), the height and weight of them gradually returned to normal. Recently there were cases of liver cancer (35) in NICCD patients have been reported, it suggested that NICCD is not always harmless. Fortunately, none of the long-term complications including liver cirrhosis, hepatoadenoma or hepatocellular carcinoma, pancreatitis and so on occurred in the patients in this study. A recent study by Kido (11) found that the height and the weight of patients with NICCD were high than those with CTLN2, in the meantime long-term complications were seen in patients with CTLN2 but not in older NICCD patients. It is speculated that medical management including dietary interventions can help patients. Therefore, we emphasize here the necessity for dietary intervention in patients with NICCD to alleviate clinical manifestations and prevent long-term complications.

In summary, our study reviewed the clinical manifestations, genetic test results, and prognosis of NICCD patients from 4 unrelated families. There are 6 variants of SLC25A13 were identified, and three novel variants were found: c.1803C > G, c.1141delG, and exon 11 del. This result clarified their genetic etiology and provided a strong basis for the clinical diagnosis of patients. At the same time, the detection of the novel variants enriched the variant spectrum of the SLC25A13, which further laid the foundation for the correlation study of the genotype and phenotype of NICCD.