Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency in Korean Infants

The SLC25A13 gene on chromosome 7q21.3 is known to be responsible for adult-onset type II citrullinemia (CTLN2). The SLC25A13 gene consists of 18 exons and encodes a liver-type mitochondrial aspartate-glutamate carrier named citrin (1, 2), which plays an important role in malate-aspartate NADH shuttling, urea synthesis, and gluconeogenesis. CTLN2 is characterized by late-onset hyperammonemia and neuropsychiatric symptoms, such as disorientation, delirium, delusion, and disturbed consciousness (3). Ohura et al. (4) and Tazawa et al. (5) demonstrated that mutations in the SLC25A13 gene cause neonatal intrahepatic cholestasis and transient citrullinemia, which has been designated as neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD; OMIM 605814) (6). NICCD is characterized by multiple amino acidemia (involving citrulline, threonine, methionine, arginine, and tyrosine), galactosemia, hypoproteinemia, hypoglycemia, cholestasis, and fatty liver. Most NICCD patients show ameliorated symptoms within 1 yr of age and require no further special treatment other than feeding programs. However, several decades later some patients develop CTLN2 (7, 8).

Initially, NICCD was identified exclusively in the Japanese population, but recently the disease has been reported in Palestinians (9), Vietnamese (10), and Taiwanese infants (11). Furthermore, a population analysis showed that the carrier rate of mutations in SLC25A13 among Koreans was 1 in 112 (11). There has been no report of NICCD in Korea. In the present study, the authors investigated clinical findings of Korean infants with NICCD and performed mutation analysis on the SLC25A13 gene.

Among the 47 patients with neonatal cholestasis presented during the two-year period, extrahepatic biliary atresia was diagnosed in 11, congenital infection with TORCH in 4, progressive familial intrahepatic cholestasis (PFIC) in 2, ARC (arthrogryposis, renal dysfunction, cholestasis) syndrome in 2, neonatal Dubin-Johnson syndrome in 2, Alagille syndrome in 1, and non-syndromic bile duct paucity in 1. No etiology was identified in 21 patients (Table 1).

Table 1
Differential diagnosis of neonatal cholestasis

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NICCD was diagnosed in three patients. Table 2 summarizes the genotype and clinical and laboratory findings of these three patient. Birth weights ranged from 2.7 to 3.1 kg. The chief complaint was cholestatic jaundice in all patients, and all were referred for suspected neonatal hepatitis or biliary atresia. Two infants were referred at 3 months of age and one infant at 7 months. Two infants showed failure to thrive at initial presentation. Newborn tandem mass screening performed on case 2 at 1 week of age was normal. Total serum protein levels were reduced in two patients. Blood ammonia levels were slightly increased in all three, and gamma glutamyltranspeptidase and alkaline phosphatase were markedly elevated in all three, whereas alanine aminotransferase levels were slightly increased in two. Hypoglycemia was present in case 1. Alpha-fetoprotein levels were high and galactosemia was detected in all patients. Plasma amino acid analyses showed that citrulline, methionine, and arginine were significantly elevated in all three. Plasma tyrosine, threonine, and lysine were elevated in two (Table 3). No patient had a bleeding tendency or a cataract.

Table 2
Clinical and laboratory findings, and the genotypes of the 3 NICCD patients

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Table 3
Plasma amino acid and galactose levels in the 3 NICCD patients

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Liver biopsy was performed in two patients. Cholestasis and micro-macrovesicular fatty change were observed in these two, and moderate periportal fibrosis was present in case 3.

Five of the six alleles (83%) examined showed mutations. In detail, compound heterozygous 1,638-1,660 dup/S255X mutation (mutation [III]/[IV]), compound heterozygous 851del4/ S225X mutation (mutation [I]/[IV]), and heterozygous 1,638-1,660 dup mutation (mutation [III]) were found in each patient. The allele frequencies of mutations [I], [III], and [IV] were 17%, 33%, and 33%, respectively.

Lactose-free soy formula and a medium chain triglyceride (MCT) formula were fed to all patients. Fat-soluble vitamins were added. Within 2 months of diagnosis, plasma amino acid profiles were normalized, whereas liver functions were nearly normalized at the age of 9 to 13 months. At the age of 18 months, catch-up growth had been achieved by all three patients.

Citrullinemia is categorized as CTLN1 (classical neonatal onset) or as CTLN2 (adult onset). CTLN1 is an autosomal recessive disease that is caused by a deficiency of arginiosuccinate synthetase, and is characterized by a neonatal-onset and severe hyperammonemia, irritability, lethargy, poor feeding, and tachypnea (13). On the other hand, CTLN2 is an autosomal recessive disease caused by citrin deficiency, and is characterized by late onset (11 to 79 yr), frequent attacks of hyperammonemia, and mental derangement, and leads to death within a few years from the onset (3). The SLC25A13 gene, which encodes citrin, has been found to be defective in CTLN2. In the liver, citrin participates in the urea cycle by supplying aspartate from mitochondria to the cytosol for the incorporation into argininosuccinate. Lack of aspartate for argininosuccinate synthesis under citrin deficiency probably causes citrulline accumulation (14). Previous DNA analyses have shown that some infants with idiopathic neonatal hepatitis harbor the same mutations in SLC25A13 as CTLN2 patients (4, 5), and these infants had cholestasis, hyperaminoacidemia, galactosemia, hypoproteinemia, and hepatic steatosis. On newborn tandem mass screening, about a half of NICCD patients were detected because of high citrulline, galactose, phenylalanine, and/or methionine concentrations (15, 16).

In the present study, NICCD accounted for approximately 6% (3/47) of our cohort of neonatal cholestatic liver disease. Other hereditary intrahepatic cholestasis including PFIC, Alagille syndrome, ARC syndrome, and Dubin-Johnson syndrome were found in 15%. The study shows that genetic intrahepatic cholestasis is a common cause of neonatal cholestasis in Korean infants. The initial evaluations of infants with cholestasis include ultrasonography, duodenal intubation, and percutaneous liver biopsy, in order to exclude biliary atresia. Liver biopsy is also useful for detecting Alagille syndrome, Dubin-Johnson syndrome, and PFIC. Serum γ-GT is also helpful because low levels of γ-GT in the presence of cholestasis suggest PFIC or ARC syndrome. Our data suggest that plasma amino acid analysis is useful for the screening for metabolic diseases, such as NICCD.

In the present study, NICCD patients presented with cholestatic jaundice and failure to thrive. Laboratory findings showed mild hyperammonemia, hypoproteinemia, and galactosemia. Citrin deficiency blocks the malate-aspartate shuttle, which may increase the cytosolic NADH-to-oxidized nicotinamide adenine dinucleotide ratio (NADH/NAD⁺). Since uridine diphosphate-galactose epimerase, which requires NAD⁺ as a cofactor, is strongly inhibited by NADH, a high level of NADH in liver cytosol may underlie the etiology of galactosemia in NICCD (14).

Hypoglycemia was detected in one patient. Citrin functions to provide substrates for gluconeogenesis as a component of the pathway responsible for converting amino acids to glucose (3). In the present study, plasma amino acid analysis showed that the concentrations of citrulline, methionine, threonine, arginine, and/or tyrosine were elevated. One of our three patients did not show an abnormal amino acid profile on newborn mass screening at 7 days of age. Tandem mass screening data in Japan suggest that the incidence of NICCD is approximately 1 in 34,000 (15), but the frequency of homozygotes with mutated SLC25A13 has been estimated to be 1 in 19,000 (17). Tamamori et al. (16) demonstrated that NICCD patients with negative newborn mass screening findings have lower total amino acid levels, and that citrulline/total amino acids ratios can offset the effect of low total amino acids.

This is the first report of genetically confirmed NICCD in a Korean cohort. The mutation rate observed in three NICCD patients was 83%, which is similar to that reported in Taiwanese patients (18). In the present study, the allele frequencies of mutation [I], [III], and [IV] were 17%, 33%, and 33%, respectively. Screening of 12 mutations in Korean control individuals showed that mutation [I] and [II] were found to be most common, and mutation [III] accounted for 5% of total mutations, but mutation [IV] was absent (11). A Japanese study found that the frequency of mutation [IV] differs in NICCD patients and controls (6). However, since the number of patients with NICCD was comparatively small in the present study, further mutation studies are needed. To date only one adult case of CTLN2 has been reported in Korea (19); the patient presented with insomnia and lethargy at 55 yr of age. However, the theoretical frequency of homozygotes calculated from carrier rates in Koreans is 1 in 50,000 (11), which suggests that NICCD and CTLN2 are substantially under diagnosed in Korea.

The management of NICCD is directed toward treating the consequences of cholestasis and galactosemia. MCT formula and supplementation with fat-soluble vitamins have been used to prevent complications of prolonged cholestasis. It should be noted that lactose may be toxic to NICCD patients and should be avoided while cholestasis persists (20).

The prognosis of our patients was good because liver functions were normalized before 13 months of age and catch-up growth was achieved at 18 months. The amelioration of NICCD symptoms over a year suggests hepatocyte maturation and some adaptation or compensation by other mitochondrial carriers (14). One of our three patients showed moderate periportal fibrosis on liver histology. Mild to moderate portal fibrosis has been previously observed in NICCD (5, 7). Liver function was normalized in this patient, but his clinical course needs to be carefully monitored. One NICCD patient developed liver failure and underwent liver transplantation at 11 months of age (21). It should be noted that some NICCD patients develop CTLN2 at their twenties or even several decades later (7, 8). Although the prognosis of CTLN2 is poor, liver transplantation is remarkably effective (3). As yet, it cannot be determined which NICCD patients will later develop CTLN2, and thus, all NICCD patients should be followed for signs of development of CTLN2.

The present study indicates that NICCD should be considered in the differential diagnosis of cholestatic jaundice in Korean infants, and that plasma amino acid analysis should be included in the evaluation of infantile intrahepatic cholestasis. Moreover, the findings of SLC25A13 genetic studies were found to be useful for confirming the diagnosis of NICCD.