Novel diagnostic approach to citrin deficiency: Analysis of citrin protein in lymphocytes

Abstract

Citrin deficiency induces two clinical features; namely neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and adult-onset type II citrullinemia. Hypercitrullinemia is the most characteristic feature, whereas there are non-citrullinemic individuals. Diagnosis of citrin deficiency is performed by genetic analysis, although the 12 known mutations in the alleles are not detected in about 15% of cases. Thus, we aimed to examine citrin protein in lymphocytes isolated from peripheral blood as an alternative diagnostic method. We examined 38 children having an episode of cholestatic liver dysfunction, 8 heterozygotes, and 11 healthy individuals. All subjects were evaluated for citrin protein by Western blotting and for the 12 known mutations by gene analysis. Citrin protein was detected in 15 of 38 children with cholestatic liver dysfunction. Fourteen of them were negative for 12 known mutations in both alleles, whereas one patient was found to have a known mutation in one allele. Citrin protein was absent in 23 of the 38 patients. Among these 23, gene analysis diagnosed citrin deficiency in 19, whereas 2 patients were later revealed to be NICCD with novel mutations. In the remaining 2 patients, who exhibit the clinical features of NICCD, a known mutation was detected in one allele but no mutation was identified in another allele. Citrin protein was also detected in the 8 heterozygotes and 11 healthy individuals. We disclosed that citrin was deficient in lymphocytes among patients with citrin deficiency. Analysis of citrin is useful to diagnose citrin deficiency even in patients without known mutations or hypercitrullinemia.

Introduction

Adult-onset type II citrullinemia (CTLN2; OMIM 603471) is characterized by a liver-specific argininosuccinate synthetase (ASS) deficiency with no abnormalities in the ASS gene [1], [2]. Most CTLN2 patients suffer from a sudden disturbance in consciousness associated with flapping tremor, disorientation and restlessness, and the majority die within a few years of onset, mainly due to cerebral edema [1], [3].

In 1999, Kobayashi et al. identified mutations in SLC25A13 as the cause of CTLN2 and named the protein encoded by the gene citrin [4]. In 2000, Yasuda et al. found complete loss of citrin in the liver of CTLN2 patients with SLC25A13 mutations by Western blot analysis using anti-citrin antibody [5]. Citrin is an aspartate glutamate carrier (AGC) located on the inner membrane of mitochondria, and plays a role in the malate–aspartate NADH shuttle and urea synthesis. Deficiency of citrin leads to an increased NADH/NAD⁺ ratio in the cytosol, and the failure of aspartate supply from the mitochondria to the cytoplasm for synthesis of argininosuccinate in the ASS reaction, which results in high blood levels of citrulline and ammonia [6], [7], [8].

The recent establishment of genetic analysis for SLC25A13 mutations has revealed that citrin deficiency causes not only CTLN2 but also a type of neonatal hepatitis [9], [10], [11], [12], [13], [14]. This type of neonatal hepatitis presents as cholestasis with hypercitrullinemia and has been designated NICCD (neonatal intrahepatic cholestasis caused by citrin deficiency; OMIM 605814). Most cases of NICCD present as intrahepatic cholestasis with jaundice, fatty liver, hypoglycemia and aminoacidemia (involving citrulline, threonine, methionine, tyrosine, and arginine), and are usually resolved by the age of 1 year, with or without supportive treatment [14], [15], [16]. However, a few NICCD patients have a severe form of the disorder involving liver damage and require liver transplantation [14]. Moreover, some neonatal NICCD patients develop severe CTLN2 more than a decade or several decades later [7], [11].

In the differential diagnosis of cholestatic liver dysfunction, one of the most valuable biochemical marker is plasma citrulline concentration. However, hypercitrullinemia is sometimes absent in individuals with SLC25A13 mutations in both alleles [17], [18], [19]. In our previous study, we experienced a case of NICCD without hypercitrullinemia so far in his life and found that the ratio of citrulline to serine has increased inspite of normal citrulline levels [17]. We indicated that the citrulline/serine ratio may offset the influence of a reduction in total blood amino acid levels [17]. Based on the previous reports, normal citrulline levels cannot exclude the possibility of citrin deficiency. Moreover it is difficult to assess a child over 1 year of age by citrulline levels who has an episode of cholestatic liver dysfunction in infancy, because hypercitrullinemia in NICCD is generally resolved by 12 months of age. Early identification of citrin deficiency is important for appropriate management to prevent liver failure in CTLN2 and NICCD. Most recently, Lu et al. found that citrin gene analysis can identify 89% of mutant alleles in NICCD and CTLN2 by detection of 12 known mutations [20]. This suggests that gene analysis is necessary to diagnose citrin deficiency, but in actual fact is unable to identify approximately 15% of compound heterozygotes or homozygotes carrying SLC25A13 mutations in both alleles.

In this study, we aimed to establish an alternative diagnostic method by identifying citrin protein. Previous studies have reported that citrin protein is detected not only in liver but also in other tissues, such as kidney and heart [5], [21], [22], [23], [24]; however, expression of citrin protein in lymphocytes has not been previously examined. We evaluated whether citrin protein is detectable in lymphocytes by Western blot analysis and whether examination of citrin protein in lymphocytes is a practical diagnostic method for citrin deficiency.