Mutation Spectrum And Clinical Features In Infants With Intrahepatic Cholestasis Caused By Citrin Deficiency

Cholestasis is one of the commonest presentations of liver disease in infancy affecting about one in every 2500-5000 infants. Despite extensive investigations, the etiology of cholestasis in a significant proportion of newborn babies remains unknown, which lead to the unclear diagnosis, unsuitable management and poor prognosis in some patients.Neonatal intrahepatic cholestasis caused by citrin deficiency was first identified by Ohura et al in Japan. The disease causing gene is the SLC25A13 gene, which locates on chromosome 7q21.3,160 kb in length, consists of 18 exons and encodes a 3.4 kb transcript. It is expressed ubiquitously but most abundantly in the liver. To date, more than 50 mutations have been identified. All mutations except P632L are pathogenic.Citrin deficiency was thought to be restricted to the Japanese population when it was first reported in Japan. However, recent studies indicated that the disease may be distributed worldwide, especially in the East Asian region. More than 100,000 individual may be homozygous of SLC25A13 mutations in the total population of East Asia. Mutations 851de14 and IVS11+1G>A were two of the most prevalent mutations in Japan and Korea. So far, a few Chinese cases of NICCD have been reported. Details on the spectrum of SLC25A13 gene mutation in Chinese infants with intrahepatic cholestasis are still under investigation.Citrin protein, consisting of 675 amino acid residues with a molecular weight of 74 kDa and harboring four EF-hands and six mitochondrial transmembranous (TM) spanners, has been identified as a mitochondrial aspartate-glutamate carrier protein, which transports aspartate into the cytoplasm to take part in the synthesis of urea, protein and nucleotide, meanwhile citrin protein transports glutamic acid to mitochondria for the participation of malate-asparate shuttle. Mutations of SLC25A13 lead to the truncation or premature of citrin protein, affect the biochemical metabolism and cause hypoglycemia, galactosemia and fatty liver and other symptoms including intrahepatic cholestasis, mild liver dysfunction, multiple aminoacidemia, coagulation disorders and/or high levels of plasmaα-fetoprotein. Although the symptoms of most NICCD cases may be relieved by 12 months of age spontaneously or after dietary adjustment, liver failure may occur and liver transplantation was required in a small proportion of them in early life. In less fortunate cases, CTLN2 may develop one decade or more later and may lead to death if treated inappropriately. Early diagnosis of NICCD may prevent progressing to CTLN2 by dietary adjustment, or prevent serious consequences by close follow-up and timely treatment before the onset of symptoms. Because symptoms of NICCD are transitory and complex, it is not so easy to establish definite clinical diagnostic criteria, and the best diagnostic test of NICCD is the genetic test.In this study, we explored the mutation spectrum of the SLC25A13 gene and found the more prevalent mutation types in Chinese infants with intrahepatic cholestasis, which may be used as uslful sereening tool. We screened the two common mutations of the SLC25A13 gene in infants with intrahepatic cholestasis, and found that SLC25A13 gene mutation is one of the common reasons of infantile intrahepatic cholestasis. Mutation frequency has the geographical specificity in China. The clinical features of NICCD were analyzed in the present study. Various aminoacidemia, fatty liver, multiple abnormal biochemistry parameters may provide clues for diagnosis of NICCD.Part I Mutation spectrum of SLC25A13 gene in Chinese infants with intrahepatic cholestasis and aminoacidemiaBackground. SLC25A13 gene mutations cause citrin deficiency, which leads to neonatal intrahepatic cholestasis. Information on the mutation spectrum of SLC25A13 in Chinese is limited. The aim is to explore the mutation spectrum of the SLC25A13 gene in Chinese infants with intrahepatic cholestasis and aminoacidemia.Methods. Sequence analyses were performed on 39 infants with intrahepatic cholestasis and various aminoacidemia. The known large fragment mutations Exl5dup (IVS14₁5), IVS16ins3kb, and Ex16+74_IVS17-32del516 were tested as reported previously. Homology and structural predictions were analyzed for the novel mutations. Western blotting was performed in the cases with liver specimens available.Results. SLC25A13 gene mutations existed in 28 cases, including 6 homozygotes, 13 compound heterozygotes and 9 heterozygotes. At least 19 cases were confirmed as citrin deficiency by genetic tests and Western blot analysis, account for 48.7% of the total 39 subjects. Eleven types of mutation including 8 known mutations and 3 novel mutations were found. Out of 46 mutated alleles, the known mutations include 851de14 in 26 alleles (56.5%),1638ins23 in 6 alleles (13.0%), IVS6+5GA in 2 alleles (4.3%), E601K in 2 alleles (4.3%), IVS11+1G>A in 1 allele (2.1%), R184X in 1 allele (2.1%), R360X in 1 allele (2.1%) and R585H in 1 allele (2.1%). The three novel mutations were splice site change IVS6+1G>A, deletion mutation 1092₁095delT and missense mutation L85P each in 1 allele.Conclusion. SLC25A13 gene mutation is the most important cause of infantile intrahepatic cholestasis with multiple aminoacidemia including citrullinemia. Mutation spectrum of the SLC25A13 gene in Chinese is different from that of other population groups in East Asia.Partâ…¡Most common SLC25A13 mutations in Chinese infants with intrahepatic cholestasisAims To investigate the frequency of the common SLC25A13 gene mutations, geographical distribution of the mutations and the importance of SLC25A13 gene mutation in Chinese infants with intrahepatic cholestasis.Methods 361 infants with unexplained intrahepatic cholestasis from 18 provinces or municipalities in China, including 155 infants without obvious aminoacidemia and 206 infants who did not performed MS analysis according to the clinical situation, were enrolled in this study. The two common mutations,851del4 and 1638ins23, were screened by real time fluorescent Polymerase Chain Reaction with dual-labled probes and agarose gel electrophoresis, respectively. If a patient was found to be a heterozygote, then all the 18 exons and their flanking regions were tested by directed sequencing to find the second mutated allele.Results Among the infants without obvious plasma amino acid,21 mutated alleles were found, including 3 homozygotes with 851de14,2 compound heterozygotes with 851del4/1638ins23 and 851del4/1092₅delT and 11 heterozygotes with 851de14. In infants who were not performed MS analysis,8 mutated alleles including 1 homozygote 851de14,1 compound heterozygote with 851de14/1638ins23,3 heterozygotes with 851de14 and 1 heterozygotes with 1638ins23 were found. Combined the results from the part one,77 mutated alleles were detected in 800 tested alleles, the frequency of mutation was 1/11 (77/800).41,34 and 2 mutated alleles were detected in the 474 alleles from Yangtze river area,242 alleles frome the south area and 84 alleles from the north area of China, respectively.Conclusion SLC25A13 gene mutation is one of the common causes of infants with intrahepatic cholestasis. NICCD can be detected from infats without aminoacidemia. SLC25A13 gen mutation has the geographical variation in China.Partâ…¢Clinical features of 27 infants with NICCDAims To study the plasma amino acid pattern, biochemistry parameters and the pathological change of NICCD and provide clues for the clinical diagnosis of NICCD.Methods 27 cases with NICCD confirmed by gene tests or Western blotting,39 cases with biliary atresia (BA) confirmed by Hepatobiliary Imino-Diacetic Acid (HIDA) and intraoperative cholangiography and 266 cases with idiopathic neonatal hepatitis (INH) were enrolled in our study. Plasm amino acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), international normalized ratio (INR), fasting blood glucose (GLU), Total protein (TP), albumin (Alb), triglyceride (TG), total cholesterol (Tch), high density lipoprotein (HDL), low density lipoprotein (LDL) and liver histopathology were analyzed. The data were compared on NICCD and INH, NICCD and BA, respectively. Statistical analysis was performed using the stata 10.0. Descriptive statistics were used for demographic and baseline data and summarized as medians,25th and 75th percentiles. The histology study between NICCD and INH was assessed by using Fisher's exact test. Clinical and biological continuous parameters were compared with the Wilcoxon rank sum tests. A P value<0.05 was considered statistically significant.Results Compared with the BA group and INH group, NICCD group has higher plasma citrulline, tyrosine and methionine, higher ratios of citrulline, tyrosine and methionine each to the total amino acid, lower alanine aspartic acid, tryptophan, glutamic acid, lower ratios of alanine, valine, leucine, tryptophan, glutamic acid each to the total amino acid, mild elevation of ALT, AST, remarkable elevation of the ratio of AST/ALT, higher TBA, lower TB/TBA, DB/TBA, TP, Alb and Glu. The ratios of ALT to GGT and AST to GGT in NICCD is lower than INH, but the ratio of AST to GGT in NICCD was higer than BA. Percutaneous liver biopsy was performed in 15 NICCD and showed steatosis in every cases.Discussion The abnormal amino acid pattern, multiple abnormal biochemical parameters and fattey liver may provide valuable clues for the diagnosis of NICCD.CONCLUSIONS1. SLC25A13 gene mutation is the most important cause of infants with intrahepatic cholestasis and aminoacidemia. 851de14 and 1638ins23 are the common mutations. Three novel mutations were found.2. The frequency of SLC25A13 genen mutation is 1/11 in Chinese infants with intrahepatic cholestasis. NICCD can not be excluded in ifants without aminoacidemia. Patients with mutated alleles mainly exist in the border of Yangtze river and the southern region of mainland China..3. The abnormal amino acid pattern, multiple abnormal biochemical parameters and specific histopathological chang and may provide valuable clues for the diagnosis of NICCD.