Neonatal Intrahepatic Cholestasis Caused By Citrin Deficiency(NICCD): Clinical, Genetic And Functional Analysis Of A Family

Objective: Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency(NICCD) is a Mendelian disorder arising from biallelic SLC25A13 mutations. Due to the lack of well-recognized clinical/biochemical diagnostic criteria, the definite diagnosis of NICCD relies on SLC25A13 genetic analysis. However, the pathogenicity of novel missense mutations remains an unresolved issue. Although various bioinformatic tools have been utilized for the pathogenicity prediction of missense mutations, the prediction results sometimes are inconsistent when using different tools, and the results can not reflect precisely the extent to which the citrin function is impaired.This study aims to investigate the clinical, genetic and functional characteristics of a NICCD pedigree, so as to provide scientific evidences for the diagnosis of the NICCD patient and for the prenatal diagnosis in the family.Methods: The research subjects in this study were a male infant suspected to have NICCD and his parents. The history, clinical manifestations, biochemistry alterations,and screening results of urinary analysis via Gas Chromatography-Mass Spectrometry(GC-MS) as well as blood analysis using Mass Spectrometry-Mass spectrometry(MS-MS) for the NICCD infant were collected and analyzed, and were described as a case report. Genetic analysis of the family was carried out by high-frequency mutation screening and Sanger sequencing of the SLC25A13 gene. In addition,recombinant mutant plasmid, recombinant plasmid harboring the entire ORF of SLC25A13 gene, and empty vector plasmid were transformed into the yeast strain(agc1Δ) with abrogation of agc1, the gene homologous to human SLC25A13,respectively, and after 96 hours of culturing, the OD600 values of every agc1Δ strain group were measured to analyze their growth abilities. The functional effect of the missense mutation on citrin protein was explored by comparing the growth ability differences among different groups.Results: A male infant, aged 6 months and 19 days, was referred to our hospital due to prolonged jaundice over 6 months. Physical examination revealed mildly jaundiced skin and hepatomegaly. Biochemical analysis unveiled elevated serum levels ofalanine aminotransferase(ALT), aspartate aminotransferase(AST), gamma-glutamyl transpeptidase(GGT), alkaline phosphatase(ALP), total bile acids(TBA), along with total bilirubin(Tbil) and direct bilirubin(Dbil). MS-MS analysis of the blood sample revealed remarkable elevation of citrulline, tyrosine and methionine. On GC-MS analysis of the urine sample, 4-hydroxylphenyllactic acid,4-hydroxyl phenylpyruvic acid and 3-hydroxyphenyllactic acid were elevated. SLC25A13 genetic analysis uncovered that the infant was a compound heterozygote of the mutations c.851del4 and c.103A>G(p.M35V), which was inherited from the mother and father,respectively. Functional analysis indicated that the p.M35 V mutation eliminated the citrin function.Conclusion: In this study, the main clinical presentation of the NICCD infant was cholestasis accompanied by relatively specific metabolome changes. Genetic analysis revealed that the infant harbored a maternal mutation c.851del4 and a paternal novel missense mutation c.103A>G(p.M35V). The pathogenicity of the novel missense mutation, and the extent to which the citrin function was impaired as well, were confirmed by functional analysis. This paper further enriched the SLC25A13 mutation spectrum, providing scientific evidences for the definite diagnosis of the NICCD patient and for the prenatal diagnosis in the family.