Features of liver injury in 138 Chinese patients with NICCD.

OBJECTIVES: To find biochemical and molecular markers can assist in identifying serious liver damage of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) patients. METHODS: 138 patients under 13 days to 1.1 year old diagnosed of NICCD in our center from 2004 to 2020. Base on the abnormal liver laboratory tests, we divided 138 patients into three groups: acute liver failure (ALF), liver dysfunction, and non-liver dysfunction groups, then compared their clinical, biochemical and, molecular data. RESULTS: 96 % of 138 patients had high levels of citrulline and high ratio of threonine to serine, which is the distinctive feature of plasma amino acid profile for NICCD. A total of 18.1 % of 138 patients had evidence of ALF who presented the most severity hepatic damage, 51.5 % had liver dysfunction, and the remaining 30.4 % presented mild clinical symptoms (non-liver dysfunction). In ALF group, the levels of citrulline, tyrosine, TBIL, ALP, and γ-GT was significantly elevated, and the level of ALB and Fisher ratio was pronounced low. Homozygous mutations of 1,638_1660dup, IVS6+5G.A, or IVS16ins3kb in SLC25A13 gene were only found in ALF and liver dysfunction groups. Supportive treatment including medium-chain triglyceride supplemented diet and fresh frozen plasma could be life-saving and might reverse ALF. CONCLUSIONS: High level of citrulline, tyrosine, TBIL, ALP, γ-GT, and ammonia, low level of albumin, and low Fisher ratio were predictors to suggest severe liver damage in NICCD patients who may go on to develop fatal metabolic disorder. Early identification and proper therapy is particularly important for these patients.

Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism.

The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32Y174C/K235R, Slc25a32K235R/K235R, and Slc25a32-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid ß-oxidation and amino acid metabolism being impaired, Slc25a32-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.

A novel GTPCH deficiency mouse model exhibiting tetrahydrobiopterin-related metabolic disturbance and infancy-onset motor impairments.

BACKGROUND: GTP cyclohydrolase I (GTPCH) deficiency could impair the synthesis of tetrahydrobiopterin and causes metabolic diseases involving phenylalanine catabolism, neurotransmitter synthesis, nitric oxide production and so on. Though improvements could be achieved by tetrahydrobiopterin and neurotransmitter precursor levodopa supplementation, residual motor and mental deficits remain in some patients. An appropriate GTPCH deficiency animal model with clinical symptoms, especially the motor impairments, is still not available for mechanism and therapy studies yet. OBJECTIVES AND METHODS: To investigate whether the heterozygous GTPCH missense mutation p.Leu117Arg identified from a patient with severe infancy-onset dopa-responsive motor impairments is causative and establish a clinical relevant GTPCH deficiency mouse model, we generated a mouse mutant mimicking this missense mutation using the CRISPR/Cas9 technology. Series of characterization experiments on the heterozygous and homozygous mutants were conducted. RESULTS: The expressions of GTPCH were not significantly changed in the mutants, but the enzyme activities were impaired in the homozygous mutants. BH4 reduction and phenylalanine accumulation were observed both in the liver and brain of the homozygous mutants. Severer metabolic disturbance occurred in the brain than in the liver. Significant reduction of neurotransmitter dopamine, norepinephrine and serotonin was observed in the brains of homozygous mutants. Live-born homozygous mutants exhibited infancy-onset motor and vocalization deficits similar to the disease symptoms observed in the patient, while no obvious symptoms were observed in the young heterozygous mutant mice. With benserazide-levodopa treatment, survival of the homozygous mutants was improved but not completely rescued. CONCLUSIONS: The GTPCH p.Leu117Arg missense mutation is deleterious and could cause tetrahydrobiopterin, phenylalanine and neurotransmitter metabolic disturbances and infancy-onset motor dysfunctions recessively. This is the first GTPCH deficiency mouse model which could be live-born and exhibits significant motor impairments. The different extents of BH4 reduction and phenylalanine accumulation observed between liver and brain in response to GTPCH deficiency gives potential new insights into the vulnerability of brain to GTPCH deficiency.