A 4-year-old girl with a past history of RALF was admitted to our hospital due to nausea and vomiting with a high fever (39.5 C). She had no symptoms of abdominal pain and distention or diarrhea, no chills or convulsions, and no headache, dizziness, diplopia, or blurred vision. The girl was born at full-term and met normal developmental milestones. There was no family history of hereditary or metabolic liver diseases. Physical examination (PE) of the patient showed normal cognitive function without confusion, personality changes, disorientation, or consciousness disturbance. It also revealed mild jaundice, enlarged tonsils, and palpable hepatomegaly (1-2 cm below the costal margin) without splenomegaly. Nervous system examination revealed normal muscle tone and strength, a normal Babinski sign, and negativity for ankle clonus. Laboratory data showed abnormal liver function [direct bilirubin (DB) 24.50 mumol/L (normal range: 0-6.8), total bilirubin (TB) 35.81 mumol/L (normal range: 3.4-17.1), alanine transaminase (ALT) 7169 U/L (normal range: 5-40), aspartate transaminase (AST) 9677 U/L (normal range: 8-40), gamma-glutamyl-transferase (gamma-GGT) 40 U/L (normal range: 7-32 U/L), and albumin 14 g/L (normal range: 38-54 g/L)]. The lactate dehydrogenase [>9480 U/L (normal range: 110-290 U/L)] and blood ammonia [90 mumol/L (normal range: 11-51 mumol/L)] levels were significantly increased. Coagulopathy [prothrombin time (PT) 20.1 s (normal range: 9.8-12.1 s), international normalized ratio (INR) 1.76 (normal range: 0.82-1.25), thrombin time (TT) 22.1 s (normal range: 14-21 s)] and renal dysfunction [serum creatinine (Cre) 524 mumol/L (normal range: 18-66), blood urea nitrogen (BUN) 35.6 mmol/L (normal range: 0-8.3)] were observed. Thyroid function tests were performed, with the results supporting a diagnosis of hypothyroidism [triiodothyronine (T3) 0.81 nmol/L (normal range: 1.34-3.70), thyroxine (T4) 54.04 nmol/L (normal range: 64.30-158.70), free triiodothyronine (FT3) 2.84 pmol/L (normal range: 3.58-6.92), free thyroxine (FT4) 9.35 pmol/L (normal range: 9.60-14.50), and thyroid-stimulating hormone (TSH) 0.10 muIU/ml (normal range: 0.90-4.00)]. The detection of liver-damaging viruses, such as Epstein-Barr virus, cytomegalovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, and hepatitis C virus, was all negative. Ceruloplasmin and alpha1-antitrypsin were all normal. Tandem mass spectrometry (TMS) for metabolic disorder screening was unremarkable. Analyses of autoantibodies, including autoimmune hepatitis-related autoantibodies, were all negative. Mitochondrial gene analysis was carried out, but no abnormalities were observed. Abdominal ultrasound showed hepatomegaly and mild ascites but no splenomegaly. Echocardiography showed mild pulmonary hypertension. The electrocardiogram (ECG) was normal. In addition, no abnormalities were observed by brain magnetic resonance imaging (MRI).
Considering that no etiological diagnosis was reached despite mitochondrial gene sequencing, the patient was subjected to whole-exome sequencing (WES) using genomic DNA extracted from peripheral blood mononuclear cells (PBMCs). Exome capture was carried out using IDTxGen Exome Research Panel (IDT, USA) and paired-end sequencing was performed on HiseqX10 (Illumina, USA). Sequencing data were analyzed to identify sequence variants [single nucleotide variant (SNV), insertion/deletion (Ins/Del)] and copy number variants (CNVs) using an in-house pipeline (Fulgent genetics). A phenotype-driven gene list was created to perform a primary variant interpretation for a more targeted analysis. After data analysis, variant filtering, and prioritization, a novel compound heterozygote mutation, c.3596G> A (p.C1199Y)/ex.9del (p.216-248del) in NBAS was identified (Figure 1A). No other potentially pathogenic mutations were detected in genes predisposing to ALF (e.g., TRMU, OMIM: 613070; MARS OMIM:615486; LARS, OMIM: 615438; RINT1, OMIM: 618641) by WES of the patient. The mutations were verified by Sanger sequencing and quantitative PCR, respectively (Figures 1B,C). Genotyping of the unaffected parents showed the mutation c.3596G> A (p. C1199Y) in the father and the ex.9del (p.216-248del) mutation in the mother. The c.3596G> A (p. C1199Y) mutation has been previously reported in patients with NBAS deficiency, but the ex.9del (p.216-248del) mutation has not been described previously. In silico predictors predict that the variant c.3596G> A (p. C1199Y) is located in a highly conserved amino acid residue (Sec39 domain) that affects the structure/function of the NBAS protein and is defined as pathogenic (SIFT score: 0.000, http://sift.jcvi.org; PROVEAN score: -9.563, http://provean.jcvi.org/ index.php). The ex.9 del (p.216-248del) variant located in the beta-propeller leads to the deletion of exon 9 and is predicted to be deleterious by PROVEAN with a score of -123.936 (Figure 1D). The two variants were classified as likely pathogenic according to the guidelines of the American College of Medical Genetics and Genomics (ACMGG).
To further confirm the liver damage, electron microscopy of the liver biopsy was performed. As shown in Figure 2, hepatocytes were irregular in shape and size with an eccentric nucleus (Figure 2A, 3,000 x, scale bar: 10 mum), capillary bile ducts were dilated, and a large number of microvilli and lysosomes were visible in hepatocytes (Figure 2B, 9,000 x, scale bar: 5 mum). In addition, an increased number of mitochondria with abnormal morphology and an increased density of rough endoplasmic reticulum (ER) were observed (Figure 2C, 9,000 x, scale bar: 5 mum). The patient's clinical symptoms were improved soon by managing with supportive treatment, including renal replacement therapy (CRRT), thyroid hormone supplementation, intravenous antibiotics, and glucose infusion (Table 1). Finally, the patient was discharged with full recovery after 1 month of hospitalization. During the recent 9-month follow-up period, she had complete clinical and biochemical recovery and did not require any additional treatment.