PMC7838493_01
Identification of compound heterozygous mutations in the NBAS gene in the family. (A) c.3596G>A (p.C1199Y) mutation was found by WES. (B) Sanger sequencing showed that the mutation c.3596G> A (p.C1199Y) was inherited from her father. (C) Validation of ex.9del (p.217-249del) mutation by quantitative PCR. Genomic DNA was extracted from PBMCs of the patient, her parents, and healthy control. Reactions were set up using one set of primers upstream (Forward: 5'-GCTGTGTTGATTCCATGAGGA-3') and downstream (Reverse: 5'-AGTGTTGGAACAAATCAGAGCTA-3') of exon 9 in triplicate. The beta-actin gene served as reference gene. The 2-DeltaDeltaCT method was used to calculate the copy number. Sample with normalized ratio (NR) <0.1 denotes homozygous deletion individual, sample with NR about 0.5 denotes heterozygous deletion individual, sample with NR about 1 denotes normal individual (two copies), and sample with NR about 1.5 or more denotes copy number gain individual. (D) The location of two variants in NBAS protein: c.3596G>A (p.C1199Y) locates in C-terminal beta-propeller region, and ex.9del (p.217-249del) mutation locates in the Sec39 domain.
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F1
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). 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).
Electron microscopy of liver biopsy. (A) Hepatocytes are irregular in shape and size, and the nucleus is eccentric (3,000 x, scale bar: 10 mum). (B) Part of the capillary bile duct is dilated; a large number of microvillous and lysosomes are visible (9,000 x, scale bar: 5 mum). (C) Hyperplasia of collagen fibers can be seen in liver cells, with cross-sections (*). The picture also presents with an increased number of mitochondria with abnormal morphology ( ) and an increased density of rough endoplasmic reticulum ( ) (9,000 x, scale bar: 5 mum).
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F2
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).