The patient was a 4-year-and-11-month-old Chinese boy who was admitted to our hospital due to increased liver transaminase for over two months. About two months previously (on May 19), the patient received a physical examination, which revealed that he had an abnormal liver function with increased alanine transaminase (ALT 296 U/L), aspartate aminotransferase (AST 185 U/L), and γ-glutamyl transferase (GGT 86.7 U/L) but normal bilirubin, albumin, and globulin. He did not receive any treatment. A re-examination on July 15 showed elevated biochemical parameters (ALT 342 U/L, AST 239 U/L, and GGT 88 U/L), and normal bilirubin, albumin, and globulin. Consequently, the patient was hospitalized in a local hospital for 12 days with combination therapy including diisopropylamine dichloroacetate, glycyrrhizin, creatine phosphate sodium, and methionine and vitamin B1 for injection, However, liver function abnormalities persisted even after the treatment. He was then referred to our hospital for further treatment.
The patient’s guardians denied a history of liver disease, hepatotoxic medication, exposure to trematode epidemic areas, or a family history of metabolic disorders. Physical examination showed the patient had no yellowing of the skin and sclera all over the body. His liver was 3.5 cm below the right costal arch. The patient had no neurologic abnormality on physical examination. Laboratory tests showed elevated liver transaminase levels, mild anemia, and moderate hypouricemia (Table 1). The complete blood count revealed decreased serum hemoglobin of 105g/L, granulocyte of 35.5 × 109/L, increased lymphocyte counts of 3.36 × 109/L, and basophil levels of 1.5 × 109/L. Liver function tests revealed elevated ALT, AST, GGT, lactate dehydrogenase, α-L-fucosidase, lactic acid, and plasma uric acid levels (Table 1). His coagulation tests showed increased prothrombin time (PT) and international normalized ratio (INR) of 14.7s (normal range, 11.0-14.3s) and 1.19 (normal range, 0.08–1.15), respectively, while the prothrombin time activity (of 77.0%) was decreased. The serum ceruloplasmin level was decreased, to 0.221 m/L, and his 24-h urinary copper excretion was significantly elevated (over 600 µg/day). Serological tests were negative for viral agents (hepatitis A, B, C, and E virus antibodies) and autoimmune markers such as antinuclear antibody profile, anti-double-stranded DNA antibody, anti-neutrophil cytoplasmic antibody, and autoimmune liver disease-related antibodies were negative.
Table 1
Laboratory findings in the patient with WD in this study
White blood cell count (10^9/L) (3.5–9.5) | 6.15 | ALT (U/L) (4–41) | 367 ↑ |
% granulocyte (40–75) | 35.3 ↓ | AST (U/L) (4–40) | 235 ↑ |
# granulocyte (10^9/L) (1.8–6.3) | 2.17 | Total protein (g/L) (60–80) | 65.5 |
% lymphocyte (20–50) | 54.6 ↑ | Albumin (g/L) (38–54) | 41.9 |
# lymphocyte (10^9/L) (1.1–3.2) | 3.36 ↑ | Globulin (g/L) (20–35) | 23.6 |
% monocyte (3–10) | 5.5 | Prealbumin (mg/L) (116–282) | 281 |
# monocyte (10^9/L) (0.1–0.6) | 2.17 | Total bilirubin (umol/L) (3.4–20.5) | 7.8 |
% eosinophil (0.4-8) | 3.1 ↓ | Direct bilirubin (umol/L) (0–6.84) | 2.6 |
# eosinophil (10^9/L) (0.02–0.52) | 0.19 | Indirect bilirubin (umol/L) (≤ 13.3) | 5.2 |
% basophil (0–1) | 1.5 ↑ | ALP (U/L) (1–269) | 243 |
# basophil (10^9/L) (0-0.06) | 0.09 ↑ | GGT (U/L) (10–71) | 85 ↑ |
Red blood cell count (10^12/L) (4.3–5.8) | 4.03 ↓ | Creatine kinase (U/L) (3–190) | 100 |
Hemoglobin (g/L) (130–175) | 105 ↓ | LDH (U/L) (120–300) | 419 ↑ |
Hematocrit (%) (40–50) | 31.0 ↓ | Total bile acid (umol/L) (1–10) | 9.9 |
Mean corpusular volume (fL) (82–100) | 76.9 ↓ | 5'- Nucleotidase (U/L) (2–11.4) | 5.2 |
Mean corpusular hemoglobin (pg) (27–34) | 26.1 ↓ | α-L-fucosidase (IU/L) (4–40) | 50 ↑ |
MCHC (g/L) (316–354) | 339 | Cholinesterase (U/L) (5320–12920) | 5979 |
RDW-CV (%) (< 14.9) | 12.8 | Hypersensitive C-reactive protein (mg/L) (0.1–3) | 0.3 |
RDW-SD (fL) (39–46) | 35.7 ↓ | Lactic acid (mmol/L) (0.5–2.2) | 2.93 ↑ |
Platelet count (10^9/L) (125–350) | 300.0 | Pyruvicacid (umol/L) (20–100) | 40.5 |
Platelet distribution width (fL) (9–17) | 12.0 | Ammonia (umol/L) (18–72) | 41 |
Mean platelet volume (fL) (8–15) | 10.2 | Urea (mmol/L) (1.7–8.3) | 3.93 |
Platelet-large cell ratio (%) (13–43) | 26.8 | Crea (umol/L) (59–104) | 24 ↓ |
Plateletocrit (%) (0.1–0.25) | 0.31 ↑ | Uric acid (umol/L) (202.3–416.5) | 130.8 ↓ |
K+ (mmol/L) (3.5–5.1) | 4.11 | HCO3-(mmol/L) (22–29) | 21.7 ↓ |
Na+ (mmol/L) (136–145) | 139.3 | Total cholesterol (mmol/L) (2.9–5.2) | 3.53 |
Cl− (mmol/L) (98–110) | 101.3 | Triglyceride (mmol/L) (0.05–1.7) | 0.91 |
Ca 2+(mmol/L) (2.15–2.55) | 2.36 | High-density lipoprotein (mmol/L) (1.1–1.9) | 1.29 |
corrected Ca (mmol/L) (2.15–2.57) | 2.46 | Low-density lipoprotein (mmol/L) (0.03–3.12) | 1.90 |
Prothrombin time (s) | 14.7 | Ceruloplasmine (mg/L) (> 200) | 0.221 ↓ |
The patient’s ultrasonography showed hepatomegaly and the lower boundary of the liver was located 3.0 cm below the right costal arch, without abnormalities in the main portal vein or its branches, or the bile ducts. His color Doppler flow imaging in the portal system was normal. A liver autopsy revealed the lobular structure of the liver was roughly preserved, with extensive hydrodegeneration-like mild steatosis of hepatocytes and mild proliferation of confluent fibrous connective tissue with some chronic inflammatory cell infiltration; electron microscopy of liver tissue showed mild swelling of hepatocytes, with no significant changes in the nuclei; rough endoplasmic reticulum was decreased and smooth endoplasmic reticulum was proliferated with increased glycogen content in hepatocytes. High-density electron-dense granules were observed in some mitochondria and a variable number of neutral lipid droplets and cholestatic pigment granules were found in some hepatocytes. Collagen fibril deposition was found regionally. Gas chromatography-mass spectrometry (GC/MS) analysis of urine organic acids showed a medium-to-large amount of hippuric acid and a moderate amount of α-ketopentanoic acid and 4-hydroxyhippuric acid; hydroxylcarnitine (C5-OH), pentenoylcarnitine (C5:1), hexanoylcarnitine (C6), octanoyl carnitine (C8), decenoylcarnitine (C10:1), dodecanoylcarnitine (C12), and tetradecenoylcarnitine (C14:1) levels were higher than normal. MRI of the brain and a slit-lamp exam were unremarkable, excluding the presence of neurological impairment and KF rings. The patient was then diagnosed with liver dysfunction and hepatomegaly for unknown reasons.
Whole-exome sequencing (WES) revealed compound heterozygous mutations NM_000053.4: c.2333G > T (p.R778L) and c.2514G > T (p.K838N) in the ATP7B gene (Fig. 1A, 1B), in which R778L is a recurrent hot-spot mutation in Chinese patients, according to previous reports (Fig. 1C). ATP7B K838N, located in the actuator domain, was never documented in previous literature and databases and it was likely pathogenic (PM2 + PM3 + PM5 + PP3), according to the American College of Medical Genetics and Genomics (ACMG) clinical practice guidelines (Fig. 1C, 2A). In-silico analysis showed the mutations were evolutionarily conserved in multiple species (Fig. 2A); simulation analysis based on the three-dimensional model of ATP7B revealed altering hydrogen bonds by the mutations (Fig. 2B).
Based on the above findings, the patient was diagnosed with WD. He was then treated with a copper-restricted diet, zinc acetate, and penicillamine initiated at the time of his final diagnosis. After three months of the treatment, the patient’s liver function was normal.