Infantile ATP7B-Related End-Stage Liver Disease: An Exceptional Wilson Disease Phenotype From Consecutive Generations

Supplemental Digital Content is available in the text.


INTRODUCTION
Wilson disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B gene, encoding a copper-transporting adenosine triphosphatase responsible for the ion excess handling and biliary excretion. Copper accumulation and toxicity cause liver disease and multiorgan injury, typically presenting after 3 years of age (1).
We describe a girl with neonatal cholestasis progressed to cirrhosis and end-stage liver disease. Child-parents urgent whole exome sequencing (WES) revealed ATP7B biallelic mutations in the child and her mother, supporting the concomitant diagnosis of WD in consecutive generations. We provide clinical, biochemical, and proteomic data supporting the pathogenic role of ATP7B impairment in this patient, possibly in addition with the prenatal copper overload.

CASE REPORT
A Bosnian female infant born to nonconsanguineous parents presented with jaundice in ABO incompatibility, and developed cholestasis with hepatosplenomegaly. Family and pregnancy history were unremarkable. She had normal gamma-glutamyltranspeptidase, high serum bile acids, aspartate aminotransferase > alanine aminotransferase. A liver biopsy at 2 months of age showed diffuse giant cell transformation, canalicular cholestasis, moderate lobular activity, minimal steatosis, extramedullary hematopoiesis (Fig. 1A). She was started on ursodeoxycholic acid 20 mg/kg/d. She worsened, and was evaluated for LT at 7 months with total/ conjugated bilirubin of 22/20 mg/dL, prothrombin time international normalized ratio 1.9, aspartate aminotransferase/alanine aminotransferase ×4/×1.5 upper limit of normal, normal gamma-glutamyltranspeptidase, ascites and prerenal kidney injury. She was admitted to pediatric intensive care with multiorgan failure, severe neurological impairment (hypertone, tremor, poor spontaneous motility), and mild left ventricular myocardial hypertrophy. Laryngotracheomalacia, Coombs-negative hemolytic anemia, and central hypothyroidism were also present. After surgical closure of patent ductus arteriosus, she was listed for LT (pediatric end-stage liver disease score = 34). An extensive work-up ruled out infectious and metabolic etiologies ( Table 1). An urgent child-parents WES revealed that the girl was compound heterozygote for 2 known WD causing mutations in the ATP7B gene (c.19-20del, causing p.Gln7fs, and c.3207C>A, causing p.His1069Gln); the mother was homozygote for p.His1069Gln, and then was confirmed having WD with cirrhosis, while the father carried p.Gln7fs (Fig. 1B). Patient's copper metabolism revealed normal serum ceruloplasmin (39 mg/dL, NV > 20), increased basal cupruria (100 μg/24 h, NV < 40), and free cupremia (Fig. 1E). The brain magnetic resonance showed unspecific brain injury: focal (left occipital and periventricular) T2 hyperintensity suggesting gliosis, along with mild supratentorial white matter atrophy, while basal ganglia were normal. She was started on penicillamine 20 mg/kg/d and transplanted soon thereafter. Liver copper on hepatectomy was 350 μg/g dry weight (>5 × upper limit of normal). Histology showed cirrhosis and biliary plugs (Fig. 1C). Hepatocyte ultrastructure showed increased number of mitochondria (Fig. 1D).
She is alive and well 4 years after LT with complete neurologic recovery. The mother is well under penicillamine. A 7-year-old brother was diagnosed with asymptomatic WD by screening and started on zinc acetate.
The liver differential transcription pattern (fold change from a healthy liver) of the patient showed higher similarity to WD than that of 3 biliary atresia (BA) control patients: WD-related up-or downregulated genes were 28/61 versus 18/61 in the patient and in BA controls, respectively ( Fig. 2A, B).
In both patient and BA livers, an upregulation of gene clusters related to the extracellular matrix organization was present, due to fibrosis (Fig. 2C, D). However, the patient had higher similarity with WD as for an upregulation of the gene subsets related to cell proliferation and response to stimuli, and downregulation of those related to sterol, fatty acid, and cholesterol metabolism and xenobiotic metabolic processes (Fig. 2C-F). Methods for genetic testing (WES), pathology studies, and RNA studies are described in the Supplemental Digital Content, http://links.lww.com/PG9/A58.

DISCUSSION
Reduced fertility and spontaneous miscarriages are common in WD-untreated mothers, and anecdotal descriptions of copper accumulation in the placenta and in live birth fetuses also exist (3). This is the first report of a severe infantile liver disease possibly due to WD causing ATP7B mutations. Intrauterine copper exposure due to maternal undiagnosed WD might have contributed to this exceptional phenotype.
WD typically present after infancy, possibly as the result of the ATP7B-independent embryo-fetal and neonatal copper metabolism. At birth, the ion is mostly accumulated intracellularly bound with metallothioneins, while its mobilization toward mitochondria, binding to ceruloplasmin, and ATP7B-driven biliary excretion occur later in infancy (4).  Metabolic work-up Serum alpha-1 antitrypsin, plasma and urine amino acids, urine organic acids, serum total bile acids + mass spectrometry urine analysis, transferrin isoelectric focusing, acylcarnitine profile, pyruvic acid, lactic acid, erythrocyte galactose-1-phosphate uridyl transferase activity, urine galactose, urine fructose, chloride sweat test, beta-glucosydase, lysosomal acid lipase, and acid sphingomyelinase on dried blood spot mass spectrometry  On the x-axis, the log 10 of the fold change gene expression with regard to a healthy donor liver is displayed; On the y-axis, data are plotted according to the negative log 10 of the FDR, expressing the statistical significance of the change; the colored spots indicate the significantly up-(green) or downregulated (red) Wilson disease-related genes. C-F) Bubble plot of gene ontology enrichment analysis of statistically significant gene subsets up-or downregulation in liver tissue at hepatectomy of the case patient (C, upregulated; E, downregulated) and in 3 liver specimens at hepatectomy from patients with biliary atresia (B, upregulated; D, downregulated). On the x-axis, the log 10 of the fold enrichment (expressing the representativeness of the gene subset) with regard to a healthy donor liver tissue is displayed. On the y-axis, data are plotted according to the negative log 10 of the FDR, expressing the statistical significance of the change; gene subsets are classified as biological processes (green bubbles), cell component (orange bubbles), and molecular function (blue bubbles); bubble size is proportional to the number of gene included in the subset. The red-filled bubbles are the gene subsets up-or downregulated in gene ontology analysis in Wilson disease rodent models (2). The grey-filled bubbles are gene subsets related to liver fibrosis. FDR = false discovery rate.