The utility of whole exome sequencing in diagnosing Wilson disease: A case report

Abstract

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene, resulting in toxic copper accumulation in the body. Diagnosis is typically based on biochemistries, including low serum ceruloplasmin and elevated 24‐h urine copper excretion, with Kayser–Fleischer (KF) rings being a supportive feature. We describe a 7‐year‐old girl who presented with isolated elevation of serum alanine aminotransferase (ALT), low serum ceruloplasmin, and presence of nonspecific autoantibodies. Liver biopsy revealed elevated hepatic copper content (241 μg/g dry weight), mild macrovesicular steatosis, negative copper staining and no KF rings. Targeted genetic testing identified a single pathogenic ATP7B variant, and whole exome sequencing (WES) later revealed a second variant of uncertain significance (VUS). Repeat liver biopsy showed hepatic copper content of 258 mcg/g, meeting diagnostic criteria. The patient was started on trientine, and ALT normalized within 3 months, with increased urinary copper excretion. This case underscores how WD can mimic autoimmune hepatitis and metabolic dysfunction‐associated steatotic liver disease (MASLD), and how diagnosis may rely on integrating histologic findings, genetic data, and therapeutic response.

1. INTRODUCTION

Wilson disease (WD) is an autosomal recessive condition where mutations in the ATP7B gene lead to defective biliary copper excretion and toxic accumulation of copper in tissues such as the liver, brain, and cornea. ¹ As copper accumulates gradually, manifestations of WD including elevated liver enzymes or neurological issues develop over time. Based on the 2022 American Association for the Study of Liver Diseases (AASLD) practice guidelines, ² the combination of low serum ceruloplasmin, elevated 24‐h urinary copper excretion, and Kayser–Fleischer (KF) rings is diagnostic of WD. In children, however, diagnosis often relies on biochemical markers, as KF rings and elevated basal urinary copper excretion may be absent early in disease. When initial testing is inconclusive, hepatic copper quantification and genetic testing can assist diagnosis.

WD may mimic autoimmune hepatitis (AIH), particularly in children, where overlapping features, such as positive antinuclear antibodies (ANA), antismooth muscle antibodies (ASMA) and nonspecific histology, can obscure the diagnosis. ² Genetic testing is helpful, but variant classification and reporting differ among laboratories. In some instances, variants of uncertain significance (VUS) may be omitted from reports. Whole exome sequencing (WES), interpreted in consultation with a geneticist, can identify disease‐causing variants. ³ We present a 7‐year‐old girl with elevated liver enzymes, low serum ceruloplasmin, and no KF rings, whose initial gene sequencing identified a single, likely pathogenic ATP7B variant, and a second variant initially classified as likely benign and not reported. Persistent diagnostic concern led to WES at a different laboratory, where the second variant was classified as a VUS, prompting re‐evaluation for mild, nonclassic WD.

2. CASE REPORT

A 7‐year‐old female with a body mass index (BMI) in the 99th percentile was referred for evaluation of mildly elevated alanine aminotransferase (ALT) 39 U/L (normal < 35 U/L) with normal aspartate aminotransferase. She was clinically well, with no jaundice, fatigue, pruritus, easy bruising, or neuropsychiatric complaints, and was performing well in school. On physical exam, there was no hepatosplenomegaly and neurologic exam was unremarkable. Initial evaluation included low serum ceruloplasmin (9 mg/dL), positive ANA (1:1280), and positive ASMA (1:40), raising suspicion for AIH. Alpha‐1 antitrypsin phenotype was MM, and hemoglobin was normal. An abdominal ultrasound showed mildly prominent extrahepatic bile ducts for age but was otherwise unremarkable.

Given the elevated nonspecific autoantibodies and low serum ceruloplasmin, a liver biopsy was performed. Histology revealed mild macrovesicular steatosis involving approximately 5% of hepatocytes, few glycogenated nuclei, and no interface hepatitis, portal inflammation, or significant fibrosis (Figure 1). Copper staining was negative. Hepatic copper content was 241 μg/g dry weight (normal < 50; diagnostic > 250), and the sample was adequate. An ophthalmologic exam revealed no KF rings. Three 24‐h urine copper collections were obtained, showing values of 1.6, 17.5, and 57.8 mcg/24‐h (normal 10‐30 mcg/24 h). d‐penicillamine challenge yielded <1600 μg/24‐h, which was not diagnostic. All samples were judged complete based on urine creatinine.

Figure 1.

From first biopsy. (A) Liver with retained architecture, mild macrovesicular steatosis, and no significant fibrosis, 100x. (B) Few glycogenated nuclei, 400x. (C) Minimal portal inflammation, 200x. (D) Negative copper stain, 100x.

ATP7B sequencing revealed a heterozygous pathogenic variant (c.3155 C > T, p.P1052L). Given the diagnostic uncertainty and rising ALT, a repeat liver biopsy was performed 7 months later, showing similar histologic features and hepatic copper content of 258 μg/g dry weight.

WES confirmed the pathogenic ATP7B variant and revealed a second maternally inherited missense VUS (c.1995G>A, p.M665I). The initial lab had not reported this variant due to its classification at the time as likely benign. She was diagnosed with WD and started on trientine. Her liver enzymes normalized within 3 months, and urinary copper excretion increased, consistent with a therapeutic response.

3. DISCUSSION

WD is an autosomal recessive disorder caused by mutations in ATP7B, leading to impaired copper transport and accumulation in tissues. ¹ , ⁴ According to the 2022 AASLD practice guidelines, ² diagnosis is based on low serum ceruloplasmin, presence of KF rings, and elevated 24‐h urine copper excretion. A liver biopsy with hepatic copper content > 250 μg/g dry weight is diagnostic, while levels < 50 μg/g essentially rule out the disease. ⁴ In pediatric patients, KF rings and elevated basal urinary copper excretion may be absent, making hepatic copper quantification and longitudinal assessment particularly important.

Our patient initially presented with positive autoantibodies, prompting consideration of AIH. However, the initial liver biopsy lacked interface hepatitis and instead showed mild macrovesicular steatosis and a few glycogenated nuclei, findings not typical of AIH. This overlap in initial presentation between WD and AIH can delay diagnosis if serology is overemphasized.

The patient's elevated BMI and histologic steatosis raised concern for metabolic dysfunction‐associated steatotic liver disease (MASLD). However, the absence of portal or lobular inflammation or ballooning degeneration made MASLD less likely, though lifestyle counseling was provided.

The initial hepatic copper content of 241 μg/g dry weight was just below the diagnostic threshold. The decision to defer chelation therapy reflected the non‐diagnostic additional testing, including the d‐penicillamine challenge, ophthalmologic examination, and genetic analysis. The 7‐month re‐evaluation was planned to allow time for ALT trends, genetic re‐interpretation, and possible histologic progression. Repeat biopsy confirmed a hepatic copper content of 258 μg/g, now exceeding the diagnostic threshold.

We hypothesized that the patient's young age may have contributed to insufficient copper accumulation, explaining the borderline hepatic copper level, lack of clinical symptoms, and negative d‐penicillamine challenge. However, Ferenci et al. found no correlation between age and hepatic copper concentration. ⁵ The clinical heterogeneity of WD may reflect incomplete penetrance or modifying genetic and environmental factors, which may explain this patient's milder biochemical phenotype.

Copper staining was negative on both biopsies despite elevated parenchymal copper concentration. In early WD, this finding is expected as rhodamine stains copper after it accumulates within lysosomes, whereas early disease is characterized by predominantly cytoplasmic copper deposition, resulting in false negative stains. ⁶ Electron microscopy was not performed, though it could have revealed characteristic mitochondrial changes seen in WD. Liver biopsy remains valuable in cases where biochemical and genetic testing are inconclusive, with attention to copper‐free instruments.

Initial genetic testing was targeted ATP7B gene sequencing, which revealed a paternally inherited heterozygous pathogenic variant (c.3155 C > T). WES, performed later through a different laboratory, identified a second maternally inherited variant (c.1995G > A, p.M665I), classified as a VUS. The initial lab had detected this variant but did not include it in their report due to its previous classification as likely benign. This discrepancy underscores the importance of how genetic testing laboratories report variants, and how variant classification thresholds may vary. Discussion with a clinical geneticist was critical in interpreting these findings.

WES offered more than just a second look at ATP7B as it helped exclude other mimicking genetic disorders. Although metallothionein immunohistochemistry could be faster and less costly, ⁷ WES allowed for a broader differential to be explored and was clinically justified given the complexity of the patient's presentation.

The Leipzig scoring system ⁸ integrates clinical, biochemical, and genetic data to estimate the likelihood of WD, with a score of ≥4 considered “highly likely.” Using the initial values—ceruloplasmin 9 mg/dL (<0.1 g/L), abnormal hepatic copper of 241 µg/g and one pathogenic ATP7B variant—the patient's initial score was 4. This placed her at the diagnostic threshold, but the concurrent features suggesting AIH and MASLD highlighted the limitations of relying on the score alone. Ultimately, diagnosis required integration of histologic findings, biochemical data, and subsequent clinical response to trientine.

Treatment with trientine led to normalization of serum ALT and increased urinary copper excretion within 3 months, further supporting the diagnosis of WD and demonstrates the benefit of early intervention.

4. CONCLUSION

This case emphasizes that WD can mimic autoimmune or MASLD particularly in children. It illustrates how early histologic clues, genetic interpretation, and close clinical monitoring can guide diagnosis when classic features are absent. When the genotype consists of a pathogenic variant alongside a VUS, clinical correlation is essential, and follow‐up with repeat liver biopsy or therapeutic trial may be warranted. WES offers a broader diagnostic scope, especially when conventional sequencing is non‐diagnostic, and should be considered in complex or ambiguous cases. The case also highlights inter‐laboratory variability in genetic variant reporting. Resources like ClinVar provide insights into how labs classify the same variant and their rationale, ⁹ emphasizing the need to integrate genetic results with clinical context.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

Patient's parents provided informed consent for publication of the case details.