Direct Measurement of ATP7B Peptides Is Highly Effective in the Diagnosis of Wilson Disease

Abstract

Background & aims: Both existing clinical criteria and genetic testing have significant limitations for the diagnosis of Wilson disease (WD), often creating ambiguities in patient identification and leading to delayed diagnosis and ineffective management. ATP7B protein concentration, indicated by direct measurement of surrogate peptides from patient dried blood spot samples, could provide primary evidence of WD. ATP7B concentrations were measured in patient samples from diverse backgrounds, diagnostic potential is determined, and results are compared with biochemical and genetic results from individual patients.

Methods: Two hundred and sixty-four samples from biorepositories at 3 international and 2 domestic academic centers and 150 normal controls were obtained after Institutional Review Board approval. Genetically or clinically confirmed WD patients with a Leipzig score >3 and obligate heterozygote (carriers) from affected family members were included. ATP7B peptide measurements were made by immunoaffinity enrichment mass spectrometry.

Results: Two ATP7B peptides were used to measure ATP7B protein concentration. Receiver operating characteristics curve analysis generates an area under the curve of 0.98. ATP7B peptide analysis of the sequence ATP7B 887 was found to have a sensitivity of 91.2%, specificity of 98.1%, positive predictive value of 98.0%, and a negative predictive value of 91.5%. In patients with normal ceruloplasmin concentrations (>20 mg/dL), 14 of 16 (87.5%) were ATP7B-deficient. In patients without clear genetic results, 94% were ATP7B-deficient.

Conclusions: Quantification of ATP7B peptide effectively identified WD patients in 92.1% of presented cases and reduced ambiguities resulting from ceruloplasmin and genetic analysis. Clarity is brought to patients with ambiguous genetic results, significantly aiding in noninvasive diagnosis. A proposed diagnostic score and algorithm incorporating ATP7B peptide concentrations can be rapidly diagnostic and supplemental to current Leipzig scoring systems.

Keywords: ATP7B; Immuno-SRM; Leipzig Score; Wilson disease.

Figure 1:

Patient cohort characteristics. Analysis of the genotypes of patients (A) and the characteristics of the variants present (B) show the diversity of variants and variant combinations present.

Figure 2:

Diagnostic performance of ATP7B peptide analysis. Comparison of ATP7B peptide measurements for ATP7B 1056 (A) and ATP7B 887 (B) peptides in normal control patients (NC), patients, homozygotes (H), compound heterozygotes (CH) and carriers. Dotted lines represent diagnostic cut-offs for each peptide. ROC curves show the diagnostic performance of ATP7B 1056 (C) and ATP7B 887 (D). WD patients with genetic test results are readily identified even in subgroups where genetic results are ambiguous (E).

Figure 3:

ATP7B peptide concentrations in patients with common variants are often reduced and variants causing false negatives are rare. Patients homozygous and heterozygous for p.H1069Q (A), R778L (B), M645R (C) and E1064A (D) are largely reduced. False negatives within the patient cohort are possible with the presence of specific variants including G710S (E) and R969Q (F). Patients can have variable peptide concentrations depending on the second variant. H = Homozygote, CH = Compound Heterozygote. Dotted Lines represent peptide diagnostic cutoffs.

Figure 4:

ATP7B peptide concentration analysis can provide clear results where ceruloplasmin results and genetic analysis are ambiguous. Patients with significantly (A), moderately (B), and normal (C) Cp were readily identified. ATP7B concentrations are reduced regardless of variant status including in patients with two pathogenic or likely pathogenic variants (D), at least one VUS (E), or where no second variant was found (F). Cp = Ceruloplasmin. Dotted Lines represent peptide diagnostic cutoffs. Patients with liver copper above (G) or below 256 ug/g (H) are shown. In three samples from non-WD patients with elevated liver copper, ATP7B concentrations are normal (I).

Figure 5:

Proposed Wilson Disease Diagnostic Algorithm