Targeted Proteomic Analysis of Extremely Low Abundance Signature Peptide Biomarkers for Potential Newborn Screening in Wilson's Disease

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Project Summary Wilson Disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B gene, which encodes an intracellular copper-transporting ATPase, and results in copper accumulation within the body. WD has an estimated incidence of 1 in 30,000 individuals. WD is present and asymptomatic at birth. Patients, when untreated, develop irreversible brain damage and liver cirrhosis. Early treatment is effective in preventing these negative sequelae and greatly improves patient quality of life. This makes early detection and treatment of WD, ideally in the neonatal period, of paramount importance to achieving the best long-term clinical outcomes. Newborn screening (NBS) to identify infants with treatable congenital disorders is carried out worldwide and has led to a significant increase in early diagnosis of many diseases which require early intervention. Unfortunately, no cost-effective newborn screening methods are currently available for early detection of WD. We recently demonstrated that peptide immunoaffinity enrichment coupled to SRM (immuno-SRM) can quantify peptide biomarkers in dried blood spots (DBS) and that the assay can readily distinguish affected cases from controls (R01AI123135-01) [1]. This immuno-SRM approach has opened up the possibility of using a multiplexed immuno-SRM approach to screen a variety of congenital disorders using proteins as biomarkers in DBS. Our ultimate goal is to develop a high-throughput quantitative assay for NBS of WD using DBS. We recently developed an immuno-SRM assay to quantify a low level target peptide for ATP7B, a potential marker protein for WD, in DBS [1]. The objective of this application is to enrich our current assays with additional WD biomarkers, develop quality control (QC) measures, and validate the assay in a large cohort of patients and carriers from a broad spectrum of mutations, regions and ethnicities. Our central hypothesis is that in most of these genetically confirmed WD patients, the levels of ATP7B protein in DBS would be absent or reduced; and that the immuno-SRM assay can readily differentiate affected patients from controls or proven carriers. Our specific aims are to: 1: Enhance the sensitivity and specificity by multiplexing crucial marker peptides of ATP7B with the existing well-characterized immuno-SRM assay. Monitoring multiple peptides of ATP7B protein by immuno-SRM using multiple monoclonal antibodies will help increase sensitivity and specificity. 2. Assess the ability of a multiplexed immuno-SRM to identify WD patients in a large set of clinical samples with a broad spectrum of mutations, regions and ethnicities. Here, we will validate QC process measures through the establishment of reference ranges for existing QC peptides. We will examine the hypothesis that the immuno-SRM can correctly identify patients with WD and suitability for NBS. We will (1) by establish reference ranges from a total of 400 controls with various age groups and (2) analyze patient and carrier samples (n=220) obtained from 4 domestic and 2 foreign participating centers.