Advancing Newborn Screening in Washington State: A Novel Multiplexed LC-MS/MS Proteomic Assay for Wilson Disease and Inborn Errors of Immunity

Abstract

For many genetic disorders, there are no specific metabolic biomarkers nor analytical methods suitable for newborn population screening, even where highly effective preemptive treatments are available. The direct measurement of signature peptides as a surrogate marker for the protein in dried blood spots (DBSs) has been shown to successfully identify patients with Wilson Disease (WD) and three life-threatening inborn errors of immunity, X-linked agammaglobulinemia (XLA), Wiskott-Aldrich syndrome (WAS), and adenosine deaminase deficiency (ADAD). A novel proteomic-based multiplex assay to detect these four conditions from DBS using high-throughput LC-MS/MS was developed and validated. The clinical validation results showed that the assay can accurately identify patients of targeted disorders from controls. Additionally, 30,024 newborn DBS samples from the Washington State Department of Health Newborn Screening Laboratory have been screened from 2022 to 2024. One true presumptive positive case of WD was found along with three false positive cases. Five false positives for WAS were detected, but all of them were premature and/or low-birth-weight babies and four of them had insufficient DNA for confirmation. The pilot study demonstrates the feasibility and effectiveness of utilizing this multiplexed proteomic assay for newborn screening.

Keywords: Wilson Disease; Wiskott–Aldrich syndrome; X-linked agammaglobulinemia; adenosine deaminase deficiency; mass spectrometry; newborn screening; peptide; proteomics.

Figure 1

(A) Peptide concentrations between genders (male/female) (**** = p ≤ 0.0001, ns = no significance). (B) Mean peptide concentrations between genders, differences between the means and significance.

Figure 2

(A) Peptide concentrations between different birth weight groups (<1500 g, 1500 g–2500 g, and >2500 g) (**** = p ≤ 0.0001, ns = no significance). (B) Statistical significances in the average peptide concentrations between different birth weight groups (<1500 g, 1500 g–2500 g, and >2500 g). The p-value was calculated using the Welch ANOVA test between the <1500 g and >2500 g groups, and 1500 g–2500 g and >2500 g groups.

Figure 3

(A) Peptide concentrations between ethnicities (* = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001). (B) Mean concentration differences between ethnicities in pmol/L.

Figure 4

(A) Peptide concentrations in different groups of age of newborn sample collection in days (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001). (B) Mean peptide concentrations in different age groups in pmol/L.