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Front. Immunol. | doi: 10.3389/fimmu.2018.02756

Rapid Multiplexed Proteomic Screening for Primary Immunodeficiency Disorders from Dried Blood Spots

 Christopher J. Collins1, Irene J. Chang2, Sunhee Jung1, Remwilyn Dayuha1, Jeffrey R. Whiteaker3, Gesmar R. Segundo4, Troy R. Torgerson1, 2, 5,  Hans D. Ochs1, 2, 5,  Amanda G. Paulovich3 and  SI HOUN HAHN1, 2, 5*
  • 1Seattle Children's Research Institute, United States
  • 2University of Washington, United States
  • 3Fred Hutchinson Cancer Research Center, United States
  • 4Federal University of Uberlandia, Brazil
  • 5Seattle Children's Hospital, United States

Background: Primary immunodeficiency disorders (PIDD) comprise a group of life-threateningcongenital diseases characterized by absent or impaired immune responses. Despite the fact that effective, curative treatments are available with optimal clinical outcomes when diagnosed early, newborn screening does not exist for the majority of these diseases due to the lack of detectable, specific biomarkers or validated methods for population-based screening. Peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) is a sensitive proteomic assay that allows for concurrent quantification of multiple analytes, conferring promising potential for newborn screening of PIDDs that lead to diminished or absent target proteins in the majority of cases.

Objective: To determine and evaluate if a multiplex assay based on immuno-SRM is able to reliably and precisely distinguish affected patients with X-linked agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), and CD3ɛ-associated severe combined immunodeficiency (SCID) from one another and from unaffected normal control dried blood spot (DBS) samples.

Methods: We performed a blinded, multiplexed analysis of proteolytically-generated peptidesfrom WASp, BTK, and CD3ɛ (for WAS, XLA, and SCID, respectively) in DBS samples from 42PIDD patients, 40 normal adult controls, and 62 normal newborns. The peptide of ATP7B 1056 was simultaneously monitored for quality assurance purpose.

Results: The immuno-SRM assays reliably quantified the target peptides in DBS and accurately distinguished affected patients from normal controls. Analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case ((WAS and BTK: p = 0.0001, SCID: p = 0.05). Intra and inter-assay precision ranged from 11 - 22% and 11 - 43% respectively; linearity (1.39 – 2000 fmol peptide), and stability (≤0.09% difference in 72 h) showed high precision for the multiplexed assay. Inter-laboratory assay comparison showed high concordance for measured peptide concentrations, with R2 linearity ≥ 0.97 for the WAS 274, CD3e 197, BTK 407 and ATP7B 1056 peptides.

Conclusion: Immuno-SRM-based quantification of proteotypic peptides from WASp, BTK, and CD3ɛ in DBS distinguishes relevant PIDD cases from one another and from controls, raising the possibility of employing this approach for large-scale multiplexed newborn screening of selective PIDDs.

Keywords: Primary immunodeficiency disorders, PIDD, Wiskott-Aldrich Syndrome, WAS, X-linked Agammaglobulinemia, XLA, Severe combined immunodeficiency, SCID, newborn screening, NBS, dried blood spot, DBS, Peptide immunoaffinity enrichment coupled to SRM, immuno-SRM

Received: 30 Aug 2018; Accepted: 08 Nov 2018.

Edited by:

Mirjam Van Der Burg, Leiden University Medical Center, Netherlands

Reviewed by:

Jolan E. Walter, University of South Florida, United States
Tomohiro Morio, Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University, Japan  

Copyright: © 2018 Collins, Chang, Jung, Dayuha, Whiteaker, Segundo, Torgerson, Ochs, Paulovich and HAHN. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: MD, PhD. SI HOUN HAHN, University of Washington, Seattle, United States, SIHAHN@UW.EDU