MSACL 2016 US Abstract

Dried Blood Spot Screening for Wilson Disease Using Immuno-SRM

Sunhee Jung (Presenter)
Seattle Children

Bio: Sunhee Jung is interested in biomedical application of mass spectrometry (MS) and quantitative analysis. Her current research focuses on developing MS-based screening tests to measure potential protein markers in the blood for genetic diseases such as primary immunodeficiency diseases, cystinosis and Wilson disease. She acquired a solid background in proteomics using mass spectrometry as well as molecular and cellular biology while earning her doctorate at the University of Washington.

Authorship: Sunhee Jung (1), Jeffrey R. Whiteaker (2), Lei Zhao (2), Han-Wook Yoo (3), Beom Hee Lee (3), Amanda G. Paulovich (2), and Si Houn Hahn (1,4)
1Seattle Children’s Hospital Research Institute, Seattle, WA; 2Fred Hutchison Cancer Research Center, Seattle, WA; 3Asan Medical Center, Ulsan University College of Medicine; 4Department of Pediatrics

Short Abstract Wilson Disease (WD), a copper transport disorder caused by a genetic defect in the ATP7B gene, has been a long time candidate for newborn screening because of proven interventions that give better results when carried out early in life, preventing life-long neurological disability and/or liver cirrhosis. WD presents with absent or significantly diminished ATP7B protein that is localized in the transmembrane. ATP7B has enormous potential for screening of WD if the protein can be identified from dried blood spot (DBS) samples. We herein report a proof-concept study demonstrating that the immuno-SRM platform can detect ATP7B in DBS and the assay readily distinguishes affected cases from normal controls. Our promising data opens up the great potential of a multiplexed immuno-SRM assay for screening a variety of congenital disorders lacking specific protein markers in DBS.

Long Abstract

Introduction: Newborn screening (NBS) has proven to be highly effective at reducing healthcare costs, improving outcomes, and avoiding long-term disability in affected children. Current NBS methods are limited to identify only disorders that lead to accumulation of specific small molecule metabolites (amino acids or acylcarnitines) in the serum. Unfortunately, many attractive candidates for NBS are characterized by the absence or modification of proteins for which there are currently no cost-effective screening methods. Wilson Disease (WD) is an autosomal recessive disorder of abnormal copper transport caused by mutations in the gene encoding the copper transporting ATPase (ATP7B). Early detection of WD is critical because effective medical treatments such as chelating agents and zinc salts can prevent life-long neurological disability and/or liver cirrhosis. As WD presents with absent or significantly diminished ATP7B that is localized in the transmembrane, ATP7B has enormous potential for screening of WD if the protein can be identified from blood-based samples. We hypothesized that peptide immunoaffinity enrichment coupled with Liquid Chromatography-Selected Reaction Monitoring-Mass Spectrometry (immuno-SRM) can be utilized as a rapid, inexpensive approach to detect ATP7B, a very low abundance protein, in Dried Blood Spots (DBS). The results demonstrate proof-of-concept for applying immuno-SRM to simultaneously screen for a variety of congenital disorders lacking specific protein markers.

Methods: Candidate peptides for ATP7B were screened by In Silico trypsin digestion followed by BLAST search to insure the sequences are unique within the human genome. Several candidates were selected based on detectability and response in LC-MS/MS analyses of tryptic digests of HepG2 cells. Among these, we purposely chose ATP7B 1056 peptide for polyclonal antibody generation because this peptide includes the most common mutation, p.H1069Q. The antibody was used to enrich the corresponding peptide from tryptic digests of DBS. Peptides were analyzed in SRM mode with 6500 QTRAP (ABSCIEX). Fresh blood samples from WD patients were collected after consent was obtained. Blood samples were spotted on filter paper, dried and stored at -80 oC until use.

Preliminary Results: Our preliminary results of testing of blinded DBS samples from 5 controls and 7 confirmed WD patients showed that the assay readily distinguished affected from nonaffected individuals. While we were able to reliably detect endogenous ATP7B ranged from 105 to 708 pmol/L in normal controls, the analyte response from WD patients was either not detected or below 47 pmol/L. In addition, there was no ATP7B 1056 peptide detected in either of the WD patients who carried p.H1069Q mutation, as predicted. Performance metrics of the assay were determined by generating a 5-point response curve using synthetic standard peptides. The assay showed a linear response (r2 = 0.99) for all peptide amounts tested, spanning the peptide concentration of 27 to 16,765 pmol/L (0.7 to 417 femtomoles). The median CV for all points on the response curve was 7.8%. We also assessed intra- and interassay imprecision by analyzing 2 samples (1. DBS from a normal control and 2. DBS pooled from two affected siblings) on 3 different days each using 3 replicates of each of the 2 samples. Intra- and interassay imprecision from the normal control were 8.89% CV and 7.57% CV, respectively. The assay imprecision from the pooled WD patients was not calculated because most peaks observed were below the LOD. The stability of the protein marker in DBS at various temperatures and duration is currently under investigation.

Conclusions: This is very compelling evidence that immuno-SRM assays can be used as a highly sensitive platform for DBS protein analysis in the low-picomolar range. When multiplexed, there is a significant potential to screen various congenital disorders lacking specific protein markers such as Primary Immunodeficiencies, Cystinosis, and WD. Further optimization efforts are underway to enable immuno-SRM as a viable platform for NBS. Our findings open up an innovative approach for NBS, facilitating the development of multiplexed screening tests for a broader range of genetic disorders.

References & Acknowledgements:

This study was supported by the grant from NIH

(R21HD069890-01A1).

Financial Disclosure

IP Royalty: no

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no