Measurement of psychosine in dried blood spots — a possible improvement to newborn screening programs for Krabbe disease

Abstract

Background

Newborn screening (NBS) for Krabbe disease (KD) in New York and Missouri is conducted by measuring galactocerebrosidase (GALC) activity using tandem mass spectrometry (MS/MS). These NBS efforts have shown that the incidence of KD is unexpectedly low (1:400,000) while many individuals (ca. 1:6000) with reduced GALC activity and genotypes of uncertain significance are detected and subjected to follow up testing. Psychosine (PSY) is a putative marker of KD progression and can be measured in dried blood spots (DBS). We sought to determine the role that PSY levels play in NBS for KD, follow up, and treatment monitoring.

Methods

PSY was eluted from DBS with methanol containing N,N-dimethyl-D-erythro-sphingosine as internal standard (IS). Liquid chromatography-MS/MS was conducted over 17 minutes in the multiple reaction monitoring positive mode to follow the precursor to product species transitions for PSY and IS. Separation of the structural isomers PSY and glucosylsphingosine was accomplished by hydrophilic interaction liquid chromatography.

Results

Pre-analytical and analytical factors were studied and revealed satisfactory results. PSY was also measured in DBS collected from controls (range: <8 nmol/L, N = 220), KD patients at various disease stages (range: 8–112, N = 26), and GALC mutation carriers (range: <15 nmol/L, N = 18).

Conclusions

PSY measurement in DBS could serve as a 2nd tier assay in NBS for KD, simplify and reduce the cost of follow up protocols, help determine disease progression, and be used to monitor KD patients following hematopoietic stem cell transplantation. However, additional chronological measurements of PSY in KD patients are required to confirm these possibilities.

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Fig. 1
Fig. 2

Abbreviations

GALC:

Galactocerebrosidase

DBS:

Dried blood spots

HSCT:

Hematopoietic stem cell transplantation

KD:

Krabbe disease

LC-MS/MS:

Liquid chromatography-tandem mass spectrometry

NBS:

Newborn screening

PSY:

Psychosine

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Acknowledgments

The authors would like to thank Amy Barczykowski and Erin Connors (Hunter James Kelly Research Institute, Buffalo, NY), Anna Grantham, Andrea Moran and Jacque Waggoner (Hunter’s Hope Foundation, Buffalo, NY; www.huntershope.org) as well as Dr. Michele Poe (University of Pittsburgh, Pittsburgh, PA) for assisting in the retrieval of patient samples, and the California Department of Public Health (Richmond, CA) for providing random, de-identified NBS control samples as part of a related study (#HHSN275201000017C) which was funded in part by The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services; The Newborn Screening Translational Research Network; and The Legacy of Angels Foundation.

Details of funding

Thomas J. Langan receives a salary from the University of Buffalo with a small portion of salary support from the HJKRI, funded from a grant from HHF. The HJKRI and the HHF are administratively distinct, and less than 5% of direct costs of the HJKRI are provided by the HHF.

Compliance with ethical guidelines

Conflict of interest

None.

Human and animal rights and informed consent

This article does not contain any studies with human or animal subjects performed by the any of the authors.

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Corresponding author

Correspondence to Dietrich Matern.

Additional information

Communicated by: Bridget Wilcken

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure

Comparison of psychosine (PSY) concentrations to GALC enzyme activity levels as determined in NBS samples from asymptomatic late onset KD cases (closed circles, panels A and B; N=6; same as group F in Fig. 2), from asymptomatic early infantile KD cases (+, panels A and C; N=10; same as group H in Figure 2), and DBS collected from KD patients within 1 year from onset of symptoms (open circles, panels A and D; N=7; same as group I in Fig. 2). There is no apparent correlation of PSY concentrations to GALC activity (bivariate correlation of linear fit ≤ 0.45). The dashed horizontal line indicates the 99th percentile of the controls’ PSY concentrations (N=220; groups A + B of Fig. 2) and the dashed vertical line the 1st percentile of the controls’ GALC activity levels [N=~88,000]. (PPTX 102 kb)

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Turgeon, C.T., Orsini, J.J., Sanders, K.A. et al. Measurement of psychosine in dried blood spots — a possible improvement to newborn screening programs for Krabbe disease. J Inherit Metab Dis 38, 923–929 (2015). https://doi.org/10.1007/s10545-015-9822-z

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Keywords

  • Hematopoietic Stem Cell Transplantation
  • Newborn Screening
  • Hydrophilic Interaction Liquid Chromatography
  • Twitcher Mouse
  • Determine Disease Progression