Hsuan-Chieh Liao (Presenter)
University of Washington
Bio: I am Hsuan-Chieh Liao, a visiting scholar in Michael H. Gelb's Lab at University of Washington. I worked in the newborn screening center (Chinese Foundation of Health, CFOH) as a medical technologist in Taiwan for five years and was a manager in the research and development department. During my school and career, I received the champion of Excellent Poster/Oral Award at the National Yang-Ming University and the Eto Prize (the Young Investigator Award) at the 3rd Asian Congress for Inherited Metabolic Disease (ACIMD). In the past few years, I was invited as a speaker and presented my projects about newborn screening and diagnosis of rare diseases at several international conferences.
Authorship: Hsuan-Chieh Liao, Zdenek Spacil, Arun Kumar, Michael Gelb
University of Washington
| Short Abstract Krabbe disease is an inherited autosomal recessive disorder caused by mutations in the GALC gene. Deficiency in GALC result in an abnormal accumulation of a highly cytotoxic metabolite, called psychosine. Our lab has developed mass spectrometry methods that are being used in newborn screening (NBS) of lysosomal storage diseases. Now we focus on high accuracy GALC enzyme assays using purified white blood cells from patients suspected to have Krabbe disease. Psychosine measurement in dried blood spots and cerebrospinal fluid were also developed and could serve as a second tier assay. The methods should reduce the false positive rate of NBS and help determine disease severity and monitor Krabbe patients. |
Long Abstract
Introduction
Krabbe disease (globoid cell leukodystrophy) is an autosomal recessive disorder caused by deficiency of galactosylcerebrosidase (GALC) and the consequent accumulation of galactosylceramide and psychosine. Newborn screening for Krabbe disease was developed in our lab and conducted by measuring GALC activity using tandem mass spectrometry (MS/MS). Not all patients detected in a newborn screening program will develop Krabbe disease, and a second-tier analysis is needed to help predict disease severity.
For second-tier analysis we have developed a MS/MS GALC enzymatic activity assay that is more accurate than previously reported methods. This method is based on the enormous analytical range provided by MS/MS.
The metabolite psychosine is also a putative marker for diagnosis and following Krabbe patients and can be measured in dried blood spots. Psychosine measurement by MS/MS in dried blood spots and cerebrospinal fluid were also developed and could serve as tools for confirmed diagnosis.
Method
GALC enzyme reactions in B cell and fibroblast cells were stimulated by optimal concentrations of sodium taurocholate and sodium oleate. Citrate-phosphate buffer, pH 4.2, and the glycohydrolase inhibitor 1-deoxygalactonojirimycin (1-DGJ) were used to give maximum reaction specific measurements of GALC activity and inhibition of interfering enzyme beta-galactosidase. Psychosine was eluted from DBS with methanol containing deuterated psychosine as internal standard. Ultra performance liquid chromatography-MS/MS was conducted within two minutes for GALC enzyme assay and five minutes for psychosine analysis. Separation of the structural isomers psychosine and glucosylsphingosine was accomplished by using the BEH amide UHPLC column.
Result
Using the conditions of 0.2M, ph 4.2 citrate phosphate buffer and 250 uM 1-DGJ, the GALC activity was preserved and could be specifically and accurately measured in cultured B cells. Psychosine was measured in 9 cerebrospinal fluid samples and 92 DBS collected from controls and KD patients (infantile, juvenile, and adult type). The results show that the concentration of psychosine increased significantly in Krabbe patients comparing controls.
Conclusions
The new MS/MS-based high accuracy GALC enzymatic activity assay provides a reliable method for diagnosis of Krabbe disease with clear distinction between GALC enzyme activities from wild type and disease cells. Psychosine measurement in DBS could also serve as a second tier assay as a diagnostic tool. Both of these rapid and highly sensitive methods are expected to reduce the number of false positives in NBS programs for Krabbe disease and to better predict the disease severity.
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