Rongrong Huang (Presenter)
Greenwood Genetic Center
Bio: I am currently working as a Staff Scientist in the Biochemical Genetics Laboratory in Greenwood Genetic Center, mainly interested in applying liquid chromatography and mass spectrometry (LC-MS) based techniques in the clinic field. I obtained my Ph.D degree for analytical chemistry from University of Georgia, where my doctorate research focused on structural characterization of glycosaminoglycans and its biological relevant utilizing various LC-MS instrumentations. Current work involves development and validation of diagnostic assays for measurement of disease-specific metabolites, especially for lysosomal storage diseases and some other metabolic disorders, using LC-MS/MS platform, as well as other research projects including targeted metabolomics for study of autism spectrum disorder.
Authorship: Rongrong Huang, Allison Cason, Laura Pollard, Tim Wood
Biochemical Genetics Laboratory, Greenwood Genetic Center, Greenwood, SC
| Short Abstract Several lysosomal storage diseases (LSDs), primarily the glycoproteinoses, are characterized by accumulation of free oligosaccharides as a result of impaired glycoprotein degradation. The typical method used to detect abnormal accumulation of urine oligosaccharides is thin-layer chromatography, which lacks adequate sensitivity and specificity. Our laboratory has developed a UPLC-MS/MS assay for relative quantification of seven disease-specific urine oligosaccharides using samples from 110 unaffected controls and 51 affected LSD patients. The results indicate the assay can be used for sensitive diagnosis of eight different LSDs, and potentially for treatment monitoring. Preliminary data indicates it can also be applied to other biological specimens. |
Long Abstract
Introduction
A group of lysosomal storage diseases (LSDs), primarily the glycoproteinoses, are characterized by the accumulation of specific free oligosaccharides in tissues and other biological fluid as a result of one or several deficient enzymes that are involved in the breakdown of complex carbohydrate side-chains of glycoproteins. Historically, thin-layer chromatography has been used to detect the abnormal accumulation of free oligosaccharides in urine; however, this method lacks both sensitivity and specificity. In an effort to improve the diagnosis of glycoproteinoses, our laboratory has developed a ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the detection of free oligosaccharides following reducing-end labeling derivatization. Relative quantification of seven disease-specific oligosaccharides is used to detect patients with one of the eight different LSDs. Significantly reduced oligosaccharide levels are observed in LSD patients who have received bone marrow transplantation compared to untreated patients, which demonstrates the potential use of this assay for monitoring treatment efficacy. Preliminary data also show that this assay could be applied to other biological specimens such as dried blood spots, plasma, leukocytes and fibroblasts, broadening its clinical utility.
Methods
Oligosaccharides in urine, plasma, DBS or cell lysates are derivatized by butyl-4-aminobenzoate (BAB) using a modification of a previously reported method, with ΔHexA-GlcNAc as the internal standard (IS) added to each sample prior to the BAB derivatization. Samples are then cleaned up through a Sep-Pak Aminopropyl (NH2) cartridge and subjected to LC-MS/MS analysis on a Waters Xevo TQ-S coupled with Acquity UPLC system. A linear gradient is applied on a Waters Glycan BEH Amide column (130Å, 1.7µM, 2.1*50mm) for UPLC separation, using 50mM ammonium formate in water as mobile phase A and pure acetonitrile as mobile phase B, with a total run time of seven minutes. In addition to IS, seven free oligosaccharide (FOS) compositions were selected and detected in SRM mode, including Hex1HexNAc1Asn (FOS1), Hex3HexNAc2Fuc1 (FOS2), Hex3HexNAc1 (FOS3), Hex1HexNAc1 (FOS4), Hex3HexNAc2 (FOS5), Hex2HexNAc3 (FOS6) and Neu5Ac1Hex3HexNAc2 (FOS7). The relative concentration for each FOS was calculated using the peak area ratio of each FOS versus IS and the known concentration of IS.
Results
We analyzed 51 urine samples from patients with one of eight LSDs: aspartylglucosaminuria (n=10), fucosidosis (n=4), alpha-mannosidosis (n=21), beta-mannosidosis (n=1), beta-galactosidase deficiency (n=8), Sandhoff disease (n=2), sialidosis (n=3) and galactosialidosis (n=2), which were collected either as part of the Glycoproteinoses Natural History Study or through routine diagnostic testing. Age-specific normal ranges were developed for each FOS using 110 samples from unaffected controls. The increased abundance of the disease-specific FOS in affected individuals compared to the upper limit of age-matched controls ranged from 5-3000 fold, with aspartylglucosaminuria (average 164-fold), fucosidosis (average 1380-fold), beta-galactosidase deficiency (average 82-fold) and sialidosis (average 410-fold) showing the widest dynamic range. The reproducibility of the semi-quantitative analysis was demonstrated using two samples (six inter-day prep), with relative standard deviation (%CV) ranging from 5% to 23% for IS peak area and the relative concentration for each FOS, indicating an acceptable batch to batch reproducibility.
As a proof of principle, urine samples from patients who have received bone marrow transplants were also analyzed to demonstrate the utility of the assay for treatment monitoring. The Hex3HexNAc1 level in a treated alpha-mannosidosis patient was 6-fold elevated compared with an average of 40-fold elevated in untreated patients. A fucosidosis patient and a beta-mannosidosis patient showed a 73% and 87% decrease in Hex3HexNAc2Fuc1 and Hex1HexNAc1 level respectively after receiving a bone marrow transplant. For improved accuracy and reproducibility, absolute quantification can be achieved using oligosaccharide-specific standards and internal standards, if they are available, which we have already demonstrated for aspartylglucosaminuria by quantifying GlcNAc-Asn.
Given its high sensitivity, preliminary experiments have also been conducted to apply the assay to other biological samples, including dried blood spot (DBS, n=24), plasma (n=15), leukocytes (n=14) and cultured fibroblasts (n=20). The Hex3HexNAc1 level was elevated in all sample types from alpha-mannosidosis patients: average of 5-fold in DBS, 40-fold in plasma, 80-fold in LEUKOCYTES and 30-fold in fibroblasts. Hex3HexNAc2Fuc1 was detectable in DBS and plasma from alpha-fucosidosis patients, but not from unaffected controls or patients with other LSDs. Hex3HexNAc2 was elevated in all sample types for patients with beta-galactosidase deficiency: average of 6-fold in DBS, 30-fold in plasma, 10-fold in LEUKOCYTES and 100-fold in fibroblasts. Both Hex3HexNAc2 and Neu5Ac1Hex3HexNAc2 were elevated in DBS, plasma and LEUKOCYTES from one galactosialidosis patient, but not in fibroblasts. Hex1HexNAc1Asn was detectable in DBS, plasma and LEUKOCYTES from aspartylglucosaminuria patients, but not from unaffected controls or patients with other LSDs. Hex1HexNAc1 was elevated in DBS, plasma and LEUKOCYTES from one beta-mannosidosis patient, showing a 60-95% decrease, depending on the sample type, post-bone marrow transplantation.
Conclusion
Overall, our results demonstrate that the described UPLC-MS/MS urine oligosaccharide assay can be used not only to screen for eight different LSDs but also for monitoring treatment efficacy, as treatments for these disorders become available. The potential application of this assay to different specimen types also expands its clinical utility. Furthermore, it utilizes a triple quadrupole tandem mass spectrometer rather than a MALDI-TOF instrument, making the assay applicable to more clinical laboratories. Future work will involve developing true quantitative assays for individual disease-specific oligosaccharides to improve the accuracy and reproducibility of the assay for use in clinical trials, as well as further investigation of its application in blood-based and cellular sample types.
References & Acknowledgements:
References:
1.Young et al (2003) Anal Biochem. 316: 175-180.
2.Xia et al (2013) Clin Chem. 59: 1357-1368.
Acknowledgments
We thank Dr. Sara Cathey and the NIH Rare Disease Clinical Research Network (Lysosomal Disease Network Consortium) for providing precious patient samples.
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