Francyne Kubaski (Presenter)
University of Delaware/ Nemours
Bio: I am a Bachelor in Biomedical analysis. I received my master`s degree in Medical Sciences and It was during my master`s degree under the orientation of Dr Sandra Leistner-Segal and Dr Roberto Giugliani when I started to learn about inborn errors of metabolism. In 2013, I started my PhD at the University of Delaware under the orientation of Dr Shunji Tomatsu and Dr Robert W. Mason, where a major project that I have been working on is to develop a newborn screen (NBS) for Mucopolysaccharidoses (MPS) using liquid chromatography tandem mass spectrometry (LC/MS/MS). This technology is currently in use to measure acylcarnitines and amino acids in NBSs for metabolic diseases but has not yet been developed to measure GAGs as a NBS for MPS. An innovative aspect of this research is that multiple GAGs will be measured simultaneously to detect all forms of MPS from a single sample.
Authorship: Francyne Kubaski 1,2, Robert W. Mason 1,2, Junji Hanai 3, Li Xie1, Naomi N. van Vlies4, Heather Church5, Seiji Yamaguchi6, Yasuyuki Suzuki7, Tadao Orii8, Adriana M. Montaño9, and Shunji Tomatsu1, 8
1Nemours/Alfred I. duPont Hospital for Children, Wilmington, DE; 2Department of Biological Sciences, University of Delaware, Newark, DE; 3Sapporo City Institute of Public Health, Sapporo, Japan; 4Lab
| Short Abstract Mucopolysaccharidoses (MPSs) are progressive disorders where early diagnosis allows early treatment that provides better outcomes and prognosis. We have developed a pilot study using dried blood spots (DBS) from 2640 random newborn samples and 13 newborn MPS samples. All DBS were evaluated by liquid chromatography tandem mass spectrometry to determine levels of heparan sulfate (0S, NS) and keratan sulfate (mono-KS, di-KS and ratio di-KS/total KS). Cutoffs were established for HS to diagnose MPS I, II and III. HS-0S showed 98% specificity and 77% sensitivity and it is a potential biomarker for newborn screening of MPSs. |
Long Abstract
Mucopolysaccharidoses (MPSs) have an estimated combined incidence of 1 in 22,000 live births. Specific treatment is already available for several MPSs, and there is evidence that better outcome of therapy occurs when it is started early. Thus, early diagnosis is extremely relevant to allow better treatment and improve prognosis. This pilot study aims to determine whether LC/MS/MS analysis of GAGs in dried blood spots (DBS) can distinguish MPS patients from unaffected controls. Dried blood spots from 2640 newborn controls and 13 newborn MPS patients (MPS I= 6, MPS II= 2, MPS III= 5) were evaluated with LC/MS/MS to measure levels of heparan sulfate (0S, NS) and keratan sulfate (mono-KS, di-KS and ratio di-KS/total KS). Disks (3.3mm) were cut from DBS samples using a DBS puncher (PerkinElmer®; Waltham, MA) and placed into a 96 well omega 10K filter plate with 100 μL of 0.1% BSA on a 96 well receiver plate. Samples were incubated for 15 min and then centrifuged for 15 min at 2200 g. The filter plate was transferred to a new receiver plate and a cocktail mixture (130 μL) that consisted of: 50 mM Tris/HCl, pH 7.0; internal standard, chondrosine (5 µg/mL); chondroitinase B, heparitinase, keratanase II (each 1 mU in 1% BSA) at a ratio of 90:10:10:10:10, was added to each DBS. Samples were incubated in a 37 °C water bath overnight. The next day samples were centrifuged for 15 min at 2200 g. The filter plate was discarded and receiver plates stored at -20°C until injection of samples into the LC-MS/MS. For control samples, most of the disaccharides were below the LLOQ (59% for HS-0S, 94% for HS-NS, 98% for mono-sulfated KS, and 99% for di-sulfated KS). False positive samples were defined as controls that were above the cutoff values, and false negatives were defined as newborn MPS samples that were below the cutoffs. Imprecision was calculated by replicated analysis of three different concentrations of ΔDiHS-0S and ΔDiHS-NS standard. For HS-0S CV intra was 5%, 12%, and 14% and CV inter was 7%, 12%, 14% (std 8, std 5, and std 3 respectively). For HS-NS CV intra was 9%, 12%, 16% and CV inter was 11%, 25%, and 16% (std 8, std 5, and std 3 respectively). Cutoffs were established for HS. The average values for each subclass of HS in controls was HS-0S= 37 ng/ml (+50), HS-NS= 12 ng/ml (+28), mono-KS= 154 ng/ml (+ 168), di-KS=35 ng/ml (+38), ratio di-KS in total KS=18% (+9). Average for MPS patients for HS-0S=201 ng/ml (+57), HS-NS: 60 ng/ml (+17), mono-KS=206 ng/ml (+125), di-KS=64 ng/ml (+35), ratio di-KS in total KS=26% (+12). Cutoffs from newborn control samples were HS-0S>137ng/ml and HS-NS>67ng/ml (cutoffs could not be defined for KS since this is primarily elevated in MPS IV patients for which newborn DBS were not available). On average, newborn samples in MPS patients had higher values than the control newborns for all GAGs. Newborn MPS I, II and III patients accumulated KS at higher levels than newborn controls (p=0.02), even though this would not be predicted from the enzymes deficient in these patients. Indirect elevation of levels of other GAGs appears to occur frequently in MPS. In control newborns, 2% had HS-0S values higher than the cutoffs, 6% had HS-NS values higher than the cutoffs, 0.3% had mono-KS values more than 2SD higher than the average, 2.6% had di-KS values more than 2SD higher than the average, and 4% had a ratio of di-KS in total KS that were more than 2SD higher than the average. Due to the low incidence of MPS, it is unlikely that the elevated levels of disaccharides in control samples are from MPS patients and that these most likely represent false positives that could be caused by unrelated conditions that elevate GAGs in blood. Screening for MPS can use a two-tier process in which the first-tier LC/MS/MS method detects samples with high levels of GAGs and a second-tier enzyme assay method determines whether this elevation is caused by an MPS. We conclude that HS-0S (98% specificity and 77% sensitivity) and the ratio di-KS to total KS are biomarkers with potential use in newborn screening of newborn for MPS I, II, and III.
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