Identification of glycosaminoglycans for newborn screening and therapeutic monitoring of mucopolysaccharidoses

ABSTRACTIdentification of glycosaminoglycans for newborn screening and therapeutic monitoring of mucopolysaccharidoses Mucopolysaccharidoses (MPS) are a group of 11 rare inherited metabolic diseases, each caused by a deficiency in a specific enzyme necessary for the breakdown of complex sugars termed glycosaminoglycans (GAGs). The accumulation of GAGs in various tissues causes a spectrum of health problems, including heart, bone, joint, and nervous system complications, which progressively worsen and lead to restricted mobility and premature death. Therefore, it is critical that affected newborns are identified at birth through newborn screening (NBS). However, only assays for MPS I and II are currently available in the U.S., and not all states are presently screening for MPS diseases. A methodology that can screen for MPS disorders, and identify by subtype, would be of tremendous value to the NBS community; future pilot data from a longitudinal study with such a platform would provide evidence needed to recommend uniform NBS for MPS disorders with approved therapies. The majority of currently available treatments result in a reduction in GAG accumulation and therefore monitoring of GAG levels is a crucial component of MPS treatment regimes. Standard tests for total GAG measurement are currently performed on urine samples using tandem mass spectrometry (MS/MS) or dimethyl methylene blue and have several limitations including: poor sensitivity, large sample volumes, and long turnaround times (rt3 days). To overcome these challenges in NBS and therapeutic monitoring for MPS, we propose to develop a novel high-throughput digital microfluidic (DMF) platform for identification of GAGs that can be leveraged to screen for disease AND monitor disease treatment. We will use a combination of novel (patent pending) enzyme modulation assays to measure 1) total GAG levels (to identify if MPS disease is present during NBS) and 2) specific GAG categories (to determine which specific GAG is elevated during NBS). Application of targeted next generation sequencing (tNGS) as a 3rd test will determine which specific MPS/subtype is present. The GAG modulation assays will be performed on our automated DMF cartridge and platform, which can perform the GAG analyses either on dried blood spot (screening) or whole blood sample (therapeutic monitoring). The tNGS analysis will be developed in our in-house CLIA-certified laboratory. We previously demonstrated feasibility of GAG microtiter plate assays for heparan, dermatan, and keratan sulfates. We will develop assays for total GAGs and chondroitin sulfate, and translate all GAG assays to the DMF platform. The tNGS gene panel will also be developed for use during NBS. Preliminary analytical performance of the assay panel will be assessed, and a method comparison is planned against the gold standard assays to demonstrate platform equivalence. Our innovative and groundbreaking solution for identification of GAGs will dramatically increase the rate of uptake of NBS for more MPSs in state public health laboratories. The versatility of our platform to also monitor individual GAG levels during treatment will add significant value proposition during commercialization.