P006: Genetic insights from a large-scale exome sequencing study of type 1 diabetes in Ukraine

MPS II has recently met the Recommended Uniform Screening Panel (RUSP) requirements and is being added to US newborn screening (NBS) panels.Several other MPSs, including MPS IVA, VI, and VII, have been identified as candidates for addition to public health NBS; however, evidence is lacking for a RUSP nomination.This is partly because it is unknown whether existing tests can reliably identify babies with these conditions.This validation assesses the performance of the assay for NBS in DBS and approximates the number of newborns who will subsequently require confirmatory testing and clinical follow-up.The results of this study provide important information to support public health policy decisions regarding NBS for these conditions.Methods: This assay is a laboratory-developed test for enzyme activity in DBS.Newborn DBS are incubated with a cocktail solution containing enzyme substrate and internal standard (IS) for each enzyme of interest.After incubation, the samples are extracted, dried, and reconstituted in mobile phase then analyzed via positive-ion LC-MS/MS.The peak area ratios of each enzyme-product and IS are used to calculate enzyme activity.Results: This validation assessed the enzyme activity of I2S, GALNS, GUSB, and ARSB in DBS to determine analytical specificity, sensitivity, linearity, precision, carryover, and stability of reagents and enzyme activity in DBS.Enzyme product curves were linear with R 0.99 for I2S, R 0.95 for GUSB, and R 0.98 for GALNS and ARSB.The intra-day, inter-day, inter-analyst, and inter-instrument precision of the method was CV < 20% for all analytes.In addition to these tests, a retrospective screen of ~5,000 de-identified NBS specimens from North Carolina newborns was performed to establish an initial reference range specific to the screened population.The 10% median cutoffs correspond to the following activities (units): I2S = 1.47,GUSB = 2.86, GALNS = 0.209, ARSB = 0.659.The accuracy of the cut-off established in the population study was tested against known positive specimens for each condition.Using a cutoff of 10% of the daily median for all analytes, all known positives were accurately identified by the assay. Conclusion:The multiplex method validated in this study successfully measured the enzyme activities of I2S, ARSB, GUSB, and GALNS in DBS using a single DBS punch.The 10% cutoffs accurately differentiated patient specimens from presumed unaffected individuals.The expected rate of false positive/pseudodeficiency results is 1/5,000.Data from prospective NBS are needed to demonstrate the feasibility of large-scale screening and clinical follow-up for infants who screen positive.This research highlighted the need for automation in sample preparation and data processing of multiplex assays for high-throughput screening.