D.O.1 A two-tiered approach to newborn screening for Duchenne muscular dystrophy (DMD) using dried blood spots for sequential CK and DNA analysis

Abstract

Historically, newborn screening for DMD has been challenging in part due to elevations in serum CK related to birth trauma, resulting in false-positive results when used as a screening test. Nevertheless, the rationale for newborn screening is supported by several lines of argument. One is that new techniques for molecular diagnostics hold promise for improved rapid mutational analysis from small amounts of starting material. Another is that new potential therapeutics are entering clinical trials, and these or future therapies may significantly impact disease course if offered in infancy. A complementary argument regards clinical trial readiness; studies addressing efficacy in children of this age cannot be performed if a reliable method of early detection is unavailable. We have established and validated a two-tier system of screening and molecular diagnosis that uses a dried blood spot obtained within the first 48h to first assess CK via a fluorometric assay as the first step. Preliminary studies established a population-based range of CK in newborns using 30,547 deidentified anonymous dried blood spot samples. Mutation analysis used genomic DNA extracted from the dried blood spot for whole genome amplification followed by MLPA and/or direct sequencing methods. DMD gene mutations (all exonic deletions) were found in 6 of 37,649 newborn males, all of whom had CK levels >2000U/l. In three newborns with CK >2000U/l in whom DMD gene abnormalities were not found, we identified limb-girdle muscular dystrophy gene mutations affecting the DYSF, SGCB, and FKRP genes. This data is currently in press elsewhere, but here we will review this path for screening that fits the US health care system, minimizes false-positive testing, and uses predetermined levels of CK on dried blood spots to predict DMD gene mutations, and we will discuss prospects and challenges for widespread utilization.