Multiplexed Genotyping of β-Globin Variants from PCR-amplified Newborn Blood Spot DNA by Hybridization with Allele-specific Oligodeoxynucleotides Coupled to an Array of Fluorescent Microspheres

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Abstract
Detection of hemoglobinopathies in newborns is critical for the identification of those infants in need of follow-up care [reviewed in Ref. (1)]. For example, infants homozygous for the sickle cell mutation are at greater risk for developing fatal pneumococcal infections and sepsis, which can be prevented by prophylactic antibiotic therapy. At present, the majority of newborn screening for hemoglobin (Hb) variants is done by electrophoresis, isoelectric focusing, or HPLC (2) using Hb extracted from dried blood spots. Nevertheless, detection of adult Hb variants often is complicated by the presence of fetal Hb in neonatal blood. Alternative approaches to these protein-based methodologies have been developed that directly detect the presence of hemoglobinopathy-associated mutations in newborn DNA (3), and some have been adapted to use blood spots (4)(5). Using the S and E mutations in the β-globin gene as examples, we have developed a multiplexed, high-throughput methodology that uses an array of allele-specific fluorescent beads and the Luminex100 analyzer (Luminex Corporation, Austin, TX; www.luminexcorp.com). This methodology distinguishes between the S and E alleles and their wild-type counterparts, HbA and non-E, of the β-globin gene in each specimen, making it possible to determine the genotype at each locus.