Design, Validation, and Clinical Implementation of a Gap-Polymerase Chain Reaction Method for -Thalassemia Genotyping Using Capillary Electrophoresis

alpha -Thalassemia (-thal) is genetically heterogeneous with most cases caused by variably sized deletions of the HBA1 and/or HBA2 loci. In this report, we describe the development, validation, and implementation of a novel gap-polymerase chain reaction (gap-PCR)/capillary electrophoresis (CE). method. This assay utilizes two multiplex reactions and CE to detect the following deletions: -(3.7) (rightward), -(4.2) (leftward), -()(20.5), - -(SEA) (Southeast Asian), - -(MED), - -(FIL) and - -(THAI). Validation studies using 36 previously characterized patient samples and plasmid controls demonstrated 100.0% accuracy. Following clinical implementation, 423 patients were analyzed over 24 months. Two hundred and twenty-seven cases (46.0%) showed abnormal results including heterozygous -(3.7) (n=114, 27.0%), homozygous -(3.7) (n=96, 23.0%), heterozygous - -(SEA) (n=9, 2.0%), heterozygous - (4.2) (n=5, 1.0%), heterozygous - -(MED) (n=1, <1.0%), and compound heterozygous -(3.7)/-(4.2) (n=2, <1.0%) deletions. Correlation with red blood cell (RBC) parameters showed that patients with a deletion of two or more genes were associated with significantly lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) levels than patients with wild-type results. This novel multiplex gap-PCR protocol reliably detects the seven most common deletions giving rise to -thal. Use of the fluorescently labeled CE method provides for a high throughput workflow suitable to a clinical diagnostic laboratory serving a multiethnic population.