ALPHA-THALASSEMIA SUBTYPING AND THE DETECTION OF SILENT MUTATIONS BY HIGH-RESOLUTION FRAGMENT ANALYSIS AND DNA-SEQUENCING

Background: Sets of polymerase chain reaction (PCR) primers were used that make it possible to study subtypes of the most prevalent mutation s that cause alpha-thalassemia (-alpha 3.7 deletions). These primers ( Dev and C3, Dev and C9c) can be used in PCR to amplify regions that ar e characteristic of the three -alpha 3.7 deletion subtypes (-alpha 3.7 i, alpha 3.7ii, alpha 3.7iii). These subtypes are important because th e type of alpha-chain deletion can affect the level of alpha-globin pr oduction. Methods and Results: The PCR products were screened using au tomated high resolution electrophoresis on a DNA sequencer. Subtypes w ere then identified by automated DNA sequencing. During the screening for subtypes with high-resolution fragment analysis, new fragments wer e detected that were slightly different from the expected size. DNA se quencing of these fragments demonstrated the presence of three mutatio ns that had not been described or fully characterized before. Conclusi ons: High-resolution fragment analysis is a convenient way to detect a lpha-thalassemia subtypes, and DNA sequencing of silent mutations can lead to a better understanding of the cause of these mutations.