Breakpoint characterization of a rare alpha(0)‐thalassemia deletion using targeted locus amplification on genomic DNA

INTRODUCTION: The high‐sequence homology of the α‐globin‐gene cluster is responsible for microhomology‐mediated recombination events during meiosis, resulting in a high density of deletion breakpoints within a 10 kb region. Commonly used deletion detection methods, such as multiplex ligation‐dependent probe amplification (MLPA) and Southern blot, cannot exactly define the breakpoints. This typically requires long‐range PCR, which is not always successful. Targeted locus amplification (TLA) is a targeted enrichment method that can be used to sequence up to 70 kb of neighboring DNA sequences without prior knowledge about the target site. METHODS: Genomic DNA (gDNA) TLA is a technique that folds isolated DNA, ensuring that adjacent loci are in a close spatial proximity. Subsequent digestion and religation form DNA circles that are amplified using fragment‐specific inverse primers, creating a library that is suitable for Illumina sequencing. RESULTS: Here, we describe the characterization of a rare 16 771 bp deletion, utilizing gDNA TLA with a single inverse PCR primer set on one end of the breakpoint. Primers for breakpoint PCR were designed to confirm the deletion breakpoints and were consequently used to characterize the same deletion in 10 additional carriers sharing comparable hematologic data and similar MLPA results. CONCLUSIONS: The gDNA TLA technology was successfully used to identify deletion breakpoints within the alpha‐globin cluster. The deletion was described only once in an earlier study as the ‐‐(gb), but as it was not registered correctly in the available databases, it was not initially recognized as such.