Shooter C, Rooks H, Thein SL, Clark B
Genetic testing for hemoglobinopathies is required for prenatal diagnosis, understanding complex cases where multiple pathogenic variants may be present or investigating cases of unexplained anemia. Characterization of disease causing variants that range from single base changes to large rearrangements, may require several different labor-intensive methodologies. Multiplex ligation probe amplification analysis is the current method used to detect indels, but the technique does not characterize the breakpoints or detect balanced translocations. Here we describe a next generation sequencing (NGS) method that is able to identify and characterize a novel rearrangement of the HBB cluster responsible for εγδβ thalassemia in an English family. The structural variant involved a 59.0 kb inversion encompassing HBG2 exon 3, HBG1, HBD, HBB and OR51V1, juxtaposed by a deletion of 122.6 kb including 82 bp of the inverted sequence, HBG2 exon 1 and 2, HBE, and the β-locus control region. Identification of reads spanning the breakpoints provided to-the-base resolution of the rearrangement, subsequently confirmed by gap-PCR and Sanger sequence analysis. The same rearrangement, termed Inv-Del English V εγδβ thalassemia (HbVar 2935), was identified in two other unrelated English individuals with a similar hematological phenotype. Our NGS approach should be applicable as a diagnostic tool for other disorders.