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 theHBBcluster responsible for εγδβ thalassemia in an English family. The structural variant involved a 59.0 kb inversion encompassingHBG2exon 3,HBG1,HBD,HBB, andOR51V1, juxtaposed by a deletion of 122.6 kb including 82 bp of the inverted sequence,HBG2exon 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.
Genetic testing for inherited disorders is pre-requisite for prenatal diagnosis and counseling. Next generation sequencing (NGS) has enabled complete characterization of a novel inversion-deletion β thalassemia, that eluded characterization using routine genetic techniques. Our NGS approach not only identified the rearrangement but permitted to-the-base characterization; the same rearrangement was identified in 2 other unrelated families. The NGS approach should be applicable as a diagnostic tool for other disorders.