Next Generation Sequencing Identifies a Novel Rearrangement in theHBBCluster Permitting to-the-Base Characterization

Abstract

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.

Beta thalassaemia intermedia due to co‐inheritance of three unique alpha globin cluster duplications characterised by next generation sequencing analysis

“…Another mechanism of increasing α globin output is through segmental duplication of the whole α globin complex (Harteveld et al , ) but breakpoints of the reported duplications have not been fully characterised due to technological limitations. Here, we applied a previously described next generation sequencing (NGS) methodology (Shooter et al , ,b) to characterise three α globin cluster duplications, permitting to‐the‐base resolution in two of the three cases.…”

“…Genomic DNA was extracted using the Qiagen Symphony whole blood midi kit and eluted into 200 μl ATE buffer. NGS was performed as previously described (Shooter et al , ). DNA (5 μg) was sheared using a Covaris E220 to a mean size of 500 bp and purified by double size selection using Agencourt SPRIselect beads (Beckman Coulter, Brea, CA, USA).…”

First reported duplication of the entire beta globin gene cluster causing an unusual sickle cell trait phenotype

“…Normal and positive controls with known genetic rearrangements were analysed in parallel with the test samples. All data analyses were carried out using NextGene (SoftGenetics, State College, PA, USA) as described (Shooter et al , ). A normalized measure of the coverage across the sequenced region, known as repeats per kilobase exon model per million mapped reads (RPKM), was calculated for each 120 bp region covered in the in‐solution bait capture.…”

“…A next generation sequencing (NGS) assay and bioinformatic strategy was used to identify any potential rearrangement in the HBA and HBB globin gene loci that could explain the unusually low HbS (Shooter et al , ). Leucocyte‐extracted DNA (3 μg) was sheared to 500 bp using a Bioruptor (Diagenode, Denville, NJ, USA) and used to construct a sequencing library with minimal polymerase chain reaction (PCR) amplification.…”

Hemoglobin Subunit Beta Gene Polymorphism rs33949930 T>C and Risk of Sickle Cell Disease—A Case Control Study from Tabuk (Northwestern Part of Saudi Arabia)

“…Several studies indicated that the BCL11A, HMIP, and β-globin regions were associated with increased HbF levels in different populations; and investigation of these genotypes with respect to pain crisis is warranted in different population, which may help in prognostication, as also a genome-wide association study, which may help uncover new loci controlling HbF levels [14] [15].…”