Improved Characterization of Complex β-Globin Gene Cluster Structural Variants Using Long-Read Sequencing

Complex insertion-deletion (indel) events in the globin genes manifest in widely variable clinical phenotypes. Many are incompletely characterized because of a historic lack of efficient methods. A more complete assessment enables improved prediction of clinical impact, which guides emerging therapeutic choices. Current methods have limited capacity for breakpoint assignment and accurate assessment of mutation extent, especially in cases containing duplications or multiple deletions and insertions. Technology, such as long-read sequencing, holds promise for significant impact in the characterization of indel events because of read lengths that span large regions, resulting in improved resolution. Four known complex β-globin gene cluster indel types were assessed using single-molecule, real-time sequencing technology and showed high correlation with previous reports, including the Caribbean locus control deletion (g.5,305,478_5,310,336del), a large β-gene duplication containing the Hb S mutation (g.4,640,335_5,290,171dup with g.5,248,232T>A, c.20A>T; variant allele fraction, 64%), and two nested variants (double deletions with intervening inversion): the Indian Gγ(Aγδβ)0-thalassemia (g.5,246,804-5,254,275del, g.5,254,276_5,269,600inv, and g.5,269,601_5,270,442del) and the Turkish/Macedonian (δβ)0 thalassemia (g.5,235,064_5,236,652del, g.5,236,653_5,244,280inv, and g.5,244,281_5,255,766del). Our data confirm long-read sequencing as an efficient and accurate method to identify these clinically significant complex events. Limitations include high-complexity sample preparation requirements, which hinder routine use in clinical laboratories. Continued improvements in sample and data workflow processes are needed to accommodate volumes in a tertiary clinical laboratory.