Breakpoint characterization of a rare alpha⁰‐thalassemia deletion using targeted locus amplification on genomic DNA

Abstract

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.

Characterization of New Alpha Zero (α0) Thalassaemia Deletion (−−GB) among Malays in Malaysian Population

“…The exact breakpoint of this deletion was determined by Gap-PCR and direct sequencing (described in the methodology). A similar deletion was previously described in a Dutch individual of mixed ethnicity, Indonesian and Arabic [24,32,33], respectively.…”

“…From the analysis of a total of 1054 patients from 2012 to 2018 we found 29 cases with the −− GB deletion with a frequency of 2.8%. The deletion breakpoint was successfully characterized using MLPA, based on the breakpoint characterization with a set of Gap-PCR and sequencing methods described previously [24,32,33].…”

“…The −− GB deletion was successfully characterized in a Dutch individual of Arabic and Indonesian ethnicity, based on a previous report by [24,32,33]. Recently, Hottentot et al raised the issues regarding an inaccurate deletion annotation as the −− GB deletion with a 15.2 kb deletion by Mota et al, with correspondence to the IthaID: 3294, and without determining the exact breakpoint [33,34].…”

Detection of hemoglobin H disease by long molecule sequencing

“…The ‐α 27.6 is a rare deletional mutation type and was first reported in a Chinese family, outside the routine method's test scope 15 . In fact, there were some occasional reports of similarly rare large deletion of α‐chain 16–18 . Since the normal control primers of the routine genetic analysis are designed on the HBA2 gene, it will be detected as a SEA homozygote if combined with a SEA heterozygote as long as the large fragment deletional sample involves this region.…”

“…15 In fact, there were some occasional reports of similarly rare large deletion of α-chain. [16][17][18] Since the normal control primers of the routine genetic analysis are designed on the HBA2 gene, it will be detected as a SEA homozygote if combined with a SEA heterozygote as long as the large fragment deletional sample involves this region. However, we considered Hb H disease combined with the comprehensive screening results.…”