Application of Next Generation Sequencing In the Characterisation of Variants Causing Haemoglobinopathies

Abstract

Haemoglobinopathies are a group of diseases caused by abnormal structure, function or quantity of the globin chain subunits that form haemoglobin. They are a global health problem with 7% of the population being asymptomatic carriers. The UK has implemented new-born screening and antenatal screening to identify affected individuals. A wide variety of variants causing haemoglobinopathies have been documented, from single base substitutions to large insertions, deletions and complex rearrangements. Next Generation Sequencing (NGS) could streamline laboratory diagnosis as it has the potential to identify both single nucleotide changes and large rearrangements in a single assay. With this objective in mind, we evaluated NGS and its ability to detect all varieties of causative variants in thalassaemia. DNA samples from persons with known thalassaemia-causing mutations and unknown novel variants were selected for NGS. DNA samples were fragmented and prepared for sequencing. The two genomic regions that are affected by variants causing thalassaemia, were enriched for using in-solution bait capture and sequenced on an Illumina platform. Sequence ‘reads’ of the DNA fragments were aligned to a reference sequence of the human genome using NextGene software (SoftGenetics) and sequence indels and small variants were reported. Dosage changing events were identified by changes in coverage between test and negative control samples. The breakpoints of structural variants could be identified by the presence of sequences that spanned the breakpoint region. The method was honed by analysis of samples with known, previously characterised variants and tested in samples with unknown novel variants. The findings were confirmed by Gap-PCR and sanger sequencing. We conclude that NGS is a technique that represents an improvement on the current diagnostic standard. An examination of the features of the structural rearrangements identified in this study revealed that multiple mutagenic mechanisms contribute to the range of variants that affect the alpha and beta globin gene loci.

Date of Award	2016
Original language	English
Awarding Institution	King's College London
Supervisor	Swee Lay Thein (Supervisor)