The Molecular Biology of Sickle Cell Anaemia

Abstract

Sickle cell anaemia (SCA) is a haemolytic anaemia that reduces life expectancy and places a great burden on healthcare systems worldwide. Despite being a monogenic disorder, the phenotypic severity varies greatly between patients, ranging from patients that experience multiple strokes and organ failure during childhood, to those that live largely unaffected lives. Some genetic variants that affect globin gene expression are known to influence phenotype severity, but most of this variation remains unaccounted for.
We conducted whole exome sequencing analyses, comparing SCA patients with mild and severe clinical phenotypes, with the aim of identifying novel genetic modifiers of the disease. SCA patient exomes were sequenced from a cohort at King’s College Hospital, and combined with publicly available SCA exomes recruited in the United States. Nine candidate variants were identified in genes with plausible mechanisms to influence the pathophysiology of the disease. The genes identified in this study affected nitric oxide signalling, haematopoietic regulation, globin gene expression and recovery from ischaemic injury.
In order to evaluate these variants, a CRISPR genomic editing pipeline was established and tested on two previously identified candidate modifiers of SCA, in the genes ASH1L and KLF1. These variants were successfully introduced into erythroleukaemic cells and provide a pathway for testing the novel modifier genes identified in the exome sequencing analysis. Preliminary studies indicate that both ASH1L and KLF1 variants alter globin gene expression.
In addition to genetic factors, we also hypothesised that epigenetic factors affect the SCA phenotype, and play a role in the therapeutic mechanism of hydroxyurea treatment. We optimised a method for isolating CD45+CD71+GPA- nucleated erythroid progenitors from small volumes of SCA peripheral blood. This was undertaken to evaluate the role of the epigenome in SCA phenotype severity and drug action, but for which patient sample collection proved too challenging within our clinical cohort.

Date of Award	2018
Original language	English
Awarding Institution	King's College London
Supervisor	Rebecca Oakey (Supervisor) & Swee Lay Thein (Supervisor)