Long-Read Sequencing-Based Resolution of Structural Variants in Sickle Cell Disease Patient Genomes: A Cohort from Enugu State

Sickle cell disease (SCD) severity is modulated by a range of genetic factors beyond the canonical HbS point mutation, including coinherited structural variants (SVs) in globin gene cluster regions that affect fetal hemoglobin (HbF) expression. Long-read sequencing platforms such as Oxford Nanopore and PacBio offer superior resolution of complex SVs compared to short-read methods. This study employed long-read sequencing to resolve structural variants in the beta-globin locus and identify HbF-regulating SVs in SCD patients from Enugu State University Teaching Hospital (ESUTH), Southeast Nigeria. Peripheral blood DNA was obtained from 40 HbSS patients and 15 HbAA controls. High-molecular-weight DNA was extracted using a bead-based long-read extraction protocol. Sequencing was performed on the Oxford Nanopore PromethION platform. SV calling was conducted using Sniffles2, and the BCL11A and MYB regulatory loci were examined for variant presence. Five previously unreported deletions within the HBG1/HBG2 promoter regions were identified, three of which were predicted to disrupt BCL11A repressor binding sites and were associated with elevated HbF levels by HPLC (greater than 20% HbF). One patient harbored a compound heterozygous arrangement with a novel 3.2 kb deletion at the 3-prime end of the beta-globin locus cluster. These long-read-derived SV data contribute novel genetic modifier candidates relevant to SCD severity prediction in the Southeast Nigerian patient population. Keywords: sickle cell disease, long-read sequencing, structural variants, HbF, Enugu State.