Abstract
Hemoglobin genotype and HBB haplotype are established genetic factors that modify the clinical phenotype in sickle cell disease (SCD). Current methods of establishing these two factors are cumbersome and/or prone to errors. The throughput capability of next generation sequencing (NGS) makes it ideal for simultaneous interrogation of the many genes of interest in SCD. This study was designed to confirm the diagnosis in patients with HbSS and Sβ-thalassemia, identify any ß-thal mutations and simultaneously determine the ßS HBB haplotype. Illumina Ampliseq custom DNA panel was used to genotype the DNA samples. Haplotyping was based on the alleles on five haplotype-specific SNPs. The patients studied included 159 HbSS patients and 68 Sβ-thal patients, previously diagnosed using high performance liquid chromatography (HPLC). There was considerable discordance between HPLC and NGS results, giving a false +ve rate of 20.5% with a sensitivity of 79% for the identification of Sβthal. Arab/India haplotype was found in 81.5% of βS chromosomes, while the two most common, of the 13 β-thal mutations detected, were IVS-1 del25 and IVS-II-1 (G>A). NGS is very versatile and can be deployed to simultaneously screen multiple gene loci for modifying polymorphisms, to afford personalized, evidence-based counselling and early intervention.
Highlights
Hemoglobinopathies are the most common monogenic disorders worldwide, with an estimated >300,000 affected infants born annually [1]
The clinical phenotype of sickle cell disease (SCD) is characterized by recurrent vaso-occlusion and chronic hemolytic anemia
This becomes an issue in parts of the world, especially the Mediterranean and the Middle East, where both the HbS and β-thal traits are prevalent and compound heterozygotes form a significant proportion of the SCD population [6,7]
Summary
Hemoglobinopathies are the most common monogenic disorders worldwide, with an estimated >300,000 affected infants born annually [1]. When HbA2 is estimated with HPLC in patients with Sβ-thal, it tends to be spuriously high because it co-migrates with glycated HbS ([5]) This becomes an issue in parts of the world, especially the Mediterranean and the Middle East, where both the HbS and β-thal traits are prevalent and compound heterozygotes form a significant proportion of the SCD population [6,7]. This is mandatory for prenatal diagnosis, whereby parental mutations are ascertained, for definitive screening of a fetus, using several DNA methods, ranging from PCR to sequencing What makes these molecular studies more fascinating is the discovery of genetic modifiers, which modulate the SCD phenotype and predispose it to, or protect it from, different complications. This paper is limited to only the role of NGS in diagnosis and haplotyping
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