Identifying genetic variants and pathways associated with extreme levels of fetal hemoglobin in sickle cell disease in Tanzania

Abstract

Background: Sickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common genetic variants which differ across populations and hence do not fully account for HbF variation.

Methods: We investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A, MYB, HOXA9, HBB, HBG1, HBG2, CHD4, KLF1, MBD3, ZBTB7A and PGLYRP1.

Results: Results revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified frameshift deletions in individuals with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis.

Conclusions: This study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD interrogating multiple genomic variants associated with HbF in SCD.

Keywords: Fetal hemoglobin; Genetic disorder; Hemoglobinopathy; Sickle cell disease; Tanzania.

Fig. 1

Workflow of the data analysis. Describes the bioinformatics pipelines from alignment of DNA reads, variants calling to in silico mutation prioritization

Fig. 2

Characterization of SCD gene function and exome map from the targeted next generation sequencing: This included the exon and full regions for validated and novel fetal hemoglobin-associated loci, including B-cell lymphoma/leukemia 11A (BCL11A), proto-oncogene, transcription factor (MYB), Homeobox A9 (HOXA9), Hemoglobin subunit beta (HBB), hemoglobin subunit gamma 1 (HBG1), hemoglobin subunit gamma 2 (HBG2), chromodomain helicase DNA binding protein 4 (CHD4), Kruppel like factor 1 (KLF1), methyl-CpG binding domain protein 3 (MBD3), zinc finger and BTB domain containing 7A (ZBTB7A), Peptidoglycan recognition protein 1 (PGLYRP1) on chromosomes 2, 6, 7, 11, 12 and 19, respectively. a gene functions from patients with high HbF levels and b gene functions from patients with low HbF levels

Fig. 3

Biological sub-network of the candidate mutant gene and identified modifier genes in 14 SCD patients from Tanzania. a sub-networks of the mutant gene and identified candidate genetic modifiers include CHD4 and MBD3. b description of the top most significant pathways, GO biological process, and Human Phenotypes associated with the identified variants

Fig. 4

Biological sub-network of the candidate mutant gene and identified modifier genes in 14 SCD patients from Tanzania. a sub-networks of our target sequencing variants include ZBTB7A, BCL11A, MYB, HBB, HOXA9, HBG2, CHD4, KLF1, MBD3.b description of the top most significant pathways, GO biological process, and Human Phenotypes associated with the identified variants