Genetic determinants of HbF in Saudi Arabian and African Benin haplotype sickle cell anemia

To the Editor: Each of the major haplotypes of the HBB gene cluster in sickle cell anemia are associated with different fetal hemoglobin (HbF) levels. Four of these HBB haplotypes originated in Africa (Benin, Central African Republic, Cameroon, and Senegal) while the Arab-Indian haplotype arose independently in the Arabian Peninsula or in India. Although HbF is the major modulator of disease severity, the genetic elements that underlie the association of HbF and HBB haplotypes are not fully understood. Saudi sickle cell patients from the Southwestern Province, whose HBB gene cluster is of African origin, have HbF levels of about 10%; African origin patients with the Benin haplotype have HbF levels of about 6%. Saudi patients have a genetic population structure similar to other Arabs, which does not resemble African-origin patients. We hypothesized that while Saudi and African American Benin haplotype homozygotes have similar HBB clusters, there might be common variants in the Saudi Benin patients that are associated with their increased HbF; conversely, African American Benin patients might have common variants that are associated with reduced HbF relative to Saudi patients. To study the genetic differences between Saudi and African American Benin haplotype patients that might be associated with HbF, we imputed genome-wide association study (GWAS) data from the Cooperative Study of Sickle Cell Disease (CSSCD) and patients from the Southwestern Province of Saudi Arabia to 1000 Genomes Phase 3 v5 reference panel. Preimputation quality control was performed using PLINK, and imputation was carried out through the Michigan Imputation server and phased imputed data was obtained using Eagle. We classified haplotypes using haplotypeClassifier available from https://github.com/eshaikho/haplotypeClassifier. Only homozygous Benin haplotype cases were selected for downstream analysis. We then removed related samples and outliers (based on their genetic markers) in the first 20 principal components (PCs) using King and EIGENSOFT, respectively. There were 293 African American patients not taking hydroxyurea, 153 males, and 140 females, aged between 2 and 68 years with an average HbF of 6.38%, and 63 Saudi Benin haplotype patients 27 of them are taking hydroxyurea, 36 males and 27 females, aged between 4 and 43 years, with an average HbF of 10.38%. A linear model adjusted for age and sex to predict the effect of PCs HbF level showed insignificance of the first 20 components indicating absence of population substructure within each population; the first five PCs were used in the final GWAS analysis to account for any potential bias due to these components. Log10 HbF levels were employed as a quantitative phenotype to find the most significant SNPs in each cohort. Efficient and Parallelizable Association Container Toolbox (EPACTS; https://github.com/statgen/EPACTS) adjusted for sex and the first five PCs were used for the final analysis. To avoid false associations due to small sample size we only considered SNPs with minor allele frequency (MAF) greater than 0.05. Including age as covariate does not improve the goodness of the model fit, thus it was excluded from the final model. Both cohorts were analyzed separately, and subjected to the same analytical methods except for hydroxyurea adjustment in Saudi Benin patients. In African American Benin haplotype patients, only rs1427407 in BCL11A met GWAS significance levels for association with HbF (Figure 1). Six intronic SNPs in BCL11A had marginal genome-wide significance; 3 intronic SNPS in LARGE1, NEDD9 and PAK2 also showed marginal GWAS significance with p-values between 8.97E-07 and 2.61E-07 (Table 1). In Saudi Benin cases, there were no associations with HbF meeting GWAS significance levels; however the small sample size reduced the statistical power of the study to detect an association. The allele frequencies for the top 10 associated SNPs in African American patients were similar to that in Saudi patients except for rs6706648 and rs7606173 where the MAFs were 0.4 and 0.44 in African American and 0.11 and 0.25 in Saudi cases. To examine the effect of rs1427407, rs6706648,