Sickle cell disease in the Eastern Province of Saudi Arabia: Clinical and laboratory features
Authors: Amein K. Al‐Ali, Ahmed Alsulaiman, Mohammed Alfarhan, Surinder Safaya, Chitti Babu Vatte, Waleed M. Albuali, Hatem O. Qutub, Alhusain J. Alzahrani, Jacqueline N. Milton, Martin H. Steinberg
Journal: American Journal of Hematology (2021)

Epicenters of sickle cell disease exist in the Eastern and Southwestern Provinces of Saudi Arabia.1 In the Eastern Province, the sickle hemoglobin gene (HbS; β7glu-val, GAG-GTG) is usually associated with the Arab-Indian (AI) haplotype; in Southwestern Province patients the Benin haplotype predominates. Fetal hemoglobin (HbF, α2γ2) inhibits deoxyHbS polymerization. It is the major modulator of sickle cell disease severity.2 AI haplotype sickle cell anemia was initially believed to be a “mild” form of disease that was attributed to patients' high levels of HbF. However, “mildness” was a feature of childhood when HbF totaled ~30% of hemoglobin. With aging, HbF declined to 15%–20% of hemoglobin. Correspondently, the disease phenotype began to resemble that of patients of African descent.3 We present hematologic, clinical and genetic data in a large cohort of adults homozygous for AI haplotype sickle cell anemia. Patients from the Eastern Province of Saudi Arabia, aged 2 to 82 years were recruited from 2016 to 2019 at either King Fahd Hospital, Al-Ahsa or King Fahd Hospital of the University, Imam Abdulrahman Bin Faisal University, Dammam. Clinical complications were defined by standard criteria. This study was approved by the Ethics Committee of King Faisal University, Al-Ahsa (IRB-9/31/A/60). Homozygosity for HbS gene was ascertained by ARMS-PCR. The Xmn1 restriction site 158 bp 5′of HBG2 (rs7482144) was identified by PCR-RFLP. The HBD promoter SNP (−68 C > T) was determined by di-deoxy sequencing. Homozygotes for the minor allele (T) of rs7482144, who were homozygous for the −68 HBD SNP, were deemed AI haplotype homozygotes; rs7482144 homozygotes who were heterozygotes for the −68 HBD SNP were compound heterozygotes for AI and Senegal haplotypes. Deletions in the α-globin gene cluster were detected using 7-deletion multiplex polymerase chain reaction assay. The SNPs of rs766432 in BCL11A and rs9399137 in MYB, were genotyped by nuclease allelic discrimination assay with target-specific forward and reverse primers. The HbF levels were determined by HPLC. Analyses used SAS version 9.4 (SAS Institute, Cary, NC). The mean and standard deviation or median and interquartile range were computed for continuous characteristics for normal and non-normal distributions, respectively. Frequencies and percentages were computed for non-continuous characteristics. Linear regression was performed to examine the association between HbF and age where slopes, 95% confidence intervals and P values were reported. Multivariable linear regression was performed to examine the variability in HbF explained by rs766432 and rs9399137. Features of the study population are shown in Table 1. Less than 20% used hydroxyurea. Regardless of haplotype, there were few differences in the prevalence of the major clinical complications. Vasoocclusive complications like acute chest syndrome, avascular necrosis of bone and acute painful episodes in AI haplotype patients were similar to those in patients of African origin; stroke appeared to be less common. The HBB haplotypes were available for 780 HbS homozygotes; 619 were homozygous for the AI haplotype. One hundred sixty-one additional HbS homozygotes had diverse other haplotypes that included 98 homozygotes or compound heterozygotes for Benin, Bantu, Cameroon and Senegal haplotypes, or compound heterozygotes for these and AI or atypical haplotypes.4 Sixty-three patients were compound heterozygotes for AI and Senegal haplotypes. Except for the lower hemoglobin level of 8.6 g/dL in nine CAM haplotype homozygotes, and the expected HbF difference in patients with different combinations of African haplotypes, there were few hematologic or laboratory differences among these patients. Further details are found in.4 More than half of all patients were heterozygous or homozygous for gene deletion α thalassemia. Note, HbF was not associated with age. In AI haplotype homozygotes, regression of HbF on age showed β = 0.02, 95% CI −0,03, 0.07, p = .36. The mean age in all our haplotype groups was nearly 30 years; HbF in AI haplotype patients stabilizes at ~10 years of age. The genotype frequencies of sentinel SNPs in BCL11A and MYB were similar to those reported previously (Table 1).5 AI haplotype homozygotes with minor alleles of rs766432 in BCL11A or rs9399137 in MYB had higher HbF than individuals with wild-type alleles (p < .001). In the Eastern Province of Saudi Arabia, ~1 in 80 people have sickle cell disease. Herein, we present details of the largest cohort yet reported of patients with sickle cell anemia homozygous for the AI haplotype. Both BCL11A and MYB are the major known quantitative trait loci (QTL) modulating HbF gene expression. The frequency of two sentinel minor alleles of these QTL, rs766432 in BCL11A and rs9399137 in MYB were 0.28 and 0.08, respectively; together they explained only 4.2% of HbF variance. In 2040 sickle cell anemia patients of African descent the minor allele frequencies of rs766432 and rs9399137 were simllar but explained 14.7% of HbF variance.6 Perhaps these difference are due to the prevailing effects of cis-acting HbF modulation in the AI haplotype. For example, disruption by CRISPR-Cas9 of the rs7482144 site in the HBG2 promoter in sickle cell CD34+ cells was associated with ~25% γ globin and 55% ± 5% F-cells suggesting that this is a binding motif for an uncharacterized transcription factor or complex.2 In homozygotes for rs7482144 and sickle cell anemia, HBG2 makes up 66% to 75% of γ globin compared with ~40% in other adults. Our new genetic data allows further speculation on the basis of high HbF in the AI haplotype. The AI and Senegal haplotypes both contain the −158 C-T SNP (rs7422144), yet the average HbF in the AI haplotype is about 30% higher than in the Senegal haplotype. This might be an effect of a previously described AI sub-haplotype.7 Upon this “baseline” HbF, the minor alleles of rs766432 in BCL11A or rs9399137 in MYB further increase HbF. In addition, less well defined HbF repressors might also be extant in the Arab population. One such variant in ANTXR1 was linked to the AI haplotype in Saudi but not Indian patients and explained ~10% of HbF variability. Like BCL11A and MYB, this appeared to be a HbF repressor. African-American sickle cell anemia patients with HbF levels between 17% and 20% had a larger number of minor alleles of HbF QTL than patients with HbF between 3% and 5%.8 Levels of HbF might not be similar across cohorts of AI haplotype patients with different geographical origins. Kuwaiti Arabs had a mean HbF of 22%; in two Indian cohorts HbF was 23%. Although inhomogeneity among cohorts might account for these differences, each population could have unique modulators of HbF expression in known or novel QTL. Regardless of the haplotype of the HbS gene, compared with patients of African descent, stroke is less common and leg ulcers are rare in our cohort (Tables 1 and 2). This might result from their lower rates of hemolysis. High HbF and α thalassemia diminish the intensity of hemolysis.9 Fifty-seven percent our patients had α thalassemia. Hemolysis, usually ascertained indirectly rather than by measuring erythrocyte lifespan, is reflected by reticulocyte count, bilirubin, AST and LDH levels. Intravascular hemolysis, estimated by serum LDH, correlates with more direct markers of hemolysis like plasma hemoglobin and erythrocyte microparticles. In 415 patients with sickle cell disease, a high hemolytic rate was associated with an increased the risk of death. Among patients followed for a median of 2.4 years, the top tercile of hemolysis had more than three-times the risk of death as did the lower two terciles (hazard ratio 3.44; 95% CI: 1.2–9.5).10 When clinical complications that were associated with hemolysis were aggregated in a high vascular risk profile, this was associated with a five-fold increased risk of mortality.10 Closely associated with hemolysis are the vasculopathic complications of this disease. In our cohort, as in other reports, stroke was less common than in patients of African descent, while leg ulcers were absent (Tables 1 and 2). We lack mortality information for this cohort. HbF and hemolysis correlate with mortality.9, 10 This suggests that in AI haplotype sickle cell anemia, lifespan might be extended compared with patients of African descent. A longitudinal study of a birth cohort can best confirm this hypothesis. We extend our gratitude for the laboratory and technical work of Mr. Abdulrazag Al-Wabary, Mr. Ibrahim Al-Sager, and Mr. Hani Al-Farhan and the nurses involved in the project in both hospitals. A.K. A-A. and M.H.S. designed the study, analyzed results and prepared the first and final drafts of the manuscript, J.N.M. performed statistical analysis, all other authors recruited patients, examined data, performed laboratory analysis and edited the paper. This study was supported by Alomran Scientific Chair for Hematological Diseases in Al-Ahsa, King Faisal University, and partially by King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia, grant number [12-MED2224-46]. The data that support the findings of this study are available from the first authora,b upon reasonable request. The authors declare no conflict of interest. The data that support the findings of this study are available from the 1st author upon reasonable request.