Zaki Naserullah

Fetal hemoglobin in sickle cell anemia: genetic studies of the Arab-Indian haplotype.

Sickle cell anemia is common in the Middle East and India where the HbS gene is sometimes associated with the Arab-Indian (AI) β-globin gene (HBB) cluster haplotype. In this haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are 3-4 fold higher than those found in patients with HbS haplotypes of African origin. Little is known about the genetic elements that modulate HbF in AI haplotype patients. We therefore studied Saudi HbS homozygotes with the AI haplotype (mean HbF 19.2±7.0%, range 3.6 to 39.6%) and employed targeted genotyping of polymorphic sites to explore cis- and trans- acting elements associated with high HbF expression. We also described sequences which appear to be unique to the AI haplotype for which future functional studies are needed to further define their role in HbF modulation. All cases, regardless of HbF concentration, were homozygous for AI haplotype-specific elements cis to HBB. SNPs in BCL11A and HBS1L-MYB that were associated with HbF in other populations explained only 8.8% of the variation in HbF. KLF1 polymorphisms associated previously with high HbF were not present in the 44 patients tested. More than 90% of the HbF variance in sickle cell patients with the AI haplotype remains unexplained by the genetic loci that we studied. The dispersion of HbF levels among AI haplotype patients suggests that other genetic elements modulate the effects of the known cis- and trans-acting regulators. These regulatory elements, which remain to be discovered, might be specific in the Saudi and some other populations where HbF levels are especially high.

BCL11A enhancer haplotypes and fetal hemoglobin in sickle cell anemia

BACKGROUND Fetal hemoglobin (HbF) levels in sickle cell anemia patients vary. We genotyped polymorphisms in the erythroid-specific enhancer of BCL11A to see if they might account for the very high HbF associated with the Arab-Indian (AI) haplotype and Benin haplotype of sickle cell anemia. METHODS AND RESULTS Six BCL112A enhancer SNPs and their haplotypes were studied in Saudi Arabs from the Eastern Province and Indian patients with AI haplotype (HbF ~20%), African Americans (HbF ~7%), and Saudi Arabs from the Southwestern Province (HbF ~12%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) and their haplotypes were consistently associated with HbF levels. The distributions of haplotypes differ in the 3 cohorts but not their genetic effects: the haplotype TCAG was associated with the lowest HbF level and the haplotype GTAC was associated with the highest HbF level and differences in HbF levels between carriers of these haplotypes in all cohorts were approximately 6%. CONCLUSIONS Common HbF BCL11A enhancer haplotypes in patients with African origin and AI sickle cell anemia have similar effects on HbF but they do not explain their differences in HbF.

A novel HBA2 gene conversion in cis or trans: "α12 allele" in a Saudi population.

Thalassemia and sickle cell disease are the most prevalent hemoglobin disorders in the populations of Dammam, Al-Qatif and Al-Ahsa regions in the Eastern Province of Saudi Arabia where our study cases originated. Increased HbF can modify these disorders. Direct sequencing of the HBA2 and HBA1 genes from 157 Saudi subjects revealed a new HBA2 gene conversion in cis or trans in 5.7% of the total. We refer to this new HBA2 gene convert as an α12 (HBA12) allele due to its combination of α1 (HBA1) and α2 (HBA2) sequences. Three genotypes, homozygous (-α12(3.7)/α1α12), heterozygous (α1α2/α1α12) and hemizygous (α1- (4.2)/α1α12) for the α12 allele were observed. The majority of individuals who were positive for the α12 allele had a reduction in the percentage of HbA2. Further studies are necessary to evaluate the possible effect of these changes on globin gene expression.

Co-inheritance of novel ATRX gene mutation and globin (α & β) gene mutations in transfusion dependent beta-thalassemia patients

α-Thalassemia X-linked mental retardation syndrome is a rare inherited intellectual disability disorder due to mutations in the ATRX gene. In our previous study of the prevalence of β-thalassemia mutations in the Eastern Province of Saudi Arabia, we confirmed the widespread coinheritance of α-thalassemia mutation. Some of these subjects have a family history of mental retardation, the cause of which is unknown. Therefore, we investigated the presence or absence of mutations in the ATRX gene in these patients. Three exons of the ATRX gene and their flanking regions were directly sequenced. Only four female transfusion dependent β-thalassemia patients were found to be carriers of a novel mutation in the ATRX gene. Two of the ATRX gene mutations, c.623delA and c.848T>C were present in patients homozygous for IVS I-5(G→C) and homozygous for Cd39(C → T) β-thalassemia mutation, respectively. While the other two that were located in the intronic region (flanking regions), were present in patients homozygous for Cd39(C → T) β-thalassemia mutation. The two subjects with the mutations in the coding region had family members with mental retardation, which suggests that the novel frame shift mutation and the missense mutation at coding region of ATRX gene are involved in ATRX syndrome.

A candidate transacting modulator of fetal hemoglobin gene expression in the Arab-Indian haplotype of sickle cell anemia.

Fetal hemoglobin (HbF) levels are higher in the Arab–Indian (AI) β‐globin gene haplotype of sickle cell anemia compared with African‐origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes—seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34+ cells. As CD34+ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118–1122, 2016.

Homozygosity for a Haplotype in the HBG2‐OR51B4 Region is Exclusive to Arab‐Indian Haplotype Sickle Cell Anemia

Homozygosity for rs334 (GAG-GTG, glu6val) or sickle cell anemia is common in Saudi Arabia. In the Eastern Province the sickle hemoglobin (HbS) gene is usually on the autochthonous Arab Indian (AI) β-globin gene (HBB) haplotype [1]. The higher fetal hemoglobin (HbF) level in the AI haplotype is often associated with milder disease compared with other HbS-associated haplotypes, especially in children [2, 3]. The Xmn1 G-A (C-T) polymorphism (rs7482144) is present in both the AI haplotype and the Senegal haplotype. Rs10128556, which is in linkage disequilibrium (LD) with rs7482144, could be the functional SNP of the Senegal haplotype [4]. The minor alleles of these SNPs also characterize the AI haplotype where adults had ~20% HbF compared with ~10% HbF in the Senegal haplotype. This suggests that elements linked to rs10128556 and rs7482144 in the AI but not the Senegal haplotype might affect HbF gene expression. We compared the genome-wide distribution of SNPs in Saudi patients with the AI haplotype and Benin haplotype. Variants distinguishing these populations were limited to chromosome 11p15.5. Annotation of these SNPs narrowed the selection to 3 that we analyzed jointly using haplotype analysis. Homozygosity for a T/A/T haplotype of rs16912979 (in HS-4 of the LCR), rs7482144 and rs10128556 was exclusive to the AI haplotype. The AI haplotype might include a functionally important sub-haplotype that accounts for high HbF.

KLF1 gene and borderline hemoglobin A2 in Saudi population

Introduction Elevated HbA2 (hemoglobin A2) level is considered the most reliable hematological parameter for the detection of β-thalassemia carriers. However, some carriers are difficult to recognize because the level of HbA2 is not in the distinctive carrier range, i.e. 4.0–6.0%; instead, some carriers have HbA2 levels between normal and carrier levels, i.e. borderline HbA2 (HbA2 = 3.1–3.9%). Studies have shown that variations in the erythroid Krüppel-like factor (KLF1) gene lead to borderline HbA2 in β-thalassemia carriers from various populations. The incidence of borderline HbA2 in Saudis is high. Material and methods To confirm the influence of variations in KLF1, HBA1, HBA2 and HBB genes for the reduction of the level of HbA2 in Saudi β-thalassemia carriers, we performed a direct sequence analysis of KLF1, HBA1, HBA2 and HBB genes from 212 healthy Saudis (88 subjects: HbA2 < 3; 72 subjects: HbA2 = 3.1 to 3.9; 52 subjects HbA2 > 4.3). Results The presence of the borderline HbA2 level is not specific to any type of β-thalassemia variation or β+-thalassemia variations in Saudis. Two exonic (c.304T>C and c.544T>C) and two 3′ untranslated region (3′UTR) (c.*296G>A and c.*277C>G) variations have been identified in the KLF1 gene for the first time from an Arab population. None of these four variations in KLF1 genes are significantly associated with the Saudis with borderline HbA2. α Globin genotype, –α2 3.7/α1α2, is found to be the most frequent (55.55%) among healthy Saudis with borderline HbA2 compared with the other groups (HbA2 < 3 = 20.45%; HbA2 > 4.3 = 13.51%). Conclusions Further studies are necessary to determine the influence of other factors on the presence of borderline HbA2 in 41.67% of Saudis.

Hemoglobin A2 (HbA2) has a measure of unreliability in diagnosing β-thalassemia trait (β-TT)

Abstract Introduction: Detection of β-thalassemia trait or carriers (β-TT) depends significantly on an increase in Hemoglobin A2 (HbA2) levels, which is found at low levels (<3%) in normal healthy individuals and elevated levels (≥3.5%) in β-TT individuals. The study was designed to evaluate the reliability of the diagnostic parameter HbA2 in the differentiation of β-TT and non-β-TT in Saudis. Methods: The widely used high performance liquid chromatography (Variant II Bio-Rad) was used to measure HbA2 levels in blood. Sanger sequencing was used to screen the variation in globin genes (HBB, HBD, HBA1, and HBA2). All the study subjects were divided into βTT and non-βTT (wild) categories based on the presence or absence of HBB variations and further sub-divided into false positive, true positive, false negative, and true negative, based on HbA2 values. Results: Out of 288 samples, 96 had HBB gene mutations. Of the 96 β-TT samples, sickle cell trait (SCT) samples (n = 58) were excluded, while the remaining (38 β-TT) were included in the detailed analysis: seven subjects with the HBB mutation had normal HbA2 (<3%), and three were borderline (3.1–3.9%). The remainder (n = 28) had an elevated HbA2 level (>4%). Based on HbA2 analysis alone, both these groups would be incorrectly diagnosed as normal. Similarly, of the 189 non-β-TT samples, 179 had normal HbA2, eight had borderline HbA2, and two had a HbA2 level above 4%. Based on HbA2 analysis alone, borderline and >4% HbA2 individuals, negative for β-TT, can be incorrectly diagnosed as carriers. Conclusion: Given the percentage of samples falling in the HbA2 “borderline” and “normal” categories, it can be concluded that HbA2 has a measure of unreliability in the diagnosis of β-thalassemia carriers.

The ‑α3.7 deletion in α‑globin genes increases the concentration of fetal hemoglobin and hemoglobin A2 in a Saudi Arabian population

The regions of Al‑Qatif and Al‑Ahssa in the Eastern Province of Saudi Arabia are known for their high prevalence of hemoglobinopathies, including β‑thalassemia and sickle cell anemia. Previously, the α‑gene deletion has been demonstrated as highly prevalent among populations residing in these two regions. The present study was conducted in order to investigate the implications of the α‑globin gene deletion on fetal hemoglobin (HbF) and hemoglobin α2 (HbA2) concentrations in patients with transfusion‑dependent β‑thalassemia. A total of 166 Saudi patients with transfusion‑dependent β‑thalassemia and 337 healthy Saudi patients were included in the study. The ‑α3.7, ‑α4.2, -‑FIL, -‑SEA, -‑MED and -‑(20.5) gene deletions were identified using multiplex α‑globin deletion polymerase chain reaction. The present study revealed that the ‑α3.7 gene deletion is the most prevalent (43.5%) in the Saudi populations that were analyzed and is characterized by the deletion of 3,804 base pairs. Numerous genotypes, namely ‑3.7α2/α1α2, ‑3.7α2/α1α12, ‑3.7α2/‑3.7α2, ‑3.7α2HphI/α1α2HphI, ‑3.7α2/α1‑4.2, ‑3.7α2/α1polyA‑1α2, ‑3.7α12/α1α12, ‑‑FIL/‑3.7α2 and ‑3.7α2/‑3.7α2Hb Villiers le Bel were also identified in the investigated population. Furthermore, a gradual increase in the concentration of HbF and HbA2 in patients with β‑thalassemia and the number of α‑gene deletions was demonstrated; whereas in healthy patients the level of HbA2 was demonstrated to decrease as the number of α‑gene deletions increased. Therefore, it can be concluded that the high HbF concentration in the present study is predominantly associated with other mutations associated with β‑thalassemia rather than α‑globin deletions. Furthermore, the results of the present study also revealed novel α‑gene deletion genotypes prevalent in the population studied, namely α1α2/α1α2HphI, α1α2HphI/α1α2HphI, α1α2/α1α2Hb Handsworth, ‑3.7α2HphI/α1α2HphI, ‑3.7α2/‑3.7α2Hb Villiers le Bel and ‑-MED/α1α2HphI.