Ahmed M. Al-Suliman

Sickle cell disease in Saudi Arabia: the phenotype in adults with the Arab-Indian haplotype is not benign.

Sickle cell disease (SCD) in Saudi patients from the Eastern Province is associated with the Arab‐Indian (AI) HBB (β‐globin gene) haplotype. The phenotype of AI SCD in children was described as benign and was attributed to their high fetal haemoglobin (HbF). We conducted a hospital‐based study to assess the pattern of SCD complications in adults. A total of 104 patients with average age of 27 years were enrolled. Ninety‐six per cent of these patients reported history of painful crisis; 47% had at least one episode of acute chest syndrome, however, only 15% had two or more episodes; symptomatic osteonecrosis was reported in 18%; priapism in 17%; overt stroke in 6%; none had leg ulcers. The majority of patients had persistent splenomegaly and 66% had gallstones. Half of the patients co‐inherited α‐thalassaemia and about one‐third had glucose‐6‐phosphate dehydrogenase deficiency. Higher HbF correlated with higher rate of splenic sequestration but not with other phenotypes. The phenotype of adult patients with AI SCD is not benign despite their relatively high HbF level. This is probably due to the continued decline in HbF level in adults and the heterocellular and variable distribution of HbF amongst F‐cells.

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 functional promoter polymorphism of the δ‐globin gene is a specific marker of the Arab‐Indian haplotype

Most sickle cell anemia (SCA) patients indigenous to the Eastern Province of Saudi Arabia have their HbS gene on the Arab-Indian (AI) HBB gene cluster haplotype. Their fetal hemoglobin (HbF) levels are near 20% and they have milder disease compared with SCA where the HbS gene is on African origin HBB haplotypes [1–9]. The AI haplotype is characterized by an Xmn1 restriction site at position 2158 50 to HBG2 (rs7482144), a Hinc2 site 50 to HBE (rs3834466) and other polymorphisms [10]. The causal elements that modify HbF might be in linkage disequilibrium with the b globin gene in this Saudi population. We first performed homozygosity mapping using genome-wide single nucleotide polymorphisms (SNPs) in AI HbS homozygotes [11,12] and identified a single large autozygous region including the HBB cluster and surrounding genes. By next generation sequencing, we examined this region in these same individuals and identified several variants that included a SNP in the HBD promoter region at position 268 bp 50 to HBD (CCAAC > TCAAC). We found this SNP only when the HbS gene was on an AI haplotype and not in SCA with other haplotypes. This SNP was functional in reporter assays in K562 cells and is an AI haplotype-specific marker. Table I summarizes the patient characteristics. Using genome-wide SNP data from a limited number of cases, a region of autozygosity was found only in AI HbS homozygotes on chromosome 11 (coordinates 5,196,450– 5,323,071). The region contains HBD, HBG1, HBG2, HBE1, and the Xmn1 50 HBG2 restriction site (rs7482144). By targeted deep sequencing of 400 kb of chromosome 11 (coordinates 5,143,424–5,543,424; average coverage 42x) in 4 AI patients 1,195 variants were found. A homozygous C-T variant 268 bp 50 HBD with high genotyping and mapping quality that was not in dbSNP build 135 or 1,000 Genomes, was present. Resequencing of 15.9 kb of chr11 (coordinates 5,253,531–5,269,435) by Sanger sequencing detected three new SNPs of which one was the 268 C > T SNP. We focused on this SNP because of its location within the Corfu deletion region and its location in the HBD promoter. The C > T SNP in the HBD promoter was found only in individuals with the AI haplotype. Saudi sickle cell trait carriers with the AI haplotype were heterozygous for this SNP; while siblings without HbS did not carry this mutation. Among 25 AI HbS-b thalassemia patients, 16 were heterozygous at this site (C/T) and 9 were homozygous (T/T). All AI HbS-b thalassemia patients who were homozygous T/T were also homozygous for the AI haplotype (Table I). Fifteen African American SCA patients with unusually high HbF, 54 Saudi SCA patients from the Southwestern Province (SW)—mainly Benin but including subjects with the Senegal haplotype—19 SW HbS-b thalassemia patients, 16 SW sickle cell trait cases, and 25 normal Saudi controls did not carry the 268 HBD SNP. This SNP was not found in 1,094 individuals in 1,000 Genomes May 2011 release. It is important to note that hemoglobin electrophoresis results in Table I were performed using different methods, so direct comparison of HbF and HbA2 between different groups will not be accurate. In addition, the effect of coinheritance of a-thalassemia, or presence of iron deficiency anemia on Hb A2 level was not assessed. Finally, HbA2 levels are artifactually high when HbS is present because of the co-elution of minor HbS species. For these reasons, it is not possible to estimate the effects of the 268 C-T SNP on these subjects HbA2 levels. Reduced expression of HBD relative to HBB in normal individuals is partly a result of a degenerate CCAAT box in the HBD promoter (CCAAC). The CCAAC motif is the site of the 268 C > T SNP (TCAAC) [13–15]. When we compared the activity of the wild-type HBD promoter with the promoter containing the 268 C > T SNP the variant promoter was associated with a significant decrease in the expression of a reporter construct suggesting that it could further impair already enfeebled HBD expression (Fig. 1). Although HBG is expressed at high levels in K562 cells, endogenous HBD is also expressed [16]. The expression studies were designed solely to test the hypothesis that the 268 C > T SNP downregulates the expression of the HBD promoter. The literature provides further evidence for a functional role of the 268 C > T SNP. Its presence was associated with d thalassemia in one individual with reduced HbA2 of 2% and a slightly increased HbF of 1.3% [13]. Moreover, mutations at positions 230, 231, 236, 255, 265, 276, and 277 in the HBD promoter were reported in HbVar database (http://globin.cse.psu.edu/) to cause d thalassemia [14,17–19], and HbF levels of 3.3–4.7% have been noted in some hematologically normal individuals with homozygous d thalassemia [19,20]. A mechanism for increased HbF in the presence of less common HBD promoter mutations is unknown. Any role for the 268 C-T SNP as a modifier of HbF in AI haplotype HbS sickle cell disease is unknown. Perhaps HBD promoter SNPs reduce the interaction of the locus control region and the transcription apparatus with this promoter permitting enhanced interactions with HBG promoters [21]. The paradox of the Corfu deletion first suggested the potential of the HBD-HBG1 intergenic area, the site of the 268 C-T SNP, as a silencer of HBG expression [22]. One potential functional area is the polypyrimidine (PYR) binding site about 960 bp upstream of HBD; however, polymorphisms

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.

Chronic myelogenous leukemia in sickle cell/beta 0-thalassemia.

Sickle cell/beta (0)-thalassemia (S/β(0)-thal) is a compound heterozygous state for βS and β(0) thalassemia. There are rare reported cases of patients with sickle cell disease who developed hematological neoplasms including myeloid and lymphoid conditions; however, to the best of our knowledge, chronic myelogenous leukemia (CML) occurring in S/β(0) -thal has been reported in one case and this is the second such report.

Mutations in the β‐globin gene from a Saudi population: an update

Sir, b-thalassemia is highly prevalent in the Al-Ahssa and Al-Qatif regions of the Eastern Province of Saudi Arabia [1–5] with extremely diverse clinical manifestations reflecting the heterogeneity of mutations at the b-globin locus. Approximately 200 mutations that lead to b-thalassemia have been identified in various populations, with each population characterized by specific common and rare mutations [6]. Approximately 50 b-thalassemia mutations have been identified in Arab populations, which again reflects their heterogeneity [1, 7]. Fourteen of these b-thalassemia mutations have been identified in the Eastern Province, with five accounting for more than 80%, which can be attributed to the high rate (56.2– 58%) of consanguineous marriages [8]. Our objective was to update and complete the molecular basis of b-thalassemia mutations in the Eastern Province to facilitate more accurate and effective premarital genetic counseling, contribute to the understanding of the relationship between phenotype to genotype and support the establishment of a severity index. The population of the Eastern Province of Saudi Arabia, which totals approximately 1.8 million, comprises the indigenous population and an influx of populations from other provinces, with little admix from other countries due to the high rate of consanguineous marriages [8]. Patients included in this study are all from the indigenous population, which is known for its high prevalence of bthalassemia. Over a period of 2 years (2013 and 2014), 75 (44 males and 31 females) newly clinically diagnosed transfusion-dependent b-thalassemia patients (aged 1– 3 years) from the main population centers of Dammam, Al-Qatif and Al-Ahssa regions were recruited (Table 1). After obtaining signed, informed consent, peripheral blood samples were collected in EDTA vacutainers. Fetal hemoglobin and other blood parameters were determined using MD2 hematological analyzer. The b-globin gene (HBB) was amplified as previously described [5]. In summary, the amplicons were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and the purified PCR products were cycle sequenced using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems , Foster City, CA, USA). The cycle sequenced products were then purified and electrophoresed in Genetic Analyzer 3500 (Life Technologies Corporation, Carlsbad, CA, USA). Sequencing Analysis Software Version 5.4 (Applied Biosystems ) was used for data analysis. Three b-thalassemia mutations identified in six patients are being reported here for the first time in the Saudi population, with the remaining mutations having been previously reported (Table 2) [1, 2]. Two of these newly identified mutations are HBB:c.2T>C and HBB: c.46delT, which are point mutations previously reported in populations in Russia, Croatia, Japan, and Iran [9–12]. The third mutation is 25-bp deletion (HBB:c9323_94del), which is two-bp downstream to the 25-bp deletion of HBB:c.93-21_96del, which is the deletion commonly reported (Figure 1). This anomaly prompted us to verify the HBB:c.93-21_96del mutation in 16 patients with b-thalassemia identified by the use of a commercially available Strip assay from ViennaLab and reported in our previous study [1, 2]. The sequencing results indicated that in all 16 patients, the 25-bp deleTable 1. Clinical characteristics of the study group

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.

Utilizing Whole-Exome Sequencing to Characterize the Phenotypic Variability of Sickle Cell Disease

BACKGROUND Sickle cell disease (SCD) is a monogenic disease that has wide variety of phenotypes with both and environmental factors contributing to its severity. METHODS We performed whole-exome sequencing (WES) in 22 Saudi SCD patients to identify variants that could explain differences in disease phenotypes. All variants, except those that were benign and likely benign, described in the ClinVar database, were considered in our analysis. Gene-based association testing using sequence kernel association optimal unified test (SKAT-O) with small sample adjustment was performed to evaluate the effect of multiple variants in genes on SCD phenotypes. RESULTS The mean age of participants was 28 (range, 10-48 years). All patients were homozygous for the sickle cell mutation. The Benin haplotype was present in 15 patients and the Arab-Indian haplotype in 7 patients. One patient who had both SCD and CHARGE association was heterozygous for pathogenic mutation p.Arg987Ter in the CHD7 gene. One SCD individual who had a stroke was a carrier of the pathogenic variant p.Asp36Tyr in the VKORC1 gene which is, associated with warfarin resistance. Two patients with steady hemoglobin levels of 7.5 and 7.1 g/dL were carriers of the pathogenic mutation p.Gly140Ser in the RPL5 gene that is associated with Diamond-Blackfan anemia. None of the patients were transfusion dependent. A heterozygous pathogenic mutation in the LDLR gene associated with autosomal dominant familial hypercholesterolemia was present in one patient with deep venous thrombosis, although their cholesterol level was normal. One individual with stroke was a carrier for the p.Arg284Ter variant in the NLRP12 gene, which is associated with familial cold autoinflammatory syndrome 2. Another patient with stroke and a pulmonary embolism was heterozygous for the p.Pro106Leu variant of the MPL gene, which has been associated with thrombocytosis. Coding variants in the GOLGB1, ENPP1, and PON1 genes showed no association with stroke in our study. SKAT-O analysis did not explain SCD heterogeneity. CONCLUSION WES provided limited information to explain the severity of SCD. Whole genome sequencing, epigenetic studies, and assessment of environmental factors might expand our knowledge of SCD heterogeneity.