Co-inheritance of alpha globin gene deletion lowering serum iron level in female beta thalassemia patients.

In the Eastern province of Saudi Arabia, thalassemia is highly common. Data on the effect of alpha globin gene variation on the concentration of iron on transfusion dependent Saudis are scanty. A total of 166 transfusions dependent ß-thalassemia were included in this study to understand association between the alpha globin gene variation and concentration of iron. Using multiplex PCR, the alpha globin gene deletions were identified. Also, HBA1 and HBA2 genes were sequenced by Sanger sequencing. Saudi transfusion dependent female ß-thalassemia patients with wild alpha globin genotype (αα/αα) were observed with iron level beyond the normal range. However, normal range of iron was observed in transfusion dependent Saudi female beta thalassemia patients co-inherited with double (-α3.7/-α3.7, or --Fil/αα or --MED/αα or - (α) 20.5/αα) or double heterozygosity (- -/-α3.7) alpha globin gene deletions, which is significantly (p < 0.0001) less compared to the Saudi transfused female with wild alpha globin genotype (αα/αα). The co-inheritance alpha globin gene deletions in female beta thalassemia patients were significantly lowering serum iron. Detailed studies can be taken forward to identify the molecular pathways involved in globin gene deletion as modulator.

Demands and challenges for patients with sickle-cell disease requiring hematopoietic stem cell transplantation in Saudi Arabia.

Allogeneic HSCT is the only curative treatment for SCD. In this study, we estimated the number of Saudi patients with SCD who are candidates for HSCT. We used the presence of overt stroke, recurrent ACS, and frequent severe pain crisis as indications for HSCT. We calculated the frequencies of these complications among a Saudi SCD cohort of 376 patients with SCD, 250 from SW and 126 from Eastern (E) provinces. We found that 59 (23.6%) of SW patients were transplant candidates compared to 22 (17.4%) from E province. It is estimated that about 61 000 patients with SCD live in Saudi Arabia. Thus, the projected number of Saudi patients with SCD who are candidates for HSCT is 10 536 patients. Of those, 2148 are children. The burden of SCD on HSCT centers in Saudi Arabia is substantial and is difficult currently to meet the demand. We recommend recruiting/training more transplant physicians and nurses, expand current capacity of centers if feasible, and open new transplant centers to make HSCT a practical therapeutic option for patients with severe SCD in Saudi Arabia.

Co-inheritance of novel ATRX gene mutation and globin (α & ß) 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 ß-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 ß-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) ß-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) ß-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.

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) ß-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.

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.

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 ß-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 ß-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 ß-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 ß-thalassemia variation or ß+-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, -α23.7/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.

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

The regions of Al­Qatif and Al­Ahssa in the Eastern Province of Saudi Arabia are known for their high prevalence of hemoglobinopathies, including ß­thalassemia and sickle cell anemia. Previously, the α­gene 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 α­globin gene deletion on fetal hemoglobin (HbF) and hemoglobin α2 (HbA2) concentrations in patients with transfusion­dependent ß­thalassemia. A total of 166 Saudi patients with transfusion­dependent ß­thalassemia 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 α­globin 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/α1­4.2, ­3.7α2/α1polyA­1α2, ­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 ß­thalassemia and the number of α­gene deletions was demonstrated; whereas in healthy patients the level of HbA2 was demonstrated to decrease as the number of α­gene deletions increased. Therefore, it can be concluded that the high HbF concentration in the present study is predominantly associated with other mutations associated with ß­thalassemia rather than α­globin deletions. Furthermore, the results of the present study also revealed novel α­gene 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.