Amein Al-Ali

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.

Concept and design of a genome-wide association genotyping array tailored for transplantation-specific studies

BackgroundIn addition to HLA genetic incompatibility, non-HLA difference between donor and recipients of transplantation leading to allograft rejection are now becoming evident. We aimed to create a unique genome-wide platform to facilitate genomic research studies in transplant-related studies. We designed a genome-wide genotyping tool based on the most recent human genomic reference datasets, and included customization for known and potentially relevant metabolic and pharmacological loci relevant to transplantation.MethodsWe describe here the design and implementation of a customized genome-wide genotyping array, the ‘TxArray’, comprising approximately 782,000 markers with tailored content for deeper capture of variants across HLA, KIR, pharmacogenomic, and metabolic loci important in transplantation. To test concordance and genotyping quality, we genotyped 85 HapMap samples on the array, including eight trios.ResultsWe show low Mendelian error rates and high concordance rates for HapMap samples (average parent-parent-child heritability of 0.997, and concordance of 0.996). We performed genotype imputation across autosomal regions, masking directly genotyped SNPs to assess imputation accuracy and report an accuracy of >0.962 for directly genotyped SNPs. We demonstrate much higher capture of the natural killer cell immunoglobulin-like receptor (KIR) region versus comparable platforms. Overall, we show that the genotyping quality and coverage of the TxArray is very high when compared to reference samples and to other genome-wide genotyping platforms.ConclusionsWe have designed a comprehensive genome-wide genotyping tool which enables accurate association testing and imputation of ungenotyped SNPs, facilitating powerful and cost-effective large-scale genotyping of transplant-related studies.

Vitamin D level among patients with sickle cell anemia and its influence on bone mass.

Vitamin D status varies with age, sex, body mass index (BMI), race, skin color, latitudes, sun exposure, and dietary intake [1]. A level between 52 and 72 nmol/l (21-29 ng/ml) has been defined as a state of insufficiency and a level of <50 nmol/l (<20 ng/ml) as vitamin D deficiency [2]. Vitamin D deficiency has been found to be common among the healthy Saudi population [3,4]. Homozygous sickle cell disease (SCD-SS) is also common in Saudi Arabia, affecting up to 5.27% of the population [5]. Young Saudi patients with homozygous sickle cell disease were found to have high prevalence of low bone mass [6,7]. We hypothesize that low 25(OH)D could be one of the influencing factors among Saudi patients with homozygous SCD for decreased bone density.

Spectrum of β-thalassemia mutations in the eastern province of Saudi Arabia.

β-Thalassemias comprise a group of heterogeneous hemoglobin (Hb) disorders characterized by the absence or reduced synthesis of the β-globin chain with a variable clinical presentation. The Al-Qatif and Al-Ahsa oases in the Eastern Province of Saudi Arabia are regions known for the high prevalence of these disorders. This study was conducted to provide a more precise picture of the β-thalassemia (β-thal) mutations prevalent in these regions and to estimate their frequencies. One hundred and 96 subjects with transfusion-dependent β-thalassemia (β-thal) disease were included in this study. A total of 14 β-thal mutations were identified with five mutations accounting for more than 80% of the total β-thal mutations identified. Of the 196 patients, 164 were homozygous for a β-thal mutation, while 32 were compound heterozygotes. We report here the novel identification of two mutations, namely, the Tunisian splice site IVS-I-130 (G→C) and the Mediterranean cryptic splice site IVS-I-110 (G→A), which have not been previously reported in the population of the Eastern Province. However, 15 patients (46.9%) with compound heterozygosities carried one of the β-thal mutations and the sickle cell mutation [Hb S or β6(A3)Glu→Val]. These patients were less frequently transfused than the patients who were homozygous for the β-thal mutations and presented with fewer complications. A more comprehensive overview of the genetic heterogeneity of the β-thal mutations in the Eastern Province of Saudi Arabia is presented in this article. This study will contribute to the establishment of an effective prevention program, including premarital screening.

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

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.

A Comprehensive, Ethnically Diverse Library of Sickle Cell Disease-Specific Induced Pluripotent Stem Cells

Summary Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities, β-globin gene (HBB) haplotypes, and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Here, we describe this unique resource for the study of sickle cell disease, including novel haplotype-specific polymorphisms that affect disease severity, as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell- and gene-based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation.

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.

IκΒα inhibits apoptosis at the outer mitochondrial membrane independently of NF‐κB retention

IκBα resides in the cytosol where it retains the inducible transcription factor NF‐κB. We show that IκBα also localises to the outer mitochondrial membrane (OMM) to inhibit apoptosis. This effect is especially pronounced in tumour cells with constitutively active NF‐κB that accumulate high amounts of mitochondrial IκBα as a NF‐κB target gene. 3T3 IκBα−/− cells also become protected from apoptosis when IκBα is specifically reconstituted at the OMM. Using various IκBα mutants, we demonstrate that apoptosis inhibition and NF‐κB inhibition can be functionally and structurally separated. At mitochondria, IκBα stabilises the complex of VDAC1 and hexokinase II (HKII), thereby preventing Bax recruitment to VDAC1 and the release of cytochrome c for apoptosis induction. When IκBα is reduced in tumour cells with constitutively active NF‐κB, they show an enhanced response to anticancer treatment in an in vivo xenograft tumour model. Our results reveal the unexpected activity of IκBα in guarding the integrity of the OMM against apoptosis induction and open possibilities for more specific interference in tumours with deregulated NF‐κB.

The impact of common polymorphisms in CETP and ABCA1 genes with the risk of coronary artery disease in Saudi Arabians

BackgroundCoronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Many genetic and environmental risk factors including atherogenic dyslipidemia contribute towards the development of CAD. Functionally relevant mutations in the dyslipidemia-related genes and enzymes involved in the reverse cholesterol transport system are associated with CAD and contribute to increased susceptibility of myocardial infarction (MI).MethodBlood samples from 990 angiographically confirmed Saudi CAD patients with at least one event of myocardial infarction were collected between 2012 and 2014. A total of 618 Saudi controls with no history or family history of CAD participated in the study. Four polymorphisms, rs2230806, rs2066715 (ABCA1), rs5882, and rs708272 (CETP), were genotyped using TaqMan Assay.ResultsCETP rs5882 (OR = 1.45, P < 0.005) and ABCA1 rs2230806 (OR = 1.42, P = 0.017) polymorphisms were associated with increased risk of CAD. However, rs708272 polymorphism showed protective effect (B1 vs. B2: OR = 0.80, P = 0.003 and B2B2 vs. B1B1: OR = 0.68, P = 0.012) while the ABCA1 variant rs2066715 was not associated.ConclusionThis study is the first to report the association of these polymorphisms with CAD in the population of the Eastern Province of Saudi Arabia. The rs5882 polymorphism (CETP) showed a significant association and therefore could be a promising marker for CAD risk estimation while the rs708272 polymorphism had a protective effect from CAD.