Fahad Al-Muhanna

MOLECULAR BASES OF BETA-THALASSEMIA IN THE EASTERN PROVINCE OF SAUDI ARABIA

β-thalassemia is a group of heterogeneous recessive disorders common in many parts of the world. Al-Qatif and Al-Hassa oases in the Eastern Province of Saudi Arabia are regions known for high frequency of these disorders. Using two molecular methods, based on multiplexing-amplification refractory system and reverse hybridization principles, the spectrum of β-thalassemia in the region was studied. Sixty-nine subjects with known β-thalassemia disease and volunteers with high hemoglobin A2(HbA2) and low mean corpuscular volume (MCV) were included in this study. Ten mutations were detected in 91% of the subjects under study. Six of these mutations had previously been observed while the other four mutations are reported here for the first time. In addition, four of the mutations accounted for 76.8% of the subjects studied. IVSII-1 (G > A), IVSI-5 (G > A), and codon 39 (C > T) mutations were found to be the most frequent. However, the frequencies of different mutations reported here are slightly different from those reported earlier. A number of these mutations were also found in the neighboring countries, which can be explained in terms of gene flow.

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.

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.

Elevated Fibroblast Growth Factor 23 Concentration: Prediction of Mortality among Chronic Kidney Disease Patients

Background: The osteocyte-derived hormone, fibroblast growth factor 23 (FGF23), regulates the phosphorus metabolism and suppresses 1,25-dihydroxyvitamin D production, thereby mitigating hyperphosphatemia in patients with renal disorders. An elevated FGF23 level is suggested to be an early biomarker of altered phosphorus metabolism in the initial stages of chronic kidney disease (CKD) and acts as a strong predictor of mortality in dialysis patients. In the Saudi population, there is no report on the FGF23 level in CKD patients to date. This study aims to estimate the plasma FGF23 levels in the Saudi population and to correlate it with its clinical manifestations in order to ascertain its role in the pathogenesis of CKD patients. Methods: The FGF23 level in the plasma samples was determined using ELISA in a diverse cohort of 89 cases with stage 3-5 CKD and 100 healthy subjects. The plasma FGF23 level was correlated with other biochemical parameters. Results: The results revealed that the FGF23 level was markedly elevated among CKD patients compared to the control group, and a significant inverse correlation was observed between the FGF23 level and glomerular filtration rate. FGF23 elevation was approximately 40-fold among stage 5 patients compared to the control, while the elevation of phosphate, parathyroid hormone (PTH) and alkaline phosphatase was 2-, 3- and 8-fold in this stage, respectively. Conclusion: Elevated FGF23 levels may have a strong correlation with the disease pathogenesis. In addition, FGF23 might be a future therapeutic target to intervene against the progression of CKD as well as to increase patient survivability.

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.

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.

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.

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

Investigation of KIF6 Trp719Arg gene polymorphism in a case-control study of coronary artery disease and non-fatal myocardial infarction in the Eastern Province of Saudi Arabia.

BACKGROUND Kinesin-like protein 6 (KIF6), a member of the kinesin superfamily, is involved in intracellular transport. A few prospective studies have shown the KIF6 variant Trp719Arg (rs20455) to be associated with coronary artery disease (CAD) in Caucasian populations. However, recent genome-wide association studies on CAD have not proven these associations. OBJECTIVES Since the role of KIF6 719Arg allele in other ethnic populations is largely unknown, we sought to determine whether the KIF6 719Arg allele is associated with CAD in an ethnic population of Saudi Arabia. DESIGN Case-control study. SETTING CAD patients and control subjects from King Fahd Hospital of the University, Al-Khobar, Saudi Arabia. PATIENTS AND METHODS The study population included angiographically defined CAD patients (n=1002) and controls (n=984) with a normal electrocardiogram. MAIN OUTCOME MEASURE(S) Association of KIF6 Trp719Arg mutation with CAD. RESULTS The KIF6 Trp719Arg polymorphism was not associated with CAD (OR 0.976, 95% CI 0.861–1.105; P=.704). In addition, KIF6 Trp719Arg polymorphism showed a lack of association even in stratified myocardial infarction patients (n=802) (OR 1.006, 95% CI 0.881–1.148; P=.929) in comparison to controls. CONCLUSIONS The absence of Trp719Arg polymorphism association with CAD and CAD in stratified myocardial infarction cases indicates that the polymorphism is not associated with an increased risk among CAD patients from the Eastern Province of Saudi Arabia. Further studies in different provinces are required to unravel biological mechanisms underlying CAD in patients from Saudi Arabia. LIMITATIONS Unavailability of data on statin usage among the patient population.