J. Francis Borgio

Spectrum of α-Thalassemia Mutations in Transfusion-Dependent β-Thalassemia Patients from the Eastern Province of Saudi Arabia

Both α- and β-thalassemia (α- and β-thal) are highly prevalent in the population of the Al-Qatif and Al-Ahsa regions in the Eastern Province of Saudi Arabia. This study provides a more precise picture of the α-thal mutations prevalent in 104 transfusion-dependent β-thal patients in the Eastern Province. Detection of α-thal mutations was carried out using the α-globin StripAssay kit. A total of 12 α-thal mutations (21 genotypes) were identified in 33.7% of the chromosomes (46 patients). The heterozygous and homozygous –α3.7 (α+) deletion mutations were the most prevalent in the β-thal patients (21.7%). We identified three α0 deletions [– –MED, – –FIL and –(α)20.5] that have not been previously reported for the population of Saudi Arabia. The seven point mutations identified in the β-thal patients were: codon 14 [TGG>TAG (α1)], codon 59 [GGC>GAC (α1)] (Hb Adana), polyadenylation signal site (polyA1) [AATAAA>AATAAG (α2)], codon 142 [TAA>TCA (α2)] (Hb Koya Dora), codon 59 [GGC>GAC (α2)] (Hb Adana), initiation codon [ATG>ACG (α2)] and the αααanti 3.7 gene triplication. The Hb Koya Dora mutation occurred at the highest frequency (15.38%). Comparison of the clinical phenotype of β-thal patients, with and without an α-thal mutation, showed that patients with β-thal alone had a significantly elevated level of alanine transaminase (ALT) (mean 72.5 IU/L) and aspartate transaminase (AST) (mean 71.8 IU/L) (p <0.005). In addition, the β-thal patients without an α-thal mutation had a higher percentage of osteoporosis (16.6%), fractures (12.5%), and splenectomies (58.3%). This confirms previous data that the co-inheritance of α-thal in β-thal patients results in the amelioration of the clinical phenotype of β-thal patients. Moreover, the high frequency of α- and β-thal in the Eastern Province of Saudi Arabia and their coinheritance, necessitates the inclusion of α-thal testing in the current pre marital testing program to highlight the risk to the offspring of affected individuals.

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.

Intronic Polymorphisms in the CDKN2B-AS1 Gene Are Strongly Associated with the Risk of Myocardial Infarction and Coronary Artery Disease in the Saudi Population

Recent genome-wide association studies identified single nucleotide polymorphisms (SNPs) on the chromosome 9p21.3 conferring the risk for CAD (coronary artery disease) in individuals of Caucasian ancestry. We performed a genetic association study to investigate the effect of 12 candidate SNPs within 9p21.3 locus on the risk of CAD in the Saudi population of the Eastern Province of Saudi Arabia. A total of 250 Saudi CAD patients who had experienced an myocardial infarction (MI) and 252 Saudi age-matched healthy controls were genotyped using TaqMan assay. Controls with evidenced lack of CAD provided 90% of statistical power at the type I error rate of 0.05. Five percent of the results were rechecked for quality control using Sanger sequencing, the results of which concurred with the TaqMan genotyping results. Association analysis of 12 SNPs indicated a significant difference in the genotype distribution for four SNPs between cases and controls (rs564398 p = 0.0315, χ2 = 4.6, odds ratio (OD) = 1.5; rs4977574 p = 0.0336, χ2 = 4.5, OD = 1.4; rs2891168 p = 1.85 × 10 − 10, χ2 = 40.6, OD = 2.1 and rs1333042 p = 5.14 × 10 − 9, χ2 = 34.1, OD = 2.2). The study identified three protective haplotypes (TAAG p = 1.00 × 10 − 4; AGTA p = 0.022 and GGGCC p = 0.0175) and a risk haplotype (TGGA p = 2.86 × 10 − 10) for the development of CAD. This study is in line with others that indicated that the SNPs located in the intronic region of the CDKN2B-AS1 gene are associated with CAD.

Molecular nature of alpha-globin genes in the Saudi population

Alpha-thalassemia (α-thal) is a disorder caused by the deletion of single or double α-globin genes, and/or point mutations in the α-globin genes. There are 2 common types of α-globin genes; HBA2 and HBA1. Recently, it has been discovered that the HBA2 gene is replaced by a unique HBA12 gene convert in 5.7% of the Saudi population. The α-globin genes have been emerging as a molecular target for the treatment of β-thalassemia (β-thal). Hence, it is essential to understand the molecular nature of α-globin genes to treat the most prevalent hemoglobin disorders, such as sickle cell disease, α-thal, and β-thal prevalent in the Kingdom of Saudi Arabia. Thirty-two different α-globin genotypes have been observed in the Saudi population. This review outlines the classification of the α-globin genes on the basis of their molecular nature and complex combinations of α-globin genes, and their variants predominant in Saudis.

In-Silico Computing of the Most Deleterious nsSNPs in HBA1 Gene.

Background α-Thalassemia (α-thal) is a genetic disorder caused by the substitution of single amino acid or large deletions in the HBA1 and/or HBA2 genes. Method Using modern bioinformatics tools as a systematic in-silico approach to predict the deleterious SNPs in the HBA1 gene and its significant pathogenic impact on the functions and structure of HBA1 protein was predicted. Results and Discussion A total of 389 SNPs in HBA1 were retrieved from dbSNP database, which includes: 201 non-coding synonymous (nsSNPs), 43 human active SNPs, 16 intronic SNPs, 11 mRNA 3′ UTR SNPs, 9 coding synonymous SNPs, 9 5′ UTR SNPs and other types. Structural homology-based method (PolyPhen) and sequence homology-based tool (SIFT), SNPs&Go, PROVEAN and PANTHER revealed that 2.4% of the nsSNPs are pathogenic. Conclusions A total of 5 nsSNPs (G60V, K17M, K17T, L92F and W15R) were predicted to be responsible for the structural and functional modifications of HBA1 protein. It is evident from the deep comprehensive in-silico analysis that, two nsSNPs such as G60Vand W15R in HBA1 are highly deleterious. These “2 pathogenic nsSNPs” can be considered for wet-lab confirmatory analysis.

Association of MBL2 Gene Polymorphism with Dental Caries in Saudi Children

The high prevalence of dental caries in children worldwide is a major oral health problem which requires early intervention. Dental caries is mainly caused by the action of acids produced by bacteria in addition to many other factors. Recent genetic studies have reported that a number of genes are associated with the susceptibility to dental caries. The majority of these genes are associated with inflammation, increased susceptibility to infection, and dentine matrix formation. Using the TaqMan assay and direct DNA sequencing, the prevalence of 6 single-nucleotide polymorphisms (SNPs) in MMP9, MBL2, MMP2, and TIMP2 genes was determined in 102 children with caries and in 100 age-matched caries-free controls. Out of the 6 SNPs tested in the 4 selected genes, only rs11003125 in the MBL2 gene was shown to be associated with a high prevalence of caries in our cohort. In addition, haplotype analysis of the 6 SNPs tested revealed that certain haplotypes, namely GT of rs11003125G and rs7501477T and GT of rs7096206G and rs7501477T, were found to be associated with a high prevalence of dental caries in our cohort, while haplotype AG of rs17576A and rs7501477G was found to have a protective effect against dental caries. In conclusion, the data indicate that rs11003125 in the MBL2 gene was shown to be associated with a high prevalence of caries in our cohort, and 2 haplotypes are also involved in the increased susceptibility to dental caries.

Existence of HbF Enhancer Haplotypes at HBS1L-MYB Intergenic Region in Transfusion-Dependent Saudi β-Thalassemia Patients

Background and Objectives. β-Thalassemia and sickle cell disease are genetic disorders characterized by reduced and abnormal β-globin chain production, respectively. The elevation of fetal hemoglobin (HbF) can ameliorate the severity of these disorders. In sickle cell disease patients, the HbF level elevation is associated with three quantitative trait loci (QTLs), BCL11A, HBG2 promoter, and HBS1L-MYB intergenic region. This study elucidates the existence of the variants in these three QTLs to determine their association with HbF levels of transfusion-dependent Saudi β-thalassemia patients. Materials and Methods. A total of 174 transfusion-dependent β-thalassemia patients and 164 healthy controls from Eastern Province of Saudi Arabia were genotyped for fourteen single nucleotide polymorphisms (SNPs) from the three QTL regions using TaqMan assay on real-time PCR. Results. Genotype analysis revealed that six alleles of HBS1L-MYB QTL (rs9376090C p = 0.0009, rs9399137C p = 0.008, rs4895441G p = 0.004, rs9389269C p = 0.008, rs9402686A p = 0.008, and rs9494142C p = 0.002) were predominantly associated with β-thalassemia. In addition, haplotype analysis revealed that haplotypes of HBS1L-MYB (GCCGCAC p = 0.022) and HBG2 (GTT p = 0.009) were also predominantly associated with β-thalassemia. Furthermore, the HBS1L-MYB region also exhibited association with the high HbF cohort. Conclusion. The stimulation of HbF gene expression may provide alternative therapies for the amelioration of the disease severity of β-thalassemia.

Optimal pcr primers for rapid and accurate detection of Aspergillus flavus isolates

Aspergillus flavus is among the most devastating opportunistic pathogens of several food crops including rice, due to its high production of carcinogenic aflatoxins. The presence of these organisms in economically important rice strip farming is a serious food safety concern. Several polymerase chain reaction (PCR) primers have been designed to detect this species; however, a comparative assessment of their accuracy has not been conducted. This study aims to identify the optimal diagnostic PCR primers for the identification of A. flavus, among widely available primers. We isolated 122 A. flavus native isolates from randomly collected rice strips (N = 300). We identified 109 isolates to the genus level using universal fungal PCR primer pairs. Nine pairs of primers were examined for their PCR diagnostic specificity on the 109 isolates. FLA PCR was found to be the optimal PCR primer pair for specific identification of the native isolates, over aflP(1), aflM, aflA, aflD, aflP(3), aflP(2), and aflR. The PEP primer pair was found to be the most unsuitable for A. flavus identification. In conclusion, the present study indicates the powerful specificity of the FLA PCR primer over other commonly available diagnostic primers for accurate, rapid, and large-scale identification of A. flavus native isolates. This study provides the first simple, practical comparative guide to PCR-based screening of A. flavus infection in rice strips.

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.