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Frequency of common HFE variants in the Saudi population: a high throughput molecular beacon-based study.

ABSTRACT: Hereditary Hemochromatosis (HH) is an autosomal recessive disorder highlighted by iron-overload. Two popular mutations in HFE, p.C282Y and p.H63D, have been discovered and found to associate with HH in different ethnic backgrounds. p.C282Y and p.H63D diagnosis is usually made by restriction enzyme analysis. However, the use of this technique is largely limited to research laboratories because they are relatively expensive, time-consuming, and difficult to transform into a high throughput format.Single nucleotide variations in target DNA sequences can be readily identified using molecular beacon fluorescent probes. These are quenched probes with loop and hairpin structure, and they become fluorescent upon specific target recognition. We developed high throughput homogeneous real-time PCR assays using molecular beacon technology, to genotype p.C282Y and p.H63D variants. Representative samples of different genotypes for these variants were assayed by restriction enzyme analysis and direct sequencing as bench mark methods for comparison with the newly developed molecular beacon-based real-time PCR assay.Complete concordance was achieved by all three assay formats. Homozygotes (mutant and wildtype) and heterozygotes were readily differentiated by the allele specific molecular beacons as reported by the associated fluorophore in the real-time assay developed in this study. Additionally, these assays were used in a high throughput format to establish the allele frequency of C282Y and H63D in Saudis for the first time.These assays may be reliably applied as a diagnostic test or large scale method for population screening.

SUBMITTER: Alsmadi OA

PROVIDER: S-EPMC1468397 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Frequency of common HFE variants in the Saudi population: a high throughput molecular beacon-based study.

Alsmadi Osama A OA Al-Kayal Fadi F Al-Hamed Mohamed M Meyer Brian F BF

BMC medical genetics 20060503

<h4>Background</h4>Hereditary Hemochromatosis (HH) is an autosomal recessive disorder highlighted by iron-overload. Two popular mutations in HFE, p.C282Y and p.H63D, have been discovered and found to associate with HH in different ethnic backgrounds. p.C282Y and p.H63D diagnosis is usually made by restriction enzyme analysis. However, the use of this technique is largely limited to research laboratories because they are relatively expensive, time-consuming, and difficult to transform into a high ...[more]

PMID: 16672055

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Project description:Hereditary HFE-linked hemochromatosis is a frequent recessive disorder among individuals of northern European ancestry. The clinical characteristic of this disease is the gradual accumulation of iron in internal organs, which ultimately may lead to organ damage and death. Three allelic variants of HFE gene have been correlated with hereditary hemochromatosis: C282Y is significantly associated with hereditary hemochromatosis in populations of Celtic origin, H63D and S65C are associated with milder form of iron overload. In this study we performed mutation analysis to identify allele frequency of the three variants of HFE gene in Jordanian Arab population, to assess deviations of these frequencies from those detected elsewhere, and to determine if there is an increased frequency of these variants in a diabetic population (Type 2 diabetes) from the same area. DNA was extracted from blood samples of 440 individuals attending King Abdullah University Hospital for ambulatory services. We used polymerase chain reaction (PCR) to amplify exons 2 and 4 of the HFE gene then restriction fragment length polymorphism (RFLP) method to detect the variants. There were neither homozygous nor heterozygous for C282Y variant. For the H63D variant, 0.68% were homozygous and 21.1% were heterozygous. For the S65C variant, there were no homozygous and 0.23% were heterozygous. Allelic frequencies were, 0%, 11.25%, and 0.11% for C282Y, H63D, and S65C, respectively. Our samples were subdivided into two categories of type 2 diabetic (89 cases) and controls (blood donors, 204 cases) and compared with regard to the H63D variant. Both groups did not have homozygous H63D variant. H63D heterozygous in diabetics were 23.60% and in blood donor controls 22.55%. Allelic frequency of the mutant H63D allele was 11.80% in diabetics and 11.27% for the blood donor controls. This is the first study to show the frequency of the three hemochromatosis gene variants in Jordan with the interesting finding of no C282Y allele detected in 440 samples. Additionally, no significant difference was observed in H63D variant frequency in type 2 diabetics as compared to controls.

Project description:Human leukocyte antigens (HLA) diversity has a tremendous impact on shaping the transplantation practices, transfusion-associated graft versus host disease prevention strategies, and host–pathogen interactions. Here, we conducted a retrospective study of HLA class I and class II homozygosity at allelic and haplotype levels in unrelated individuals genotyped from 2012 to 2016 in a tertiary hospital in the capital of Saudi Arabia. Among 5,000 individuals, 2,773 individuals meet inclusion criteria and were retrospectively analyzed for HLA-A, -B, -C–DRB1, and -DQB1 homozygosity at allelic and haplotype levels. HLA molecular typing was performed using a commercial reverse sequence-specific oligonucleotide (rSSO) kit. We were able to identify 15 HLA-A, 20 HLA-B, 11 HLA-C, 13 HLA-DRB1, and five HLA-DQB1 homozygous alleles demonstrating a very low genetic diversity in the Saudi population. The highest homozygosity in HLA class I was found in locus C followed by A and B (20.3% > 16.1% > 15.5%; p < 0.001) where the most homozygote alleles were A*02 (9.2%), B*51 and B*50 (5.7% and 3.7%), and C*07, C*06, and C*15 (7.2%, 5.48%, and 3.3%) and in HLA class II, the highest homozygosity was found in locus DQB1 compared to DRB1 (31.71% > 19.2%; p < 0.001), with the most common homozygote alleles being DRB1*07 and DRB1*04 (5.33% and 4.2%) and DQB1*02, DQB1*06, and DQB1*03 (13.55%, 7.92%, and 7.64%). The frequency of finding an individual with one homozygote allele was (24.6%), two homozygote alleles (13.5%), three homozygote alleles (4.7%), four homozygote alleles (3.4%), and five alleles were (4.8%). The most frequent homozygote haplotypes are A*23∼C*06∼B*50∼DRB1*07∼DQB1*02 and A*02∼C*06∼B*50∼DRB1*07∼DQB1*02. This study shows low diversity of both class I and II alleles and haplotypes in the Saudi population, which would have a significant impact on shaping the transplantation practices, transfusion-associated graft versus host disease prevention strategies, and host–pathogen interactions.

Project description:IntroductionStroke is a multifactorial and heterogeneous disorder, correlates with heritability and considered as one of the major diseases. The prior reports performed the variable models such as genome-wide association studies (GWAS), replication, case-control, cross-sectional and meta-analysis studies and still, we lack diagnostic marker in the global world. There are limited studies were carried out in Saudi population, and we aim to investigate the molecular association of single nucleotide polymorphisms (SNPs) identified through GWAS and meta-analysis studies in stroke patients in the Saudi population.MethodsIn this case-control study, we have opted gender equality of 207 cases and 207 controls from the capital city of Saudi Arabia in King Saud University Hospital. The peripheral blood (5 ml) sample will be collected in two different vacutainers, and three mL of the coagulated blood will be used for lipid analysis (biochemical tests) and two mL will be used for DNA analysis (molecular tests). Genomic DNA will be extracted with the collected blood samples, and specific primers will be designed for the opted SNPs (SORT1-rs646218 and OLR1-rs11053646 polymorphisms) and PCR-RFLP will be performed and randomly DNA sequencing will be carried out to cross check the results.ResultsThe rs646218 and rs11053646 polymorphisms were significantly associated with allele, genotype and dominant models with and without crude odds ratios (OR's) and Multiple logistic regression analysis (p < 0.05). Correlation between lipid profile and genotypes has confirmed the significant relation between triglycerides and rs646218 and rs1105364 6polymorphisms. However, rs11053646 polymorphism was correlated with HDLC (p = 0.04). Genotypes were examined in both males' vs. males and females' vs. females in cases and control and we concluded that in rs11053646 polymorphisms with male subjects compared between cases and controls found to be associated with dominant model heterozygote genotypes (p < 0.05).ConclusionThe results of the current study confirmed the SORT1 and OLR1 SNPs were associated in the Saudi population. The current results were in the association with the prior study results documented through GWAS and meta-analysis association. However, other ethnic population studies should be performed to rule out in the human hereditary diseases.

Dataset Information

Frequency of common HFE variants in the Saudi population: a high throughput molecular beacon-based study.

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Frequency of common HFE variants in the Saudi population: a high throughput molecular beacon-based study.

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