Polymorphism in methylenetetrahydrofolate reductase, plasminogen activator inhibitor-1, and apolipoprotein E in hemodialysis patients.

The methylenetetrahydrofolate reductase (MTHFR) gene polymorphism, apolipoprotein E (apo epsilon4) gene polymorphism and polymorphism of plasminogen activator inhibitor-1 (PAI-1) have been shown to be associated with end-stage renal disease (ESRD). To determine the prevalence of these mutations in Saudi patients with ESRD on hemodialysis, we studied the allelic frequency and genotype distribution in patients receiving hemodialysis and in a control group, all residing in the Eastern Province of Saudi Arabia. The genotypes were determined using allele specific hybridization procedures and were confirmed by restriction fragment length polymorphism. The T allele frequency and homozygous genotype of MTHFR in ESRD patients were 14% and 2.4%, respectively compared to 13.4% and 0%, respectively in the control group. The allele frequency and homozygous genotype of 4G/4G PAI-1 gene polymorphism were 46.4% and 4.8% respectively in ESRD patients compared to 57.1% and 32% respectively in the control group. The apo s4 allele frequency and homozygous genotype distribution in hemodialysis patients were 7% and 2.4%, respectively compared to 13% and 2% in the control group. Although allele frequency of C677T of MTHFR was statistically similar in the hemodialysis patients and in the control group, the homozygotes T allele genotype was over represented in the hemodialysis group compared to normal. The prevalence of PAI-1 4G/4G polymorphism in ESRD patients was lower when compared to the control group. The prevalence of apo s4 allele did not differ significantly between the two groups. The present results demonstrate that all three studied polymorphic mutations are present in our population and that they may contribute to the etiology of the disease in our area.

Increased Prevalence of Glycoprotein IIb/IIIa Leu 33 Pro Polymorphism in End Stage Renal Disease Patients on Hemodialysis.

Traditional atherosclerosis risk factors cannot elucidate the increased prevalence of cardiovascular events in end stage renal disease (ESRD) patients on hemodialysis. A previous study has indicated a strong association of the PI(A1/A2) polymorphism with myocardial infarction, diabetes and renal allograft rejection. In this investigation, we determined the prevalence of the PI(A1/A2) polymorphism of platelet glycoprotein (GP) IIb/IIIa in ESRD patients on hemodialysis in the Eastern Province of Saudi Arabia. The PI(A1/A2) polymorphism was determined in 42 ESRD patients receiving hemodialysis and in 49 subjects without current or past history of renal disease. Genotypes were determined by a reverse-hybridization assay and were confirmed by restriction fragment length polymorphism procedures. The PI(A2) allele frequency among the control sample was 28.6% (2 were homozygous for PI(A2), 23 were homozygous for PI(A1), and 24 were heterozygous PI(A1/A2)). The PI(A2) allele frequency among the hemodialysis sample was 50% (2 were homozygous for PI(A2), 2 were homozygous for PI(A1) and 38 were heterozygous for PI(A1/A2)). The PI(A2) allele frequency among the hemodialysis patients was significantly higher than that in the control group [Odds ratios 2.5 (1.35-4.61), p<0.003; Adjusted odds ratios of 2.21 (1.05-4.65), p<0.036 after adjustment for the presence of diabetes; Simultaneously adjusting the odds ratios for the presence of standard risk factors (diabetes and hypertension) gave an adjusted OR of 6.87 (1.54-30.71), p=0.064]. These results suggest that the PI(A2) polymorphism may contribute toward the etiology of cardiovascular diseases in ESRD patients. A further study with a larger sample size is needed to confirm above results.

Vitamin D deficiency and rickets in the Eastern Province of Saudi Arabia.

BACKGROUND: Nutritional rickets remains prevalent in many developing countries, despite the availability of ample sunlight. The aim of this study was to investigate the clinical features and chemical pathology in a group of children with rickets and to compare them with a control group. SUBJECTS AND METHODS: In a case-control study over a 1-year period (March 2004 to February 2005), children clinically diagnosed with rickets (n=61) were age- and sex-matched with controls (n=58). In addition to routine chemical pathology, 25 (OH) vitamin D3 and parathormone (PTH) were determined. Controls were children without clinical rickets attending hospital for other blood investigations. RESULTS: The mean age of children with rickets was 14.8 mths and of controls was 16.5 mths. Mean (SD) body mass index of the children with rickets [16.8 (1.86)] was not significantly different from that of the controls [17.02 (3.16)]. Mean (SD) head circumference of rachitic children [45.41 (3.64) cm] was greater than that of controls [44.39 (5.07) cm, p=0.03]. Eighty per cent of the children with rickets were breastfed compared with 67% of controls. Thirty per cent of children with rickets were hypocalcaemic vs <7% of controls, 89% had phosphorus values <1.5 mmol/L vs 34.5% of controls and 75% had alkaline phosphatise levels >500 IU/L vs 28% of controls. Seventy-five per cent of children with rickets had serum 25 (OH) D3 <20 nmol/L vs 25% of controls. Mean (SD) PTH level was 23.59 (19.03) pmol/L in the rachitic group and 1.9 (1.05) pmol/L in controls (p<0.05). Lack of exposure to sunlight was recorded in 90% of the children with rickets and in 37% of the controls. CONCLUSION: Apparently healthy children living in areas where rickets is prevalent have risk factors for rickets and a small proportion will have evidence of biochemical rickets.

Validation of a reverse-hybridization StripAssay for the simultaneous analysis of common alpha-thalassemia point mutations and deletions.

BACKGROUND: alpha-Thalassemia is a worldwide disease and considered to be a major public health problem in countries within the so-called thalassemia belt. The complex genetics of alpha-thalassemias requires diagnostic methods with the capacity to screen rapidly and accurately for common causative mutations. METHODS: We developed and validated a reverse-hybridization assay (Alpha-Globin StripAssay) for the rapid and simultaneous detection of 21 alpha-globin mutations: two single gene deletions (-alpha(3.7); -alpha(4.2)), five double gene deletions [--(MED); --(SEA); --(THAI); --(FIL); -(alpha)(20.5)], alpha alpha alpha(anti-3.7) gene triplication, two point mutations in the alpha1 gene (cd 14 G>A; Hb Adana) and 11 point mutations in the alpha2 gene (initiation cd T>C; cd 19 -G; IVS1 -5nt; cd 59 G>A; Hb Quong Sze; Hb Constant Spring; Hb Icaria; Hb Pakse; Hb Koya Dora; polyA-1; polyA-2). RESULTS: Reliable genotyping of recombinant mutant clones and reference DNA samples was achieved by means of two corresponding test strips presenting parallel arrays of allele-specific oligonucleotides. The entire procedure from blood sampling to the identification of mutations required less than 6 h, and hybridization/detection was manual or automated. The diagnostic potential of this Alpha-Globin StripAssay was carefully evaluated on 272 pre-typed samples in a multicenter validation study. In 96.14% of the cases, StripAssay typing was completely concordant with the reference methods. CONCLUSIONS: The Alpha-Globin StripAssay proved to be a fast, easy-to-perform and reliable screening method to identify >90% of alpha-globin mutations in endemic areas worldwide.

Frequency of methylenetetrahydrofolate reductase C677T polymorphism in patients with cardiovascular disease in Eastern Saudi Arabia.

OBJECTIVE: Homozygosity for the C677T mutation in the gene of the thermolabile enzyme 5,10 methylenetetrahydrofolate reductase (MTHFR) associates with reduced enzyme activity, leading to mild hyperhomocysteinemia. We now know that an elevated level of homocysteine is an important risk factor for cardiovascular disease (CVD). The objective of this study was to determine the prevalence of the C677T mutation in Saudi patients diagnosed with CVD. METHODS: Over a period of 2 years (2003-2004) in a case control study, we determined the prevalence of the C677T mutation in 83 CVD patients and in 40 age and gender-matched controls in the Eastern Province of Saudi Arabia. We determined the MTHFR genotype by restriction fragment length polymorphism and allele specific hybridization procedures. RESULTS: The CVD group showed over representation of the C677T allele frequencies (20.5%) compared with unaffected controls (15%) (p=0.3). Furthermore, the genotypic data indicated that the prevalence of homozygosity for the C677T mutation was dramatically higher in the CVD patients (10.8%) when compared with normal (0%) (p=0.058). CONCLUSION: These results suggest that the MTHFR C677T variant mildly influences CVD. However, we require further investigation in large independent samples.

Molecular bases of beta-thalassemia in the Eastern Province of Saudi Arabia.

beta-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 beta-thalassemia in the region was studied. Sixty-nine subjects with known beta-thalassemia disease and volunteers with high hemoglobin $A(2)(HbA(2))$ 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.

Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in the Eastern Province of Saudi Arabia.

The level of activity of the enzyme glucose-6-phosphate dehydrogenase (G6PD) was determined in 154 unrelated Saudi males and females with G6PD deficiency who were residing in the Eastern Province of Saudi Arabia. DNA was extracted from blood samples and analyzed for known G6PD mutations by polymerase chain reaction (PCR) and restriction fragment length polymorphism techniques. Two different polymorphic mutations were identified which accounted for 90% of the samples analyzed. Of 114 G6PD-deficient males, 96 had G6PD Mediterranean, nine had African deficient variant G6PD A- and in nine the mutation has not been identified. Of the 40 G6PD-deficient females, 34 were homozygous for the G6PD Mediterranean mutation and six were genetic compound, G6PD Mediterranean/G6PD A-. The data indicate that the G6PD Mediterranean mutation is the most common (84%) in the Eastern Province, followed by G6PD A- (5.8%). Seventy one subjects who suffered from favism were found to carry the Mediterranean mutation.