The association of HBG2, BCL11A, and HMIP polymorphisms with fetal hemoglobin and clinical phenotype in Iraqi Kurds with sickle cell disease.

INTRODUCTION: Fetal hemoglobin (HbF) is the major modifier for sickle cell disease (SCD) severity. HbF is modulated mainly by three major quantitative trait loci (QTL) on chromosomes 2, 6, and 11. METHODS: Five SNPs in the three QTLs (HBG2, rs7482144; BCL11A, rs1427407 and rs10189857; and HBS1L-MYB intergenic region, rs28384513 and rs9399137) were investigated by multiplex PCR and reverse hybridization, and their roles in HbF and clinical phenotype variability in Iraqi Kurds with SCD were assessed. RESULTS: HBG2 rs7482144 with minor allele frequency (MAF) of 0.133 was the most significant contributor to HbF variability, contributing 18.1%, followed by rs1427407 (MAF of 0.266) and rs9399137 (MAF of 0.137) at 14.3% and 8.8%, respectively. The other two SNPs were not significant contributors. Furthermore, when the cumulative numbers of minor alleles in the three contributing SNPs were assessed, HbF% and hemoglobin concentration increased with increasing number of minor alleles (P < 0.0005 and 0.001, respectively), while serum lactic dehydrogenase, reticulocytes, leukocytes, transfusion, and pain frequencies decreased (P = 0.003, 0.004, <0.0005, <0.0005, and 0.017, respectively). CONCLUSIONS: It was demonstrated that SNPs in all three major HbF QTLs contribute significantly to HbF and clinical variability in Iraqi Kurds with SCD and that the cumulative number of minor alleles at contributing SNPs may serve as a better predictor of such variability in this population.

Genetic Modifiers in ß-Thalassemia Intermedia: A Study on 102 Iraqi Arab Patients.

To determine the molecular basis of ß-thalassemia intermedia (TI) and the contribution of the three hemoglobin F (HbF) quantitative trait loci (QTLs) on chromosomes 11, 2, and 6 to the milder phenotype, a total of 102 Iraqi Arab patients with TI were studied. The ß and α genotypes as well as HBG2 g. 158 C>T (rs7482144), BCL11A (rs1427407 and rs10189857), and HBS1L-MYB (rs28384513 and rs9399137) by multiplex polymerase chain reaction and reverse hybridization were studied. A total of 21 different ß-thalassemia mutations arranged in 35 different genotypes were identified. The genotypes encompassed ß(+)/ß(+) mutations in 33 cases, ß(+)/ß(0) in 17 cases, ß(0)/ß(0) in 47 cases, ß(0)/wild type in 3 and ß(0)/Hb E in 2 cases. The most common was IVS-II-1 (G>A)/IVS-II-1 (G>A), followed by IVS-I-6 (T>C)/IVS-I-6 (T>C) and IVS-I-110 (G>A)/IVS-I-110 (G>A), in 31.4%, 17.6%, and 6.9%, respectively. Alpha-thalassemia mutations were found in 15.2% of those homozygous for the ß-mutations, while α gene triplication was identified in all three heterozygotes. Of the five QTLs tested, only rs7482144 and rs10189857 were significantly associated with ß(0)/ß(0) when compared to ß(+)/ß(+), with odds ratios of 6.4 (95% confidence interval [CI] 2.9-14.0) and 3.2 (95% CI 1.2-8.6), respectively. In conclusion, this study has demonstrated that among Iraqi patients with thal intermedia, the main contributors to the milder phenotype were ß(+) alleles, XmnI polymorphism, and BCL11A (rs10189857), while other QTLs on chromosomes 2 and 6, as well as alpha-thalassemia, were not significantly relevant.

VKORC1 -1639G>A and CYP2C9*3 are the major genetic predictors of phenprocoumon dose requirement.

PURPOSE: Phenprocoumon, similar to other coumarin-derived anticoagulants, is associated with a large variation in the individual dose requirement to achieve stable anticoagulation. Polymorphisms in the vitamin K epoxide reductase complex subunit 1 (VKORC1) and the liver enzyme cytochrome P450 2C9 (CYP2C9) effectively account for the variability in warfarin and acenocoumarol response but are less well-defined pharmacogenetic predictors in phenprocoumon therapy. METHODS: A retrospective study was performed on 185 outpatients attending anticoagulation clinics in Austria and Germany. These patients were genotyped for the VKORC1 -1639G>A and 3730G>A polymorphisms as well as for the CYP2C9 *2 and *3 polymorphisms using a reverse hybridisation-based teststrip assay. RESULTS: The VKORC1 -1639A allele, which was present at a frequency of 41.4% in the study cohort, significantly reduced the mean weekly phenprocoumon dose by 3 mg (19%) in the heterozygous and by 6.7 mg (43%) in the homozygous state compared to wild-type carriers (15.5 +/- 6.8 mg, p < 0.0001). A stepwise multiple regression analysis revealed that VKORC1 -1639G>A, age and CYP2C9*3 were the major independent determinants of phenprocoumon dose, accounting for 14.2, 9.1 and 4.7% of its variability, respectively (p A genotype had no additional predictive power for individual dose variability. CONCLUSION: Similar to warfarin and acenocoumarol, the VKORC1 -1639G>A polymorphism had the highest impact on the maintenance dose of phenprocoumon. The factor age was the second most important predictor and explained a greater percentage of the variability than CYP2C9 genotype.

Association of endothelial protein C receptor haplotypes, factor V Leiden and recurrent first trimester pregnancy loss.

OBJECTIVES: We evaluated whether the endothelial protein C receptor (EPCR) haplotypes A1 and A3 exert effects on the development of recurrent pregnancy loss (RPL) in association with factor V Leiden. DESIGN AND METHODS: We determined the EPCR haplotypes A1 and A3 and factor V Leiden in 49 women with a history of RPL and 48 parous controls. RESULTS: In carriers of factor V Leiden the A1 haplotype decreased the relative risk for RPL from 2.2 to 1.0. CONCLUSIONS: The EPCR A1 haplotype tends to modulate the risk for RPL in carriers of factor V Leiden.

Measurement of lipid transfer protein in 88 apple cultivars.

BACKGROUND: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars. OBJECTIVE: Determination of the levels of LTP in a wide range of apple cultivars. METHODS: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC). RESULTS: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson's correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4-253.2 (sELISA), 2.7-120.2 (cELISA) and 0.4-47.3 microg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels. CONCLUSIONS: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.

Elucidating the spectrum of alpha-thalassemia mutations in Iran.

Alpha thalassemia (alpha-thal) is one of the most common hemoglobin (Hb) disorders in the world. Alpha-globin genes are located on chromosome 16. The majority of alpha-thal mutations are deletions but point mutations are found as well. Since the Iranian population is a mixture of different ethnic groups, frequency and distribution of alpha-globin mutations in various regions of the country need to be clarified. These findings can contribute to a wider understanding of this disorder.

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.

MdAP, a novel protein in apple, is associated with the major allergen Mal d 1.

Mal d 1, an 18-kDa intracellular pathogenesis-related protein (PR-10), has been known since long as the major apple allergen in Middle and Northern Europe. However, its biological function, as that of many other PR-10 proteins, is still unknown. In order to identify proteins putatively interacting with Mal d 1, an expression library of Malus domestica was screened using the yeast-two-hybrid (Y2H) system. A novel protein binding to two isoforms of Mal d 1 being used as 'bait' was isolated. The deduced amino acid sequence from the corresponding full-length cDNA of the predicted Mal d 1-Associated-Protein (MdAP) do not display any homology to known proteins, but shares 45% identity with a 'hypothetical protein' in Arabidopsis thaliana. Southern analysis of the apple genome indicated that MdAP, comprising 190 amino acids, is encoded by a single gene. The expression pattern of the 1-kb MdAP transcript resembled the expression profile of the different Mal d 1 isoforms in various apple organs, however at a much lower level. Furthermore, a huge variation in transcription levels of Mal d 1 isoforms was observed in apple tissue. For both, Mal d 1 and MdAP highest amounts of mRNAs were measured in ripe fruits and significantly lower amounts in vegetative tissue by quantitative reverse transcription-polymerase chain reaction (RT-PCR).