Genetic variants associated with fetal hemoglobin levels show the diverse ethnic origin in Colombian patients with sickle cell anemia / Las variantes genéticas asociadas con niveles de hemoglobina fetal señalan diversos orígenes étnicos en pacientes colombianos con anemia falciforme.

Introduction: Fetal hemoglobin is an important factor in modulating the severity of sickle cell anemia. Its level in peripheral blood underlies strong genetic determination. Associated loci with increased levels of fetal hemoglobin display population-specific allele frequencies. Objective: We investigated the presence and effect of known common genetic variants promoting fetal hemoglobin persistence (rs11886868, rs9399137, rs4895441, and rs7482144) in 60 Colombian patients with sickle cell anemia. Materials and methods: Four single nucleotide polymorphisms (SNP) were genotyped by restriction fragment length polymorphisms (RFLP) and the use of the TaqMan procedure. Fetal hemoglobin (HbF) from these patients was quantified using the oxyhemoglobin alkaline denaturation technique. Genotype frequencies were compared with frequencies reported in global reference populations. Results: We detected genetic variants in the four SNPs, reported to be associated with higher HbF levels for all four SNPs in the Colombian patients. Genetic association between SNPs and HbF levels did not reach statistical significance. The frequency of these variants reflected the specific ethnic make-up of our patient population: A high prevalence of rs7482144-'A' reflects the West-African origin of the sickle cell mutation, while high frequencies of rs4895441-'G' and rs11886868-'C' point to a significant influence of an Amerindian ethnic background in the Colombian sickle cell disease population. Conclusion: These results showed that in the sickle cell disease population in Colombia there is not a unique genetic background, but two (African and Amerindian). This unique genetic situation will provide opportunities for a further study of these loci, such as fine-mapping and molecular-biological investigation. Colombian patients are expected to yield a distinctive insight into the effect of modifier loci in sickle cell disease.

Introducción. La hemoglobina fetal es un importante factor modulador de la gravedad de la anemia falciforme, cuya expresión está muy condicionada por el factor genético. Los loci asociados con el incremento de la hemoglobina fetal pueden presentar frecuencias alélicas específicas para cada población. Objetivo. Investigar la presencia y el efecto de las variantes genéticas rs11886868, rs9399137, rs4895441 y rs7482144 asociadas con la persistencia de hemoglobina fetal, en 60 pacientes colombianos con anemia falciforme. Materiales y métodos. Se hizo la genotipificación de los polimorfismos de nucleótido simple ( Single Nucleotide Polymorphisms, SNP) mediante la técnica de polimorfismos de longitud de fragmentos de restricción ( Restriction Fragment Length Polymorphisms, RFLP) y el procedimiento TaqMan. La hemoglobina fetal (HbF) se cuantificó utilizando la técnica de desnaturalización alcalina de la oxihemoglobina. Las frecuencias genotípicas se compararon con las reportadas en poblaciones de referencia global. Resultados. Se observaron variantes genéticas ya reportadas para aumento de HbF en los cuatro SNP. La asociación genética entre los SNP y el incremento de la HbF no alcanzó significancia estadística. La frecuencia de estos alelos reflejó la siguiente composición específica en esta muestra de pacientes colombianos: una gran prevalencia de rs7482144-'A', lo que indica que el origen de la mutación para la anemia falciforme es África occidental, y una gran frecuencia de rs4895441-'G' y rs11886868-'C', lo que denota la influencia significativa del origen genético amerindio. Conclusión. Los resultados evidenciaron que la población con anemia falciforme de Colombia no tiene un único origen genético, sino que existen dos (africano y amerindio). Esta situación genética única ofrece la oportunidad de llevar a cabo un estudio más amplio de estos loci a nivel molecular. Se espera que el estudio de pacientes colombianos permita una visión diferente del efecto de los loci modificadores en esta enfermedad.

[Evaluation of knocking down of the RNAi mediated gamma globin repressor into K562 cells, for gene therapy of beta-thalassemia]

Background and Aim: Beta-thalassemia is a genetic disorder manifested by the presence of anemia in adult patients. One approach to treatment of beta-thalassemia is induction of the fetal gamma-globin gene. One of the gamma-globin repressors is a complex called DRED [Direct repeat erythroid-definitive]. DRED is composed of TR2 and TR4 DNA binding subunits. The aim of this study was to set up the RNAi system to increase the expression of the gamma-globin gene

Materials and Methods: In this study, lipofectamin[Trade Mark] 2000 was used to transfect siRNA molecules and pSV-beta-Galactosidase vector was used as a reporter to monitor transfection efficiency. Real-time PCR method was used to measure TR4 knockdown and gamma-globin expression levels

Results: Our findings showed that K562 cells were transfected by 40% using Lipofectamine[Trade Mark] 2000. Also, the level of TR4 expression decreased by 44% after using TR4siRNA, even though the expression of gamma-globin gene was induced by 1.18 times

Conclusion: Despite a 44% knockdown of TR4, no increase in gamma-globin mRNA was observed. Two factors may count for this observation; first, TR4 knockdown may have been limited by our transfection efficiency. Second, simultaneous knockdown of TR2 and TR4 may lead to increased gamma-globin levels. In conclusion, although knocking down of TR4 expression occurred by using RNAi system, this can not be a solitary efficient way to induce the gamma-globin expression

Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients

Fetal hemoglobin (HbF), encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their ƒÀS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104) and an Afro-descendant one (N = 98), to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP) located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C¨T in the HBG1 promoter region were associated with the Central African Republic ƒÀS-globin haplotype. In contrast, the -369 C¨G and 309 A¨G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

[Determination of gamma-globin induction levels by different concentrations of hydroxyurea in K 562 cell lines for beta thalassemia treatment

Hydroxyurea [HU] is currently used for beta thalassemia treatment through induction of fetal gamma-globin, In this study, effects of different concentrations of hydroxyurea on induction of gamma-globin gene in K562 cells, and inhibitory effect of siRNA against candidate gene which may be involved in HU mediated gamma-globin induction were assessed. In this eperimantal study, K562 cells were treated by different concentrations of HU [0, 50, 100 and 200 microM]. siRNA against candidate gene in HU mediated gamma-globin gene induction was transfected to K562 cells using lipofectamine 2000. The level of gammal-globin gene induction and inhibition were determined by quantitavie real-time PCR. There were 1.75-, 2.45- and 2.55- fold inductions in gamma-globin gene using 50, 100 and 200 microM HU, respectively. There has been 79.5% down regulation on candidate gene in siRNA transfected K562 cells. This study showed that gamma-globin induction in 100 microM HU is similar to 200 micro M HU treated K562 cells. There was also efficient inhibitory effect on candidate gene which may be involved in HU mediated gamma-globin induction