TNF-alpha and IL-8: serum levels and gene polymorphisms (-308G>A and -251A>T) are associated with classical biomarkers and medical history in children with sickle cell anemia.

Sickle cell anemia (SCA) is a disorder characterized by a heterogeneous clinical outcome. In the present study, we investigated the associations between Tumor Necrosis Factor-alpha (TNF-alpha) -308G>A and Interleukin 8 (IL-8) -251A>T gene polymorphisms, medical history and classical biomarkers in children with steady-state SCA. In total, 210 SCA patients aged 2-21 years and 200 healthy controls were studied. Gene polymorphisms, betaS-globin haplotypes and a 3.7-kb deletion in alpha2-thalassemia (α2-thal3.7 kb) were investigated by PCR/RFLP analysis, and cytokine levels were determined by ELISA. Splenomegaly (p=.032) was more prevalent among children younger than 5 years of age. The A allele of the TNF-alpha -308G>A gene polymorphism and the presence of α2-thal3.7 kb were associated with an increase risk of splenic sequestration events (p=.001; p=.046), while the T allele of the IL-8 -251A>T gene polymorphism was considered to be a protective factor for splenomegaly events (p=.032). Moreover, the A allele of the TNF-alpha -308G>A gene polymorphism was associated with high TNF-alpha levels (p=.021), and the hemoglobin F and hemoglobin S haplotypes were correlated with serum levels of IL-8. The logistic regression analysis showed significant effects of the TNF-alpha and IL-8 gene polymorphisms, beta(S)-globin gene haplotypes and α2-thal3.7 kb on the occurrence of splenic sequestration events. Our study emphasizes that the identification of new genetic and immunological biomarkers and their associations with classical markers is an important strategy to elucidate the underlying causes of different SCA phenotypes and their effects on patient outcome.

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.

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 betaS-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 betaS-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.

Evaluating glutathione S-transferase (GST) null genotypes (GSTT1 and GSTM1) as a potential biomarker of predisposition for developing leukopenia.

Glutathione S-transferase (GST) enzymes protect cells against xenobiotics and oxidative stress products through an electrophilic conjugation process. We investigated the theta (GSTT1) and mu (GSTM1) null genotypes in a group of leukopenic subjects and normal subjects from Northeast Brazil, evaluating their use as biomarkers of susceptibility for developing leukopenia. In a sample-based case-control study, we analysed white blood cell (WBC) counts and GSTT1 and GSTM1 genotypes. A total of 278 subjects were analysed: 91 with leukopenia and 187 controls. GSTT1 null genotype conferred a 5.92-fold risk for occurrence of leukopenia [odds ratios (OR) = 5.92, CI(MLE): 1.64-26.72, P(MLE) = 0.002] and a 3.90-fold risk of neutropenia (OR = 3.90; CI(MLE): 1.05-13.66; P(MLE) = 0.02), while GSTM1 null genotype conferred a 1.78-fold risk for leukopenia (OR = 1.75; CI(MLE): 1.04-3.06, P(MLE) = 0.017) and no risk of neutropenia (OR = 1.71; CI(MLE): 0.88-3.35; P(MLE) = 0.06). The GSTT1, but not the GSTM1 null genotype, was found to be associated with leukopenia and neutropenia. More cellular and molecular studies are needed to evaluate the existence of genotype interactions, and to confirm the appropriateness of using the GSTT1 and/or GSTM1 null genotypes as biomarkers of susceptibility to white blood-cell deficiencies.

Sequence change in the HS2-LCR and Ggamma-globin gene promoter region of sickle cell anemia patients.

The fetal hemoglobin (HbF) levels and betaS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Ggamma- and Agamma-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR) of patients with HbF level disparities as compared to their betaS haplotypes. Sixty-four (51.2%) patients had CAR/Ben genotype; 36 (28.8%) Ben/Ben; 18 (14.4%) CAR/CAR; 2 (1.6%) CAR/Atypical; 2 (1.6%) Ben/Cam; 1 (0.8%) CAR/Cam; 1 (0.8%) CAR/Arab-Indian, and 1 (0.8%) Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

Sequence change in the HS2-LCR and Gg-globin gene promoter region of sickle cell anemia patients

The fetal hemoglobin (HbF) levels and betaS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Gg- and Ag-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR) of patients with HbF level disparities as compared to their ßS haplotypes. Sixty-four (51.2 percent) patients had CAR/Ben genotype; 36 (28.8 percent) Ben/Ben; 18 (14.4 percent) CAR/CAR; 2 (1.6 percent) CAR/Atypical; 2 (1.6 percent) Ben/Cam; 1 (0.8 percent) CAR/Cam; 1 (0.8 percent) CAR/Arab-Indian, and 1 (0.8 percent) Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

alpha-Thalassemia 2, 3.7 kb deletion and hemoglobin AC heterozygosity in pregnancy: a molecular and hematological analysis.

alpha-Thalassemia is a synthesis hemoglobinopathy with a worldwide distribution. alpha-thalassemia-23.7kb (alpha-Thal23.7kb) was investigated by PCR and standard hematologic analysis techniques in 106 pregnant women - 53 heterozygous for hemoglobin (Hb) A and C (AC) and 53 homozygous for the normal Hb A (AA) with similar ages and race ancestry. Eleven (21%) of AC women were alpha-Thal23.7kb heterozygous and 1 (2%) was homozygous, while 12 AA women (23%) were heterozygous. In the AA group, the MCV differed among those with normal alpha genes and those with alpha-Thal23.7kb (P = 0.031). Statistical analysis of AC group patients with normal alpha genes and alpha-Thal23.7kb carriers showed differences in MCV (P = 0.001); MCH (P = 0.003) and Hb C concentrations (P = 0.011). Analysis of AA and AC group patients with normal alpha genes showed differences in RBC (P = 0.033), Hb concentration (P = 0.003) and MCHC (P < 0.0001). There were no statistically significant differences for any hematologic parameters between AC and AA group patients with the alpha-Thal23.7kb genotype. The AC alpha-Thal23.7kb homozygous women had low hematologic parameters. Serum ferritin levels were normal among the groups studied. These results emphasize the importance of diagnosis and follow-up of patients with hemoglobinopathy carriers during pregnancy in order to administer adequate therapy and avoid further complications for mothers and newborns.