Isa Menezes Lyra

Clinical and molecular characteristics of sickle cell anemia in the northeast of Brazil

Beta S-globin gene (βS-globin) haplotypes, markers for severe sickle cell anemia (SCA), and the alpha-thalassemia 2 gene 3.7 kb deletion (-α2 3.7 kb thal) along with demographic and clinical data were investigated in SCA outpatients (n = 125, 63 female and 62 male) in the Brazilian state of Bahia, which has a high prevalence SCA. PCR-RFLP showed that the Central African Republic/Benin (CAR/BEN, 51.2%) haplotype was most frequent, followed by the Benin/Benin (Ben/Ben, 28.8%). At least one CAR haplotype was present in every outpatient with a history of cerebrovascular accident. The Cameroon (Cam), Senegal (Sen) and Arab-India haplotypes occurred in small numbers, as did atypical haplotypes. Fetal hemoglobin (HbF, %) was unevenly distributed. Compared to those > 18 y, those aged ≤ 18 y had had fewer erythrocyte transfusions and high HbF levels (12.3% ± 7.01 to 7.9% ± 4.36) but a higher frequency of spleen sequestration and pneumonia. Compared with normal α - genes carriers values, the outpatients with -α2 3.7 kb thal (determined by PCR analysis) had significantly higher mean hemoglobin concentration (Hb) (8.3 ± 1.34 g/dL, p = 0.018) and packed cell volume (PCV = 27.1% ± 4.26, p = 0.019) but low mean corpuscular volume (MCV = 86.1 fL = 10-15 L ± 9.56, p = 0.0004) and mean corpuscular hemoglobin (MCH = 26.6% ± 4.60, p = 0.039).

The beta-globin gene cluster haplotypes in sickle cell anemia patients from Northeast Brazil: a clinical and molecular view.

The βS‐globin haplotypes were studied in 78 sickle cell Brazilian patients from Bahia, Northeast Brazil, that has a large population of African origin. Hemoglobin (Hb) profiles were developed by high‐performance liquid chromatography (HPLC), and βS‐globin gene haplotypes were determined by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) techniques. We identified 44 (55.0%) patients with the CAR/Ben (Central African Republic/Benin) genotype, 16 (20.0%) Ben/Ben, 13 (16.2%) CAR/CAR and seven (8.8%) with other genotypes. Analyses of the phenotypes showed clinical differences related only to Hb F levels and blood transfusion therapy; the presence of − α− 3.7‐thalassemia (thal) demonstrated statistical significance when associated with hematocrit (p = 0.044), MCV (p = 0.0007), MCH (p = 0.012) and spleen sequestration events. The haplotype diversity found in the present study can be justified by information about the origin of the slave traffic period in Bahia during the 19th century. The specific characteristics described among the Bahian sickle cell patients could be confirmed by increasing the number of patients with specific genotypes and further studies of genetic markers.

Ocular lesions in sickle cell disease patients from Bahia, Brazil

Objective: The present study aims to describe ocular alterations in sickle cell disease patients in Bahia, a Northeast state, with the highest prevalence of the disease in Brazil. Methods: We carried out a cross-sectional study in a group of 146 (292 eyes) sickle cell disease patients (90 HBSS and 56 HBSC). Ophthalmologic examination including indirect binocular ophthalmoscopy was performed. Examination was completed by fluorescein angiography to detect retinal lesions. Results: The most frequent ocular lesions identified were “vascular tortuosity” and “black sunburst”. Proliferative retinopathy was found in 22 (12.2%) eyes of HBSS patients and 25 (22.3%) eyes of HBSC patients (OR=2.06; CI95%: 1.5-4.06, p=0.022); Its frequency was higher among HBSS patients aged 20-39 years, while in HBSC patients, it peaked after 40 years (35.7% and 42.8%) and dropped sharply afterwards. Conclusion: Proliferative retinopathy was described as early as 10 years of age in both patients groups. Proliferative sickle retinopathy can result in blindness and the knowledge of the most prevalent ocular alterations and age risk will be important to establish a protocol of ophthalmologic follow-up, in order to prevent a severe visual loss and increase patients life quality.

Laboratory and Genetic Biomarkers Associated with Cerebral Blood Flow Velocity in Hemoglobin SC Disease

Reference values for cerebral blood flow velocity (CBFV) in hemoglobin SC disease (HbSC) have not been established. We aimed to investigate associations between laboratory and genetic biomarkers associated with CBFV in HbSC children. Sixty-eight HbSC children were included; CBFV was analyzed by transcranial Doppler, and the time-averaged maximum mean velocity (TAMMV) was estimated. Hematological, biochemical, immunological, and genetic analyses were performed. TAMMV was negatively correlated with red blood cell count (RBC) count, hemoglobin, hematocrit, and direct bilirubin (DB), yet positively correlated with monocytes and ferritin. We found that children with TAMMV ≥ 128 cm/s had decreased red blood cell distribution width (RDW) and nitric oxide metabolite (NOx) concentration. Children with TAMMV ≥ 143.50 cm/s had decreased hemoglobin and hematocrit, as well as increased ferritin levels. Decreased hemoglobin, hematocrit, RDW, and NOx and increased ferritin were detected in children with TAMMV ≥ 125.75 cm/s. The CAR haplotype was associated with higher TAMMV. In association analyses, RBC, hemoglobin, hematocrit, RDW, monocyte, DB, NOx, and ferritin, as well as the CAR haplotype, were found to be associated with higher TAMMV in HbSC children. Multivariate analysis suggested that high TAMMV was independently associated with hematocrit, RDW, and NOx. Additional studies are warranted to validate the establishment of a cutoff value of 125.75 cm/s associated with elevated TAMMV in HbSC children.

Genetic modulation of HbF in Brazilians with HbSC disease and sickle cell anemia

Sickle cell anemia (homozygosity for HbS) and HbSC disease (compound heterozygosity for HbS and HbC) are the most common genotypes of sickle cell disease (SCD). Fetal hemoglobin (HbF), the major genetic modulator of SCD, interferes with deoxyHbS polymerization [1]. Patients with HbSC disease have about half the number of complications as do HbS homozygotes [1], however, their mean HbF levels were 2.4% compared with about 6% in patients with sickle cell anemia [2]. Single nucleotide polymorphisms (SNPs) in quantitative trait loci cis and trans to the β-globin gene cluster are associated with HbF. We analyzed the association of SNPs in BCL11A, (chromosome 2p), in the HBS1L-MYB intergenic region (HMIP) (chromosome 6q), and in the 5′ olfactory receptor (OR) gene cluster of chromosome 11p with HbF in Brazilian patients with SCD. 622 patients were studied. They were not treated with hydroxyurea and were aged ≥6 years. Hemoglobin was analyzed by HPLC. SNPs rs766432 and rs6732518 in BCL11A; rs35959442 and rs11759553 in HMIP; and rs4910755 and 7483122 located in OR51B5-OR51B6 were genotyped by ABI TaqMan Assays. The βS and βC globin haplotypes were investigated with PCR and restriction fragment length polymorphism techniques. Mean HbF was compared according to genotype using the additive genetic model adjusting for age, gender and HbS homozygotes versus HbSC. A dominant genetic model adjusting for age, gender and SCD genotype was used to test the association SNPs with HbF. We tested to see if there was an association between HbF and SNPs in the OR gene cluster region after adjusting for age, gender, SCD genotype and the HBB gene cluster haplotype (Central African Republic [CAR] or Bantu versus absence of a CAR haplotype). To determine if there was a synergistic effect between the SNPs or if the effect exerted on HbF was independent, we developed multivariable linear regression models adjusting for age, gender and SCD disease status and ran stepwise linear regression analyses where we added the most significant SNPs from BCL11A, HMIP one-at-a-time. Data analysis was performed using R version 2.14.1 and the cubic root transformation of HbF [3]. 392 patients were HbS homozygotes (HbF 7.4±4.4%) and 230 had HbSC disease (HbF 2.5±2.3%). The association between HbF and SNPs in the BCL11A, HMIP and the OR gene cluster for all patients combined and for HbS homozygotes and HbSC patients separately are shown in Table I a-c (on-line supplementary material). In the combined group, there is a statistically significant association between HbF levels and both BCL11A SNPs and with HMIP SNP rs11759553 after adjusting for age, gender and the SCD genotype. As the number of minor alleles increase, so does HbF level; in HbS homozygotes a similar result was found; in HbSC disease both SNPs in BCL11A and HMIP were associated with HbF. The association between HbF levels and SNPs in the OR genes, after adjusting fo r HBB haplotype was not significant (Table II, on-line supplementary material). In a stepwise linear regression analysis adding the SNPs to a multivariable linear regression model as described, after adjusting for age, gender and SCD genotype rs35959442 is still significantly associated with HbF indicating that its effect on HbF is independent of rs766432 (Table III, on-line supplementary material). We dichotomized the patients according to the HBB gene cluster haplotype. Brazilians with SCD have a higher prevalence of the CAR haplotype compared with African Americans. This haplotype is associated with lower levels of HbF than other haplotypes [4][5]. Sickle cell anemia patients were heterozygous or homozygous for the CAR haplotype chromosome or lacked a CAR haplotype chromosome; HbSC disease patients had or did not have a CAR haplotype chromosome. Most patients with a CAR haplotype had the minor allele for rs4910755 and rs7483122, both of which are located in the 5′ OR gene cluster of chromosome 11p and could be in linkage disequilibrium with SNPs characterizing this haplotype. HbSC disease patients usually have low HbF levels. Nevertheless, it is a milder condition than sickle cell anemia even though patients are more susceptible to clinical events related to high bloodviscosity. Although HbF in HbSC disease is lower than in HbS homozygotes, BCL11A and HMIP modulate HbF in both genotypes.

Inflammatory mediators in sickle cell anaemia highlight the difference between steady state and crisis in paediatric patients

Instituto Gonc alo Moniz- Fundacao Oswaldo Cruz (IGM-FIOCRUZ) and the Hospital da Crianc a das Obras Sociais Irma Dulce (HCOSID) approved this study, with protocol number 0016.0.225.000-09.

Heme-mediated cell activation: the inflammatory puzzle of sickle cell anemia

ABSTRACT Introduction: Hemolysis triggers the onset of several clinical manifestations of sickle cell anemia (SCA). During hemolysis, heme, which is derived from hemoglobin (Hb), accumulates due to the inability of detoxification systems to scavenge sufficiently. Heme exerts multiple harmful effects, including leukocyte activation and migration, enhanced adhesion molecule expression by endothelial cells and the production of pro-oxidant molecules. Area covered: In this review, we describe the effects of heme on leukocytes and endothelial cells, as well as the features of vascular endothelial cells related to vaso-occlusion in SCA. Expert commentary: Free Hb, heme and iron, potent cytotoxic intravascular molecules released during hemolysis, can exacerbate, modulate and maintain the inflammatory response, a main feature of SCA. Endothelial cells in the vascular environment, as well as leukocytes, can become activated via the molecular signaling effects of heme. Due to the hemolytic nature of SCA, hemolysis represents an interesting therapeutic target for heme-scavenging purposes.