Maria Stella Figueiredo

Effect of α‐thalassemia and β‐globin gene cluster haplotypes on the hematological and clinical features of sickle‐cell anemia in Brazil

To compare the features of sickle‐cell anemia in Brazil with those in other locales, we studied the effects of the β‐globin‐like gene cluster haplotype and α‐thalassemia upon the clinical and hematological features in 85 patients. The distribution of haplotypes differed from that in the United States and Jamaica. The Central African Republic (CAR) haplotype predominated; 34% of patients were CAR haplotype homozygotes, 45% CAR/Benin homozygotes, and 11% Benin homozygotes. No Senegal haplotype chromosomes were observed. α‐thalassemia was present in 17.5% of patients. HbF levels were higher in Benin homozygotes, compared with the other two groups (P < 0.05). Nearly half the patients with a CAR haplotype had leg ulcers, compared to 12.5% of the Benin homozygote group; stroke did not occur in α‐thalassemia carriers, but neither result was statistically significant. As in other studies, our results indicate that the CAR haplotype may be associated with more severe disease.

Clinical, hematological, and molecular characterization of sickle cell anemia pediatric patients from two different cities in Brazil.

This study focused on clinical, hematological, and molecular aspects of sickle cell anemia pediatric patients from two different cites in Brazil. Seventy-one patients from São Paulo and Salvador, aged 3 to 18 years, were evaluated. Hematological analyses, betaS globin gene haplotypes, and alpha2 3.7kb-thalassemia were performed. Numbers of hospitalizations due to vaso-occlusive crises, infections, stroke, and cholelithiasis were investigated. São Paulo had more hospitalizations from vaso-occlusion, cholelithiasis, and stroke than Salvador. The Ben/CAR genotype predominated in both cities. Alpha2 3.7kb-thalassemia had a frequency of 28.2% in Salvador, mostly with Ben/CAR genotype (45.0%), while São Paulo had 22.5% with similar frequencies of the Ben/ CAR and CAR/CAR genotypes. Sickle cell anemia patients from São Paulo also had more episodes of stroke, which was observed among CAR/CAR, atypical, and BEN/CAR haplotypes. In Salvador stroke was only observed in the Ben/CAR genotype. Cholelithiasis had similar frequencies in the two cities. These data suggest a milder phenotype among patients in Salvador, possibly due to genetic, environmental, and socioeconomic factors. Further studies are needed to elucidate modulating factors and phenotype association.

Renal dysfunction in patients with sickle cell anemia or sickle cell trait

Patients with sickle cell anemia (Hb SS) or sickle cell trait (Hb AS) may present several types of renal dysfunction; however, comparison of the prevalence of these abnormalities between these two groups and correlation with the duration of disease in a large number of patients have not been thoroughly investigated. In a cross-sectional study using immunoenzymometric assays to measure tubular proteinuria, microalbuminuria, measurement of creatinine clearance, urinary osmolality and analysis of urine sediment, we evaluated glomerular and tubular renal function in 106 adults and children with Hb SS (N = 66) or Hb AS (N = 40) with no renal failure (glomerular filtration rate (GFR) > 85 ml/min). The percentage of individuals with microalbuminuria was higher among Hb SS than among Hb AS patients (30 vs 8%, P < 0.0001). The prevalence of microhematuria was similar in both groups (26 vs 30%, respectively). Increased urinary levels of retinol-binding protein or beta 2-microglobulin were detected in only 3 Hb SS and 2 Hb AS patients. Urinary osmolality was reduced in patients with Hb SS or with Hb AS; however, it was particularly evident in Hb SS patients older than 15 years (median = 393 mOsm/kg, range = 366-469) compared with Hb AS patients (median = 541 mOsm/kg, range = 406-722). Thus, in addition to the frequently reported early reduction of urinary osmolality and increased GFR, nondysmorphic hematuria was found in 26 and 30% of patients with Hb SS or Hb AS, respectively. Microalbuminuria is an important marker of glomerular injury in patients with Hb SS and may also be demonstrated in some Hb AS individuals. Significant proximal tubular dysfunction is not a common feature in Hb SS and Hb AS population at this stage of the disease (i.e., GFR > 85 ml/min).

Brain Magnetic Resonance Imaging Abnormalities in Adult Patients With Sickle Cell Disease Correlation With Transcranial Doppler Findings

Background and Purpose— Brain imaging abnormalities were reported in up to 44% of children with sickle cell disease (SCD). The prevalence of neuroimaging abnormalities in adult patients with SCD and their relationship to transcranial Doppler is still unclear. Our objectives were to study the frequency of MRI and MR angiography abnormalities in adults with SCD and to define what transcranial Doppler velocities are associated with intracranial stenoses detected by MR angiography. Methods— We examined all adult patients (>16 years) with SCD followed in the hematology outpatient clinic at our university hospital with MRI, MR angiography, and transcranial Doppler. Results— We evaluated 50 patients. The overall prevalence of MRI abnormalities was 60%. Abnormal MRI findings were more frequent when vessel tortuosity or stenoses were present on MR angiography (P<0.01). Patients with intracranial stenoses had significantly higher time-averaged maximum mean velocities (P=0.01). A time-averaged maximum mean velocity of 123.5 cm/s allowed the diagnosis of middle cerebral artery or internal carotid artery intracranial stenosis with sensitivity of 100% and specificity of 73% with an area under the receiver operator characteristic curve of 0.91 (CI, 0.79 to 1.00). Conclusions— The frequency of brain imaging abnormalities detected by MRI/MR angiography in adults with SCD was higher than that described for children. Transcranial Doppler velocities in adult patients with intracranial stenoses were lower than those described for the pediatric population with SCD.

Hereditary hemochromatosis: mutations in genes involved in iron homeostasis in Brazilian patients.

BACKGROUND p.C282Y mutation and rare variants in the HFE gene have been associated with hereditary hemochromatosis (HH). HH is also caused by mutations in other genes, such as the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1). The low rate homozygous p.C282Y mutation in Brazil is suggestive that mutations in non-HFE genes may be linked to HH phenotype. AIM To screen exon-by-exon DNA sequences of HFE, HJV, HAMP, TFR2 and SLC40A1 genes to characterize the molecular basis of HH in a sample of the Brazilian population. MATERIALS AND METHODS Fifty-one patients with primary iron overload (transferrin saturation ≥50% in females and ≥60% in males) were selected. Subsequent bidirectional DNA sequencing of HFE, HJV, HAMP, TFR2 and SLC40A1 exons was performed. RESULTS Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n=11, 21.6%). In addition, heterozygous HFE p.S65C mutation was found in combination with p.H63D in two patients and homozygous HFE p.H63D was found in two patients as well. Sequencing in the HJV and HAMP genes revealed HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Molecular and clinical diagnosis of juvenile hemochromatosis (homozygous form for the HJV p.G320V) was described for the first time in Brazil. Three TFR2 polymorphisms (p.A75V, p.A617A and p.R752H) and six SLC40A1 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704, rs11568346) and the novel mutation SLC40A1 p.G204S were also found. CONCLUSIONS The HFE p.C282Y in homozygosity or in heterozygosity with p.H63D was the most frequent mutation associated with HH in this sample. The HJV p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G204S mutation may also be linked to primary iron overload but their role in the pathophysiology of HH remain to be elucidated.

Allelic polymorphisms of human Fcγ receptor IIa and Fcγ receptor IIIb among distinct groups in Brazil

BACKGROUND: The FcγRIIA gene is expressed in two polymorphic forms, R131 and H131, which differ by the replacement of histidine by arginine at position 131. The FCGR3B (FcγRIIIB) gene exists in two allelic isoforms, known as FCGR3B1 (FcγRIIIB‐NA1) and FCGR3B2 (FcγRIIIB‐NA2), which differ in nucleotides 141, 147, 227, 277, and 349. An additional polymorphism is the SH antigen that is associated with the FCGR3B3 (FcγRIIIB‐SH) allele.

Expression levels of CD47, CD35, CD55, and CD59 on red blood cells and signal-regulatory protein-α,β on monocytes from patients with warm autoimmune hemolytic anemia

BACKGROUND: Animal models have shown that CD47‐deficient mice develop severe autoimmune hemolytic anemia (AIHA) because the binding of red blood cell (RBC) CD47 to signal‐regulatory protein (SIRP‐α) on macrophages contributes to the inhibition of phagocytosis. In contrast, complement‐inhibitory proteins such as CD35, CD55, and CD59 may protect RBCs against the lysis by complement.

Analysis of polymorphism at site -174 G/C of interleukin-6 promoter region in multiple myeloma

It is well established that interleukin-6 (IL-6) is an essential growth factor for multiple myeloma (MM) and patients with increased IL-6 levels have a poor prognosis. In healthy subjects, the presence of the C allele at a polymorphic site (-174 G/C) of the IL-6 gene is related to low IL-6 levels. In view of the potential association of this particular polymorphism with IL-6 concentration, and the relevance of IL-6 in MM pathogenesis, the objective of the present study was to investigate the prevalence of IL-6 (-174 G/C) promoter polymorphism and its association with development of MM in Brazilian individuals. We investigated the prevalence of these alleles in 52 patients and 60 healthy subjects (matched by age, sex, and race) of a Brazilian population. Thirty patients were male (42.4%), 24 (46.2%) were white and the median age at diagnosis was 58.5 years (range: 28 to 84 years). To determine the IL-6 (-174 G/C) polymorphism, molecular analysis was performed by polymerase chain reaction followed by endonuclease restriction digestion. The genotype distributions observed in the group of patients were 4% CC, 42% GC and 54% GG. The C allele frequency was 0.25. These results were similar to the control group, suggesting no impact of this polymorphism on the susceptibility to MM.

The clinical impact of MTHFR polymorphism on the vascular complications of sickle cell disease

Sickle cell disease (SCD) is one of the most common inherited diseases in the world and the patients present notorious clinical heterogeneity. It is known that patients with SCD present activation of the blood coagulation and fibrinolytic systems, especially during vaso-occlusive crises, but also during the steady state of the disease. We determined if the presence of the factor V gene G1691A mutation (factor V Leiden), the prothrombin gene G20210A variant, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism may be risk factors for vascular complications in individuals with SCD. We studied 53 patients with SCD (60% being women), 29 with SS (sickle cell anemia; 28 years, range: 13-52 years) and 24 with SC (sickle-hemoglobin C disease; 38.5 years, range: 17-72 years) hemoglobinopathy. Factor V Leiden, MTHFR C677T polymorphism, and prothrombin G20210A variant were identified by PCR followed by further digestion of the PCR product with specific endonucleases. The following vascular complications were recorded: stroke, retinopathy, acute thoracic syndrome, and X-ray-documented avascular necrosis. Only one patient was heterozygous for factor V Leiden (1.8%) and there was no prothrombin G20210A variant. MTHFR 677TT polymorphism was detected in 1 patient (1.8%) and the heterozygous form 677TC was observed in 18 patients (34%, 9 with SS and 9 with SC disease), a prevalence similar to that reported by others. No association was detected between the presence of the MTHFR 677T allele and other genetic modulation factors, such as alpha-thalassemia, beta-globin gene haplotype and fetal hemoglobin. The presence of the MTHFR 677T allele was associated with the occurrence of vascular complications in SCD, although this association was not significant when each complication was considered separately. In conclusion, MTHFR C677T polymorphism might be a risk factor for vascular complications in SCD.

Genetic analysis of beta-thalassemia major and beta-thalassemia intermedia in Brazil.

The development of methodologies to identify the molecular lesions responsible for different types of beta-thalassemia has made it possible to correlate these data with clinical and hematological severity. We examined DNA from 35 patients with beta-thalassemia, residents of the State of São Paulo, Brazil, for some types of genetic modifying factors: beta-thalassemia mutations, the upstream Xmnl GY-globin gene polymorphisms, and alpha-globin gene deletions. Additionally, the beta-like gene cluster haplotypes and the presence of the AYT variant were studied. The following mutations were present in the 70 chromosomes studied: 54.3% codon 39 (C-->T) (beta degree); 18.6% IVS-I-6 (T-->C) (beta+); 18.6% IVS-I-110 (G-->A) (beta+), and 4.3% IVS-I-1 (G-->T) (beta degree). Haplotype II was associated with the nonsense mutation at codon 39, haplotype I with the IVS-I-110 and codon 39 mutations, and haplotypes VI and VII with the IVS-I-6 mutation. The Xmnl polymorphism was detected in three out of 31 patients studied. No alpha-thalassemia was detected among the thalassemia intermedia patients. The AYT variant was present in 87.1% of 31 thalassemia patients and was associated with the codon 39/haplotype II and IVS-I-6/haplotype VI mutations. This is the first study of the Brazilian population that has analyzed the beta-thalassemia mutations and other molecular variants, and has correlated them with the clinical manifestations.