Aderson S. Araújo

DNA polymorphisms at the BCL11A, HBS1L-MYB, and β-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease

Sickle cell disease (SCD) is a debilitating monogenic blood disorder with a highly variable phenotype characterized by severe pain crises, acute clinical events, and early mortality. Interindividual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. Common single nucleotide polymorphisms (SNPs) at the BCL11A and HBS1L-MYB loci have been implicated previously in HbF level variation in nonanemic European populations. We recently demonstrated an association between a BCL11A SNP and HbF levels in one SCD cohort [Uda M, et al. (2008) Proc Natl Acad Sci USA 105:1620–1625]. Here, we genotyped additional BCL11A SNPs, HBS1L-MYB SNPs, and an SNP upstream of Gγ-globin (HBG2; the XmnI polymorphism), in two independent SCD cohorts: the African American Cooperative Study of Sickle Cell Disease (CSSCD) and an SCD cohort from Brazil. We studied the effect of these SNPs on HbF levels and on a measure of SCD-related morbidity (pain crisis rate). We strongly replicated the association between these SNPs and HbF level variation (in the CSSCD, P values range from 0.04 to 2 × 10−42). Together, common SNPs at the BCL11A, HBS1L-MYB, and β-globin (HBB) loci account for >20% of the variation in HbF levels in SCD patients. We also have shown that HbF-associated SNPs associate with pain crisis rate in SCD patients. These results provide a clear example of inherited common sequence variants modifying the severity of a monogenic disease.

Molecular Variations Linked to the Grouping of β- and α-Globin Genes in Neonatal Patients with Sickle Cell Disease in the State of Pernambuco, Brazil

Various factors have been described as phenotypic modulators of sickle cell disease, such as levels of fetal hemoglobin (Hb F), presence of α-thalassemia (thal), and haplotypes of the β-globin genes. In order to characterize and determine the frequency of the βS and βC mutations and the prevalence of −α3.7-thal, 74 patients with sickle cell disease detected during neonatal screening in the State of Pernambuco, Brazil, were studied. The haplotypes of the β gene and −α3.7-thal were determined using polymerase chain reaction (PCR), and specific restriction endonucleases were used to establish the polymorphic sites of the haplotypes. The results showed the high frequency of the Central African Republic (CAR) or Bantu haplotype in the State of Pernambuco, Brazil. The low frequency of the Benin haplotype recorded in this study, in comparison with other states in northeast Brazil, suggests the diversity of origins of Afro-Brazilians in this region.

Elevated plasma levels and platelet‐associated expression of the pro‐thrombotic and pro‐inflammatory protein, TNFSF14 (LIGHT), in sickle cell disease

Chronic vascular inflammation and endothelial activation may initiate vaso‐occlusion in sickle cell disease (SCD). TNFSF14 (CD258; LIGHT), a recently‐identified pro‐thrombotic and pro‐inflammatory tumour necrosis factor (TNF)‐superfamily cytokine, has a potent activating effect on endothelial cells. We evaluated whether TNFSF14 production is altered in SCD and whether platelets contribute to this production. TNFSF14 was measured in platelet‐free plasma from healthy‐control individuals (CON), steady‐state sickle cell anaemia (SCA), SCA on hydroxycarbamide therapy (SCAHC) and haemoglobin SC (HbSC) patients. Mean plasma TNFSF14 was significantly increased in SCA, SCAHC and HbSC, compared to CON individuals. In SCA/SCAHC patients, plasma TNFSF14, showed no correlation with haematological variables, but was significantly correlated with serum lactate dehydrogenase and inflammatory markers (CD40LG , IL8 and ICAM1). Platelet‐membrane TNFSF14 expression was significantly augmented on SCA platelets, and correlated with platelet activation; furthermore, measurement of platelet TNFSF14 release indicated that platelets may be a major source of circulating TNFSF14 in SCA. Interestingly, high plasma TNFSF14 was significantly associated with elevated tricuspid regurgitant velocity (≥2·5 m/s) in a population of SCA/SCAHC patients. The pro‐inflammatory and atherogenic cytokine, TNFSF14, could contribute to endothelial activation and inflammation in SCA; future investigations may confirm whether this protein contributes to major clinical complications of the disease, such as pulmonary hypertension, and represents a potential therapeutic target.

Association of variant alleles of MBL2 gene with vasoocclusive crisis in children with sickle cell anemia

Vasoocclusive crisis (VOC) is the major cause of morbidity and mortality in sickle cell anemia (SCA), which is caused by the occlusion of blood vessels, followed by ischemia or infarct, resulting in progressive damage to organs. However, this clinical manifestation is variable, indicating that this process could be influenced by modifier genes. The gene MBL2 which codes for mannose-binding lectin (MBL) has been associated with modifications in the progression of infectious and inflammatory vascular diseases. The aim of this study was to determine the frequency of the polymorphisms of exon 1 (alleles A/O) and promoter region -221 (alleles Y/X) of MBL2 in children with SCA and to verify their association with VOC. The determination of the polymorphism of exon 1 and the promoter region of MBL2 was performed by SYBR GREEN((R)) and Taqman((R)) system, respectively. In the patients with SCA, the frequency of the genotype related to high production of MBL was 0.46 (YA/YA) and for intermediate/low production was 0.54 (YA/XA, XA/XA, YA/YO, XA/YO, YO/YO). The frequency of the genotypes and haplotypes of MBL2 in patients with SCA did not differ from control individuals. The populations were in Hardy-Weinberg equilibrium. The patients were divided into two groups. The groups were separated by the frequency of VOC, which was defined by the total of VOC episodes divided by the age of the children at the end of this study. Since, we choose a cut point in FVOC <1 (n=48) (which we considered of mild presentation of disease) and FVOC >or=1 (n=39) (higher severity). In children with SCA, the frequency of the genotypes of MBL2 of intermediate/low expression for MBL was associated with FVOC >or=1 (p=0.0188 OR=3.15 CI=1.19-8.50). The results suggest that MBL2 polymorphism at promoter and first exon of MBL2 associated with low serum levels and structural alterations of MBL could modify the phenotype of the child with SCA related to VOC.

Erythropoiesis-driven regulation of hepcidin in human red cell disorders is better reflected through concentrations of soluble transferrin receptor rather than growth differentiation factor 15

Growth differentiation factor 15 (GDF‐15) is a bone marrow‐derived cytokine whose ability to suppress iron regulator hepcidin in vitro and increased concentrations found in patients with ineffective erythropoiesis (IE) suggest that hepcidin deficiency mediated by GDF‐15 may be the pathophysiological explanation for nontransfusional iron overload. We aimed to compare GDF‐15 production in anemic states with different types of erythropoietic dysfunction. Complete blood counts, biochemical markers of iron status, plasma hepcidin, GDF‐15, and known hepcidin regulators [interleukin‐6 and erythropoietin (EPO)] were measured in 87 patients with red cell disorders comprising IE and hemolytic states: thalassemia, sickle cell anemia, and cobalamin deficiency. Healthy volunteers were also evaluated for comparison. Neither overall increased EPO, nor variable GDF‐15 concentrations correlated with circulating hepcidin concentrations (P = 0.265 and P = 0.872). Relative hepcidin deficiency was found in disorders presenting with concurrent elevation of GDF‐15 and soluble transferrin receptor (sTfR), a biomarker of erythropoiesis, and sTfR had the strongest correlation with hepcidin (rS = −0.584, P < 0.0001). Our data show that high concentrations of GDF‐15 in vivo are not necessarily associated with pathological hepcidin reduction, and hepcidin deficiency was only found when associated with sTfR overproduction. sTfR elevation may be a necessary common denominator of erythropoiesis‐driven mechanisms to favor iron absorption in anemic states and appears a suitable target for investigative approaches to iron disorders. Am. J. Hematol. 89:385–390, 2014.

The deletion of SOX8 is not associated with ATR-16 in an HbH family from Brazil

The most common cause of a-thalassaemia are deletions in the a-globin gene cluster, taking away one or more a-globin genes. However, an increasing number of rare deletions have been described that cause monosomy of the telomeric region of chromosome 16 in individuals with a-thalassaemia only, or a-thalassaemia and a variety of developmental abnormalities and mild mental retardation, known as the ATR-16 syndrome (Daniels et al, 2001) (Horsley et al, 2001). ATR-16 is defined as a contiguous gene syndrome resulting from haploinsufficiency of the a-globin gene cluster and genes involved in mental retardation (MR). To date, only few cases have been described which result from pure monosomy for a deletion of 16p. The region on chromosome 16p for which haploinsufficiency leads to the dysmorphic features and MR typical for ATR-16, has been narrowed down to a region 0Æ9–1Æ5 Mb from the telomere (Harteveld et al, 2007), encompassing SOX8 at 0Æ97 Mb, which is generally assumed to be a candidate gene for ATR-16 (Meyer et al, 1997; Pfeifer et al, 2000; Takada & Koopman, 2003). Here we describe a family of Brazilian origin investigated by multiplex ligation dependent probe amplification (MLPA) because of a suspected large deletion based on the presence of HbH and Inclusion Bodies while only the common )a (Rightward) deletion was observed. The family consisted of three generations. The father (FI.1) was a carrier of the )a deletion and the mother (FI.2) presented with microcytic hypochromic anaemia, showing none of the seven most common a-thalassaemia deletions detected by gap-polymerase chain reaction (PCR) (Tan et al, 2001). They had six children, three were heterozygous for the )a allele (FII.3, 5, 6), one (FII.2) presented with the same microcytic hypochromic anaemia observed in the mother without any abnormalities on gap-PCR, while the two index patients (FII.1 and FII.4) with HbH disease had only the )a deletion detected by gap-PCR (Tan et al, 2001). Finally the son of propositus FII.1 (FIII.1 in Table I) had inherited the thalassaemia trait from the paternal grandmother. The MLPA analysis revealed an extensive deletion, 0Æ97– 1Æ05 Mb in size, removing the most telomeric MLPA-probe, located between position 45 799–45 856 [UCSC Genome Browser (May 2004) chromosome 16p13Æ3] up to probe 42 (at position 976 591–976 640) (Fig 1). The deletion breakpoint is located between position 976 640 and 1 053 683. The MLPA-probes 41 and 42 at the 3¢ end of the breakpoint, located in exon1 and exon3 of SOX8 respectively, were both deleted, indicating the complete absence of expression of this three-exon gene on the deletion allele. The carriers in this family had the largest a-thalassaemia deletion, without having the features of ATR-16. The proband (FII.1) is working as a security agent in a private company; his sister (FII.4), with

Association between the genetic polymorphisms of glutathione S-transferase (GSTM1 and GSTT1) and the clinical manifestations in sickle cell anemia.

The hereditary deficiency of antioxidant enzymes when associated with sickle cell anemia (SCA) further contributes to the oxidation of hemoglobin S, which increases the formation of degradation products of this hemoglobin. The glutathione S transferases play an important role in the conjugation of glutathione to endogenous products of peroxidation of lipids and protect cells from the deleterious effects of oxidative stress. We analyzed genomic DNA from 278 patients with sickle cell anemia to correlate the genotypes GSTT1 and/or GSTM1 null (determined by multiplex PCR technique) and the clinical manifestations of the disease. 27% of patients showed absence of the GSTM1 gene and 15% had absence of GSTT1. The GSTM1 and GSTT1 null genotypes were found in 11% of the population. The risk of individuals with the GSTT1 null genotype developing acute chest syndrome and aseptic necrosis of the femoral head were, respectively, 10 and 6.3 times higher when compared with those individuals who had of this gene. Patients with GSTM1 null showed a risk 3.9 times higher to develop stroke and high risk for malleolar ulcers and acute chest syndrome (OR=6.9 and 4.2, respectively). The individuals with the GSTM1 and GSTT1 null genotypes showed a higher chance of developing acute chest syndrome, malleolar ulcer and aseptic necrosis of the femoral head. The absence of GSTT1 and/or GSTM1 was an important risk factor for increasing the morbidity of SCA, especially in regard to acute chest syndrome.

Triagem familiar para o gene HBB*S e detecção de novos casos de traço falciforme em Pernambuco

OBJECTIVE To estimate the additional number of affected individuals based on the prevalence of sickle-cell syndromes among relatives of index cases. METHODS Cross-sectional study of relatives of a random sample of index cases identified through a neonatal screening program in Northeastern Brazil, between 2001 and 2005. The extended family trial model included 463 relatives of 21 index cases. Relatives were classified as nuclear family (NF: father, mother, and siblings); first degree extended family (N1: grandparents, uncles and aunts, and first cousins); second degree extended family (N2: children of first cousins); extended family (NA: NF+N1+N2); and extended nuclear family (NA1: NF+N1). The presence of HBB*S and other abnormal hemoglobins was confirmed by high-performance liquid chromatography. The association between the presence of HBB*S and other variables was calculated using prevalence ratios and their respective 95% confidence intervals, and differences between means were calculated using Students t test with a 5% significance level. RESULTS Of relatives, 81% had no knowledge of sickle-cell anemia and HBB*S was present in 114 family members. A total of 53.3% of the studied population was considered as of reproductive age, and 80% of HBB*S carriers had already had children. Frequency was higher among NF (69%), but was also high in N1 (22.8%). NA1 screening resulted in the detection of 69 carriers additional (a 172% increase). CONCLUSIONS These results indicate that family screening for the identification of sickle-cell carriers should be extended to first degree relatives.OBJECTIVE: To estimate the additional number of affected individuals based on the prevalence of sickle-cell syndromes among relatives of index cases. METHODS: Cross-sectional study of relatives of a random sample of index cases identified through a neonatal screening program in Northeastern Brazil, between 2001 and 2005. The extended family trial model included 463 relatives of 21 index cases. Relatives were classified as nuclear family (NF: father, mother, and siblings); first degree extended family (N1: grandparents, uncles and aunts, and first cousins); second degree extended family (N2: children of first cousins); extended family (NA: NF+N1+N2); and extended nuclear family (NA1: NF+N1). The presence of HBB*S and other abnormal hemoglobins was confirmed by high-performance liquid chromatography. The association between the presence of HBB*S and other variables was calculated using prevalence ratios and their respective 95% confidence intervals, and differences between means were calculated using Students t test with a 5% significance level. RESULTS: Of relatives, 81% had no knowledge of sickle-cell anemia and HBB*S was present in 114 family members. A total of 53.3% of the studied population was considered as of reproductive age, and 80% of HBB*S carriers had already had children. Frequency was higher among NF (69%), but was also high in N1 (22.8%). NA1 screening resulted in the detection of 69 carriers additional (a 172% increase). CONCLUSIONS: These results indicate that family screening for the identification of sickle-cell carriers should be extended to first degree relatives.

Reduced rate of sickle-related complications in Brazilian patients carrying HbF-promoting alleles at the BCL11A and HMIP-2 loci

The presence of high levels of fetal haemoglobin (HbF) provides well‐validated clinical benefits to patients with sickle cell anaemia (SCA). Nevertheless it has been difficult to show clear direct effects of the known genetic HbF modifiers, such as the enhancer polymorphisms for haematopoietic transcription factors BCL11A and MYB, on SCA severity. Investigating SCA patients from Brazil, with a high degree of European genetic admixture, we have detected strong effects of these variants on HbF levels. Critically, we have shown, for the first time, that the presence of such HbF‐promoting variants leads to a reduced rate of SCA complications, especially stroke.

Hidroxiuréia em pacientes com síndromes falciformes acompanhados no Hospital Hemope, Recife, Brasil

The use of hydroxyurea increases concentrations of fetal hemoglobin (Hb F) in sickle cell disease patients. It has been used in adults and in trials with children with the aim of preventing events such as episodes of pain or stokes. The objective of this study was to analyze the efficacy and side effects of Hydroxyurea in children with ages ranging from 5 to 17 years and also in young adults with SS or Sβ0 hemoglobinopathies. The patients were treated in the outpatient clinic of the Hemope Hospital. Young patients were treated with hydroxyurea at 10 mg/kg/day which was increased by 5 mg/kg/day at 8-week intervals until reaching a maximum dose of 25 mg/kg/day. For adults, the treatment started at 500 mg/day and increased until a dose of 1000 mg/day was reached. Total Hb F levels and the Mean Corpuscular Volume rose with hydroxyurea therapy and there was a reduction of events involving pain as well as the necessity of hospitalization among the pediatric patients. With the over 18year-old patients, a better clinical state was noticed together with a rise in hemoglobin levels and a reduction in the reticulocyte, leukocyte and platelet counts. No signs or symptoms in respect to drug toxicity were evidenced in either group. The use of hydroxyurea seems to be safe and effective in both children and young adults with sickle cell disease. The drug also improves theO uso de hidroxiureia promove a elevacao dos niveis de hemoglobina fetal (Hb F) em pacientes portadores de sindromes falciformes (SF) e o medicamento vem sendo estudado em varios grupos de pacientes, incluindo adultos e criancas. O presente trabalho analisou a eficacia e tolerabilidade do uso de hidroxiureia em criancas na faixa etaria entre 5 e 17 anos de idade e em adultos jovens acima de 18 anos, portadores de hemoglobinopatia SS ou Sb0 que foram acompanhados regularmente no ambulatorio do Hospital Hemope. Os pacientes pediatricos foram tratados com dose inicial de hidroxiureia de 10 mg/kg/dia, a qual era aumentada em 5 mg/kg por dia em intervalos de oito semanas, ate a dose maxima de 25 mg/kg/dia. Para os adultos, o tratamento foi iniciado com 500 mg/dia de hidroxiureia ate a dose maxima de 1g/dia. Foi observada reducao do numero de crises algicas assim como do numero de internacoes hospitalares, elevacao do nivel de Hb F e do Volume Corpuscular Medio, no grupo pediatrico. Entre os pacientes maiores de 18 anos, tambem se observou melhora clinica e significância estatistica com aumento dos valores da hemoglobina e reducao dos valores de reticulocitos, leucocitos e plaquetas. Nao foram observados sinais ou sintomas sugestivos de toxicidade medicamentosa em ambos os grupos. O uso de hidroxiureia em todos os pacientes parece ser seguro e eficaz e assegura melhora da qualidade de vida e beneficios a seus familiares. Ademais, as doses preconizadas de hidroxiureia aparentemente nao foram mielotoxicas, nao tendo sido necessaria a suspensao do tratamento em nenhum dos pacientes.