RESUMO
Bone marrow, spleen, liver and kidney proton transverse relaxation rates (R2), together with cardiac R2* from patients with sickle cell disease (SCD), paroxysmal nocturnal hemoglobinuria (PNH) and non-transfusion dependent thalassemia (NTDT) have been compared with a control group. Increased liver and bone marrow R2 values for the three groups of patients in comparison with the controls have been found. SCD and PNH patients also present an increased spleen R2 in comparison with the controls. The simultaneous measurement of R2 values for several tissue types by magnetic resonance imaging (MRI) has allowed the identification of iron distribution patterns in diseases associated with iron imbalance. Preferential liver iron loading is found in the highly transfused SCD patients, while the low transfused ones present a preferential iron loading of the spleen. Similar to the highly transfused SCD group, PNH patients preferentially accumulate iron in the liver. A reduced spleen iron accumulation in comparison with the liver and bone marrow loading has been found in NTDT patients, presumably related to the differential increased intestinal iron absorption. The correlation between serum ferritin and tissue R2 is moderate to good for the liver, spleen and bone marrow in SCD and PNH patients. However, serum ferritin does not correlate with NTDT liver R2, spleen R2 or heart R2*. As opposed to serum ferritin measurements, tissue R2 values are a more direct measurement of each tissue's iron loading. This kind of determination will allow a better understanding of the different patterns of tissue iron biodistribution in diseases predisposed to tissue iron accumulation.
Assuntos
Anemia Falciforme/metabolismo , Hemoglobinúria Paroxística/metabolismo , Compostos de Ferro/metabolismo , Talassemia beta/metabolismo , Adulto , Medula Óssea/química , Estudos de Casos e Controles , Feminino , Humanos , Compostos de Ferro/análise , Rim/química , Fígado/química , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Miocárdio/química , Baço/química , Distribuição TecidualRESUMO
HMIP-2 is a human quantitative trait locus affecting peripheral numbers, size and hemoglobin composition of red blood cells, with a marked effect on the persistence of the fetal form of hemoglobin, HbF, in adults. The locus consists of multiple common variants in an enhancer region for MYB (chr 6q23.3), which encodes the hematopoietic transcription factor cMYB. Studying a European population cohort and four African-descended groups of patients with sickle cell anemia, we found that all share a set of two spatially separate HbF-promoting alleles at HMIP-2, termed "A" and "B." These typically occurred together ("A-B") on European chromosomes, but existed on separate homologous chromosomes in Africans. Using haplotype signatures for "A" and "B," we interrogated public population datasets. Haplotypes carrying only "A" or "B" were typical for populations in Sub-Saharan Africa. The "A-B" combination was frequent in European, Asian, and Amerindian populations. Both alleles were infrequent in tropical regions, possibly undergoing negative selection by geographical factors, as has been reported for malaria with other hematological traits. We propose that the ascertainment of worldwide distribution patterns for common, HbF-promoting alleles can aid their further genetic characterization, including the investigation of gene-environment interaction during human migration and adaptation.
Assuntos
Anemia Falciforme/genética , Elementos Facilitadores Genéticos , Eritrócitos/citologia , Locos de Características Quantitativas , Alelos , População Negra/genética , Interação Gene-Ambiente , Estudos de Associação Genética , Genética Populacional , Genoma Humano , Genótipo , Haplótipos , Humanos , Proteínas Proto-Oncogênicas c-myb/genética , Análise de Sequência de DNA , População Branca/genéticaRESUMO
Spin density projection-assisted R2-magnetic resonance imaging (R2-MRI; FerriScan(®)) scans from 40 chelation-naïve sickle cell patients were used to assess renal iron load by measuring renal R2 (R-R2). Clinical data were collected retrospectively for the 2-year period preceding the scan. R-R2 showed no significant correlation with transfusional iron load (assessed by liver iron concentration), but correlated significantly with serum bilirubin (R = 0·61, P < 0·0001) and lactate dehydrogenase (R = 0·58, P < 0·0001). Mean (±standard deviation) R-R2 was higher (P = 0·02) in patients with renal hyperfiltration (29·8 ± 10·3/s) than those without (23·11 ± 6·6/s). Five patients had significantly lower signal intensity in the renal cortex, as compared to the medulla. These patients had a significantly higher (P < 0·0001) mean R-R2 than those showing no cortico-medullary difference. We postulate that the increased R-R2 is associated with haemolysis rather than transfusional iron load in sickle cell disease.
Assuntos
Anemia Falciforme/complicações , Hemólise , Sobrecarga de Ferro/etiologia , Rim/patologia , Adolescente , Adulto , Anemia Falciforme/terapia , Feminino , Humanos , Ferro/metabolismo , Sobrecarga de Ferro/diagnóstico , Rim/metabolismo , Rim/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Reação Transfusional , Adulto JovemAssuntos
Anemia Falciforme , Antidrepanocíticos/administração & dosagem , Hidroxiureia/administração & dosagem , Ácidos Nucleicos/sangue , Talassemia alfa , Anemia Falciforme/sangue , Anemia Falciforme/complicações , Anemia Falciforme/tratamento farmacológico , Feminino , Humanos , Lactente , Masculino , Talassemia alfa/sangue , Talassemia alfa/complicações , Talassemia alfa/tratamento farmacológicoRESUMO
Transfusion of red blood cells is a major therapeutic option in sickle cell disease (SCD). There is strong evidence for its efficacy, particularly in primary and secondary stroke prevention in children, however, its use in other areas remains controversial. This study assessed the patterns of transfusion in the adult cohort attending King's College Hospital over a 10-year period, from 2000 to 2009. Total blood usage has increased significantly (P = 0·006) during this time, with 78% of the blood received by only 6% of the patients. The increase is explained by increased automated red cell exchange and increased usage for planned and acute transfusions for sickle-related complications.
Assuntos
Anemia Falciforme/terapia , Transfusão de Sangue/estatística & dados numéricos , Padrões de Prática Médica/estatística & dados numéricos , Doença Aguda , Adolescente , Adulto , Idoso , Anemia Falciforme/complicações , Transfusão de Sangue/métodos , Transfusão de Sangue/tendências , Feminino , Humanos , Londres , Masculino , Pessoa de Meia-Idade , Padrões de Prática Médica/tendências , Estudos Retrospectivos , Adulto JovemRESUMO
Fetal hemoglobin (HbF, α(2)γ(2)) is a major contributor to the remarkable phenotypic heterogeneity of sickle cell anemia (SCA). Genetic variation at 3 principal loci (HBB cluster on chromosome 11p, HBS1L-MYB region on chromosome 6q, and BCL11A on chromosome 2p) have been shown to influence HbF levels and disease severity in ß-thalassemia and SCA. Previous studies in SCA, however, have been restricted to populations from the African diaspora, which include multiple genealogies. We have investigated the influence of these 3 loci on HbF levels in sickle cell patients from Tanzania and in a small group of African British sickle patients. All 3 loci have a significant impact on the trait in both patient groups. The results suggest the presence of HBS1L-MYB variants affecting HbF in patients who are not tracked well by European-derived markers, such as rs9399137. Additional loci may be identified through independent genome-wide association studies in African populations.
Assuntos
Anemia Falciforme/etnologia , Anemia Falciforme/genética , Hemoglobina Fetal/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , População Negra/genética , Criança , Pré-Escolar , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Tanzânia , Reino Unido , População Branca/genética , Adulto JovemRESUMO
BACKGROUND: Sickle cell anemia is caused by a single type of mutation, a homozygous AâT substitution in the ß globin gene. Clinical severity is diverse, partially due to additional, disease-modifying genetic factors. We are studying one such modifier locus, HMIP (HBS1L-MYB intergenic polymorphism, chromosome 6q23.3). Working with a genetically admixed patient population, we have encountered the necessity to generate haplotype signatures of genetic markers to label genomic fragments with distinct genealogical origin at this locus. With the goal to generate haplotype signatures from patients experimentally, we have investigated the suitability of an existing nanofluidic assay platform to perform phase alignment with single-nucleotide polymorphism alleles. METHODOLOGY/PRINCIPAL FINDINGS: Patient DNA samples were loaded onto Fluidigm Digital Arrays and individual DNA molecules were assayed with allele-specific probes for SNP markers. Here we present data showing the utility of the nanofluidic approach, yielding haplotype data identical to those obtained with a family-based method. We then determined haplotype composition in a group of patients with sickle cell disease, including in those where a mathematical inference approach gave ambiguous or misleading results. Experimental phasing of genotypes across 3.8 kb for rs9399137, rs9402685, and rs11759553 created unequivocal haplotype signatures for each of the patients. In 68 patients, we found 8 copies of a haplotype signature ('C-C-T'), which is known to be prevalent in Europeans but to be absent in West African populations. We have confirmed the identity of our phased allele pairs by single-molecule sequencing and have demonstrated, in principle, that three-allele phasing (using three colors) is a potential extension to this method. CONCLUSIONS/SIGNIFICANCE: Phased haplotypes yield more information than the individual marker genotypes. Procedures such as the one described here would therefore benefit genetic mapping and functional studies as well as diagnostic procedures where the identity or parental origin of short genetic fragments is of importance.
Assuntos
Anemia Falciforme/genética , Técnicas Genéticas , Polimorfismo de Nucleotídeo Único , Cromossomos Humanos Par 6/genética , Feminino , Técnicas Genéticas/instrumentação , Haplótipos , Humanos , Masculino , LinhagemRESUMO
Hepcidin is known to be a key systemic iron-regulatory hormone which has been demonstrated to be associated with a number of iron disorders. Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation-exchange magnetic nanoparticles. The basic peptides were eluted from the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI-TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70-80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within -day repeatability (CV <13%) and between-day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P < 0.000005) and elevated levels in inflammation (P < 0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adulto , Peptídeos Catiônicos Antimicrobianos/urina , Feminino , Hepcidinas , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Serum and urine levels are increased in inflammation and suppressed in hemochromatosis, and they may have diagnostic importance. This study describes the development and validation of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. A stable, isotopically labeled internal standard, [15N,13C2]Gly12,20-hepcidin, was synthesized and a standard quantity was added to urine samples. Extraction was performed using weak cation exchange magnetic nanoparticles. An ion trap mass spectrometer was used to quantify hepcidin in the samples. The hepcidin assay was validated, and good recovery of hepcidin was obtained. The assay is accurate and precise. Urinary hepcidin levels of 3 to 9 nmol/mmol creatinine(-1) were found in healthy controls, with reduced levels in hemochromatosis (P<0.00006) and elevated levels in inflammation (P<0.00035). In sickle cell disease, a wide range was found, with the mean value not differing significantly from controls (P<0.26). In summary, a validated method has been developed for the quantitation of hepcidin using a stable, isotopically labeled internal standard and applied to determine the concentrations of hepcidin in the low nanomolar range in urine samples from patients and controls.
Assuntos
Antibacterianos/urina , Peptídeos Catiônicos Antimicrobianos/urina , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Adulto , Calibragem , Feminino , Hepcidinas , Humanos , Marcação por Isótopo , Masculino , Pessoa de Meia-IdadeAssuntos
Anemia Falciforme/complicações , Proteína Morfogenética Óssea 6/genética , Osteonecrose/etiologia , Adolescente , Adulto , Anexina A2/genética , Feminino , Necrose da Cabeça do Fêmur , Predisposição Genética para Doença , Glucuronidase/genética , Humanos , Proteínas Klotho , Masculino , Pessoa de Meia-Idade , Osteonecrose/genética , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
Hydroxycarbamide (HC), although a key drug therapy in sickle cell disease (SCD), does not result in a clinical response in all patients. Increases in fetal haemoglobin (HbF) and mean corpuscular volume of erythrocytes are standard clinical measures of HC efficacy in SCD. Genetic studies have determined that the majority of HbF regulation occurs outside the beta-globin locus. Approximately 30% of SCD patients have co-inherited alpha-thalassaemia resulting in hypochromic and microcytic erythrocytes. We provide data from 30 SCD patients (10 with alpha-thalassaemia) demonstrating that co-existing alpha-thalassaemia significantly affects several standard measures of HC efficacy in SCD.
Assuntos
Anemia Falciforme/complicações , Antidrepanocíticos/uso terapêutico , Hidroxiureia/uso terapêutico , Talassemia/tratamento farmacológico , Adolescente , Adulto , Anemia Falciforme/sangue , Criança , Monitoramento de Medicamentos/métodos , Contagem de Eritrócitos , Índices de Eritrócitos , Feminino , Hemoglobina Fetal/metabolismo , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Talassemia/sangue , Talassemia/complicações , Talassemia/genética , Resultado do Tratamento , Adulto Jovem , alfa-Globinas/genéticaRESUMO
Hydroxyurea reduces the frequency of acute pain in sickle cell disease (SCD). We sought to determine if hydroxyurea therapy affects cell free DNA (cfDNA) levels in SCD. cfDNA levels fell in all 10 patients studied; before hydroxyurea, mean was 1,879 (95% CI 1,104-3,199) GE/mL; after hydroxyurea, mean was 780 (95% CI, 634-959) GE/mL (P = 0.002). Mean cfDNA level in the 10 HbSS adults prior to starting hydroxyurea was also significantly higher than that in 115 HbSS case controls who had never taken hydroxyurea (1,879 vs 975 GE/mL, P = 0.02). cfDNA levels may be useful in monitoring response to hydroxyurea therapy in SCD.
Assuntos
Anemia Falciforme/tratamento farmacológico , DNA/sangue , Hidroxiureia/uso terapêutico , Adolescente , Adulto , Idoso , Anemia Falciforme/sangue , Biomarcadores , Morte Celular/efeitos dos fármacos , Feminino , Humanos , Hidroxiureia/farmacologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Viés de SeleçãoRESUMO
Free circulating DNA is present in the plasma of healthy subjects, and is elevated in conditions characterized by increased cell death, such as cancer and physical trauma. Analysis of circulating DNA in plasma could provide a useful biomarker in sickle cell disease (SCD) in view of the increased cell turnover through chronic ongoing haemolysis, recurrent vaso-occlusion and inflammation. Plasma DNA was determined by real-time quantitative polymerase chain reaction (PCR) amplification of the beta-globin gene (HBB) in 154 patients with SCD [105 haemoglobin (Hb)SS, 46 HbSC and three HbS/beta(0) thalassaemia] and 53 ethnically matched controls. Blood samples were obtained from all patients in steady state; 21 of the 154 patients were also sampled during admission to hospital for acute pain. Median concentration of circulating plasma DNA in acute pain was more than 10-fold that in steady state and in controls - 10070 vs. 841 and 10070 vs. 933 genome equivalents/ml respectively (P < 0.0001, in both cases). During steady state, patients had plasma DNA levels similar to controls. Plasma DNA levels in SCD correlated with C-reactive protein levels (P < 0.005) and total white cell counts (P < 0.05) in steady state. The study shows that plasma DNA concentration may have potential as a biomarker in sickle cell patients.
Assuntos
Anemia Falciforme/sangue , DNA/sangue , Doença Aguda , Adolescente , Adulto , Anemia Falciforme/imunologia , Biomarcadores/sangue , Proteína C-Reativa/análise , Estudos de Casos e Controles , Feminino , Humanos , Contagem de Leucócitos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Serum bilirubin levels and predisposition to gallstones in sickle cell disease (SCD) are influenced by genetic variation in the hepatic uridine diphosphate (UDP)-glucuronosyltransferase (UGT1A1) gene, but the association is not consistent. This study investigated whether variation in the gene encoding haem oxygenase (HMOX1), a rate-limiting enzyme upstream of UGT1A in the haem catabolic pathway, and alpha-thalassaemia could explain some of the inconsistent effects. The UGT1A1 [TA](n) and HMOX1 [GT](n) promoter polymorphisms and alpha globin genotypes were determined in 263 SCD patients (199 HbSS, 5 HbS/beta(0), 59 HbSC). Detection of gallstones was based on ultrasound of the liver/biliary tree. Regression analysis showed that serum bilirubin levels and the incidence of gallstones were strongly associated with the number of UGT1A1 [TA] repeats in all subjects (P < 0.0001 and P < 0.01, respectively). While HMOX1 genotype had no effect, co-inheritance of alpha-thalassaemia reduced serum bilirubin levels in all SCD patients independently of the number of UGT1A1 [TA] repeats. Each additional [TA] repeat is associated with an increase in mean serum bilirubin levels of 21% and cholelithiasis risk of 87% in SCD.
Assuntos
Anemia Falciforme/genética , Cálculos Biliares/genética , Glucuronosiltransferase/genética , Heme Oxigenase-1/genética , Talassemia alfa/genética , Adolescente , Adulto , Idoso , Anemia Falciforme/complicações , Bilirrubina/sangue , Criança , Feminino , Cálculos Biliares/complicações , Cálculos Biliares/diagnóstico por imagem , Genótipo , Globinas/genética , Doença da Hemoglobina SC/complicações , Doença da Hemoglobina SC/genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Regiões Promotoras Genéticas , Medição de Risco/métodos , Ultrassonografia , Talassemia alfa/complicaçõesRESUMO
BACKGROUND: Glucocorticoids are the mainstay of asthma therapy; however, a proportion of patients with asthma has a severe form of the disease that fails to respond to therapy. Understanding the molecular mechanisms behind glucocorticoid-insensitive asthma is therefore of clinical importance. Evidence in glucocorticoid-unresponsive Henrietta Lack (HeLa) cells indicated that cofilin-1 could act as an inhibitor of glucocorticoid function. OBJECTIVE: To determine whether cofilin-1 expression is abnormally expressed in cells from patients with severe glucocorticoid-insensitive asthma and examine the effect of cofilin-1 overexpression on glucocorticoid function. METHODS: Peripheral blood CD4(+) T cells were purified from 16 subjects with severe glucocorticoid-insensitive asthma and 16 subjects with mild glucocorticoid-sensitive asthma, and cofilin-1 expression was determined by quantitative real-time RT-PCR and Western blotting. The effect of dexamethasone on cofilin-1 expression was determined in Jurkat T cells, and the effect of cofilin-1 overexpression on anti-CD3/CD28-stimulated IL-2 release was measured. RESULTS: Peripheral blood CD4(+) T cells from subjects with severe glucocorticoid-insensitive asthma are less responsive to dexamethasone than cells from subjects with mild glucocorticoid-sensitive asthma. Cells from these patients express significantly (P < .05) higher levels of cofilin-1 than cells from subjects with mild asthma. Dexamethasone did not affect cofilin-1 expression in Jurkat T cells. Functionally, dexamethasone suppression of anti-CD3/CD28-stimulated IL-2 was attenuated in Jurkat cells overexpressing cofilin-1. CONCLUSION: These results suggest that increased cofilin-1 expression may be important in the regulation of glucocorticoid sensitivity in peripheral blood lymphocytes of patients with severe treatment-insensitive asthma. CLINICAL IMPLICATIONS: Understanding the mechanisms of enhanced cofilin-1 expression may lead to the development of new therapies for severe treatment-insensitive asthma.
Assuntos
Asma/tratamento farmacológico , Asma/metabolismo , Cofilina 1/biossíntese , Cofilina 1/genética , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Índice de Gravidade de Doença , Adulto , Asma/fisiopatologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Dexametasona/antagonistas & inibidores , Feminino , Volume Expiratório Forçado/efeitos dos fármacos , Humanos , Imunossupressores/antagonistas & inibidores , Imunossupressores/farmacologia , Células Jurkat , Masculino , Pessoa de Meia-IdadeRESUMO
Despite the theoretical evidence of the utility of single-nucleotide polymorphisms (SNPs) for linkage analysis, no whole-genome scans of a complex disease have yet been published to directly compare SNPs with microsatellites. Here, we describe a whole-genome screen of 157 families with multiple cases of rheumatoid arthritis (RA), performed using 11,245 genomewide SNPs. The results were compared with those from a 10-cM microsatellite scan in the same cohort. The SNP analysis detected HLA*DRB1, the major RA susceptibility locus (P=.00004), with a linkage interval of 31 cM, compared with a 50-cM linkage interval detected by the microsatellite scan. In addition, four loci were detected at a nominal significance level (P<.05) in the SNP linkage analysis; these were not observed in the microsatellite scan. We demonstrate that variation in information content was the main factor contributing to observed differences in the two scans, with the SNPs providing significantly higher information content than the microsatellites. Reducing the number of SNPs in the marker set to 3,300 (1-cM spacing) caused several loci to drop below nominal significance levels, suggesting that decreases in information content can have significant effects on linkage results. In contrast, differences in maps employed in the analysis, the low detectable rate of genotyping error, and the presence of moderate linkage disequilibrium between markers did not significantly affect the results. We have demonstrated the utility of a dense SNP map for performing linkage analysis in a late-age-at-onset disease, where DNA from parents is not always available. The high SNP density allows loci to be defined more precisely and provides a partial scaffold for association studies, substantially reducing the resource requirement for gene-mapping studies.