RESUMEN
BACKGROUND AND OBJECTIVES: Wilson's disease (WD) in children and adolescents is predominantly asymptomatic or oligo-symptomatic. The symptoms are nonspecific and difficult to distinguish from other hepatic or neuropsychiatric disorders. In this study, we present the experience of a pediatric referral center for WD diagnosis and treatment. PATIENTS AND METHODS: We retrospectively analyzed clinical and laboratory data from 99 patients with WD of Sardinian origin, including physical examination, laboratory biochemical testing, liver biopsy, and genetic analysis. RESULTS: Patients were prevalently oligo-symptomatic or asymptomatic. The median age of diagnosis was 8.78 years. Ceruloplasmin values were lower than normal values in all analyzed patients. Twenty-four-hour urinary copper levels were higher than 40 µg/24-h in 92/96 patients. In all analyzed patients with the exception of one, liver copper was higher than 250 µg/g of dry weight but all had >75 µg/g of dry weight. Statistical analysis showed correlation between the age at diagnosis, serum copper, and 24-h urinary copper. Correlation was also found between serum copper and 24-h urinary copper. Molecular analysis of ATP7B gene allowed complete characterization in all the analyzed patients. CONCLUSION: A high index of clinical suspicion and biochemical tests including liver tests, serum ceruloplasmin, and basal 24-h urinary copper excretion and genotype determination are key to WD diagnosis. The long experience that a referral center for WD possesses is an important factor in making WD diagnosis a more accurate process. Studies in animal models on WD could be used as a guide to further investigate the molecular mechanisms that regulate copper metabolism and influence the natural history of WD.
RESUMEN
α-thalassemia carriers are common in Mediterranean regions, particularly in the Sardinian population. Their haematological phenotype is characterized by reduced MCV and/or MCH with normal or slightly reduced HbA2 levels and normal HbF. Krüppel-like factor 1 (KLF1) is a pleiotropic erythroid transcription factor that is essential for haematopoiesis. Mutations in the KLF1 gene trigger a series of benign human red blood phenotypes, such as an increase in HbA2 and HBF. Recently, it has been found that KLF1 mutations were a frequent cause of borderline HbA2 levels in a group of Sardinian subjects. Here, we found that KLF1 mutations modulate the phenotype in a cohort of α-thalassemia carriers.
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Hemoglobina Fetal , Hemoglobina A2 , Heterocigoto , Factores de Transcripción de Tipo Kruppel/genética , Mutación , Talasemia alfa , Adolescente , Adulto , Femenino , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Hemoglobina A2/genética , Hemoglobina A2/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Talasemia alfa/sangre , Talasemia alfa/genéticaRESUMEN
BACKGROUND: Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on ß-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning. METHODS: Erythroid precursor cells were obtained from 72 patients, mostly ß-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC. RESULTS: In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from ß-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers. CONCLUSION: The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results.
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Bancos de Muestras Biológicas , Talasemia beta/patología , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Criopreservación , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Eritropoyetina/farmacología , Hemoglobina Fetal/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Cinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: The increase in HbA2 is the most important parameter for the identification of thalassemia carriers. However, in routine screening for hemoglobinopathies, some cases are difficult to classify because the level of HbA2 is not typically elevated. In this work, we report the results of a molecular investigation on a cohort of subjects with borderline HbA2. METHODS: All subjects with a ß-thalassemia carrier partner and a borderline percentage level of HbA2 were investigated for the presence of a pathological mutation in the ß-globin gene. All negative subjects were screened for both the KLF1 mutation and the presence of ααα/ or αααα/ alleles. The subjects with reduced MCV and/or MCH were also screened for deletional and nondeletional α-globin gene defects. RESULTS: Various ß-globin mutations and KLF1 gene defects are the most common genetic determinants responsible for this phenotype in our population. CONCLUSION: KLF1 mutations are important in a screening program for hemoglobinopathies. An increase in HbF in association with borderline HbA2 levels is a useful but not exclusive marker that suggests the investigation of this gene. On the basis of our findings, we are able to suggest the molecular procedure to use in a population characterized by a high prevalence of thalassemia carriers.
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Hemoglobina A2/genética , Talasemia beta/diagnóstico , Humanos , Mutación , Globinas alfa/genética , Talasemia alfa/genética , Globinas beta/genéticaRESUMEN
Clinical and hematologic characteristics of beta(ß)-thalassemia are determined by several factors resulting in a wide spectrum of severity. Phenotype modulators are: HBB mutations, HBA defects and fetal hemoglobin production modulators (HBG2:g.-158C>T polymorphism, HBS1L-MYB intergenic region and the BCL11A). We characterized 54 genetic variants at these five loci robustly associated with the amelioration of beta-thalassemia phenotype, to build a predictive score of severity using a representative cohort of 890 ß-thalassemic patients. Using Cox proportional hazard analysis on a training set, we assessed the effect of these loci on the age at which patient started regular transfusions, built a Thalassemia Severity Score, and validated it on a testing set. Discriminatory power of the model was high (C-index=0.705; R(2)=0.343) and the validation conducted on the testing set confirmed its predictive accuracy with transfusion-free survival probability (P<0.001) and with transfusion dependency status (Area Under the Receiver Operating Characteristic Curve=0.774; P<0.001). Finally, an automatized on-line calculation of the score was made available at http://tss.unica.it. Besides the accurate assessment of genetic predictors effect, the present results could be helpful in the management of patients, both as a predictive score for screening and a standardized scale of severity to overcome the major-intermedia dichotomy and support clinical decisions.
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Variación Genética , Globinas beta/genética , Talasemia beta/diagnóstico , Talasemia beta/genética , Transfusión Sanguínea , ADN Intergénico , Femenino , Estudios de Asociación Genética , Sitios Genéticos , Humanos , Estimación de Kaplan-Meier , Masculino , Mutación , Fenotipo , Polimorfismo de Nucleótido Simple , Pronóstico , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Talasemia beta/mortalidad , Talasemia beta/terapiaRESUMEN
UNLABELLED: BACKGROUND AND RATIONALE FOR THE STUDY: Genome-wide association studies have identified host genetic variation to be critical for spontaneous clearance and treatment response in patients infected with hepatitis C virus. Recently, the role of the IFNL3 polymorphisms in influencing the spontaneous clearance of HCV, the response to interferon and the progression of liver fibrosis, was also demonstrated in patients with thalassemia major infected by genotype 1b. In the present study we retrospectively analyzed 368 anti-HCV positive patients with beta-thalassemia at two Italian major centers in Cagliari and Torino. RESULTS: C/C variant of polymorphism rs12979860 was related to response to interferon treatment and, above all, to spontaneous clearance of the virus. However, the positive predictive power was stronger for viral persistence than spontaneous clearance and in such respect the TT allele was more predictive than CC. The methylation associated polymorphism rs4803221 had independent effects with respect to rs12979860 and the haplotype tagged by SNP rs12979860 and rs4803221 significantly could improve the viral clearance prediction in infected patients. Neither necroinflammation or bilirubin values in the chronic phase of the hepatitis C were related to IFNL3 polymorphisms. No relation among IFNL3 polymorphisms and fibrosis stage directly shown by the liver biopsy was found. CONCLUSIONS: Also in thalassemia the SNPs on chromosome 19q13 closely associates with spontaneous and treatment-induced HCV clearance. The viral clearance prediction is significantly improved by the haplotype tagged by SNP rs12979860 and rs4803221. Neither necroinflammation, bilirubin values or fibrosis stage seem to be related to IFNL3 polymorphisms.
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ADN/genética , Hepatitis C Crónica/genética , Interleucinas/genética , Polimorfismo Genético , Talasemia beta/genética , Femenino , Genotipo , Hepacivirus/genética , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/metabolismo , Humanos , Interferones , Interleucinas/metabolismo , Masculino , ARN Viral/análisis , Estudios Retrospectivos , Adulto Joven , Talasemia beta/complicaciones , Talasemia beta/metabolismoRESUMEN
Increased hemoglobin A(2) (HbA(2); ie, levels > 3.9%) is the most important feature of ß-thalassemia carriers. However, it is not uncommon to find persons with borderline HbA(2) (levels, 3.3%-3.8%), who pose a relevant screening problem. Several genotypes have been associated with borderline HbA(2), but sometimes the reasons for this unusual phenotype are unknown. In this paper, we report, for the first time, that mutations of KLF1 result in HbA(2) levels in the borderline range. Six different KLF1 mutations were identified in 52 of 145 subjects with borderline HbA(2) and normal mean corpuscular volume and mean corpuscular hemoglobin. Two mutations (T327S and T280_H283del) are here reported for the first time. The prevalent mutation in Sardinians is S270X, which accounts for 80.8% of the total. The frequent discovery of KLF1 mutations in these atypical carriers may contribute significantly to the thalassemia screening programs aimed at identification of at risk couples.
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Hemoglobina A2/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Mutación , Talasemia beta/diagnóstico , Talasemia beta/genética , Índices de Eritrocitos , Femenino , Humanos , Masculino , Linaje , Talasemia beta/metabolismoRESUMEN
Variations at the KLF1 gene have been associated with a series of human erythroid phenotypes including the In-(Lu) phenotype, hereditary persistence of fetal hemoglobin, congenital dyserythropoietic anemia, borderline HbA(2) and increased red blood cell protoporphyrin. Natural mutations have shown that KLF1 regulates gamma globin gene expression and its role in the switching from fetal to adult globin expression has been suggested by experimental studies. In this paper we report that subjects with S270X KLF1 mutations show a decrease of HbF levels with increasing age, supporting in vivo the role of KLF1 in hemoglobin switching in humans.
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Envejecimiento/genética , Hemoglobina Fetal/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Hemoglobina A/genética , Factores de Transcripción de Tipo Kruppel/genética , Polimorfismo de Nucleótido Simple , Adolescente , Niño , Preescolar , Hemoglobina Fetal/metabolismo , Técnicas de Genotipaje , Hemoglobina A/metabolismo , Humanos , Lactante , Italia , Estudios Longitudinales , Mutación , Adulto Joven , gamma-Globinas/genéticaRESUMEN
BACKGROUND: The clinical and hematologic features of ß-thalassemia are modulated by different factors, resulting in a wide range of clinical severity. The main factors are the type of disease-causing mutation and the ability to produce α-globin and γ-globin chains. In the present study we investigated the respective contributions of known modifiers to the prediction of the clinical severity of ß-thalassemia as assessed by the patients' age at first transfusion. DESIGN AND METHODS: We studied the effect of seven loci in a cohort of 316 Sardinian patients with ß(0)-thalassemia. In addition to characterizing the ß-globin gene mutations, α-globin gene defects and HBG2:g.-158C>T polymorphism, we genotyped two different markers in the BCL11A gene and three in the HBS1L-MYB intergenic region using single nucleotide polymorphism microarrays, imputation and direct genotyping. We performed Cox proportional hazard analysis of the time to first transfusion. RESULTS: According to the resulting model, we were able to explain phenotypic severity to a large extent (Harrell's concordance index=0.72; Cox & Snell R(2)=0.394) and demonstrated that most of the model's discriminatory ability is attributable to the genetic variants affecting fetal hemoglobin production (HBG2:g.-158C>T, BCL11A and HBS1L-MYB loci: C-index=0.68, R(2)=0.272), while the remaining is due to α-globin gene defects and gender. Consequently, significantly distinct survival curves can be described in our population. CONCLUSIONS: This detailed analysis clarifies the impact of genetic modifiers on the clinical severity of the disease, measured by time to first transfusion, by determining their relative contributions in a homogeneous cohort of ß(0)-thalassemia patients. It may also support clinical decisions regarding the beginning of transfusion therapy in patients with ß-thalassemia.
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Proteínas Portadoras/genética , ADN Intergénico/genética , Hemoglobina Fetal/genética , Proteínas Nucleares/genética , Talasemia beta/genética , Adolescente , Adulto , Factores de Edad , Transfusión Sanguínea , Estudios de Cohortes , Dermatoglifia del ADN , Femenino , Sitios Genéticos , Marcadores Genéticos , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Modelos de Riesgos Proporcionales , Proteínas Represoras , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , Globinas alfa/genética , Globinas beta/genética , Talasemia beta/mortalidad , Talasemia beta/patologíaRESUMEN
Sardinian beta-thalassemia patients all are homozygotes for the same null allele in the beta-globin gene, but the clinical manifestations are extremely variable in severity. Previous studies have shown that the coinheritance of alpha-thalassemia or the presence of genetic variants that sustain fetal hemoglobin production has a strong impact on ameliorating the clinical phenotype. Here we evaluate the contribution of variants in the BCL11A, and HBS1L-MYB genes, implicated in the regulation of fetal hemoglobin, and of alpha-thalassemia coinheritance in 50 thalassemia intermedia and 75 thalassemia major patients. We confirm that alpha-thalassemia and allele C of single nucleotide polymorphism rs-11886868 in BCL11A were selectively represented in thalassemia intermedia patients. Moreover, allele G at single nucleotide polymorphism rs9389268 in the HBS1L-MYB locus was significantly more frequent in the thalassemia intermedia patients. This trio of genetic factors can account for 75% of the variation differences in phenotype severity.
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Alelos , Proteínas Portadoras/genética , Homocigoto , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-myb/genética , Talasemia alfa/genética , Talasemia beta/genética , Adolescente , Adulto , Proteínas Portadoras/metabolismo , Femenino , Hemoglobina Fetal/biosíntesis , Hemoglobina Fetal/genética , Humanos , Italia , Masculino , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Sitios de Carácter Cuantitativo/genética , Proteínas Represoras , Talasemia alfa/metabolismo , Talasemia beta/metabolismoRESUMEN
The persistence of high fetal hemoglobin level in adults may ameliorate the clinical phenotype of beta-thalassemia and sickle cell anemia. Several genetic variants responsible for hereditary persistence of fetal hemoglobin, linked and not linked to the beta globin gene cluster, have been identified in patients and in normal individuals. Monoallelic loss of KLF1, a gene with a key role in erythropoiesis, has been recently reported to be responsible for persistence of high levels of fetal hemoglobin. In a Sardinian family, high levels of HbF (22.1-30.9%) were present only in compound heterozygotes for the S270X nonsense and K332Q missense mutations, while the isolated S270X nonsense (haploinsufficiency) or K332Q missense mutation were associated with normal HbF levels (<1.5%). Functionally, the K332Q Klf1 mutation impairs binding to the BCl11A gene and activation of the γ- and ß-globin promoters. Moreover, we report for the first time the association of KLF1 mutations with very high levels of zinc protoporphyrin.
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Eritrocitos/metabolismo , Hemoglobina Fetal/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Mutación , Protoporfirinas/metabolismo , Adulto , Secuencia de Bases , Análisis Mutacional de ADN , Salud de la Familia , Femenino , Fluorometría , Células HEK293 , Heterocigoto , Humanos , Italia , Masculino , Persona de Mediana Edad , Linaje , Reacción en Cadena de la PolimerasaRESUMEN
beta-Thalassemia and sickle cell disease both display a great deal of phenotypic heterogeneity, despite being generally thought of as simple Mendelian diseases. The reasons for this are not well understood, although the level of fetal hemoglobin (HbF) is one well characterized ameliorating factor in both of these conditions. To better understand the genetic basis of this heterogeneity, we carried out genome-wide scans with 362,129 common SNPs on 4,305 Sardinians to look for genetic linkage and association with HbF levels, as well as other red blood cell-related traits. Among major variants affecting HbF levels, SNP rs11886868 in the BCL11A gene was strongly associated with this trait (P < 10(-35)). The C allele frequency was significantly higher in Sardinian individuals with elevated HbF levels, detected by screening for beta-thalassemia, and patients with attenuated forms of beta-thalassemia vs. those with thalassemia major. We also show that the same BCL11A variant is strongly associated with HbF levels in a large cohort of sickle cell patients. These results indicate that BCL11A variants, by modulating HbF levels, act as an important ameliorating factor of the beta-thalassemia phenotype, and it is likely they could help ameliorate other hemoglobin disorders. We expect our findings will help to characterize the molecular mechanisms of fetal globin regulation and could eventually contribute to the development of new therapeutic approaches for beta-thalassemia and sickle cell anemia.
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Proteínas Portadoras/genética , Hemoglobina Fetal/análisis , Hemoglobina Fetal/metabolismo , Ligamiento Genético , Proteínas Nucleares/genética , Talasemia beta/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Frecuencia de los Genes , Genoma Humano , Humanos , Italia , Masculino , Persona de Mediana Edad , Fenotipo , Polimorfismo de Nucleótido Simple , Proteínas RepresorasRESUMEN
We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.
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Bases de Datos Genéticas , Variación Genética , Hemoglobinopatías/genética , Secuencia de Bases , ADN/genética , Minería de Datos , Genoma Humano , Hemoglobinas/genética , Proyecto Genoma Humano , Humanos , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , EdiciónRESUMEN
We report the sixth occurrence of Hb Belfast [beta15(A12)Trp-->Arg], a mild, unstable beta chain variant, in a large family wherein nine subjects were affected. DNA analysis showed a TUG-->AGG mutation at codon 15 of the beta-globin gene, confirming a Trp-->Arg amino acid substitution. The oxygen affinity of the isolated variant was increased. The clinical phenotype is silent or very mild, the only clinical finding being an intermittent moderate jaundice.
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Sustitución de Aminoácidos/genética , Hemoglobinas Anormales/química , Hemoglobinas Anormales/genética , Oxígeno/química , Fenotipo , Mutación Puntual/genética , Adulto , Codón/genética , Familia , Femenino , Hemoglobinas Anormales/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Oxígeno/metabolismo , Linaje , Unión Proteica/genéticaRESUMEN
The clinical phenotype of homozygous beta thalassemia varies in severity from the mild thalassemia intermedia to the severe thalassemia major. This variability depends largely on the molecular heterogeneity of beta thalassemia defects. We report the first case of a homozygous state for nondeletion Sardinian delta-beta(0) thalassemia, which resulted in a symptomless clinical phenotype with a peculiar hemoglobin (Hb) pattern (99.8% Hb F and 0.2% Hb A(2)). The molecular defect was characterized by the presence of 2 nucleotide substitutions: -196C>T in the promoter of the Agamma-globin gene and beta 39C>T nonsense mutation. The absence of typical beta thalassemia clinical findings was due to the high Hb F output, which compensated for the absence of beta chains. The near absence of Hb A(2) may have resulted from either alterations in the globin gene transcriptional complex with preferential activation of gamma-globin genes and suppression of delta-globin genes or preferential survival of red blood cells with the highest Hb F content and low Hb A(2) level.