Progress in Gene Therapy of Sickle Cell Disease Based on Hemoglobin F--Review / 中国实验血液学杂志

Sickle cell disease (SCD) is a single gene genetic disease, which seriously threatens the life span and quality of patients. On the basis of the pathogenesis of SCD and the alternative therapy based on fetal hemoglobin F (HbF), the research progress of transcription factors involved in the regulation of HbF gene expression, such as BCL11A, ZBTB7A, KLF-1, c-MYB and SOX6, as well as the application of CRISPR / Cas9, TALEN, zinc finger nuclease and other gene editing technologies in this field has been made, providing a solid theoretical basis for the exploration of new treatment schemes for β- like hemoglobin diseases, such as sickle cell disease and β- thalassemia.

Genetic variants associated with fetal hemoglobin levels show the diverse ethnic origin in Colombian patients with sickle cell anemia / Las variantes genéticas asociadas con niveles de hemoglobina fetal señalan diversos orígenes étnicos en pacientes colombianos con anemia falciforme.

Introduction: Fetal hemoglobin is an important factor in modulating the severity of sickle cell anemia. Its level in peripheral blood underlies strong genetic determination. Associated loci with increased levels of fetal hemoglobin display population-specific allele frequencies. Objective: We investigated the presence and effect of known common genetic variants promoting fetal hemoglobin persistence (rs11886868, rs9399137, rs4895441, and rs7482144) in 60 Colombian patients with sickle cell anemia. Materials and methods: Four single nucleotide polymorphisms (SNP) were genotyped by restriction fragment length polymorphisms (RFLP) and the use of the TaqMan procedure. Fetal hemoglobin (HbF) from these patients was quantified using the oxyhemoglobin alkaline denaturation technique. Genotype frequencies were compared with frequencies reported in global reference populations. Results: We detected genetic variants in the four SNPs, reported to be associated with higher HbF levels for all four SNPs in the Colombian patients. Genetic association between SNPs and HbF levels did not reach statistical significance. The frequency of these variants reflected the specific ethnic make-up of our patient population: A high prevalence of rs7482144-'A' reflects the West-African origin of the sickle cell mutation, while high frequencies of rs4895441-'G' and rs11886868-'C' point to a significant influence of an Amerindian ethnic background in the Colombian sickle cell disease population. Conclusion: These results showed that in the sickle cell disease population in Colombia there is not a unique genetic background, but two (African and Amerindian). This unique genetic situation will provide opportunities for a further study of these loci, such as fine-mapping and molecular-biological investigation. Colombian patients are expected to yield a distinctive insight into the effect of modifier loci in sickle cell disease.

Introducción. La hemoglobina fetal es un importante factor modulador de la gravedad de la anemia falciforme, cuya expresión está muy condicionada por el factor genético. Los loci asociados con el incremento de la hemoglobina fetal pueden presentar frecuencias alélicas específicas para cada población. Objetivo. Investigar la presencia y el efecto de las variantes genéticas rs11886868, rs9399137, rs4895441 y rs7482144 asociadas con la persistencia de hemoglobina fetal, en 60 pacientes colombianos con anemia falciforme. Materiales y métodos. Se hizo la genotipificación de los polimorfismos de nucleótido simple ( Single Nucleotide Polymorphisms, SNP) mediante la técnica de polimorfismos de longitud de fragmentos de restricción ( Restriction Fragment Length Polymorphisms, RFLP) y el procedimiento TaqMan. La hemoglobina fetal (HbF) se cuantificó utilizando la técnica de desnaturalización alcalina de la oxihemoglobina. Las frecuencias genotípicas se compararon con las reportadas en poblaciones de referencia global. Resultados. Se observaron variantes genéticas ya reportadas para aumento de HbF en los cuatro SNP. La asociación genética entre los SNP y el incremento de la HbF no alcanzó significancia estadística. La frecuencia de estos alelos reflejó la siguiente composición específica en esta muestra de pacientes colombianos: una gran prevalencia de rs7482144-'A', lo que indica que el origen de la mutación para la anemia falciforme es África occidental, y una gran frecuencia de rs4895441-'G' y rs11886868-'C', lo que denota la influencia significativa del origen genético amerindio. Conclusión. Los resultados evidenciaron que la población con anemia falciforme de Colombia no tiene un único origen genético, sino que existen dos (africano y amerindio). Esta situación genética única ofrece la oportunidad de llevar a cabo un estudio más amplio de estos loci a nivel molecular. Se espera que el estudio de pacientes colombianos permita una visión diferente del efecto de los loci modificadores en esta enfermedad.

Comparison of in-vitro and in-vivo response to fetal hemoglobin production and γ-mRNA expression by hydroxyurea in Hemoglobinopathies.

BACKGROUND: Hydroxyurea, which induces Fetal hemoglobin (HbF) synthesis, is the only drug widely used in different hemoglobinopathies; however, the response is very variable. We compared the efficacy of hydroxyurea in-vitro in erythroid cultures and in-vivo in the same patients with different hemoglobinopathies to induce HbF production and enhance γ-messenger RNA expression. MATERIALS AND METHODS: A total of 24-patients with different Hemoglobinopathies were given hydroxyurea and their response was studied in-vivo and in-vitro on mononuclear cells collected from them simultaneously. RESULTS: A total of 57.7% of patients (responders) showed no further crisis or transfusion requirements after hydroxyurea therapy with a mean increase in fetal cells (F-cells) of 63.8 ± 59.1% and γ-mRNA expression of 205.5 ± 120.8%. In-vitro results also showed a mean increase in F-cells of 27.2 ± 24.7% and γ-mRNA expression of 119.6% ± 65.4% among the treated cells. Nearly 19.0% of the partial-responders reduced their transfusion requirements by 50% with a mean increase in F-cells of 61.2 ± 25.0% and 28.4 ± 25.3% and γ-mRNA-expression of 21.0% ± 1.4% and 80.0% ± 14.1% in-vivo and in-vitro respectively. The non-responders (15.3%) showed no change in their clinical status and there was no significant increase in F-cells levels and γ-mRNA expression in-vivo or in-vitro. CONCLUSION: Thus, this method may help to predict the in-vivo response to hydroxyurea therapy; however, a much larger study is required.

Hb E/beta-thalassaemia: a common & clinically diverse disorder.

Haemoglobin E-beta thalassaemia (Hb E/β-thalassaemia) is the genotype responsible for approximately one-half of all severe beta-thalassaemia worldwide. The disorder is characterized by marked clinical variability, ranging from a mild and asymptomatic anaemia to a life-threatening disorder requiring transfusions from infancy. The phenotypic variability of Hb E/β-thalassaemia and the paucity of long-term clinical data, present challenges in providing definitive recommendations for the optimal management of patients. Genetic factors influencing the severity of this disorder include the type of beta-thalassaemia mutation, the co-inheritance of alpha-thalassaemia, and polymorphisms associated with increased production of foetal haemoglobin. Other factors, including a variable increase in serum erythropoietin in response to anaemia, previous or ongoing infection with malaria, previous splenectomy and other environmental influences, may be involved. The remarkable variation, and the instability, of the clinical phenotype of Hb E beta-thalassaemia suggests that careful tailoring of treatment is required for each patient, and that therapeutic approaches should be re-assessed over-time.

Frequency of haemoglobinopathies and glucose-6-phosphate dehydrogenase deficiency in Basra

Basra, southern Iraq, was mapped for haemoglobinopathies and glucose-6-phosphate dehydrogenase [G6PD] deficiency. Of 1064 couples aged 14-60 years recruited from the Public Health Laboratory, 49 had beta-thalassaemia trait, 69 had sickle-cell trait, 2 had haemoglobin D trait, 2 had haemoglobin C trait and 1 had high persistent fetal haemoglobin. Carriers of major beta-globin disorders comprised 11.48%. G6PD deficiency was detected in 133 individuals [12.5%]. Only 10 couples [0.94%] were at risk of having children affected with either sickle-cell disease or beta-thalassaemia major. These defects constitute a real health problem and necessitate a management plan and public health education for early diagnosis and therapy

The Agamma-195 (C->G) mutation in hereditary persistence of fetal hemoglobin is not associated with activation of a reporter gene in vitro

Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression

Molecular mechanisms of thalassemia in southeast Asia.

Hemoglobinopathies are the most common genetic disorders in Southeast Asia. alpha-Thalassemia is most often due to a alpha-globin gene deletion. Hb Constant Spring (CS) occurs from the mutation at the termination codon of the alpha-globin gene resulting in an elongated polypeptide; alpha(CS)-globin mRNA is also unstable and only small amounts of Hb CS are produced. Thus Hb CS has an alpha-thalassemia 2-like effect. beta-Thalassemia results from a variety of molecular mechanisms, most of which are single base substitutions or deletions or insertions of one to four nucleotides. Hemoglobin E occurs from a Glu --> Lys substitution at position 26 of the beta-globin chain. The abnormal gene also results in reduced amounts of beta E-mRNA and hence of beta E-globin chains. Therefore, Hb E has a mild beta + thalassemia phenotype. Homozygous beta-thalassemia and beta-thalassemia/Hb E are the major beta-thalassemic syndromes in Southeast Asia. In spite of seemingly identical genotypes, severity of beta-thalassemia/Hb E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta-thalassemia. A number of genetic factors have been shown to determine the differences in severity of anemia in beta-thalassemia/Hb E, including co-inheritance of alpha-thalassemia determinants and co-inheritance of other determinants which elevate Hb F expression. A correlation between the extent of beta E-globin mRNA cryptic splicing and the severity of anemia in beta(zero)-thalassemia/Hb E patients has been observed. Complete characterization of mutations causing hemoglobinopathies will help to bolster the establishment of prenatal diagnosis of these genetic disorders in the region.

Molecular analysis of the human fetal-to-adult globin switching.

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by continued expression of the gamma-globin gene in adult life. Analysis of a Japanese HPFH family had revealed that the C-T transition at position -114 within the distal CCAAT box of the gamma-globin gene associated with the HPFH allele. In the vicinity of the distal CCAAT box, other two mutations (-117 C-T, 13 bp del) had been identified in individuals with a HPFH phenotype. Functional analysis of these mutant promoters in erythroid cell lines suggested that the distal CCAAT box works positively in the fetus but negatively in the adult on the expression of the gamma-globin gene. Further study on transgenic mice showed that the -114 mutation was responsible for the elevated expression of the gamma-globin gene in the adult. To elucidate the molecular mechanism underlying the persistent expression of the gamma-globin genes associated with the HPFH mutations, interaction of the mutant promoters with nuclear factors was analyzed. Relevance of the nuclear factor, NFE3, to the gamma-globin regulation was suggested by the affected binding of NFE3 to the altered distal CCAAT boxes with HPFH mutations (-117, -114, 13 bp del).

A case of homozygous sickle cell disease in Sri Lanka.

An 11 year old Muslim boy with a 2 month history of fever, loss of appetite, pallor and abdominal distension, had hepato-splenomegaly. Haemoglobin electrophoresis showed the presence of haemoglobins S and F, with complete absence of haemoglobin A. The sickling test was positive. Marital consanguinity was present. In both parents, the sickling test was positive and haemoglobin electrophoresis showed the presence of haemoglobins A and S. This is the first report of homozygous sickle cell disease in Sri Lanka.

Hiperglicemia e hemoglobina fetal / Hyperglycemia and fetal hemoglobin

O presente trabalho se refere a importancia da hiperglicemia na síntese de hemoglobina F. Foram estudados 1027 indivíduos normais e 226 com hiperglicemia (diabéticos descompensados). A hemoglobina F foi dosada pela técnica da Desnaturaçäo alcalina de Betke e cols (1969). Os nossos resultados mostraram que: 1) nos indivíduos com hiperglicemia os níveis de hemoglobina F estäo diminuidos quando comparados com os níveis apresentados pelos indivíduos normais; 2) os níveis diminuídos de hemoglobina F näo estiveram relacionados com determinadas condiçöes que estäo presentes nos diabéticos - faixa etária, tipo de diabete I e II, período da diabete no indivíduo (meses, anos) e evoluçäo clínica da diabete (presença ou ausência de coma). Discute-se sobre a açäo da eritropoietina e da hiperglicemia na eritropoiese: a) acredita-se que a eritropoietina e a hiperglicemia atuam de modo diferente na celula precursora eritróide (BFU-E); b) a eritropoietina estimulando as divisöes celulares tendo como consequencia maior ou menor número de hemácias circulantes; c) a hiperglicemia interferindo na diminuiçäo da produçäo de hemoglobina F. Conclue-se admitindo-se que, o padräo de formaçäo de hemoglobina F no tecido adulto hematopoiético pode ser reversivel na dependência do ambiente metabólico celular, e que, a elucidaçäo destes mecanismos pode ser de particular importância para ajudar no diagnóstico e ou tratamento de variadas disordens hereditárias e adquiridas que envolvem alteraçöes da hemoglobina F

Estigma para hemoglobina S e hemoglobina fetal / Stigma for hemoglobin S and fetal hemoglobin

Säo analisados os resultados de hemoglobina fetal apresentados por 112 indivíduos com estigma para hemoglobina S e comparados com os resultados de hemoglobina fetal encontrados em 112 indivíduos com homozigose para hemoglobina A. Os estigmas para hemoglobina S foram pareados com os homozigotos de hemoglobina A através do sexo, idade, dosagem de hemoglobina total e hematócrito. A hemoglobina fetal com valores elevados (acima de 1,3%) somente foi encontrada naqueles que possuíam a seguinte associaçäo: estigma para hemoglobina S e dosagem de hemoglobina total igual ou acima de 13,7 g/100 ml para os homens e 12,4 g/100 ml para as mulheres