RESUMO
Abnormal hemoglobin anti-Lepore Hong Kong is a rare ßδ fusion variants resulting from non-homologous crossover during meiosis. Anti-Lepore Hong Kong is known to consistently exhibit significantly increased level of HbA2. In this study, we used multiplex ligation-dependent probe amplification (MLPA) and single molecular real-time (SMRT) sequencing, as well as Sanger sequencing, to identify variants in five unrelated families with abnormal elevated HbA2 level. All probands in these five families were found to be heterozygous for anti-Lepore Hong Kong. Among them, two families showed co-occurrence of ß0-thalassemia and α-thalassemia (-SEA/ or αCSα/). Heterozygotes for anti-Lepore Hong Kong displayed an average HbA2 level of 17.7% and behaved normal. However, when combined with ß0-thalassemia and α-thalassemia, the probands exhibited higher HbA2 level (30.2-40.8%) and behaved with ß-thalassemia trait. Furthermore, determination of the α/ß-mRNA ratio revealed a slight downregulation of ß-globin, similar to that of ß-thalassemia minor. Our study is the first to identify compound heterozygotes for anti-Lepore Hong Kong, ß0-thalassemia and α-thalassemia, provide valuable information for prenatal counseling.
Assuntos
Hemoglobinas Anormais , Talassemia alfa , Talassemia beta , Humanos , Gravidez , Feminino , Talassemia alfa/genética , Hemoglobinas Anormais/genética , Talassemia beta/genética , Globinas beta/genéticaRESUMO
To assess the effectiveness of three-level prevention and control of thalassemia, we routinely collect samples from transfusion-dependent individuals and perform genetic analysis. Here, we report on a 10-year-old boy requiring blood transfusions with routine thalassemia gene test results of αα/αα, and ßCD41/42/ßN, but he had thalassemia-like changes in his appearance and a high need for frequent blood transfusions, suggesting a case of thalassemia major in childhood. Given these equivocal results, samples from the family members were collected for further analysis. A multiplex ligation-dependent probe amplification assay was used to detect a multicopy number variant of the α globin gene cluster in the proband. The variant was detected as a long fragment repeat of 380 Kb using CNV assay technique, which contains the entire α globin gene cluster, describing it as αααα380/αα. Analysis of family members suggested that both the brother and mother of the proband carried the variant, and both MCV and MCH values were reduced in carriers. Individuals carrying multiple copy number variants of the α globin gene cluster exist in the population. Individuals carrying such variants who are also heterozygous for the ß0 thalassemia variant result in an imbalance in the α/ß chain ratio, potentially leading to the creation of individuals with a severe anemia genotype. Most secondary prevention and control laboratories currently do not include variants with increased α gene copy number in their testing, which is one of the blind spots of prevention and control efforts. In order to provide more accurate genetic counseling to test subjects, especially in regions with high rates of thalassemia carriage, testing laboratories should pay attention to individual genotype-phenotype matches to avoid the under-detection of such variants.
Assuntos
Talassemia , Talassemia alfa , Talassemia beta , Masculino , Humanos , Genótipo , Talassemia/genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Talassemia beta/terapia , Fenótipo , Família Multigênica , Transfusão de Sangue , alfa-Globinas/genética , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Talassemia alfa/epidemiologiaRESUMO
Hb Tacoma [ß30(B12)ArgâSer] is a missense variant that is caused by either an AGG>AGT or AGG>AGC substitution at codon 30 of the HBB gene. Currently, the latter is classified as a rare cause of ß0-thalassemia (ß0-thal). We propose that HBB: c.93G>C has been incorrectly assigned as ß0-thal and discuss whether HBB: c.93G>T or HBB: c.93G>C should be classified as ß+-thal instead, or as ß-globin variants without thalassemic effect. We present several subjects who are heterozygous for Hb Tacoma, one with HBB: c.93G>T and two with HBB: c.93G>C, to support our conclusions.
Assuntos
Hemoglobinas Anormais , Talassemia beta , Hemoglobinas Anormais/genética , Humanos , Mutação de Sentido Incorreto , Globinas beta/genética , Talassemia beta/diagnóstico , Talassemia beta/genéticaRESUMO
INTRODUCTION: ß-thalassemia is a severe hereditary hemolytic anemia. Due to the diversity of mutations spectrum, ß-thalassemia manifests a highly heterogeneous clinical severity. We noted that a previous report characterized HBB:c.313delA, at the end of exon 2, as a ß-thalassemia trait rather than dominant ß-thalassemia, the classification given to similar mutations. We further explored the impact of this functional variant on globin structure through larger pedigree analysis and in vitro studies. METHODS: Hematological analysis and molecular genotyping were conducted on the proband and his family members. We evaluated functional effects of the variant on ß-globin gene in the proband's nucleated erythrocytes and transfected HEK-293T cells. Three-dimensional construction of protein structure was carried out in silico to demonstrate amino acid changes. RESULTS: The thalassemia major proband was identified as a compound heterozygote of HBB:c.313delA and HBB:c.126_129delCTTT. Three family members with heterozygotes of HBB:c.313delA displayed microcytic hypochromic anemia. Molecular characterization demonstrated that the frameshift mutation could give rise to retro-positioning of the termination codon, resulting in an elongated ß-globin chain with an extension of 10 amino acids. Clinical phenotype and functional experiments indicated that HBB:c.313delA led to ß0 -thalassemia phenotype. CONCLUSION: We concluded that the phenotype of HBB:c.313delA was mainly related to the stability of mutant mRNA, the degradation of mutant proteins, and production of inclusion bodies according to a systematic description of clinical phenotype and a series of molecular experiments.
Assuntos
Alelos , Fenótipo , Deleção de Sequência , Globinas beta/genética , Talassemia beta/sangue , Talassemia beta/genética , Substituição de Aminoácidos , Índices de Eritrócitos , Eritrócitos/metabolismo , Eritrócitos/patologia , Éxons , Mutação da Fase de Leitura , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos , Genótipo , Heterozigoto , Humanos , Corpos de Inclusão , Talassemia beta/diagnósticoRESUMO
Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of ß-thalassaemia. The homozygous mutation c.-196 C>T in the Aγ-globin (HBG1) promoter, which causes Sardinian δß0 -thalassaemia, is able to completely rescue the ß-major thalassaemia phenotype caused by the ß0 39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from ß0 -thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the -196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous ß0 39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the -196 position has the potential to induce therapeutic levels of HbF in patients with most types of ß-thalassaemia irrespective of the ß-globin gene (HBB) mutations.
Assuntos
Hemoglobina Fetal/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Talassemia beta/genética , Sistemas CRISPR-Cas , Células Cultivadas , Células HEK293 , Humanos , Células K562 , Regulação para CimaRESUMO
Hemoglobin D-Punjab is a common hemoglobin variant in India but very rare in Morocco. Often, its presence has minimal or no clinical impact. Its heterozygous association with ß-thalassemia is exceptional. The purpose of the study is to describe the epidemiological, diagnostic and prophylactic aspects of hemoglobinosis D-Punjab from a family case study. MATERIAL AND METHODS: Case study of hemoglobinosis D-Punjab in a Moroccan family, diagnosed at the Laboratory of Biochemistry-Toxicology of the Mohammed V Military Teaching Hospital. The biological study was based on iron and hemolysis checkups, hemogram and study of hemoglobin (electrophoresis in alkaline and acid medium, high performance liquid chromatography). The index patient also benefited from sequencing by molecular biology. RESULTS: The index patient was heterozygous D-Punjab/ß0-thalassemia, confirmed by molecular biology. Two of her sisters had the same hemoglobin profile. At electrophoresis, all three had hemoglobin D-Punjab higher than 90%, hemoglobin A less than 1% and hemoglobin A2 higher than 6%. The results of the three hemograms showed similar abnormalities (pseudo-polycythemia, hypochromia, microcytosis, anisopoikilocytosis). Six other members of the family had a thalassemic trait and another three had heterozygous hemoglobinosis D-Punjab. CONCLUSION: Hemoglobin D-Punjab remains extremely rare in Morocco and very poorly documented in the literature. The number of reported cases is expected to raise due to increasing migration. Biologist advisory services require a precise diagnosis in order to give correct genetic counseling.
Assuntos
Hemoglobinas Anormais/genética , Talassemia beta/genética , Adolescente , Adulto , Criança , Família , Feminino , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Marrocos , Linhagem , Talassemia beta/sangueRESUMO
Several types of thalassemia (including ß039-thalassemia) are caused by nonsense mutations in genes controlling globin production, leading to premature translation termination and mRNA destabilization mediated by the nonsense mediated mRNA decay. Drugs (for instance, aminoglycosides) can be designed to suppress premature translation termination by inducing readthrough (or nonsense suppression) at the premature termination codon. These findings have introduced new hopes for the development of a pharmacologic approach to cure this genetic disease. In the present review, we first summarize the principle and current status of the chemical relief for the expression of functional proteins from genes otherwise unfruitful for the presence of nonsense mutations. Second, we compare data available on readthrough molecules for ß0-thalassemia. The examples reported in the review strongly suggest that ribosomal readthrough should be considered as a therapeutic approach for the treatment of ß0-thalassemia caused by nonsense mutations. Concluding, the discovery of molecules, exhibiting the property of inducing ß-globin, such as readthrough compounds, is of great interest and represents a hope for several patients, whose survival will depend on the possible use of drugs rendering blood transfusion and chelation therapy unnecessary.
RESUMO
Hemoglobin (Hb) D. Punjab [ß121(GH4) GluâGln; HBB: C.364G>C] and ß0-thalassemia 3.4 kb deletion are very rare in the Thai population. For the first time, the coinheritance of HbD-Punjab with ß0-thalassemia 3.4 kb deletion was reported in a 7-year-old Thai girl. She had mild anemia (Hb 115.0 g/L and mean corpuscular hemoglobin 18.1 pg) with red blood cell microcytosis (mean corpuscular volume 52.5 fL). By capillary electrophoresis (CE), HbD-Punjab was found at a migration position of 180 s with the value of 81.9% while the level of HbA2 was 7.3%. Based on the elevated HbA2, the molecular analysis for detection of ß0-thalassemia mutations was performed. The 490 bp amplified fragments from ß0-thalassemia 3.4 kb deletion was observed. Thus, the coinheritance of HbD-Punjab with ß0-thalassemia can be found in the Thai population. The HbA2 measured on CE is a reliable parameter for differentiating the homozygote of HbD-Punjab and compound heterozygote of HbD-Punjab and ß0-thalassemia.
RESUMO
Hb Q-Thailand [α74(EF3)AspâHis (α1); HBA1: c.223 G>C] is an abnormal hemoglobin (Hb), variant found mainly in China and Southeast Asian countries. The association of the αQ-Thailand allele with other globin gene disorders has important implications in diagnosis. Here, we report a hitherto undescribed condition of patients with a double heterozygosity for Hb Q-Thailand with α0-thalassemia (α0-thal) and in combination with ß0-thalassemia (ß0-thal) in a Chinese family. Our study will provide some clinical manifestations, laboratory diagnosis and genetic counseling for complex hemoglobinopathies.
Assuntos
Hemoglobinas Anormais/genética , Mutação , alfa-Globinas/genética , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Adulto , Povo Asiático/genética , Criança , China , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Hemoglobinopatias/diagnóstico , Hemoglobinopatias/genética , Heterozigoto , Humanos , Masculino , Fenótipo , Talassemia alfa/sangue , Talassemia beta/sangueRESUMO
We report the molecular and hematological feature of a Thai woman who had clinical diagnosis of ß-thalassemia intermedia (ß-TI). Hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis identified Hb A (64.4%), Hb F (12.3%) and Hb A2/E (15.9%) with small peaks of Hb Bart's (γ4) and Hb H (ß4). She was initially diagnosed as EA Bart's disease, which occurs from combination of Hb H disease and Hb E (HBB: c.79G > A) trait. However, the Hb analysis using capillary electrophoresis (CE) demonstrated no Hb E, 68.5% Hb A, 15.5% Hb F and 16.0% Hb A2. DNA analysis showed a compound heterozygosity for (ß(+)) -31 (A > G) (HBB: c.-81A > G) and (ß(0)) codon 17 (A > T) (HBB: c.52A > T) mutations and deletional Hb H (- -(SEA)/-α(3.7)). Thus, she was finally diagnosed with a combination of Hb H disease and compound heterozygosity of ß(+)/ß(0)-thalassemia (ß(+)/ß(0)-thal). The ß-globin mutations could affect not only hematological parameters but also elevate the Hb A2 levels. These effects could not be ameliorated by the coinheritance of Hb H disease. Therefore, a better understanding of the effects of this combination on hematological analysis data will be useful for providing accurate diagnosis, genetic counseling, prevention and control programs of ß-thalassemia major (ß-TM).
Assuntos
Códon , Hemoglobina A2/genética , Hemoglobina A2/metabolismo , Heterozigoto , Mutação , alfa-Globinas/genética , Globinas beta/genética , Adolescente , Análise Mutacional de DNA , Índices de Eritrócitos , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Talassemia beta/sangue , Talassemia beta/diagnóstico , Talassemia beta/genéticaRESUMO
A novel ß(0)-thalassemia (ß-thal) frameshift mutation, HBB: c.209delG; p.Gly70Valfs*20, is described in a 21-year-old African American female with ß-thalassemia major (ß-TM) due to compound heterozygosity for the ß(0)-thal mutation HBB: c.92+2T>C [formerly known as IVS-I-2 (T>C)] and HBB: c.209delG. The combination of these mutations demonstrates a complete lack of ß-globin chain synthesis, evidenced by the proband having no Hb A present.