Analysis of a Novel Mexican Variant of the HBB Gene Associated with ß-Thalassemia Using Bioinformatic Tools.

We present a study performed on 54 unrelated subjects, with and without thalassemic features. Two primer pairs were proposed to perform Sanger sequencing of the complete HBB gene. The bioinformatic analysis was performed taking advantage of the availability of free online tools. In the sample, we found 11 variants, 10 reported, and one novel. Among the variants found, six are clinically important: three encode a premature stop codon [codon 39 (C>T) (HBB: c.118C>T); IVS-II-1 (G>A) (HBB: c.315+1G>A), and one not reported], a double substitution within the same allele [Hb Borås (HBB: c.266T>G) and Hb Santa Giusta Sardegna (HBB: c.282T>C)], and one whose pathogenicity is not yet defined [Hb Fannin-Lubbock I (HBB: c.359G>A)]. Even though the variants Hb Borås and Hb Santa Giusta Sardegna have been described, there is no report of their combined occurrence on the same allele, which could cause hemolytic anemia. Although the p.Leu88Arg and p.Cys93Trp variants do not alter the final length of the protein, the bioinformatic results suggest that there are differences in the tertiary structure of ß-globin genes, mainly affecting helices E and F, being the motifs of interaction with the heme group. The novel variant is a 4 bp insertion that modifies the open reading frame, changing the last amino acid residue and causing a premature stop codon (HBB: c.291-294insGCAC). The variant was associated with ß-thalassemia (ß-thal). Bioinformatic analysis made it possible to predict the consequences that the new variant of the HBB gene caused on the ß-globin tertiary structure.

Genetic Regulation of Redox Balance in ß-Thalassemia Trait.

ß-Thalassemia (ß-thal) trait is a heterogeneous group of genetic defects leading to decreased ß-globin production, ineffective erythropoiesis, and oxidative stress. The aim is to evaluate the cytoprotective response, at transcriptional and systemic levels, of the variations of global redox balance in ß-thal trait patients. Sixty-six subjects (40 healthy and 26 with ß-thal trait) were analyzed at the Universidad Nacional de Tucumán, Tucumán, Argentina, between 2016 and 2017. The following parameters were evaluated: complete blood count, iron status, hemoglobin (Hb) electrophoresis, Hb A2, thiobarbituric acid reactive species (TBARS), serum catalase (CAT), and superoxide dismutase (SOD) activity, FOXO3a, NRF2, SOD, PRDX2, CAT, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) gene expression. The ß-thal trait group showed a decrease in Hb levels, MCV, and MCH with higher TBARS levels. The SOD activity was significantly increased by 32.0% in ß-thal trait patients respect to the control group. Relative expression of NRF2 was 4.7-fold higher in ß-thal trait than in the control group, while FOXO3a expression was similar in both groups. The SOD, PRDX2, and proinflammatory cytokines transcriptional expression was significantly upregulated in ß-thal trait patients. This is the first study on the genetic regulation of redox balance in ß-thal trait patients in which interesting modifications were observed in the transcript levels of some redox regulators that could be associated with changes in the erythrocyte proteome in this disorder. A better understanding of the pathophysiological mechanisms present in these heterozygous patients would allow adequate therapy in situations such as growth, pregnancy, or high performance sports, favoring a personalized treatment.

ß-Thalassemia Mutations in Jamaica: Geographic Variation in Small Communities.

Over the last 43 years, surveys of over 200,000 subjects in Jamaica have identified ß-thalassemia (ß-thal) mutations. In most, these genes were detected at birth in patients with sickle cell-ß-thal and so the prevalence and distribution would not be influenced by subsequent clinical course. There were two newborn populations, 100,000 deliveries in the corporate area between 1973-1981 and 84,940 in south and western Jamaica between 2008-2016. A third population, which derived from the Manchester Project in central Jamaica, screened 16,612 secondary school children, aged predominantly 15-19 years, and identified 150 students with the ß-thal trait and 11 with sickle cell [Hb S (HBB: c.20A>T)]- or Hb C (HBB: c.19G>A)-ß-thal. The latter patients may have been subject to symptomatic selection, but this should not have affected those with ß-thal trait. Of the 24 different molecular mutations, ß0-thal genes accounted for 10.0-27.0% of these groups and most common was IVS-II-849 (A>G) (HBB: c.316-2A>G). Of the ß+ mutations, seven subjects had severe genes with low levels of ß chain synthesis but the majority were benign mutations in the promoter region. The -29 (A>G) (HBB: c.-79A>G) mutation dominated in the newborn study in Kingston, similar to experiences in Guadeloupe and African Americans but the -88 (C>T) (HBB: c.-138C>T) mutation was more common among school students in central Jamaica. Caribbean populations are genetically heterogeneous but variations within different parts of Jamaica is of potential importance for prenatal diagnosis and genetic counseling. This information may also be useful among the large Jamaican diaspora.

Mutational Profile of Homozygous ß-Thalassemia in Rio de Janeiro, Brazil.

ß-Thalassemia (ß-thal) is a hemolytic anemia that is caused by point mutations in most cases. The Brazilian population is highly heterogeneous and knowledge of the mutations that make up the genotypic profile of individuals can contribute information about the formation of the population and clinical condition of patients. In this study, we evaluated the mutations present in homozygous ß-thal patients from Rio de Janeiro, Brazil. We analyzed 24 samples of peripheral blood of patients with homozygous ß-thal. To identify the mutations, we carried out allele-specific-polymerase chain reaction (AS-PCR) and DNA sequencing. We found 11 different mutations on the ß-globin gene. Among the most frequent mutations observed were HBB: c.92 + 6T>C, followed by HBB: c.93-21G>A, HBB: c.118C>T and HBB: c.92 + 1G>A. We also identified the rare mutation HBB: c.75T>A that was reported in an individual carrying Hb S (HBB: c.20A>T)/ß-thal (HBB: c.75T>A) but not in Brazilian thalassemic patients, thus, this is the first report of this mutation in Brazilian ß-thal patients. For its multiethnic character, Brazil has different mutations that cause ß-thal and that are distributed with different frequencies according to the regions of the country. Our findings contribute to the description of the mutational profile of Brazilian thalassemic patients, showing wide heterogeneity and genetic variability.

The Spectrum of ß-Thalassemia Mutations in a Population from the Brazilian Amazon.

The spectrum of ß-thalassemia (ß-thal) mutations was investigated for the first time in a cohort of 33 unrelated patients from the Brazilian Amazon attending the Center for Hemotherapy and Hematology of the Pará Foundation (HEMOPA), in Belém, the state capital of Pará, Northern Brazil. Identification of the ß-thal mutations was made by direct genomic sequencing of the ß-globin gene. Mutations were identified in all patients, corresponding to a spectrum of 10 different point mutations and a total of 37 alleles studied. HBB: c.92 + 5G > A [IVS-I-5 (G > A)], was the most common ß-thal mutation, followed by HBB: c.118C > T [codon 39 (C > T)], HBB: c.-138C > T [-88 (C>T)], HBB: c.92 + 1G > A [IVS-I-1 (G > A)] and HBB: c.92 + 6T > C [IVS-I-6 (T > C)] mutations. These five mutations (four Mediterranean origin and one African origin) accounted for 86.5% of the ß-thal alleles. The profile of ß-thal mutations found in northern Brazil is different from those described in other regions of the country. In the southeast and south, the nonsense mutation HBB: c.118C > T is the most prevalent, followed by HBB: c.93-21G > A [IVS-I-110 (G > A)], whereas in the northeast, HBB: c.92 + 6T > C has been identified as the most common mutation, followed by HBB: c.92 + 1G > A. This heterogeneous geographical distribution is certainly related to the ancestry of Brazilian populations because they have similar genetic backgrounds (European, African and Amerindian), although with slightly different admixture proportions. Furthermore, the European contribution in the southeast and south was largely made up of immigrants of other nationalities, such as Italian and Spanish, in addition to Portuguese.

Molecular characterization and phenotypical study of ß-thalassemia in Tucumán, Argentina.

The main hereditary hemoglobin (Hb) disorder in Argentina is ß-thalassemia (ß-thal). Molecular studies performed in the center of the country exhibited a marked prevalence of the codon 39 (C > T) and IVS-I-110 (G > A) mutations. The northwest region of Argentina has a different demographic history characterized by an important Spanish influx. Seventy-one ß-thal carriers attending the Instituto de Bioquímica Aplicada, Tucumán, Argentina, were investigated for ß-globin gene mutations by real-time polymerase chain reaction (RT-PCR). To examine the genotype-phenotype relationship, mean corpuscular volume (MCV), mean corpuscular Hb (MCH) and Hb A2 were measured. In order to recognize ß-thal, Mentzer Index, Shine & Lal and Red Cell Distribution Width Index (RDWI), were calculated. The ethnic background of subjects revealed that 82.0% of the population was of Italian, Spanish and Arab origin. Seven mutations were detected: codon 39 (45.0%), IVS-I-1 (G > A) (22.5%), IVS-I-110 (16.3%), IVS-II-1 (G > A) (4.1%), IVS-I-1 (G > T) (2.0%), IVS-I-6 (T > C) (2.0%) and IVS-II-745 (G > C) (2.0%). In three families (6.1%), ß-thal mutations were not determined. These results differed from other Argentinian studies because at present codon 39 and IVS-I-1 are the most prevalent; MCV, MCH and Hb A2 did not correlate with the type of mutation (ß(0)/ß(+)). Values of MCV (67.0 fL) and Hb A2 (4.85%) were unable to discriminate between them. Significant differences (p < 0.05) in MCV, MCH and Shine & Lal were observed between the undetermined group and the three most common mutations. These data show different patterns of ß-thal mutations in the center and northwest regions of Argentina. Differences might represent the influence of Spanish immigration.