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1.  β-Globin chain abnormalities with coexisting α-thalassemia mutations 
Introduction
The frequency of hemoglobinopathies is still high in Adana, the biggest city of the Cukurova Region that is located in the southern part of Turkey. Our aim was to identify the concomitant mutations in α- and β-globin genes which lead to complex hemoglobinopathies and to establish an appropriate plan of action for each subject, particularly when prenatal diagnosis is necessary.
Material and methods
We studied the association between the β-globin gene and α-thalassemia genotypes. The reverse hybridization technique was employed to perform molecular analysis, and the results were confirmed by amplification refractory mutation system (ARMS) or restriction fragment length polymorphism (RFLP) technique.
Results
We evaluated 36 adult subjects (28 female and 8 male; age range: 18-52 years) with concomitant mutations in their α- and β-globin genes. The –α3.7/αα deletion was the commonest defect in the α-chain as expected, followed by α3.7/–α3.7 deletion. Twenty-five of 36 cases were sickle cell trait with coexisting α-thalassemia, while seven Hb S/S patients had concurrent mutations in their α-genes. The coexistence of αPolyA-2α/αα with Hb A/D and with Hb S/D, which is very uncommon, was also detected. There was a subject with compound heterozygosity for β-globin chain (–α3.7/αα with IVSI.110/S), and also a case who had –α3.7/αα deletion with IVSI.110/A.
Conclusions
Although limited, our data suggest that it would be valuable to study coexisting α-globin mutations in subjects with sickle cell disease or β-thalassemia trait during the screening programs for premarital couples, especially in populations with a high frequency of hemoglobinopathies.
doi:10.5114/aoms.2012.28723
PMCID: PMC3460491  PMID: 23056075
a-globin gene; b-globin gene; mutation; S-a-thalassemia; sickle cell anemia
2.  Application of diagnostic methods and molecular diagnosis of hemoglobin disorders in Khuzestan province of Iran 
BACKGROUND:
The hemoglobinopathies refer to a diverse group of inherited disorders characterized by a reduced synthesis of one or more globin chains (thalassemias) or the synthesis of structurally abnormal hemoglobin (Hb). The thalassemias often coexist with a variety of structural Hb variants giving rise to complex genotypes and an extremely wide spectrum of clinical and hematological phenotypes. Hematological and biochemical investigations and family studies provide essential clues to the different interactions and are fundamental to DNA diagnostics of the Hb disorders. Although DNA diagnostics have made a major impact on our understanding and detection of the hemoglobinopathies, DNA mutation testing should never be considered a shortcut or the test of first choice in the workup of a hemoglobinopathy.
MATERIALS AND METHODS:
A careful three-tier approach involving: (1) Full blood count (2) Special hematological tests, followed by (3) DNA mutation analysis, provides the most effective way in which to detect primary gene mutations as well as gene-gene interactions that can influence the overall phenotype. With the exception of a few rare deletions and rearrangements, the molecular lesions causing hemoglobinopathies are all identifiable by PCR-based techniques. Furthermore, each at-risk ethnic group has its own combination of common Hb variants and thalassemia mutations. In Iran, there are many different forms of α and β thalassemia. Increasingly, different Hb variants are being detected and their effects per se or in combination with the thalassemias, provide additional diagnostic challenges.
RESULTS:
We did step-by-step diagnosis workup in 800 patients with hemoglobinopathies who referred to Research center of Thalassemia and Hemoglobinopathies in Shafa Hospital of Ahwaz Joundishapour University of medical sciences, respectively. We detected 173 patients as iron deficiency anemia (IDA) and 627 individuals as thalassemic patients by use of different indices. We have successfully detected 75% (472/627) of the β-thalassemia mutations by using amplification refractory mutation system (ARMS) technique and 19% (130/627) of the β-thalassemia mutations by using Gap-PCR technique and 6% (25/627) as Hb variants by Hb electrophoresis technique. We did prenatal diagnosis (PND) for 176 couples which had background of thalassemia in first pregnancy. Result of PND diagnosis in the first trimester was 35% (62/176) affected fetus with β-thalassemia major and sickle cell disease that led to termination of the pregnancy.
CONCLUSION:
Almost all hemoglobinopathies can be detected with the current PCR-based assays with the exception of a few rare deletions. However, the molecular diagnostic service is still under development to try and meet the demands of the population it serves. In the short term, the current generation of instruments such as the capillary electrophoresis systems, has greatly simplified DNA sequence analysis.
doi:10.4103/0971-6866.32028
PMCID: PMC3168155  PMID: 21957335
α and β-thalassemia; hemoglobin variants; hemoglobinopathies; iron deficiency anemia; polymerase chain reaction; prenatal diagnosis
3.  Hemoglobinopathy: Molecular Epidemiological Characteristics and Health Effects on Hakka People in the Meizhou Region, Southern China 
PLoS ONE  2013;8(2):e55024.
Background
Hemoglobinopathies are the most common inherited diseases in southern China. However, there have been only a few epidemiological studies of hemoglobinopathies in Guangdong province.
Materials and Methods
Peripheral blood samples were collected from 15299 “healthy” unrelated subjects of dominantly ethnic Hakka in the Meizhou region, on which hemoglobin electrophoresis and routine blood tests were performed. Suspected cases with hemoglobin variants and hereditary persistence of fetal hemoglobin (HPFH) were further characterized by PCR, DNA sequencing, reverse dot blot (RDB) or multiplex ligation-dependent probe amplification (MLPA). In addition, 1743 samples were randomly selected from the 15299 subjects for thalassemia screening, and suspected thalassemia carriers were identified by PCR and RDB.
Results
The gene frequency of hemoglobin variants was 0.477% (73/15299). The five main subgroups of the ten hemoglobin variants were Hb E, Hb G-Chinese, Hb Q-Tahiland, Hb New York and Hb J-Bangkok. 277 cases (15.89%, 277/1743) of suspected thalassemia carriers with microcytosis (MCV<82 fl) were found by thalassemia screening, and were tested by a RDB gene chip to reveal a total of 196 mutant chromosomes: including 124 α-thalassemia mutant chromosomes and 72 β-thalassemia mutant chromosomes. These results give a heterozygote frequency of 11.24% for common α and β thalassemia in the Hakka population in the Meizhou region. 3 cases of HPFH/δβ-thalassemia were found, including 2 cases of Vietnamese HPFH (FPFH-7) and a rare Belgian Gγ(Aγδβ)0–thalassemia identified in Chinese.
Conclusions
Our results provide a detailed prevalence and molecular characterization of hemoglobinopathies in Hakka people of the Meizhou region. The estimated numbers of pregnancies each year in the Meizhou region, in which the fetus would be at risk for β thalassemia major or intermedia, Bart’s hydrops fetalis, and Hb H disease, are 25 (95% CI, 15 to 38), 40 (95% CI, 26 to 57), and 15 (95% CI, 8 to 23), respectively.
doi:10.1371/journal.pone.0055024
PMCID: PMC3562339  PMID: 23383304
4.  Translation of β-Globin m-RNA in β-Thalassemia and the S and C Hemoglobinopathies 
Journal of Clinical Investigation  1972;51(2):364-372.
Genetic and biochemical evidence indicates that in β-thalassemia there is impaired synthesis of the β-globin chains of hemoglobin A. In patients heterozygous for the hemoglobinopathies, hemoglobin S and hemoglobin C, the mutant β-chain is produced in smaller amounts than normal βA. Defective m-RNA translation has been suggested as a possible cause of decreased β-globin polypeptide synthesis in thalassemia and the hemoglobinopathies. In the present study, the ribosomal assembly of β-globin chains was examined in the peripheral, nucleated red blood cells and reticulocytes of patients with Cooley's anemia, thalassemia intermedia, sickle thalassemia, sickle cell anemia, hemoglobin C disease, and in hemolytic anemias not associated with a hemoglobinopathy. The translation times of βA, βS, and βC did not differ significantly (average times; βA = 75 sec, range 43-114, βS = 69 sec, βC = 92 sec). In thalassemia, no evidence was found for a delay in translation as the cause of the marked impairment of β-globin synthesis. In several specimens of peripheral blood from thalassemic patients, the translation time of the β-chain was even shorter than in nonthalassemic specimens (average time = 45 sec, range 35-59). The results suggest that the defect in β-globin synthesis in β-thalassemia is due to impaired initiation of β-globin chain assembly or a quantitative deficiency in m-RNA.
PMCID: PMC302135  PMID: 5009120
5.  Reticulocyte hemoglobin equivalent to detect thalassemia and thalassemic hemoglobin variants 
Introduction
Thalassemia and iron deficiency may both result in hypochromic microcytic anemia. Hematological algorithms that differentiate the two are mainly established in adult selected diagnostic groups. We aimed at creating an algorithm applicable in the presence of children, hemoglobin variants, and iron deficiency.
Methods
Our study material constituted blood samples referred during 1 year for routine diagnostics of hemoglobinopathy. We included 443 samples, of which 37% were from children 3 months or older. We found β-thalassemia trait (n = 100), α-thalassemia (n = 75), combined α-/β-thalassemia (n = 14), hemoglobin variants (n = 42), and no-hemoglobinopathy (n = 207), of whom 107 had a ferritin at or below 20 μg/L. We included reticulocyte hemoglobin equivalent, ferritin, and erythrocyte count in our algorithm.
Results
Our algorithm differentiated β-thalassemia trait from no-hemoglobinopathy with a sensitivity of 99% at 83% specificity. It performed better than other published algorithms when applied to all patient samples, while equally or moderately better in the 63% adult samples. Our algorithm also detected the clinically significant α-thalassemias, and most of the combined α-/β-thalassemias and thalassemic hemoglobin variants.
Conclusion
Our algorithm efficiently differentiated thalassemia and thalassemic hemoglobin variants from iron deficiency in children and adults.
doi:10.1111/j.1751-553X.2012.01442.x
PMCID: PMC3533779  PMID: 22765164
Thalassemia; hemoglobinopathy; reticulocytes; hemoglobin; RBC; ferritin
6.  Novel therapeutic candidates, identified by molecular modeling, induce γ-globin gene expression in vivo☆ 
Blood cells, molecules & diseases  2011;47(2):107-116.
The β-hemoglobinopathies and thalassemias are serious genetic blood disorders affecting the β-globin chain of hemoglobin A (α2βA2). Their clinical severity can be reduced by enhancing expression of fetal hemoglobin (γ-globin), producing HbF (α2γ2,). In studies reported here, γ-globin induction by 23 novel, structurally-unrelated compounds, which had been predicted through molecular modeling and in silico screening of a 13,000 chemical library, was evaluated in vitro in erythroid progenitors cultured from normal subjects and β-thalassemia patients, and in vivo in transgenic mice or anemic baboons. Four predicted candidates were found to have high potency, with 4- to 8-fold induction of HbF. Two of these compounds have pharmacokinetic profiles favorable for clinical application. These studies thus effectively identified high potency γ-globin inducing candidate therapeutics and validated the utility of in silico molecular modeling.
doi:10.1016/j.bcmd.2011.04.008
PMCID: PMC3148318  PMID: 21641240
Butyrate; fetal hemoglobin; hemoglobinopathy; small molecules
7.  Prenatal diagnosis of hemoglobinopathies: evaluation of techniques for analysing globin-chain synthesis in blood samples obtained by fetoscopy. 
Three techniques for analysing hemoglobin synthesis in blood samples obtained by fetoscopy were evaluated. Of the fetuses studied, 12 were not at risk of genetic disorders, 10 were at risk of beta-thalassemia, 2 were at risk of sickle cell anemia and 1 was at risk of both diseases. The conventional method of prenatal diagnosis of hemoglobinopathies, involving the separation of globin chains labelled with a radioactive isotope on carboxymethyl cellulose (CMC) columns, was compared with a method involving globin-chain separation by high-pressure liquid chromatography (HPLC) and with direct analysis of labelled hemoglobin tetramers obtained from cell lysates by chromatography on ion-exchange columns. The last method is technically the simplest and can be used for diagnosing beta-thalassemia and sickle cell anemia. However, it gives spuriously high levels of adult hemoglobin in samples containing nonlabelled adult hemoglobin. HPLC is the fastest method for prenatal diagnosis of beta-thalassemia and may prove as reliable as the CMC method. Of the 13 fetuses at risk for hemoglobinopathies, 1 was predicted to be affected, and the diagnosis was confirmed in the abortus. Of 12 predicted to be unaffected, 1 was aborted spontaneously and was unavailable for confirmatory studies, as were 3 of the infants; however, the diagnosis was confirmed in seven cases and is awaiting confirmation when the infant in 6 months old in one case. Couples at risk of bearing a child with a hemoglobinopathy should be referred for genetic counselling before pregnancy or, at the latest, by the 12th week of gestation so that prenatal diagnosis can be attempted by amniocentesis, safer procedure, with restriction endonuclease analysis of the amniotic fluid cells.
PMCID: PMC1862227  PMID: 7139502
8.  Hemoglobin Disorders in South India 
ISRN Hematology  2011;2011:748939.
Cation exchange-high performance liquid chromatography (CE-HPLC) is increasingly being used as a first line of investigation for hemoglobinopathies and thalassemias. Together with a complete blood count, the CE-HPLC is effective in categorizing hemoglobinopathies as traits, homozygous disorders and compound heterozygous disorders. We carried out a one year study in Apollo Hospitals, Chennai (Tamil Nadu, South India) during which 543 abnormal chromatogram patterns were seen. The commonest disorder we encountered was β-thalassemia trait (37.9%), followed by HbE trait (23.2%), homozygous HbE disease (18.9%), HbS trait (5.3%), HbE β-thalassemia (4.6%), HbS β-thalassemia (2.5%), β-thalassemia major (2.3%), HbH (1.6%), homozygous HbS (1.4%), HbD trait (0.7%). The average value of HbA2 in β-thalassemia minor was 5.4%. β-thalassemia major had an average HbF of 88% and in HbH the mean A2 was 1.4%. Among the HbE disorders the HbA2 + HbE was 30.1% in the heterozygous state, 90.8% in the homozygous state and 54.8% in HbE β-thalassemia. In the sickle cell disorders, HbS varied from 30.9% in the trait to 79.9% in the homozygous state to 65.6% in HbS β-thalassemia.
doi:10.5402/2011/748939
PMCID: PMC3199938  PMID: 22084704
9.  Prenatal Diagnosis of Different Polymorphisms of β-globin Gene in Ahvaz 
Background
Hemoglobinopathy and thalassemia are prevalent genetic disorders throughout the world. Beta thalassemia is one of these disorders with high prevalence in Iran, especially in Khuzestan province. In this study, the rate of different mutations in β-globin gene for prenatal diagnosis in fetal samples was evaluated.
Materials and methods
In this experimental pilot study, 316 fetal samples (chorionic villus or amniotic fluid) suspicious to hemoglobin disorders were enrolled. Afterwards, DNA was extracted and PCR and DNA sequencing were used for evaluation of different mutations in β-globin gene.
Results
Amongst 316 samples evaluated for prenatal diagnosis, 180 cases (56.8%) were carrying at least one mutated gene of β-thalassemia. In addition, results showed that CD 36-37 (- T) and IVS II-1 (G > A) polymorphisms are the most prevalent polymorphisms of β-thalassemia in Ahvaz city with 13.9% and 10.1% rates, respectively.
Conclusion
Using molecular tests for prenatal diagnosis is considered an efficient approach for reducing the birth of children with hemoglobinopathy and identification of prevalent mutations in each region.
PMCID: PMC3913140  PMID: 24505523
Hemoglobinopathy; β-thalassemia; Prenatal diagnosis; Polymorphism
10.  Gene Therapy in Thalassemia and Hemoglobinopathies 
Sickle cell disease (SCD) and ß-thalassemia represent the most common hemoglobinopathies caused, respectively, by the alteration of structural features or deficient production of the ß-chain of the Hb molecule. Other hemoglobinopathies are characterized by different mutations in the α- or ß-globin genes and are associated with anemia and might require periodic or chronic blood transfusions. Therefore, ß-thalassemia, SCD and other hemoglobinopathies are excellent candidates for genetic approaches since they are monogenic disorders and, potentially, could be cured by introducing or correcting a single gene into the hematopoietic compartment or a single stem cell. Initial attempts at gene transfer of these hemoglobinopathies have proved unsuccessful due to limitations of available gene transfer vectors. With the advent of lentiviral vectors many of the initial limitations have been overcame. New approaches have also focused on targeting the specific mutation in the ß-globin genes, correcting the DNA sequence or manipulating the fate of RNA translation and splicing to restore ß-globin chain synthesis. These techniques have the potential to correct the defect into hematopoietic stem cells or be utilized to modify stem cells generated from patients affected by these disorders. This review discusses gene therapy strategies for the hemoglobinopathies, including the use of lentiviral vectors, generation of induced pluripotent stem cells (iPS) cells, gene targeting, splice-switching and stop codon readthrough.
doi:10.4084/MJHID.2009.008
PMCID: PMC3033156  PMID: 21415990
11.  Beta-globin Gene Mutations in Turkish Children with Beta-Thalassemia: Results from a Single Center Study 
Introduction
The beta thalassemias are common genetic disorders in Turkey and in this retrospective study our aim was to evaluate β-globin chain mutations and the phenotypic severity of β-thalassemia patients followed-up in our hospital, a tertiary center which serves patients from all regions of Turkey.
Materials and Methods
106 pediatric patients were analysed for β-globin gene mutations by using DNA analysis. Patients were classified as having β-thalassemia major or β-thalassemia intermedia based on age at diagnosis, transfusion frequency and lowest hemoglobin concentration in between transfusions.
Results
There were 106 patients (52.8% female and 47.2% male) with a mean age of 11.2±5 years (1.6 – 22.3 years). Eighty-four (79.2%) patients had β-thalassemia major, whereas the remaining 22 patients (20.8%) were identified as having β-thalassemia intermedia. Overall, 18 different mutations were detected on 212 alleles. The most frequently encountered mutation was IVS I.110 (G>A) (35.3%), followed by Codon 8 del-AA (10.4%), IVS II.1 (G>A) (8%), IVS I.1 (G>A) (7.5%), Codon 39 (C>T) (7.1%) and Codon 5 (−CT) (6.6%), which made up 79.4% of observed mutations. According to present results, IVS I.110 (G>AA) was the most frequent mutation observed in this study, as in other results from Turkey. Evaluation of β-thalassemia mutations in 106 patients with 212 alleles, revealed the presence of homozygous mutation in 85 patients (80.2%) and compound heterozygous mutation in 21 patients (19.8%). The mutations detected in patients with homozygous mutation were IVS I.110 (G>A) (38.8%), Codon 8 del –AA (11.8%), IVS II.1 (G>A) (8.2%) and IVS I.1 (G>A) (8.2%). Observed mutations in the compound heterozygotes were Codon 39 (C>T)/Codon 41–42 (−CTTT) (14.3%), IVS I.110 (G>A)/Codon 39(C>T) (14.3%), IVS I.110 (G>A)/Codon 44(−C) (14.3%), and IVS II.745 (C>G)/5′UTR + 22 (G>A) (9.5%).
Conclusion
Our hospital is a tertiary referral center that provides care to patients from all over the country, and thus the distribution of mutations observed in the current study is significant in term of representing that of the country as a whole.
doi:10.4084/MJHID.2013.055
PMCID: PMC3787702  PMID: 24106605
12.  The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells 
Human Gene Therapy  2011;23(1):15-31.
Abstract
To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the Aγ-globin gene with the −117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of Aγ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34+ hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with β-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro.
Papanikolaou and colleagues describe a novel locus control region (LCR)-free self-inactivating (SIN) γ-globin-insulated lentiviral vector for gene therapy in β-thalassemia. The authors show that this vector has a mean transduction efficiency of 40.3% and is able to increase the production of γ-globin by 32.9% in CD34+ hematopoietic stem cells isolated from patients with β-thalassemia.
doi:10.1089/hum.2011.048
PMCID: PMC3260446  PMID: 21875313
13.  The thalassemias and related disorders 
The thalassemias, sickle cell disease, and other hemoglobinopathies represent a major group of inherited disorders of hemoglobin synthesis. The abnormal hemoglobins were reviewed in the July 2006 issue of Baylor University Medical Center Proceedings. Because of immigration patterns and population flow, these disorders are becoming increasingly more prevalent in the USA. In this article, the clinical aspects of the more common thalassemia syndromes are reviewed. For most symptomatic patients with thalassemia, there is no definite cure; only supportive management of the anemia is possible. A very limited number of patients with thalassemia may be cured by bone marrow transplantation from HLA-identical donors. Other tentative approaches to management include stimulation of fetal hemoglobin synthesis and attempts at somatic cell gene therapy. Prevention of disease transmission by carrier screening programs along with prenatal diagnosis remain of paramount importance in the reduction of these diseases worldwide.
PMCID: PMC1769530  PMID: 17256039
14.  Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies 
Nucleic Acids Research  2004;32(Database issue):D537-D541.
HbVar (http://globin.cse.psu.edu/globin/hbvar/) is a relational database developed by a multi-center academic effort to provide up-to-date and high quality information on the genomic sequence changes leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Extensive information is recorded for each variant and mutation, including sequence alterations, biochemical and hematological effects, associated pathology, ethnic occurrence and references. In addition to the regular updates to entries, we report two significant advances: (i) The frequencies for a large number of mutations causing β-thalassemia in at-risk populations have been extracted from the published literature and made available for the user to query upon. (ii) HbVar has been linked with the GALA (Genome Alignment and Annotation database, available at http://globin.cse.psu.edu/gala/) so that users can combine information on hemoglobin variants and thalassemia mutations with a wide spectrum of genomic data. It also expands the capacity to view and analyze the data, using tools within GALA and the University of California at Santa Cruz (UCSC) Genome Browser.
doi:10.1093/nar/gkh006
PMCID: PMC308741  PMID: 14681476
15.  Comparison of Capillary Electrophoresis with Cellulose Acetate Electrophoresis for the Screening of Hemoglobinopathies 
Background
β-thalassemia is primarily found in individuals of Mediterranean and Southeast Asian ancestry. With rapid growth in the Southeast Asian segments of the Korean population, the geographic distribution of hemoglobinopathies is expected to become significantly different from what it is today. In this study, Hb fractions were measured in patients with hypochromic microcytosis to detect thalassemia and Hb variants. To evaluate the feasibility of replacing cellulose acetate electrophoresis (CA) with capillary electrophoresis (CE) in a clinical laboratory, both techniques were performed and the outcomes were compared.
Methods
To evaluate hemoglobinopathies, complete blood cell counts (CBC), CA, and CE were carried out on samples from healthy and microcytic hypochromic groups. The microcytic hypochromic group consisted of 103 patients whose mean corpuscular volume (MCV) was less than 75 fL and mean corpuscular hemoglobin (MCH) was less than 24 pg. Quantitative analysis of Hb fractions was performed on 143 whole blood samples.
Results
There was a good correlation for measurements of HbA (r=0.9370, P<0.0001), HbA2 (r=0.8973 P<0.0001), and HbF (r= 0.8010, P=0.0304) between the two methods. In the microcytic hypochromic group, there were 29 cases (28.2%) with decreased HbA2, 2 cases (1.9%) with increased HbA2, 3 cases (2.9%) with increased HbF, and 2 cases (1.9%) with increased HbA2 and HbF.
Conclusions
CE is comparable to CA for reliable measurement of Hb fractions. It is suitable for screening of hemoglobinopathies in many clinical laboratories.
doi:10.3343/kjlm.2011.31.4.238
PMCID: PMC3190001  PMID: 22016676
Capillary electrophoresis; Thalassemia; Hemoglobinopathies
16.  Updates of the HbVar database of human hemoglobin variants and thalassemia mutations 
Nucleic Acids Research  2013;42(D1):D1063-D1069.
HbVar (http://globin.bx.psu.edu/hbvar) is one of the oldest and most appreciated locus-specific databases launched in 2001 by a multi-center academic effort to provide timely information on the genomic alterations leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Database records include extensive phenotypic descriptions, biochemical and hematological effects, associated pathology and ethnic occurrence, accompanied by mutation frequencies and references. Here, we report updates to >600 HbVar entries, inclusion of population-specific data for 28 populations and 27 ethnic groups for α-, and β-thalassemias and additional querying options in the HbVar query page. HbVar content was also inter-connected with two other established genetic databases, namely FINDbase (http://www.findbase.org) and Leiden Open-Access Variation database (http://www.lovd.nl), which allows comparative data querying and analysis. HbVar data content has contributed to the realization of two collaborative projects to identify genomic variants that lie on different globin paralogs. Most importantly, HbVar data content has contributed to demonstrate the microattribution concept in practice. These updates significantly enriched the database content and querying potential, enhanced the database profile and data quality and broadened the inter-relation of HbVar with other databases, which should increase the already high impact of this resource to the globin and genetic database community.
doi:10.1093/nar/gkt911
PMCID: PMC3964999  PMID: 24137000
17.  Negative epistasis between the malaria-protective effects of α+-thalassemia and the sickle cell trait 
Nature genetics  2005;37(11):1253-1257.
The hemoglobinopathies, disorders of hemoglobin structure and production, protect against death from malaria1. In sub-Saharan Africa, two such conditions occur at particularly high frequencies: presence of the structural variant hemoglobin S and α+-thalassemia, a condition characterized by reduced production of the normal α-globin component of hemoglobin. Individually, each is protective against severe Plasmodium falciparum malaria2–4, but little is known about their malaria-protective effects when inherited in combination. We investigated this question by studying a population on the coast of Kenya and found that the protection afforded by each condition inherited alone was lost when the two conditions were inherited together, to such a degree that the incidence of both uncomplicated and severe P. falciparum malaria was close to baseline in children heterozygous with respect to the mutation underlying the hemoglobin S variant and homozygous with respect to the mutation underlying α+-thalassemia. Negative epistasis could explain the failure of α+-thalassemia to reach fixation in any population in sub-Saharan Africa.
doi:10.1038/ng1660
PMCID: PMC3521056  PMID: 16227994
18.  Genotype-phenotype correlation of β-thalassemia spectrum of mutations in an Indian population 
Hematology Reports  2012;4(2):e9.
Coexistence of thalassemia, hemoglobinopathies and malaria has interested geneticists over many decades. The present study represents such a population from the eastern Indian state of Orissa. Children and their siblings (n=38) were genotyped for β-thalassemia mutations and genotype-phenotype correlation was determined. The major genotype was IVS 1.5 mutation: 26% homozygous (n=10) and 37% (n=14) double heterozygous with other mutations or hemoglobinopathies. Sickle hemoglobin was the major associated hemoglobinopathy (n=12, 32%). Other mutations found were Cd 8/9, HbE and Cd 41/42. The study population did not contain any IVS 1.1 mutations which is the second major Indo-Asian genotype. Genotype-phenotype correlation revealed that genotypes of IVS 1.5, Cd 8/9 Cd 41/42 alone or in association, exhibit severe, moderate and mild severity of thalassemia, respectively. Identification of the mutation at an early age as a part of new born screening and early intervention may help reduce the thalassemia-related morbidity.
doi:10.4081/hr.2012.e9
PMCID: PMC3401138  PMID: 22826799
β-thalassemia mutations; genotype-phenotype correlation; HbS; IVS 1.5 associated genotypes; malaria.
19.  Detection of Common Deletional Alpha-Thalassemia Spectrum by Molecular Technique in Kelantan, Northeastern Malaysia 
ISRN Hematology  2012;2012:462969.
Thalassemia is a hereditary blood disorder that results from genetic defects causing deficient synthesis of hemoglobin polypeptide chains. Although thalassemia mostly affects developing countries, there is limited knowledge of its accurate frequency and distribution in these regions. Knowing the prevalence of thalassemia and the frequency of responsible mutations is therefore an important step in the prevention and control program as well as treatment strategies. This study was performed to determine the prevalence and to study the spectrum of gene deletions that are responsible in α-thalassemia in Kelantan, located in northeastern Malaysia. A total 400 first-time blood donors from multiple areas of donation centre were chosen randomly. The presence of three types of α-thalassemia gene deletion in southeast Asian population which were -SEAdeletion, -α3.7 rightward deletion, and -α4.2 leftward deletion was detected by using multiplex PCR method. 37 (9.25%) of blood donors were confirmed to have α-thalassemia deletion types. 34 (8%) were heterozygous for α3.7 deletion, 1 (0.25%) was heterozygous for α4.2 deletion, and 2 (0.5%) were heterozygous for SEA type deletion. Alpha-thalassemia-2 with 3.7 deletion was the most common determinant detected in Kelantan Malay compared to other ethnic groups. It has been noted that alpha-thalassemia-2 with 3.7 deletion is the most common type of α-thalassemia throughout the world.
doi:10.5402/2012/462969
PMCID: PMC3408647  PMID: 22888447
20.  Frequency and spectrum of hemoglobinopathy mutations in a Uruguayan pediatric population 
Genetics and Molecular Biology  2013;36(3):316-322.
Hemoglobinopathies are the most common recessive diseases worldwide but their prevalence in Uruguay has not been investigated. In this study, 397 unrelated outpatient children from the Pereira Rosell Hospital Center (CHPR), as well as 31 selected patients with microcytic anemia and 28 β-thalassemia carriers were analyzed for hemoglobinopathies by using biochemical and molecular biology methods. Parametric and non-parametric methods were used to compare the hematological indices between groups of genotypes. Of the 397 patients in the first group, approximately 1% (0.76% HbS and 0.25% β-thalassemia) had a mutation in the HBB gene and 3.3% had β-thalassemia. These mutations had a heterogeneous distribution that varied according to individual ancestry. HbS was found exclusively in individuals with declared African ancestry and had a carrier frequency of 2.2%. The frequency of α-thalassemia carriers in outpatients of European and African ancestry was 1.2% and 6.5%, respectively. In contrast, the frequency of α-thalassemia carriers in patients with microcytic anemia was 25.8%, significantly higher (p < 0.01) than that observed in the sample as a whole and in Afro-descendants and Euro-descendants. Significant differences were observed in the hematological parameters between individuals with thalassemia genotypes and those with a normal genotype. These results indicate that hemoglobinopathies are a relevant health problem in Uruguay.
doi:10.1590/S1415-47572013005000027
PMCID: PMC3795163  PMID: 24130436
alpha-globin; beta-globin; hemoglobinopathies; Uruguayan population
21.  Hereditary hemolytic anemia in Korea from 2007 to 2011: A study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology 
Blood research  2013;48(3):211-216.
Background
The number of patients diagnosed with hereditary hemolytic anemia (HHA) has increased since the advent of novel diagnostic techniques that accurately identify this disorder. Here, we report data from a survey on the prevalence and characteristics of patients diagnosed with HHA in Korea from 2007 to 2011.
Methods
Information on patients diagnosed with HHA in Korea and their clinical and laboratory results were collected using a survey questionnaire. Globin gene and red blood cell (RBC) enzyme analyses were performed. In addition, we analyzed data collected by pediatricians.
Results
In total, 195 cases of HHA were identified. Etiologies identified for HHA were RBC membranopathies, hemoglobinopathies, and RBC enzymopathies, which accounted for 127 (64%), 39 (19.9%), and 26 (13.3%) cases, respectively. Of the 39 patients with hemoglobinopathies, 26 were confirmed by globin gene analysis, including 20 patients with β-thalassemia minor, 5 patients with α-thalassemia minor, and 1 patient with unstable hemoglobin disease.
Conclusion
The number of patients diagnosed with hemoglobinopathies and RBC enzymopathies has increased considerably since the previous survey on HHA in Korea, dated from 1997 to 2006. This is likely the result of improved diagnostic techniques. Nevertheless, there is still a need for more sensitive diagnostic tests utilizing flow cytometry and for better standardization of test results to improve the accuracy of diagnosis of RBC membranopathies in Korea. Additionally, more accurate assays for the identification of RBC enzymopathies are warranted.
doi:10.5045/br.2013.48.3.211
PMCID: PMC3786282  PMID: 24086942
Congenital hemolytic anemia; Hereditary spherocytosis; Thalassemia; Congenital nonspherocytic anemia
22.  Induction of Fetal Hemoglobin In Vivo Mediated by a Synthetic γ-Globin Zinc Finger Activator 
Anemia  2012;2012:507894.
Sickle cell disease (SCD) and β-thalassemia patients are phenotypically normal if they carry compensatory hereditary persistence of fetal hemoglobin (HPFH) mutations that result in increased levels of fetal hemoglobin (HbF, γ-globin chains) in adulthood. Thus, research has focused on manipulating the reactivation of γ-globin gene expression during adult definitive erythropoiesis as the most promising therapy to treat these hemoglobinopathies. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the −117 region of the Aγ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene and in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients. Herein, we report that gg1-VP64 increased γ-globin gene expression in vivo, in peripheral blood samples from gg1-VP64 β-YAC double-transgenic (bigenic) mice. Our results demonstrate that ATFs function in an animal model to increase gene expression. Thus, this class of reagent may be an effective gene therapy for treatment of some inherited diseases.
doi:10.1155/2012/507894
PMCID: PMC3384929  PMID: 22778925
23.  Genetic diversity of hemoglobinopathies, G6PD deficiency, and ABO and Rhesus blood groups in two isolates of a primitive Kharia Tribe in Sundargarh District of Northwestern Orissa, India 
Journal of community genetics  2010;1(3):117-123.
Tribal communities constitute about 8.2% of the total population of India. Their health needs are even larger than elsewhere in India; this study investigates the genetic diversity in relation to hemoglobinopathies, G6PD deficiency and, ABO and Rhesus (D) blood groups in two sects, i.e. Dudh (converted Christian) and Dhelki (Hinduised) Kharia, a primitive tribe in Sundargarh district of Orissa in Central-Eastern India. A randomized screening of 767 Kharia tribals (377 males and 390 females) belonging to all age groups and both sexes was done. Laboratory analysis was carried out following the standard methodology and techniques. Contrasting differences were observed in the frequency of hematological genetic disorders such as β-thalassemia, sickle cell, hemoglobin E, G6PD deficiency, ABO and Rhesus (D) blood groups between the two subgroups. Dudh Kharia had no hemoglobin variant allele other than the high prevalence of β-thalassemia trait (8.1%), whereas, their counterpart Dhelki Kharia had the high prevalence of sickle cell allele (12.4%), hemoglobin E allele (3.2%), and β-thalassemia allele (4.0%). Frequency distribution of hemoglobin variants between Dudh and Dhelki Kharia tribe was statistically highly significant (p < 0.001). High G6PD deficiency was detected 19.2% and 30.7% in Dudh Kharia and Dhelki Kharia, respectively (p < 0.001), the average being 24.4% in Kharia tribe. Kharia tribes show a trend for replacement of sickle cell gene with G6PD-deficiency gene as the clinical manifestations of G6PD deficiency are mild (do not result in a complete loss of enzyme activity) against the sickle cell disease with high morbidity and mortality. Rhesus (D)-negative blood group was 1.1% in Dudh Kharia and absent in Dhelki Kharia (p < 0.05). This study showed genetic isolation of the two sects of Kharia tribe. Antimalarial drugs administration needs to be done with caution. Hematological disorders pose a major health challenge having multifaceted implications in public health genetics.
doi:10.1007/s12687-010-0016-y
PMCID: PMC3185996  PMID: 22460244
Genetic isolates; Dhelki Kharia; Dudh Kharia; Hemoglobin variants; G6PD deficiency; India
24.  α-Thalassemia in the American Negro 
Journal of Clinical Investigation  1972;51(2):412-418.
In Italian and Chinese patients with the α-thalassemia syndromes the production of α-chain of normal hemoglobin is decreased relative to that of β-chain in reticulocytes. In this study the relative rates of α- and β-chain synthesis were determined in members of three Negro families with α-thalassemia. Two of the families had members with hemoglobin H disease and α-thalassemia trait, while the mother of several children with α-thalassemia trait in the third family was doubly heterozygous for α-thalassemia and an α-chain mutant. The α/β ratios of globin synthesis in the patients with hemoglobin H disease and α-thalassemia trait indicated less severe biochemical defects in the peripheral blood than those previously determined in Italian and Chinese patients. In the third family, there was a heterogeneity of expression of the gene for α-thalassemia, including patients with normal red cell indices and synthesis ratios. These findings differ from those previously described in patients with α-thalassemia from other racial groups. Hydrops fetalis due to homozygous α-thalassemia may not occur in the Negro because of the relatively mild thalassemic defect.
PMCID: PMC302140  PMID: 5061833
25.  Hemoglobinopathies in South Gujarat population and incidence of anemia in them 
Indian Journal of Human Genetics  2012;18(3):294-298.
OBJECTIVE:
To Screen of South Gujarat population for determination of prevalence of different hemoglobinopathies particularly beta thalassemia trait (BTT) and sickle cell trait (SCT) and find out the incidence of anemia in them.
MATERIAL AND METHODS:
The present study screened 32,857 samples of students from different school and colleges in South Gujarat. Blood samples were initially tested for solubility test and complete hemogram on hematology analyzer. Samples having MCV (≤78), MCH (≤28) and/or positive solubility test were investigated for Hb electrophoresis on cellulose acetate membrane (pH 8.6). Hb A2 level ≥3.5% was considered as diagnostic for BTT. High performance liquid chromatography on Biorad Hb variant system was done on samples having doubtful results.
RESULT:
Overall prevalence of BTT and SCT in South Gujarat was 4.4% and 1.3% respectively. Gamit, Vasava, Chaudhary, and Mahyavanshi castes had high prevalence of BTT (15.9%, 13.6%, 12.6%, and 6.9%) as well as SCT (22.2%, 15.2, 22.3, and 4.2%) respectively. Other communities like Lohana (10.8%), Sindhi (10.2%), Prajapati (6.3%), and Ghanchi (6.2%) also showed higher prevalence of BTT. Incidence of mild to moderate anemia was higher in BTT and SCT compared to non-BTT or non-SCT subjects.
CONCLUSION
Study suggests that BTT is the most prevalent hemoglobinopathy in South Gujarat. β-thalassemia and Sickle cell anemia are highly prevalent in Mahyavanshi, Chaudhary, Gamit, Vasava and Rohit. Prajapati, Lohana, Leva Patel, and Ghanchi have β- thalassemia risk. SCT is more frequently detected in Dhodia Patel and Kukanas.
doi:10.4103/0971-6866.107979
PMCID: PMC3656517  PMID: 23716936
Hb D; Hb E; hemoglobinopathy; sickle cell trait; South Gujarat; β-thalassemia trait

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