Normal HbA2 Levels Research Articles

S Estimation of HbA2 Alone Sufficient for Screening Beta Thalassaemia Carriers: A Case in Perspective

Beta thalassaemia is one of the most common inherited haemoglobinopathies, characterised by reduced or absent production of the beta globin chain. In India, the carrier frequency of thalassaemia is estimated to be 3-4%. The prevention of Beta thalassaemia is the best strategy, and this can be achieved through carrier screening and prenatal diagnosis. Carriers of beta thalassaemia can be easily identified using haematological parameters such as complete blood count and High Performance Liquid Chromatography (HPLC) for haemoglobin analysis. The characteristic findings observed in thalassaemia carriers include microcytosis, hypochromia, with a Mean Corpuscular Volume (MCV) of less than 80 fL and Mean Corpuscular Haemoglobin (MCH) of less than 28 pg. They also present with elevated levels of HBA2 (α2δ2) ≥3.5%. Carrier screening for beta thalassaemia primarily relies on the observation of elevated HbA2 levels. However, in rare cases, some carriers can have normal HbA2 levels, leading to false-negative screening results. In a case involving a married couple who underwent routine preconceptional screening by complete blood count and HPLC for thalassaemia screening, the male partner had elevated HbA2 levels (5.2%), while the female partner had normal HbA2 levels (1.6%). Molecular testing revealed that the male partner was heterozygous for the Intervening Sequence (IVS) 1-5 (G>C) mutation, while the female partner was found to be heterozygous for the CD41-42 (-CTTT) mutation. It is important to consider molecular testing of the HBB gene in couples, even if one partner is a carrier and the other partner has normal or borderline HbA2 levels.

Detection of BCL11A, HMIP, and XmnI Polymorphisms among Anemic Pregnant Women in Hospital Universiti Sains Malaysia

BACKGROUND: Anemia is one of the most common conditions in women during pregnancy. Fetal hemoglobin (HbF) levels are usually <1.0%. There are several genetic loci that have a significant influence on HbF levels. AIMS AND OBJECTIVES: The aim of this study is to determine the association of HbF level and DNA polymorphism at BCL11A rs1186868, HMIP rs9376090, and XmnI rs7482144 in anemic pregnant women. MATERIALS AND METHODS: Blood samples were collected from 164 anemic pregnant women at Obstetrics and Gynecology Clinic, Hospital Universiti Sains Malaysia. High-performance liquid chromatography was used to determine the HbF and HbA2 levels. RESULTS: Multiplex amplification-refractory mutation system-polymerase chain reaction (PCR) and gap-PCR were performed for 44 samples with a high HbA2 level (>3.2%) and normal HbA2 level (≤3.2%) to detect mutation at b-globin gene cluster. Allelic discrimination for rs1186868, rs9376090, and rs7482144 was performed using the real-time PCR technique for samples with no mutation detected. The mean age of patients was 31 ± 5.9 years. The HbF and Hb levels showed a strong positive correlation (r = 0.61). Out of 22 samples, 15 mutations at the b-globin gene and no mutation at the δβ-globin gene were detected. The mean difference of HbF level of patients with and without b-globin gene cluster was 1.07 ± 0.6 and 1.79 ± 3.5, respectively. The minor allele frequency (MAF) for the studied single nucleotide polymorphisms as follows: rs1186868 (MAF = 0%), rs9376090 (MAF = 19%), and rs7482144 (MAF = 32.8%). CONCLUSION: In conclusion, the baseline HbF levels were unaltered by BCL11A, HMIP, and XmnI genetic polymorphisms.

Co-Inheritance of Heterozygous β0-Thalassemia with Single Functional α-Globin Gene: Challenges of Carrier Detection in Pre-Marital Screening Program for Thalassemia

This is a report of a couple with abnormal hematological indices who were investigated for α & β-thalassemia mutations. Based on CBC and capillary hemoglobin electrophoresis results, the male and female subjects were β & α-thalassemia carriers, respectively. Multiplex-Gap-PCR and Sanger sequencing techniques were used for the identification of mutations on α and β-globin genes. The DNA test showed the presence of c.315 + 1 G > A mutation on β-globin gene of male subject while the female case had – MED double gene deletion and c.427T > C mutation on α-globin and, interestingly, she was also a carrier for c.315 + 1 G > A mutation on β-globin gene. Cases with the coinheritance of heterozygous β0-thalassemia with one functional α-globin gene have normal HbA2 levels that may lead to their being misdiagnosed as β-thalassemia carriers, especially in premarital screening programs for thalassemia. Therefore, β-globin gene sequencing is recommended in cases with normal Hb electrophoresis and reduced hematological indices in premarital screening programs for thalassemia, especially in regions with a high frequency of β-globin mutations, in order to identify all the β-thalassemia carriers.

Compound heterozygous alpha-thalassemia 3.7 kb deletion and hemoglobin adana: a case report

Introduction: A point mutation of codon 59 (GGC ➞ GAC) of the α2-globin gene, known as haemoglobin (Hb) Adana, contributes to various kinds of α-thalassemia syndrome. This case report described a patient who had heterozygous alpha-thalassemia 3.7 kb deletion and hemoglobin adana. Case report: We report a case of a 17-year-old boy who was referred for the investigation of persistent anemia. His peripheral blood film was consistent with mild hemolytic anemia, without HbH inclusions. Normal HbA2 levels without HbH and HbBart peaks. Both his parents had normal Hb levels, but his mother presented with mild microcytosis. DNA analysis revealed a compound heterozygote for one gene deletion (α-3.7) thalassemia and CD59 (GGC→GAC) mutation in the α2-globin gene. The mother was heterozygous for Hb Adana, and the father was heterozygous for α-3.7. Conclusion: This case report emphasizes the need to consider Hb Adana detection in the absence of HbH inclusions and normal Hb analysis. Therefore, DNA analysis is strongly suggested to confirm the diagnosis and improve the management of thalassemia patients.

A Compound Heterozygous HBB: C.79G>A Associated with HBB: C.-78A>G Mutation of HBB Gene Leading to Β-Thalassemia Intermedia in A Vietnamese Patient: A Case Study

β-thalassemia is an inherited blood disorder that is the most common single-gene disorder worldwide and in Vietnam. This study reports an intermedia β thalassemia patient in a family from Northern Vietnam. The mother has compound heterozygous thalassemia presenting mutation of hemoglobin HBB: c.79G>A associated with HBB: c.-78A>G. The result indicates that the determination of molecular characterization of subjects presenting normal HbA2 level associated with abnormal red cell indices is needed for accurate diagnosis and improved genetic counseling.

Elevated Hb A2 is Not Always Indicative of β-Thalassemia

Hb A2 levels are usually high in carriers of β-thalassemia (β-thal). These levels also provide a sensitive marker for the identification of hemoglobin (Hb) variants. In this study, we aimed to examine two patients from two Chinese families who showed elevated Hb A2 levels but did not show any signs of β-thal. The HBB variants were analyzed using direct sequencing of HBB and in silico prediction analysis. Moreover, the family’s genetic history was investigated. We examined two probands from different Chinese families with elevated Hb A2 levels who were not afflicted with β-thal, although several nucleotide changes were found at codon 81 (CTC>CTA) (HBB: c.246C>A) in Family 1 and a compound heterozygosity for codon 40 (AGG>AAG) (HBB: c.122G>A) and IVS-II-478 (C>A) (HBB: c.316-373C>A) in Family 2. After investigating the genetic history of both families including the β-thal aspect, we found that these mutations were not responsible for the elevated Hb A2 levels. It is rarely reported that high Hb A2 level is not indicative of β-thal. In contrast, low or normal Hb A2 level is always found with β-thal due to other molecular defects that mask their β-thal genotype. Our results highlight the importance of considering the genetic factors related and unrelated to β-thal to improve the accuracy of future genetic counseling.

β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia

Mutations in the β-globin gene cluster can lead to β-thalassaemia, δβ-thalassaemia, hereditary persistence of foetal haemoglobin (HPFH) and some of the haemoglobin variants. The clinical and haematological spectrum of thalassaemia range from benign to pathogenic conditions including severe transfusion dependent thalassaemia. Therefore, it is important to accurately diagnose β-globin gene cluster mutations to prevent thalassaemia major or intermedia offspring. The objective of this study is to detect β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia. This study involved 144 anaemic patients with HbF level >1.0%. High-Performance Liquid Chromatography (HPLC) was used to determine the HbF and HbA2 levels. Multiplex-ARMS (ARMS)-PCR and gap-PCR were performed for those patients with high HbA2 level (>3.2%) and normal HbA2 level (≤3.2%) to detect β-globin gene cluster mutations and large deletions respectively. The majority of patients were Malays (99.3%) with mean age of 19.99 ± 1.64 years and female 61.1% predominance. Out of 87 samples tested using multiplex ARMS-PCR against eight targeted single mutation; Cd 41/42, IVS 1–5, Cd 26, Cd 17, Cd 71/72, IVS 1–1, Cd 8/9 and -28 mutations, 65 (74.7%) patients were detected which comprises of Cd 26 (56.3%), Cd 41/42 (11.5%), compound Cd 26 and Cd 41/42 (3.4%) and IVS 1–1 (3.4%). Meanwhile, for multiplex gap-PCR which detect four types of large deletions; Thai (δβ)o-thalassaemia, HPFH-6, Siriraj J and Hb Lepore, one out of 57 patients (1.8%) was found positive with Thai (δβ)o-thalassaemia. There was a significant difference between the mean of HbF level, MCV level as well as MCH level of patients with and without β-globin gene mutations and large deletions (P<0.05). This study highlighted the presence of various types of β-globin gene cluster mutations detection in establishing a definitive diagnosis among this selected group of patients for the large-scale screening of the thalassaemia gene.

A Chinese Male with Normal Hematological Indices and High Hb A2 Levels in β-Thalassemia Trait

β-Thalassemia (β-thal) is a common hematological disorder in the Guangxi Zhuang Autonomous Region of Southern China. Heterozygous β-thal is usually associated with reduced hematological indices and increased Hb A2 levels. However, the preventive program of the regional government only conducts the screening of hematological indices [complete blood count (CBC)] especially at primary hospitals. In this study, we describe a male β-thal patient presenting normal hematological indices but with a high Hb A2 level. We proposed that hemoglobin (Hb) analysis and CBC should be performed together in the preventive screening program in regions with a high thalassemia incidence to avoid misdiagnosis.

Coexistence of hemoglobin Handsworth and alpha 3.7 kb deletion in Caucasian woman in Poland

AbstractBackgroundThis report presents a case of an adult Polish women of Caucasian origin who was heterozygous for the nondeletional mutation: Hb Handsworth (HBA2 or HBA1: c.55G > C, p.Gly19Arg) and deletional (-α3.7) α-thalassemia mutation.MethodsThe HbA2 and HbF levels were measured by microcolumn chromatography and alkaline denaturation procedure, respectively, while electrophoresis was used to detect pathological hemoglobin fraction. The β- and α-globin genotypes were determined by DNA sequencing, gap-polymerase chain reaction, α gene triplication and MLPA.ResultsThe HbA2 and HbF levels were normal, but hemoglobin electrophoresis on agarose gel alkaline pH showed a strong band migration in a position of hemoglobin S and faint bands in the neighborhood of band A on acid electrophoresis. Molecular analysis of the alpha globin cluster detected a point mutation at codon 19 in HBA2 (c.55G > C, p.Gl- y19Arg) and deletion -α3.7.ConclusionsOur compound heterozygosity does not produce severe clinical or hematological symptoms but it is important to say that in our part of Europe such cases do appear. Molecular analysis of the alpha globin cluster is required for correct diagnosis in patients with normal HbA2 levels. Compound heterozygosity was unmasked by molecular diagnosis only.

Gambaran Fraksi Hemoglobin Penderita Talasemia Menggunakan Metode Elektroforesis Kapiler

Thalassemia is a hereditary blood disease that is found in Indonesia. One method of examining modern thalassemia is the examination of hemoglobin fraction using the Capillary Electrophoresis. This method has a high level of accuracy and precision for quantification of hemoglobin variants.The purpose of this study was to determine the hemoglobin fraction in thalassemia patients using the Capillary Electrophoresis method. This type of research is descriptive research. The sample of this study was 3 blood specimens from thalassemia sufferers.The results showed that the first specimen, age 4 years had HbA levels of 59.9%, HbA2 levels of 4.3%, HbF levels of 14.7% and HbE levels of 21.1%. In the second specimen, 8 years of age had 88.7% HbA, 2.5% HbA2, 3.2% HbF and 5.6% HbE. In the third specimen, the age of 13 years had HbA levels of 93.8%, HbA2 levels of 4.9% and HbF levels of 1.3%.The conclusion of this study was the first specimen, HbA levels decreased, HbA2 levels increased and HbF levels increased and hemoglobin variants were found, namely HbE. In the second specimen, HbA levels decreased from the normal range, HbA2 levels were in the normal range and HbF levels increased and hemoglobin variants were found, namely HbE. In the third specimen, HbA levels decreased from the normal range, HbA2 levels and HbF levels increased, but no hemoglobin variants were found in this specimen.

The phenotypic and molecular diversity of hemoglobinopathies in India: A review of 15 years at a referral center

The hemoglobinopathies pose a significant health burden in India. Apart from the β thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15years. Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. The study includes 31075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 β thalassemia mutations. 143 β thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in β thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδβ)0 Indian inversion and the HPFH-3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (-α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of β thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon β chain variants were identified. The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India.

Phenotypes of Hb Okayama Mutation

Introduction:Since its first detection in the year of 1983 very little has been reported about Hb Okayama mutation. Hb Okayama, reported as a silent mutation (Globin Gene Server Hb Var ID: 220, dbSNP rs713040), happened to be detected in the process of HPLC analysis for the measurement of HbA1c in Japanese diabetic patient [1,2,3]. Till date only few Hb Okayama has been reported from Japanese and Austrian ethnicity [2,3,4]. Its phenotypes and co-inheritance with other beta globin gene mutation is not yet known. In this study we report phenotypes of Hb Okayama in heterozygous as well as compound heterozygous state when it is co-existing with a common beta globin gene mutation. To the best of our knowledge no such study has been reported in world literature.Methodology:Two groups of patients- beta carriers requiring blood transfusion; and patients with low MCV, MCH but normal in HPLC were further investigated by molecular analysis. ARMS PCR analysis for beta globin gene mutation detection, GAP PCR for alpha deletion and triplication analysis and DNA sequencing (ABI 3500 Genetic Analyzer) to identify rare beta mutations were carried out.Result:Among around 300 patients subjected to molecular investigation over last three years there were 118 cases of thalassaemia trait (by HPLC and confirmed to carry a heterozygous common beta mutations by ARMS PCR analysis), but were requiring blood transfusion or behaving like intermedia. They were subjected to alpha globin gene triplication analysis by GAP PCR. There were 84 cases found to carry absence of alpha triplication with heterozygous beta mutation. Beta globin gene sequencing analysis of these 84 patients revealed that there were 4 patients carrying Okayama heterozygous mutation [ Figure 1, Figure 2 ] along with a common heterozygous beta gene mutation ( IVS 1-5 G>C mutation). HPLC report of these patients carrying Hb Okayama along with IVS 1-5 mutations in compound heterozygous state showed increased HbA2 values like that of beta trait [Table 1 ]. Another group of patients with low MCV, MCH but Normal in HPLC were subjected to alpha deletion analysis after ruling out low Ferritin level. There were 72 patients showing absence of alpha deletion by GAP PCR analysis and carried low MCV, MCH value with Normal HPLC parameters were also subjected to beta globin gene sequencing which revealed the presence of Hb Okayama mutation among two patients in heterozygous state [Figure- 1, Figure 2], who had HbA2 values in normal or border line range with low MCV, MCH levels [Table-2].Discussion:HbA2 measurement is used as the marker for screening of beta trait. Silent beta-thalassaemia carriers represent normal HbA2 level which makes their identification difficult. Okayama mutation or Hb Okayama is known to be a silent mutation [1-4] with normal HPLC. Hb Okayama is structural beta variant with a change of amino acid ( His > Gln) at Codon 2 (CD 2). Change in the nucleotide sequence at 70603 position from T to A or C (CAT>CAA or CAG) of beta globin gene (NG_000007.3) causes this mutation. The phenotypic associations of Hb Okayama in thalassaemia have very little been known till date. There has been report of very high expression of HbF(70%) value in HPLC resulting from compound heterozygous mutations one of which being silent (Cap+1) [ 5] ; where as there are also reports where the phenotypes of compound heterozygous including a silent mutation showing normal HPLC parameters [ 6 ]. However, in those studies no information about the clinical history and blood transfusion is described. In this study Hb Okayama heterozygous co-inheriting with IVS1-5(G>C) heterozygous mutation showed beta trait like HPLC parameters though all the patients carrying these compound heterozygous mutations required blood transfusion. The silent feature of Hb Okayama was evident in the case of the patients carrying only Hb Okayama mutation who showed absolutely normal HPLC parameters with low MCV, MCH and none of them requiring blood transfusion.Conclusion:Beta globin gene expression analysis to understand the association of Hb Okayama mutation in heterozygous and compound heterozygous states will enable to explain the mechanism of its phenotypes. How this mutation interferes with the expression of HbF or switching of delta globin gene is also to be understood as the HbF levels in HPLC was found to be like beta traits in this study. DisclosuresNo relevant conflicts of interest to declare.

Molecular Characteristics of Hb New York [β113(G15)Val→Glu, HBB: c.341T>A] in Thailand

Hb New York or Hb Kaohsiung [β113(G15)Val→Glu (GTG>GAG), HBB: c.341T>A] has been considered a rare β hemoglobin (Hb) variant found originally in an Iranian woman and later in diverse populations but its genetic origin has not been elucidated. Here we report molecular and hematological descriptions of this variant found in the Thai population. Among 5643 subjects referred for hemoglobinopathy investigation during January 2015 to September 2017, 183 (3.2%) were found to carry several Hb variants, including β chain variants (n = 135, 2.4%), α chain variants (n = 33, 0.6%), Hb Lepore-Hollandia (NG_000007.3: g.63290_70702del) and Hb Lepore-Boston-Washington (NG_000007.3: g.63632_71046del) (δβ hybrid Hb) (n = 12, 0.2%) and δ chain variants (n = 3, 0.05%). Of patients with β chain variants, six with normal high performance liquid chromatography (HPLC) patterns, had an abnormal Hb in zone 11 of capillary electrophoresis (CE), the amounts of which ranged from 29.6-45.4% with normal levels of Hb A2 and Hb F. DNA analysis identified a heterozygous Hb New York mutation in all cases. Further screening of α-thalassemia (α-thal) identified coinheritance of α+- and α0-thal in two of them who had reduced levels of Hb New York. Haplotype analysis suggested that the Thai Hb New York was likely associated with a single β-globin haplotype [+ – – – – + +], indicating that it was of the same origin. Hematological findings and simple DNA assay based on allele-specific polymerase chain reaction (PCR) for rapid detection of Hb New York are presented.

Hb A2-Tianhe (HBD: c.323G>A): First Report in a Chinese Family with Normal Hb A2-β-Thalassemia Trait

Coinheritance of δ-globin variants along with β-globin gene defects can interfere with correct diagnosis of β-thalassemia (β-thal) trait. In this report, we present the coinheritance of a δ-globin variant, Hb A2-Tianhe [δ107(G9)Gly→Asp; HBD: c.323G>A] and a heterozygous β-thal in a Chinese individual with microcytosis, hypochromia and a normal Hb A2 level.

Phenotypic Evaluation of a Novel Nucleotide Substitution (HBD: c.442T>C) on the δ-Globin Gene

HBD: c.442T>C is a new mutation at the stop codon (TGA>CGA) of the δ-globin gene, which produces a new codon for arginine. This substitution causes a 51 nucleotides longer open reading frame determining the synthesis of a potential larger δ subunit, which is a probable target of mechanisms for the degradation of aberrant proteins as well as the defective synthesized mRNA molecules, and may also be rapidly degraded by a variety of RNA surveillance pathways. We identified this molecular defect in four patients: three women with a reduced HbA2 level and a 37-year-old male showing the typical phenotype of an α-thalassemia (α-thal) carrier with reduced values of red cell indices and normal HbA2 level (2.5%). The mutation on the δ-globin gene was found to have been coinherited with a β-globin gene defect leading to a normalized HbA2 level. These data support the necessity of investigating these cases at a molecular level, particularly if the partner is also a β-thalassemia (β-thal) carrier. The present data emphasizes the importance of a careful evaluation of correlation between genotypes resulting from DNA analysis and phenotypes, especially in cases of atypical hematological parameters, in order to carry out an adequate diagnostic process finalized to appropriate genetic counseling.

Systematic evaluation of paediatric cohort with iron refractory iron deficiency anaemia (IRIDA) phenotype reveals multiple TMPRSS6 gene variations.

Systematic screening identified patients with an iron refractory iron deficiency anaemia (IRIDA) phenotype and genotype in iron-deficient children in the Indian subcontinent. Cases of moderate to severe microcytosis and anaemia with no obvious cause and normal C-reactive protein, HbA2 and tissue transglutaminase antibody levels (n=550) were put on a trial of oral iron for 4weeks. Sixty of these 550 cases (11%) were variably refractory to oral iron therapy (<10g/l Hb rise) at 4-6weeks and were subsequently evaluated for plasma iron, ferritin and hepcidin levels. The mean age of this cohort was 2.06years. Low-normal to normal ferritin and normal to high hepcidin levels were noted in 25/60 (41.6%) and 47/60 (78.3%), respectively. An IRIDA phenotype was noted in 38.3% (23/60) based on standard criteria. TMPRSS6 gene sequencing in 20 cases with IRIDA phenotype revealed 9 potentially deleterious intronic and two benign exonic variations in 12/20 cases (60%). Of these, 4 intronic and both exonic variations were noted in multiple cases and are likely to act synergistically leading to an IRIDA phenotype. However, given that only 38% (23/60 cases) of cases with iron refractoriness had IRIDA phenotype, a balanced approach is needed and other causes for refractoriness should be investigated before genetic studies for TMPRSS6 are undertaken.

Molecular Study of Deletional and Nondeletional Mutations on the α-Globin Locus in the Azeri Population of Northwestern Iran

The aim of this study was to determine the molecular spectrum and frequency of deletional and nondeletional α-thalassemia (α-thal) mutations and the genotype–phenotype correlation in common mutations in the Azeri population of Northwestern Iran. A total of 1256 potential carriers with microcytic and hypochromic anemia and normal Hb A2 levels (<3.5%) and without iron deficiency anemia plus three fetuses were identified. Multiplex gap-polymerase chain reaction (gap-PCR) and sequencing for α-thal mutations were carried out. In 606 individuals, the α-globin gene was normal, but in 650 persons (51.6%) and three fetuses, 10 different mutations were detected. The most frequent deletional genotypes were as follows: αα/–α3.7 (61.7%), –α3.7/–α3.7 (11.9%), αα/–α4.2 (4.6%), αα/– –MED (4.3%) and αα/–(α)20.5 (3.8%). The most frequent nondeletional genotypes were αα/αIVS-I (–5 nt)α (HBA2: c.95+2_95+6delTGAGG) and αα/αPoly A2α [polyadenylation signal (polyA2) (AATAAA>AATGAA); HBA2: c.*96G>A] with frequencies of 1.08% and 0.92%, respectively. Meanwhile, 7.71% of individuals with a proven β-thalassemia (β-thal) mutation were found to also carry an α-thal mutation. Persons having two functional α-globin genes showed lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) values compared to those with one mutated α-globin gene, provided that they had normal β-globin genes. Overall, the incidence of α-thal was 2.7% in the Azeri population in Northwestern Iran. Our results showed that the variability of α-thal mutations are high in the Azeri population and that α-thal mutations are highly heterogeneous in both deletional and nondeletional genotype aspects.

Molecular analysis of complex cases of alpha- and beta-thalassemia in Mexican mestizo patients with microcytosis and hypochromia reveals two novel alpha(0) -thalassemia deletions - -(Mex1) and - -(Mex2).

Alpha-thalassemia (α-thal) is a common monogenic disorder worldwide. In mixed ethnic populations, α-thal and beta-thalassemia (β-thal) can be expected, sometimes giving complex phenotypes, which without molecular analysis are not easily explained. We performed the molecular identification of α- and β-thal alleles in 51 Mexican patients with microcytosis, hypochromia, and normal or low levels of HbA2 . Common deletional alleles (-α(3.7) , -α(4.2) , - -(SEA) , - -(MED) , - -(FIL) , - -(THAI) , -α(20.5) ) and α-triplication were studied by gap-PCR and nondeletional alleles (α(IVSI) ((-5nt)) , α2 (NcoI) , α1 (NcoI) ) by ARMS. β-thal alleles Cd39 (C>T), IVS1:1 (G>A), IVS1:110 (G>A), and Spanish δβ-thal were also investigated. DNA sequencing was performed on HBA2, HBA1, and HBB genes. Negative samples were subjected to MLPA. In 35 subjects, we identified the mutations, -α(3.7) , - -(SEA) , - -(FIL) , α(IVSI) ((-5nt)) , and ααα(anti3.7) and two novel deletion alleles - -(Mex1) (6.8-8.9 kb) and - -(Mex2) (77.6-135.7 kb). Four individuals also had a β-thal allele (Cd39/IVS1:110). No α-thal alleles were observed in 16 subjects, but three had a β-thal mutation Cd39, IVS1:110, and Spanish δβ-thal. α-thal is relatively common in Mexican patients, the combination with β-thal is sometimes unexpected, and this underlines the importance of performing molecular analysis for both α- and β-genes defects in patients showing microcytic hypochromic anemia.

A New δ-Globin Gene Variant: Hb A2-Fengshun [δ121(GH4)Glu→Lys (HBD: c.364G > A)

An elevated Hb A2 (α2δ2 level) is a diagnostic marker for heterozygous β-thalassemia (β-thal). Mutations in the δ-globin gene can cause decreased expression of Hb A2, compromising screening for heterozygous β-thal. In this report, we describe a novel missense mutation of the δ-globin [Hb A2-Fengshun or δ121(GH4)Glu→Lys, HBD: c.364G > A] in a Chinese individual who had coinherited a heterozygous β-thal with a normal Hb A2 level.

Identification and molecular characterization of a novel 163 kb deletion: The Italian (ϵγδβ)0-thalassemia

Objective and importance: To verify the presence of β-thalassemia in subjects showing hematologic phenotype of α-thalassemia, conduct normal molecular sequence analysis of the α-globin genes, and detect the absence of the most frequent α-thalassemia deletions.Clinical presentation: A patient from Apulia (Southern Italy) was referred to our institution for the occasional founding of hypochromic polyglobulia and microcytic red blood cells associated with normal levels of Hb A2 and Hb F and normal iron parameters.Intervention and technique: The patient has been investigated using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), quantitative real-time PCR, restriction analysis, and gap-PCR. A novel deletion, the Italian (ϵγδβ)0-thalassemia, has been identified. The 5′ breakpoint was within a LINE element of 80 kb 3′ of the ε-globin gene, and the 3′ breakpoint was within a 160-bp palindrome of about 30 kb 5′ of the β-globin gene. The breakpoint region was characterized by the presence of a microhomology (5′-TCT-3′) and of an insertion of 43 bp owing to the duplication of the 160-bp palindrome. Comparison of the Hb and Hb A2 values of (ϵγδβ)0-thalassemia from the literature with those of (molecularly known) thalassemia carriers indicated a higher level of Hb A2 with respect to α-thalassemia and a lower level of Hb with respect to β0-thalassemia carriers.Conclusion: In this study, we report the first (ϵγδβ)0-thalassemia case identified in Italy. To avoid misdiagnosis of β-thalassemia, we suggest verifying the presence of large deletions of the β-globin gene cluster in subjects showing a higher border line level of Hb A2 and a lower level of Hb.