Molecular characterization of a novel homozygous deletion in ß-globin cluster causing (δß)0-Thalassemia among Tunisian family.

BACKGROUND: Deletions in the ß-globin cluster are uncommon and cause thalassemia (thal) with hereditary persistence of fetal hemoglobin. They constitute a heterogenous group of disorders characterized by absent or reduced synthesis of adult hemoglobin (Hb A) and increased synthesis of fetal hemoglobin (Hb F). Although the clinical severity of these disorders are asymptomatic owing to the increased Hb F levels, the molecular basis is very heterogenous due to the large deletions in the ß-globin cluster spanning both HBD and HBB genes. Here, we describe a Tunisian family carrying a novel deletion mutation causing (δß)°-thalassemia. METHODS: The amounts of hemoglobin fractions were measured by capillary electrophoresis of hemoglobin. Amplification and sequencing of different regions on the ß-gene cluster were performed by Sanger method. RESULTS: Family study and genetic analysis revealed a large deletion mutation in the ß-globin cluster of 14.5 kb (NG_000,007.3:g. 58,253 to g.72837del14584) at the homozygous state in the patient and at heterozygous state at the other members of the family. This deletion removes the HBD and HBB genes. CONCLUSIONS: In our knowledge, this new large deletion is described for the first time in the Tunisian population and in the world, designed Tunisian(δß)0 in Ithanet database (IthaID: 3971). Therefore, it is important to identify the deletion leading to δß-thalassemia carriers at the molecular level, to highlight the importance of recognizing the clinical features and implementing appropriate testing to clarify the diagnosis and manage the condition.

Genotypic-phenotypic heterogeneity of 뫧-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) in India.

Large deletions in the ß-globin gene cluster lead to increased HbF levels by delaying the γ- to ß-globin switch process. However, these deletions when inherited as a homozygous condition or when co-inherited with ß-thalassemia result in variable clinical phenotypes. Individuals or families with a clinically presenting child, where the parents had HbF levels ≥ 10%, were further screened for the presence of large ß-globin cluster deletions. Six deletions in the ß-globin gene cluster were screened by GAP-PCR, and the uncharacterized deletions were further analyzed by gene dosage or by multiplex ligation-dependent probe amplification (MLPA). Among 192 individuals suspected for the inheritance of large deletions, 138 were heterozygous for large deletions, 45 were compound heterozygous of a large ß-globin cluster deletion and ß-thalassemia, and 9 were found to be homozygous for deletions. Among the heterozygotes, the Asian Indian inversion-deletion was found to be the most common deletion (39.9%), followed by the HPFH-3 deletion (30.0%). Other deletions 49.3 kb, δß-thalassemia (21.2%), and 32.6 kb deletion (4.4%) were also found to be prevalent in our population. Patients compound heterozygous or homozygous for HPFH-3 and 32.6 kb deletions showed a milder clinical presentation, as compared with the patients compound heterozygous or homozygous for the Asian Indian inversion-deletion and 49.3 kb δß-thalassemia. This comprehensive study highlights the mutation spectrum of large ß-globin cluster deletions and the clinical heterogeneity in the patients homozygous or compound heterozygous with ß-thalassemia, thus asserting the need for molecular characterization of these deletions.

Molecular epidemiology and hematologic characterization of 뫧-thalassemia and hereditary persistence of fetal hemoglobin in 125,661 families of greater Guangzhou area, the metropolis of southern China.

BACKGROUND: Individuals with δß-thalassemia/HPFH and ß-thalassemia usually present with intermedia or thalassemia major. No large-scale survey on HPFH/δß-thalassemia in southern China has been reported to date. The purpose of this study was to examine the molecular epidemiology and hematologic characteristics of these disorders in Guangzhou, the largest city in Southern China, to offer advice for thalassemia screening programs and genetic counseling. METHODS: A total of 125,661 couples participated in pregestational thalassemia screening. 654 subjects with fetal hemoglobin (HbF) level ≥ 5% were selected for further investigation. Gap-PCR combined with Multiplex ligation dependent probe amplification (MLPA) was used to screen for ß-globin gene cluster deletions. Gene sequencing for the promoter region of HBG1 /HBG2 gene was performed for all those subjects. RESULTS: A total of 654 individuals had hemoglobin (HbF) levels≥5, and 0.12% of the couples were found to be heterozygous for HPFH/δß-thalassemia, including Chinese Gγ (Aγδß)0-thal, Southeast Asia HPFH (SEA-HPFH), Taiwanese deletion and Hb Lepore-Boston-Washington. The highest prevalence was observed in the Huadu district and the lowest in the Nansha district. Three cases were identified as carrying ß-globin gene cluster deletions, which had not been previously reported. Two at-risk couples (0.0015%) were required to receive prenatal diagnosis. We also found 55cases of nondeletional-HPFH (nd-HPFH), including 54 with Italian nd-HPFH and one with the Aγ-197C-T heterozygous state. It is difficult to discriminate between Chinese Gγ (Aγδß)0-thal and Italian nd-HPFH carriers using hemoglobin (Hb) analysis. CONCLUSIONS: This study is the first to describe the familial prevalence of HPFH/δß-thalassemia and the high-risk rate in Greater Guangzhou Area, and the findings will support the implementation of thalassemia screening for three common deletions by gap-PCR. We also presented a systematic description of genotype-phenotype relationships which will be useful for genetic counseling and prenatal diagnostic services for ß-thalassemia intermedia.

Clinical variability and molecular characterization of Hbs/Gγ (Aγδß)0-thal and Hbs/HPFH in Indian sickle cell disease patients: AIIMS experience.

INTRODUCTION: In sickle cell disease (SCD) patients, among the predictors of survival, HbF levels play a significant role in lowering the morbidity and mortality. Coinheritance of 뫧 thalassemia and hereditary persistence of fetal hemoglobin (HPFH) may contribute to variable HbF levels in SCD patients, thus influencing their clinicopathological profile. Such cases are sparsely documented in the literature and thus, we screened the presence of 뫧 thalassemia and HPFH in 126 cases of SCD with high HbF. MATERIAL AND METHODS: A total 126 SCD individuals with raised HbF levels were the study subject. Capillary zone electrophoresis (CZE) was done for the quantitative assessment of hemoglobin variants. HbSC, HbSD, HbAS and HbSE cases were excluded. Asian Indian Gγ(Aγδß)0-thal, δß0-thal (Sicilian, 13.4 kb), (Chinese, 100 kb), HPFH-1 (Black, 106 kb), HPFH-2 (Ghanaian, 105 kb), HPFH-3 (Indian, 48.5 kb) were done by GAP-PCR. RESULTS: Out of 126, 78 cases (62%) were homozygous for SCD. The remaining 48 cases suspected to be heterozygous were furthered screened and 6/48 cases (12.5%) were found to be compound heterozygous. Out of these 6 cases,4(66.66%) had HbS/ δß- Gγ(Aγδß)0 and 2(33%) had HbS/HPFH compound heterozygous condition. None of the patients had δß0-thal (Sicilian, 13.4 kb), (Chinese, 100 kb), HPFH-1 (Black, 106 kb), HPFH-2 (Ghanaian, 105 kb). CONCLUSION: This study highlights the importance of understanding the complex patho-physiology of compound heterozygous cases of HbS/HPFH and HbS/뫧 thalassemia, as these infrequent conditions lead to change in phenotype and clinical severity of the disease. Insight into more such cases will open the window to better analyze the disease pathogenesis in these rare compound heterozygous conditions, as this will be beneficial to formulate proper management protocol in these patients.

Haemoglobinopathies that occur with decreased HbA2 levels: a gene mutation set involving the δ gene at a Spanish centre.

AIMS: Haemoglobin A2 (HbA2) consists of two globin chains, α and ß. Alterations in any of these genes influences the level of HbA2. Here, we present cases of structural Hb variants and thalassaemias which present either alone or together and reduce the level of HbA2 at varying degrees. Furthermore, we present a novel structural mutation in the δ globin gene, called Hb A2-Madrid. METHODS: The levels of HbA2 and HbF and the different haemoglobin variants were measured and analysed by ion exchange high performance liquid chromatography (HPLC, VARIANT II), the types of haemoglobins were determined by capillary zone electrophoresis (CZE) (Sebia) and the globin chains were determined by reversed-phase HPLC. Genetic analysis was performed by automatic sequencing of the α and δ genes as well as by multiple PCRs for the α globin genes. RESULTS: In α thalassaemia (n=94), the HbA2 levels ranged from 1.39% to 2.43%. Among individuals with δ thalassaemia (n=5), the HbA2 level of those with δ+ thalassaemia was 1.77%, and that of those with δ0 thalassaemia was 1.70%. Among the individuals with 뫧 thalassaemia (n=13), those who were homozygous lacked HbA2. All structural haemoglobinopathies (n=97) were heterozygous; the α chain variants (n=84) presented with an HbA2 level of 1.76%, while the δ chain variants (n=13) presented with a level of 1.75%. CONCLUSION: HbA2 is an essential parameter in the diagnostics of haemoglobinopathies. HPLC-EC and CZE allow the quantification of HbA2. Here, we show that quantification of HbA2 is critical for the identification of α, δ and ßδ thalassaemias. Structural variants are discovered by HPLC. Molecular genetics is required for the proper identification of the mutations. Only with this knowledge is genetic counselling possible.

The New -474(C→T) Substitution Discovered in the HBG2 Promoter of a Sardinian δß-Thalassemia Carrier.

During a screening for hemoglobinopathies, we found a carrier of the Sardinian δß-thalassemia condition. The proband's hematology and hemoglobin (Hb) profile agreed with those of the other carriers previously identified during our diagnostic program except for the fetal Hb (HbF) composition, which consisted of both α2Aγ2 and α2Gγ2 instead of nearly 100% α2Aγ2. In order to explain the unusual γ-chain ratio, sequencing of the Gγ promoter was carried out and revealed two nucleotide substitutions in cis: C→T at position -474 and A→G at position -309 from the Cap site. The latter had previously been observed in subjects with raised HbF levels, although it has not yet been evaluated at functional level. We used the luciferase assay to determine whether the two mutations modify the transcriptional activity of the Gγ promoter. Results indicated that the observed in vivo Gγ-globin production cannot be translated into increased in vitro promoter function, suggesting that the assessed mutations cannot be considered as functional single nucleotide polymorphisms per se; instead, a more complex regulatory mechanism might be involved.

Etiopathological mechanisms and clinical characteristics of hyperhemolysis syndrome in Spanish patients with thalassemia.

Hyperhemolysis syndrome (HHS) is characterized by severe intravascular hemolysis with a decrease in the reticulocyte count, which is triggered and aggravated by transfusion and cannot be explained by standard immunohematological studies. A nationwide study was conducted in order to retrospectively identify thalassemia patients with HHS in Spain in order to assess pre-disposing mechanisms for this syndrome. For this, the expression of adhesion (CD49, CD36) and complement-related molecules (C3a, CD59) and the levels of reticulocyte apoptosis and macrophage activation were measured in 4 thalassemia patients with HHS, 14 patients without HHS, and 10 healthy subjects. Five of the six thalassemia patients had δß-thalassemia. The patients were not alloimmunized prior to the syndrome, which was developed after the first transfusion in all but one case. Patients with δß-thalassemia did not respond to corticoids or immunoglobulins; only splenectomy was successful. The expression of CD49 (α4ß1 integrin) was far higher in patients who had experienced HHS (85.07 ± 18.46 vs. 46.28 ± 24.31; p < 0.01), and the difference remained significant after correcting by the number of molecules analyzed (Bonferroni p < 0.05). In our population, δß-thalassemia was the most common hemoglobinopathy in patients with HHS. Furthermore, the risk to develop this syndrome may be associated with an increased expression of α4ß1 integrin.

Reticulocyte parameters of delta beta thalassaemia trait, beta thalassaemia trait and iron deficiency anaemia.

AIMS: To analyse the differences in reticulocyte indices between delta beta thalassaemia trait (δß-TT), beta thalassaemia trait (ß-TT) and iron deficiency anaemia (IDA), and to correlate those differences with the physiopathological features of these three types of microcytoses. METHODS: We performed a descriptive study of 428 samples (43 δß-TT, 179 ß-TT and 206 IDA) that were run on Advia 2120 analyser (Siemens). The following reticulocyte indices were assessed: absolute reticulocyte count (ARC), percentage of reticulocytes, mean corpuscular volume of reticulocytes (MCVr), haemoglobin content of reticulocytes (CHr), mean corpuscular haemoglobin concentration of reticulocytes, red blood cell distribution width of reticulocytes (RDWr), haemoglobin distribution width of reticulocytes (HDWr) and reticulocyte subpopulations based on their fluorescence according to mRNA (low (L-R), medium (M-R) and high (H-R)), MCV ratio and MCHC ratio. Correlation between fetal haemoglobin (HbF) and RDWr in patients with thalassaemia was evaluated. RESULTS: RDWr was significantly higher in δß-TT compared with ß-TT (15.03% vs 13.82%, p<0.001), and so were HDWr (3.65% vs 3.27%, p<0.001), CHr (23.68 vs 22.66 pg, p<0.001) and MCVr (88.3 vs 85.5 fL, p<0.001). A good correlation was observed between HbF and RDWr (r=0.551, p<0.001). IDA subjects have more immature reticulocytes, but less ARC than ß-TT, suggesting a certain degree of inefficient erythropoiesis in IDA in comparison with ß-TT. CONCLUSIONS: Previously described differences between δß-TT, ß-TT and IDA in the corpuscular indices of mature red blood cell can also be observed in reticulocytes. The degree of anisocytosis in reticulocytes from patients with thalassaemia is correlated with HbF.

[Delta°-thalassemia by insertion of 27 base pairs in δ-globin gene with decreased hemoglobin A2 levels]. / Delta(0)-talasemia por inserción de 27 pares de bases en el gen δ-globina con descenso de los valores de hemoglobina A2.

BACKGROUND AND OBJECTIVE: We describe a novel delta-thalassemia mutation causing decreased hemoglobin (Hb) A2 levels associated with Hb Watts, variant Hb resulting from a trinucleotide deletion in Spain. PATIENTS AND METHOD: Hb variant analysis was performed by cation-exchange high performance liquid chromatography (HPLC) and capillary zone electrophoresis. Polymerase chain reaction and DNA sequence analyses were used to identify mutations in the δ- and α-globin genes. RESULTS: Abnormal Hb was observed on capillary zone electrophoresis in Z6 and by cation-exchange HPLC a slower peak than HbA was observed at an retention time of 4.19min. This variant Hb is called Hb Watts [α2 74(EF3)Asp->0 or α2 75(EF4)Asp->0; HBA2:c.226_228delGAC]. The decreased HbA2 percentage owes to an insertion of 27nt between nt 83 and 84 of IVS-I of the δ-globin gene. CONCLUSIONS: When analyzing a chromatogram, the possibility of the existence of delta-thalassemia or an HbA2 variant should be considered, apart from alfa-, beta-thalassemia and structural haemoglobinopathies. To this end, each of the peaks and their percentages should be considered to allow for correct interpretation and to avoid misdiagnosis as much as possible.

δß-Thalassemia trait: how can we discriminate it from ß-thalassemia trait and iron deficiency anemia?

OBJECTIVES: To analyze the differences not only in classic hematologic parameters but also in RBC subpopulations among δß-thalassemia trait (δß-TT), ß-thalassemia trait (ß-TT), and iron deficiency anemia (IDA) and to evaluate the role of fetal hemoglobin (HbF) in elevated RBC distribution width (RDW). METHODS: Samples from 553 patients with microcytosis (74 δß-TT, 272 ß-TT, and 207 IDA) were run on an Advia 2120i analyzer (Siemens Medical Solutions Diagnostics, Tarrytown, NY). Classic hematologic parameters and RBC subpopulations were assessed. The correlation between HbF and RDW in patients with thalassemia (both ß and δß) was evaluated. An independent sample t test was used to compare classic hematologic parameters and RBC subpopulations among ß-TT, IDA, and δß-TT and receiver operating characteristic curves performed in the significant comparisons. RESULTS: RDW was significantly higher in δß-TT compared with ß-TT (18.79% vs 16.04%, P < .001), as was mean corpuscular volume (66.39 vs 64.82 fL, P < .001), mean corpuscular hemoglobin (20.73 vs 20.04 pg, P < .001), and mean corpuscular hemoglobin concentration (31.16 vs 30.66 g/dL, P = .03). Pearson coefficient showed a good correlation between HbF and RDW. The values obtained for all the parameters were significantly different (P < .001) between patients with thalassemia (ß and δß) and IDA. CONCLUSIONS: RDW is the best parameter to discriminate δß-TT from ß-TT. The degree of anisocytosis in patients with ß-TT and δß-TT is strongly correlated with HbF.

Known and new δ-globin gene mutations and other factors influencing Hb A2 measurement in the Omani population.

Although δ-thalassemia (δ-thal) is not categorized as a severe disease, it is essential to know the molecular spectrum of the δ gene mutations frequently occurring in specific areas, particularly if these areas are characterized by a high rate of ß-thalassemia (ß-thal) such as Oman. This is because coinherited δ-globin gene defects can interfere with the basic diagnosis of a ß-thal carrier when this is based upon the measurement of the Hb A2 only. Because of that, we have investigated 33 patients with low Hb A2 levels, collected from different hospitals in Oman. Some cases had a second Hb A2 fraction, while others had only significantly lower Hb A2 levels. Among these patients, 20 did carry a δ-globin gene mutation, the rest were carriers of α thalassemia (α-thal) defects or could be iron depleted or both. In total, eight different known mutations and two novel δ variants were found. The characterization of the δ-globin gene mutation spectrum will improve carrier diagnostics and genetic counseling in the Omani population screened for ß-thal.

εGγAγδß0-thalassemia: a rare but clinically significant cause of hemolytic anemia in infants.

Normal hemoglobin is made of a tetramer of 2 α-globin and 2 ß-globin polypeptide chains. Deletions in the ß-globin gene cluster can range from a few hundred base pairs to loss of the entire cluster resulting in rare, but clinically significant, thalassemias. One such entity is εGγAγδß0-thalassemia, a condition that presents within the first few weeks of life as a Coombs-negative hemolytic anemia and is not identified on routine newborn screening or hemoglobin electrophoresis.

Erythrocytosis in a child due to Hb Andrew-Minneapolis [ß144(HC1)Lys→Asn (AAG>AAT or AAC)] associated with a Spanish (δß)(0)-thalassemia.

We report a rare association of δß-thalassemia (δß-thal) and a hemoglobin (Hb) variant with high oxygen affinity in a Spanish newborn. The proband had no Hb A and showed microcytosis and hypochromia; the peripheral blood smear was compatible with a thalassemia trait. Molecular studies revealed that the proband had a Spanish (δß)(0)-thal (inherited from his father) and also carried a de novo variant (Hb Andrew-Minneapolis) because from the point of hematology, his mother was quite normal. The hemoglobinopathies with high affinity for oxygen constitute an infrequent cause of secondary congenital erythrocytosis. The degree of erythrocytosis and the resulting clinical manifestations are highly variable, depending on the degree of altered oxygen affinity and the presence of thalassemic genes. Thus, when these variants are associated with ß(0)- or δß-thal, as in our case, the proportion of abnormal Hb is ∼100.0%, which may cause polycythemia, hyperviscosity, and iron deficiency. This type of association is very rare and few have been described, especially in children, as they would normally be detected in adults as the increased packed cell volume (PCV) also increases blood viscosity and causes the typical symptoms (cephalalgia, drowsiness, dizziness). The association of a high oxygen affinity Hb and a δß-thal presents a greater degree of erythrocytosis than when this same variant is associated with a ß(0)-thal, mainly because the Hb F percentage is usually greater in the δß-thal, and Hb F normally shows a greater affinity for oxygen and a reduced P(50), although one must always take into account the degree of oxygen affinity of the Hb variant. Familial erythrocytosis and an abnormal electrophoresis finding are indicative of a high affinity Hb. However, the absence of these findings does not reject the possibility of hemoglobinopathies, and in these cases, functional and molecular studies would be justified and should be mandatory for the differential diagnosis of erythrocytosis.

Co-inheritance of compound heterozygous Hb Constant Spring and a single -alpha(3.7) gene deletion with heterozygous deltabeta thalassaemia: a diagnostic challenge.

Haemoglobin Constant Spring (Hb CS) mutation and single gene deletions are common underlying genetic abnormalities for alpha thalassaemias. Co-inheritance of deletional and non-deletional alpha (alpha) thalassaemias may result in various thalassaemia syndromes. Concomitant co-inheritance with beta (beta) and delta (delta) gene abnormalities would result in improved clinical phenotype. We report here a 33-year-old male patient who was admitted with dengue haemorrhagic fever, with a background history of Grave's disease, incidentally noted to have mild hypochromic microcytic red cell indices. Physical examination revealed no thalassaemic features or hepatosplenomegaly. His full blood picture showed hypochromic microcytic red cells with normal haemoglobin (Hb) level. Quantitation of Hb using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) revealed raised Hb F, normal Hb A2 and Hb A levels. There was also small peak of Hb CS noted in CE. H inclusions was negative. Kleihauer test was positive with heterocellular distribution of Hb F among the red cells. DNA analysis for alpha globin gene mutations showed a single -alpha(-3.7) deletion and Hb CS mutation. These findings were suggestive of compound heterozygosity of Hb CS and a single -alpha(-3.7) deletion with a concomitant heterozygous deltabeta thalassaemia. Co-inheritance of Hb CS and a single -alpha(-3.7) deletion is expected to result at the very least in a clinical phenotype similar to that of two alpha genes deletion. However we demonstrate here a phenotypic modification of alpha thalassemia presumptively as a result of co-inheritance with deltabeta chain abnormality as suggested by the high Hb F level.

Evaluation of HPFH and 뫧-thalassemia mutations in a Brazilian group with high Hb F levels.

Fetal hemoglobin (Hb F) is characteristic of the fetal development period. However, in some genetic conditions, such as hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia (뫧-thalassemia), Hb F continues to be produced in adulthood. We evaluated the frequency of two mutations of HPFH, HPFH-1 and HPFH-2 African, and two mutations in 뫧-thalassemia, Sicilian and Spanish, in a Brazilian population. Peripheral blood samples were collected from adults from hospitals and blood centers in southeast and northeast Brazil. These individuals were healthy and without complaints of anemia, but had increased Hb F. Samples were submitted to electrophoretic and chromatographic analyses to quantify Hb F values and, subsequently, to molecular analyses to verify the mutations. In the molecular analysis, 16 of the 60 samples showed a heterozygous profile for the HPFH mutations, two for HPFH-1 and 14 for HPFH-2. In the same sample set, three were heterozygous for Spanish 뫧-thalassemia and none were heterozygous for Sicilian 뫧- thalassemia. The Hb F values in the HPFH-2 heterozygotes differed from those previously reported for this mutation. In this group, the HPFH mutations were more frequent than the 뫧-thalassemia mutations. The finding of these mutations in this Brazilian population reflects the mixing process that occurred during its formation.

Co-inheritance of ß- and δ-thalassemia compromising prenatal screening in a Chinese couple seeking prevention.

Although δ-globin gene mutations have no clinical implications, association of ß- and δ-thalassemia may lead to misdiagnosis. We describe the case of a Chinese woman who was assumed to have an α-thalassemia heterozygote, but was later shown to have ß-thalassemia with a normal amount of HbA(2) caused by the -77 mutation of the δ-globin gene. This study highlights the importance of considering δ-thalassemia during ß-thalassemia screening to avoid false-negative results in the detection of at-risk couples.

First report on the co-inheritance of beta-globin IVS-I-5 (G-->C) thalassemia with delta globin CD12 {Asn-->Lys (AAT-->AAA)}HbA2-NYU in Iran.

BACKGROUND: Co-inheritance of ß- and δ-globin mutations in Iran is not uncommon. This situation may interfere with correct diagnosis and genetic counseling of α- and ß-thalassemia in screening programs. Here we report the co-inheritance of ß- and δ-globin gene mutations in an individual with microcytosis, hypochromia and a normal hemoglobin A2 (HbA2) level. METHODS: Genomic DNA extraction, amplification refractory mutation system (ARMS) polymerase chain reaction and direct DNA sequencing of δ- and ß-globin genes were exploited for detection of the mutations in these two genes in an individual with low hematological indices and normal HbA2. RESULTS: ARMS-PCR technique revealed the ß(+) IVSI-5 (G to C) mutation and direct DNA sequencing of the δ-globin gene detected a previously reported delta codon 12 (AAT-->AAA) HbA2-NYU. This study reports HbA2-NYU in association with the ß IVSI-5 (G to C) mutation in Iran. DISCUSSION: This report emphasizes that normal HbA2 expression in a ß-goblin carrier is due to mutation in the δ-globin gene and may cause misdiagnosis of thalassemia.

Identification of a novel δ-globin gene mutation in an Iranian family.

δ-Thalassemia (δ-thal) has no clinical symptoms, but its coinheritance with ß-thal may cause misdiagnosis, especially in countries with a high prevalence of ß-thal where prevention programs have been implemented. The molecular basis of most ß-thal syndromes have been defined, while the spectrum of mutations causing δ-thal have not been well characterized. A couple was referred to us for thalassemia molecular screening. Since she had rather low values of Hb A2 and normal Hb F, her δ-globin gene was amplified and directly sequenced. We found two different mutations on her δ-globin genes: HBD: c.92+5G>T/HBD:c.428C>A. The c.92+5G>T mutation has not been previously reported. Two different mutations in trans may explain the reduced Hb A2 level.

Genotype-phenotype relationship of the δ-thalassemia and Hb A(2) variants: observation of 52 genotypes.

The increase of Hb A(2) (α2δ2) beyond the upper limit [2.0-2.2/3.3-3.4% of the total hemoglobin (Hb)] is an invaluable tool in the hematological screening of ß-thalassemia (ß-thal) carriers. Factors decreasing Hb A(2) percentages can hinder correct diagnosis. In order to analyze the genotype-phenotype relationship, we characterized δ-, ß- and α-globin genotypes in 190 families where the probands had Hb A(2) values of ≤2.0% or were ß-thal heterozygotes with normal Hb A(2) levels. Hb A(2) was measured with cation exchange high performance liquid chromatography (HPLC). Mutations were detected with allele-specific methods or DNA sequencing; two multiplex-ARMS (amplification refractory mutation system) assays were set up. The molecular basis underlying the decrease in Hb A(2) was extremely heterogeneous. Nineteen δ-globin alleles (Hb A(2)-S.N. Garganico was new) were detected; their interaction with α- or ß-globin alleles (10 and eight, respectively) led us to observe 52 genotypes in 261 carriers. The type of δ-globin mutations, the relative genotypes, the interaction with α(0)-thal traits, are the most important factors in decreasing the Hb A(2) percentage. These results are extremely useful in addressing the molecular diagnosis of hemoglobinopathies and thalassemias.

Molecular mechanisms underlying thalassemia intermedia in Iran.

To improve the differentiation of thalassemia intermedia from other hemoglobinopathies in Iran, four known genetic mechanisms-XmnI (G)gamma polymorphism, inheritance of mild and silent beta-thalassemia alleles, delta beta deletion, and coinheritance of alpha- and beta-thalassemia-were investigated in 52 Iranian individuals suspected to have thalassemia intermedia based on clinical and hematological characteristics. Beta-globin mutations were studied using a reverse-hybridization assay and sequencing of the total beta-globin gene. The XmnI (G)gamma polymorphism, the Sicilian delta beta deletion, and four alpha-globin mutations (-a(3.7), -a(4.2), -(MED), aaa(anti-3.7)) were studied using PCR-based techniques. The inheritance of the XmnI (G)gamma polymorphism with severe beta-thalassemia alleles in the homozygous or compound heterozygous state was the predominant mechanism observed in 27 individuals (55.3%). In five cases, this status overlapped with the -a(3.7)/aa genotype. The second most frequent cause for thalassemia intermedia (14.8%) was the inheritance of mild beta-thalassemia alleles, including IVS-I-6 (T > C), -88 (C > A), and + 113 (A > G). In three subjects (4.3%) the Sicilian delta beta deletion was identified. HbS in association with beta-zero-thalassemia was found in three patients with thalassemia intermedia phenotype. In 11 cases (21.3%) no causative genetic alteration could be identified. Our results reflect the diversity underlying thalassemia intermedia, and the limitations of the applied clinical, hematological, and molecular approaches for correct diagnosis. Some of the unresolved cases will offer an opportunity to discover additional molecular mechanisms leading to thalassemia intermedia.