Abstract
Asian Indian inversion deletion Gγ (Aγδβ)0-thalassemia is a rare entities characterized by high HbF. Due to interaction with various genetic factors, patients with Gγ (Aγδβ)0-thalassemia showed clinical variability. Here we are presenting the phenotypic expression of Gγ(Aγδβ)0 thalassemia under influence of various co-inherited factors. Patient with α-globin gene deletion had mild phenotype than the patient with β-globin mutations. Patient with alpha gene deletion were presenting clinical character like thalassemia intermedia while Gγ (Aγδβ)0-thalassemia patients with co- presence of beta thalssemia mutation clinically behaved like thalassemia major.
Keywords: Thalassemia, PCR, Delta beta thalassemia, Gap-PCR
Introduction
Delta beta (δβ) thalassemia is a heterogeneous disorders characterized by increased levels of HbF in adult life. Several types of (δβ)0 thalassemia have been characterized on the basis of molecular defects [1]. Two type of (δβ)0 thalassemia (namely, GγAγ(δβ)0 and Gγ(Aγδβ)0 have been reported in the Asian population. The Gγ (Aγδβ)0-thalassaemias are due to deletions or rearrangements that abolish expression from the Aγ, δ, and β-globin genes while leaving the Gγ-globin gene intact. Ten different Gγ (Aγδβ)0-thalassaemia deletions and rearrangements have been reported in a wide range of ethnic groups [2]. Co-inheritance of δβ-thalassemia and β-thalassemia usually results in a clinical phenotype of thalassemia intermedia because of the high HbF levels [3]. δβ-thalassemia homozygotes have hypochromic microcytic red cells with variable severity ranging from mild anemia to thalassemia major but generally milder than homozygous β-thalassemia, where there is little compensatory γ-globin chain production [4–7]. Heterozygotes for δβ-thalassemia tend to have a modest elevation of HbF (5–20 %) with hypochromic microcytic red cell indices. Individuals with these disorders exhibit milder clinical symptoms than those with typical β-thalassemia, due to the beneficial effect of HbF on red blood cell production and survival [8, 9]. Because homozygotes and compound heterozygotes for these high HbF determinants are relatively rare, their clinical features have not been well documented thus our aim was to determine the phenotypic expression of Asian Indian inversion deletion Gγ (Aγδβ)0-thalassaemias due to co-inheritance of various factor.
Materials and Methods
Seven cases were sent from hematology OPD; All India Institute of Medical Sciences New Delhi to molecular study for delta beta thalassemia. Complete blood count and red cell indices were measured by automated cell analyzer (SYSMEX K-4500, Kobe Japan). Giemsa-stained peripheral blood smears were examined for red cell morphology. Quantitative assessment of hemoglobin HbF, HbA and HbA2 were performed by high performance liquid chromatography (HPLC-Bio-Rad-Variant™Bio Rad, CA, USA). DNA extraction was done by phenol–chloroform method. Molecular study for alpha deletions and beta mutations were done according to published literatures [10–14]. Asian Indian inversion-deletion GγAγ(δβ)0-thalassemia type A and type B mutations were identified according to Craig et al. [15] by Gap-PCR. Type A and type B GγAγ(δβ)0-thalassemia mutations agarose gel picture are given in Figs. 1 and 2.
Fig. 1.
Agarose gel picture of Gγ (Aγδβ)0 thalassemia breakpoint A (5′ deletion) (Lane 1AB, 2AB, 4AB and 7AB is heterozygous and 3AB, 5AB and 6AB is normal)
Fig. 2.
Agarose gel picture of Gγ (Aγδβ)0 thalassemia breakpoint B (3′ deletion) (Lane 1AB, 3AB and 6AB is heterozygous and lane 2AB, 4AB and 5AB is normal)
Result and Discussion
A total seven patients of GγAγ(δβ)0-thalassemia were characterized. Red cells of patients were hypochromic microcytic with slight anisocytosis and poikilocytosis. Out of the seven patients, four were heterozygous for Gγ(Aγδβ)0 breakpoint A and three were heterozygous for Gγ(Aγδβ)0 breakpoint B. All the patents were >20 year old and presenting elevated HbF. Type A Gγ(Aγδβ)0 inversion deletions patients had one heterozygous alpha 3.7 kb deletions and two IVS 1–5(G–C) mutation while one patient was heterozygous alpha 3.7 kb deletion and one was IVS 1–5(G–C) positive in type B Gγ(Aγδβ)0 deletions. Patients with co-existing beta thalassemia and delta gene mutations resulting in transfusion dependent anemia. None of the patients were homozygous for Gγ(Aγδβ)0 deletion in type A and type B breakpoints. The heterozygous Gγ(Aγδβ)0 patients red cell indices were in normal range with reduced hemoglobin A2 while patients with co-existing beta thalassemia mutation had low hemoglobin. All the hematological and genotypic data of Gγ(Aγδβ)0 breakpoint A and B are given in Tables 1 and 2. The complex double deletion-inversion rearrangement Gγ(Aγ δβ)0-thalassemia appears to be a common form of δβ-thalassemia found in central Asia, including in India [16, 17], Kuwait [18], and Iran [15]. Co-inheritance of δβ-thalassemia with increased production of γ-chains has an ameliorating effect on the clinical picture of β-thalassemia disease [1, 19]. Heterozygotes for δβ thalassemia tend to have a modest elevation of Hb F (5–20 %) with hypochromic microcytic red cell indices [20]. Molecular studies of these heterogeneous disorders could be classified as deletional and non deletional [8, 11]. Co-inheritance of HPFH or δβ thalassemia with β-thalassemia and other hemoglobinopathies has an ameliorating effect on the severity of the disease and thus usually results in the thalassemia intermedia phenotype [9, 21]. In our studied cases, two patients with breakpoint A deletions in which one had heterozygous 3.7 kb deletion was presenting mild anemia and weakness while 2 patients with co-existence of IVS 1–5(G–C) β-thalassemia mutation were transfusion dependent and showed recurrent jaundice with moderate anemia. Type B Gγ(Aγ δβ)0-thalassemia patients in which one was heterozygous for alpha 3.7 kb deletion and other with normal alpha genotype had mild anemia with fever while the patient with IVS 1–5(G–C) beta thalassemia was showed moderate anemia with blood transfusion dependent. Co-existence of α-thalassemia (−3.7 kb) decreases the HbF in IVSI-6 homozygote and in δß-thalassemia trait and may ameliorate the disease in ß-thalassemia compound heterozygotes associated with one mild and one severe ß-thalassemia mutation [22]. Co- existence of δ-thalassemia with β-thalassemia may mask the diagnosis of beta thalassemia trait. Beta thalassemia patient hemoglobin A2 increase in typically in the range of 4–6 %. However the co-existence of a delta thalassemia mutation decreases the value of the hemoglobin A2 into the normal range thereby obscuring the diagnosis of beta thalassemia trait [23]. Observation of the cases study were showed the co-existence of alpha globin gene deletion or beta globin mutation in Gγ(Aγδβ)0 patients present clinical variability. Patient with alpha gene deletion and heterozygous for Gγ(Aγδβ)0 results phenotype as thalassemia intermedia while the patient with beta thalassemia and heterozygous for Gγ(Aγδβ)0 clinically behaved like thalassemia major. In conclusion of the case study, co-inheritance of various factors with Gγ(Aγ δβ)0-thalassemia lead to variable clinical phenotypes. Molecular study should be performed the HbF determinants in thalassemia that help us the exact molecular basis and clinical outcome in Gγ(Aγ δβ)0-thalassemia patients.
Table 1.
Hematological and genotypic data of patients with breakpoint A (5′ deletion)
| Hematological parameters | Patient 1 | Patient 2 | Patient 3 | Patient 4 |
|---|---|---|---|---|
| Age | 23.6 year | 27 year | 20.8 year | 24.3 year |
| Sex | Female | Male | Female | Female |
| HbA2 | 2.1 | 3.2 | 2.4 | 2.8 |
| HbF | 23.4 | 21.3 | 14.2 | 10.7 |
| HGB | 11.4 | 10.6 | 14.5 | 8.8 |
| RBCs | 4.10 | 3.28 | 5. 06 | 3.16 |
| HCT | 34.9 | 31.1 | 39.2 | 28.5 |
| MCV | 85.1 | 94.8 | 77.7 | 90.2 |
| MCH | 23.8 | 32.3 | 28.7 | 27.8 |
| MCHC | 27.37 | 34.1 | 36.9 | 30.9 |
| α genotype | −α3.7/αα | αα/αα | αα/αα | αα/αα |
| β thal. Mutation | ßA/ßA | IVS1-5/ßA | ßA/ßA | IVS1-5/ßA |
| Gγ(Aγδβ)0 | Gγ(Aγδβ)0/ßA | Gγ(Aγδβ)0/ßA | Gγ(Aγδβ)0/ßA | Gγ(Aγδβ)0/ßA |
Table 2.
Haematological and genotypic data of patients with breakpoint B (3′ deletion) 1A
| Hematological parameters | Patient 1 | Patient 2 | Patient 3 |
|---|---|---|---|
| Age | 21 year | 23.4 year | 23.6 year |
| Sex | Male | Male | Female |
| HbA2 | 1.3 | 1.5 | 2.7 |
| HbF | 17.8 | 12.5 | 20.3 |
| HGB | 11.5 | 13.9 | 9.8 |
| RBCs | 3.92 | 4.78 | 4.75 |
| HCT | 34.8 | 41.3 | 32.7 |
| MCV | 88.8 | 86.4 | 68.8 |
| MCH | 29.3 | 29.1 | 20.6 |
| MCHC | 33.0 | 33.7 | 30.0 |
| α Genotype | αα/αα | αα/αα | −α3.7/αα |
| β Thal. mutation | ßA/ßA | ßA/ßA | IVS1-5/ßA |
| Gγ(Aγδβ)0 | Gγ(Aγδβ)0/ßA | Gγ(Aγδβ)0/ßA | Gγ(Aγδβ)0/ßA |
Acknowledgments
Sincere thanks to Mr. Naval Kishore, Mr. Suresh kumar, Mr.Rajesh Kumar and Mr.Bhanu Pratap, technical staff of department of hematology AIIMS, for expert assistance.
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