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.

Background : Deletions in the β-globin gene cluster are usually rare, problematic to detect, and subsequently possess a challenge in many diagnostic laboratories. They are normally related to the heterozygous of the delta beta (δβ) thalassemia, hereditary persistence of fetalhemoglobin (HPFH) and some of the hemoglobin variants. These disorders are typically presented by elevated levels of hemoglobin F (Hb F), but with low to normal hemoglobin A2 (Hb A2). However, despite their existence, there is still a limited number of studies focusing on this area, hence no definitive diagnosis could be conclusively established. Therefore, this pilot study was carried out to fill this knowledge gap. Methods: In this study, screening of the selected deletional mutations in the β-globin gene cluster among patients with Hb F (>1%) and Hb A2 (<4%) were performed using multiplex Gap-PCR and multiplex ligation-dependent probe amplification (MLPA). Results: The results showed that out of 54 samples tested using multiplex Gap-PCR against four target deletions; Thai (δβ)°-thalassemia, HPFH-6, Siriraj J and HbLepore, one sample was found positive with Thai (δβ)°-thalassemia. Further findings from the MLPA screening on 12 randomly selected samples revealed that another patient was positive with double deletions within the region of the β-globin gene cluster. These deletions occur at the gamma-globin gene 1 (HBG1) and gamma-globin gene 2 (HBG2) in exon 3. Conclusions: In conclusion, this study highlighted the importance of these deletions’ characterization using multiplex Gap-PCR and MLPA which helps in establishing a definitive diagnosis among this selected group of patients. Bangladesh Journal of Medical Science Vol.20(3) 2021 p.618-624