Molecular and hematological studies in a cohort of beta zero South East Asia deletion (ß°-thal SEA) from Malaysian perspective.

Abstract: We report the haematological parameters and molecular characterization of beta zero (ß°) South East Asia (SEA) deletion in the HBB gene cluster with unusually high levels of Hb F compared to a classical heterozygous beta zero (ß°)-thalassaemia. Methods: Retrospective study on 17 cases of (ß°) South East Asia (SEA) deletion from 2016 to 2019 referred to Institute for Medical Research were conducted. The clinical information and haematological profiles were evaluated. The mutation was analyzed, and the results were compared with other ß°-thalassaemia groups. For HBB gene genotyping, all the cases were subjected for multiplex gap-PCR, 5 cases were subjected for HBB gene sequencing for exclusion of compound heterozygous with other beta variants. Co-inheritance of α-thalassaemia were determined using multiplex gap-PCR and multiplex ARMS-PCR. Results: Seventeen cases were positive for ß°-thal SEA deletion. Fifteen cases were heterozygous and two were compound heterozygous for ß°-thal SEA deletion. The results were compared with 182 cases of various heterozygous ß° deletions and mutations. The mean Hb for heterozygous ß°-thal SEA deletion (13.44 ± 1.45 g/dl) was normal and significantly higher than heterozygous IVS 1-1 and Codon 41/42 (post hoc test, p < 0.05). The medians for the MCV and MCH of ß°-thal SEA deletion were significantly higher than for all heterozygote ß°-thalassaemia traits (Mann Whitney test, p < 0.05). Patients with ß°-thal SEA deletion had elevated levels of Hb A2 consistent with ß-thalassaemia traits, with Hb F levels consistent with HPFH or δß-thalassaemia carriers. The median for Hb A2 was 4.00 + 1.00%, similar to that observed in other ß°-thalassaemia groups except for IVS 1-1 mutation (median 5.30 + 0.45%) and ß°-Filipino (∼45 kb deletion) deletion (median 6.00 + 0.58). Interestingly, we found that Hb F levels for ß°-thal SEA deletion were statistically higher than other ß°-thalassaemia mutations (median 19.00 + 5.50%, p < 0.05), except for the ß°-thal 3.5 kb deletion group. Conclusion: We conclude that ß°-thal SEA deletion has a unique haematological parameters of beta zero thalassaemia trait. We affirm to classifying this deletion as SEA-HPFH based on previous studies considering the phenotype features rather than the molecular defect of ß°-thal SEA deletion, as this will make it easier to offer genetic counselling to affected individuals.

LARP2 DNA Methylation in Transfusion-dependent Haemoglobin E-Beta (HbE/β) and β-Thalassaemia Major Patients

Abstract@#Introduction: The large clinical spectrum of haemoglobin E-beta (HbE/β) thalassaemia leads to the investigation of complex mechanisms involved in erythropoiesis. DNA methylation in LARP2 is one of the potential epigenetic modifiers not fully explored in HbE/β and β-thalassaemia major. This study aimed to analyse DNA methylation profile and gene expression of LARP2 using peripheral blood (PB) in nucleated red blood cells (NRBCs) for the source of DNA of HbE/β- and β-thalassaemia major patients. Methods: PB were collected from 33 transfusion-dependent thalassemia patients from Hospital USM and Hospital RPZII, Kelantan, Malaysia. DNA methylation profile and gene expression of LARP2 were examined by bisulphite sequencing PCR and quantitative real-time PCR respectively. Results: Partial DNA methylation of LARP2 was observed in 43% (9/21) HbE/β- and 17% (2/12) β-thalassaemia major patients. LARP2 expression (1.49±26.60) in HbE/β-thalassaemia was not significant against normal controls and β-thalassaemia major (p>0.05). In contrast, LARP2 expression (6.8±16.42) in β-thalassaemia major showed a significant up-regulation against normal controls (p<0.05). The association of LARP2 expression and DNA methylation profile was statistically significant (p<0.001). LARP2 expression was down-regulated in 75% (3/4) HbE/β-thalassaemia patients with CD26/IVS1-5, in contrast to up-regulation of 80% (4/5) IVS1-5/IVS1-5 β-thalassaemia major patients. DNA methylation of LARP2 in these patients were either partially methylated or unmethylated in CD26/IVS1-5 and IVS15/IVS1-5 respectively. Conclusion: DNA methylation of LARP2 may act as an additional modifier to gene mutation especially involving IVS1-5 in HbE/β-thalassaemia. Homozygous IVS1-5 in β-thalassaemia major may contribute to different disease presentation compared to those involving CD26 in HbE/β-thalassaemia.