Thalassemia is one of the most heterogeneous diseases, with more than a thousand mutation types recorded worldwide. Molecular diagnosis of thalassemia by conventional PCR-based DNA analysis is time- and resource-consuming owing to the phenotype variability, disease complexity, and molecular diagnostic test limitations. Moreover, genetic counseling must be backed-up by an extensive diagnosis of the thalassemia-causing phenotype and the possible genetic modifiers. Data coming from advanced molecular techniques such as targeted sequencing by next-generation sequencing (NGS) and third-generation sequencing (TGS) are more appropriate and valuable for DNA analysis of thalassemia. While NGS is superior at variant calling to TGS thanks to its lower error rates, the longer reads nature of the TGS permits haplotype-phasing that is superior for variant discovery on the homologous genes and CNV calling. The emergence of many cutting-edge machine learning-based bioinformatics tools has improved the accuracy of variant and CNV calling. Constant improvement of these sequencing and bioinformatics will enable precise thalassemia detections, especially for the CNV and the homologous HBA and HBG genes. In conclusion, laboratory transiting from conventional DNA analysis to NGS or TGS and following the guidelines towards a single assay will contribute to a better diagnostics approach of thalassemia.

We report the haematological parameters and molecular characterization of beta zero (β°) South East Asia (SEA) deletion in the HBB gene cluster with unusually high levels of Hb F compared to a classical heterozygous beta zero (β°)-thalassaemia.Methods: Retrospective study on 17 cases of (β°) 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 β°-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 β°-thal SEA deletion. Fifteen cases were heterozygous and two were compound heterozygous for β°-thal SEA deletion. The results were compared with 182 cases of various heterozygous β° deletions and mutations. The mean Hb for heterozygous β°-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 β°-thal SEA deletion were significantly higher than for all heterozygote β°-thalassaemia traits (Mann Whitney test, p < 0.05). Patients with β°-thal SEA deletion had elevated levels of Hb A2 consistent with β-thalassaemia traits, with Hb F levels consistent with HPFH or δβ-thalassaemia carriers. The median for Hb A2 was 4.00 + 1.00%, similar to that observed in other β°-thalassaemia groups except for IVS 1-1 mutation (median 5.30 + 0.45%) and β°-Filipino (∼45 kb deletion) deletion (median 6.00 + 0.58). Interestingly, we found that Hb F levels for β°-thal SEA deletion were statistically higher than other β°-thalassaemia mutations (median 19.00 + 5.50%, p < 0.05), except for the β°-thal 3.5 kb deletion group.Conclusion: We conclude that β°-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 β°-thal SEA deletion, as this will make it easier to offer genetic counselling to affected individuals.

Malaysia is a multicultural and multiethnic country comprising numerous ethnic groups. From the total population of 32.7 million, Malays form the bulk of the Bumiputera in Malaysia comprise about 69.9%, followed by Chinese 22.8%, Indian 6.6%, and others 0.7%. The heterogeneous population and increasing numbers of non-citizens in this country affects the heterogeneity of genetic diseases, diversity, and heterogeneity of thalassaemia mutations. Alpha (α)-thalassaemia is an inherited haemoglobin disorder characterized by hypochromic microcytic anaemia caused by a quantitative reduction in the α-globin chain. A majority of the α-thalassaemia are caused by deletions in the α-globin gene cluster. Among Malays, the most common deletional alpha thalassaemia is −α^3.7 deletion followed by −−^SEA deletion. We described the molecular characterization of a new −−^GB deletion in our population, involving both alpha genes in cis. Interestingly, we found that this mutation is unique among Malay ethnicities. It is important to diagnose this deletion because of the 25% risk of Hb Bart’s with hydrops fetalis in the offspring when in combination with another α⁰- thalassaemia allele. MLPA is a suitable method to detect unknown and uncommon deletions and to characterize those cases which remain unresolved after a standard diagnostic approach.

Poly A (AATAAA > AATA--) [HBA2:c.*93_*94delAA] is a rare α-variant reported in our population. It is caused by 2 bp deletion (--AA) in the α2 poly A sequence, leading to a significant α–thalassaemia phenotype. Background/Objectives: This study describes the haematological parameters, phenotype, and genotype characteristics of AATA(--AA) in the Malaysian population. Methods: The study was carried out on 17 177 cases referred to the Institute for Medical Research, Malaysia, for further diagnosis of α-thalassaemia in a five-year period. Alpha-Gap and ARMS-PCR were performed to detect common α-thalassaemia, followed by HBA1 and HBA2 genes sequencing and multiplex ligation-dependent probe amplification (MLPA). Haematological parameters among various groups with the AATA(--AA) allele were presented in this study. Results: Thirty-two patients with AATA(--AA) displaying an α–thalassaemia-like phenotype were analysed. They comprised 22 (68.75%) AATA(--AA) carriers, 2 (6.25%) compounds with 3.7 deletion, 2 (6.25%) compounds with --SEA deletion, 1 (3.12%) AATA(--AA) homozygote, and 3 (9.37%) compounds of Hb Adana, Hb CS, and Hb Pakse with co-inheritance Hb E, respectively. Most of the patients with AATA(--AA) compounds with the α-variant exhibited a significant phenotype between moderate to severe thalassaemia, especially cases with compound α^−AAα/α^Adanaα. Conclusions: AATA(--AA) is a significant pathogenic variant that should be diagnosed to prevent significant thalassaemia phenotype or transfusion-dependent thalassaemia.

This study assesses haematological parameters as screening tools for identifying α zero (α⁰) thalassaemia candidates for DNA analysis. The Malaysia Thalassaemia Diagnosis Code (MTDC) uses mean corpuscular haemoglobin (MCH) < 25 pg to screen for suspected α⁰ thalassaemia carriers. Six haematological parameters from 304 cases genotyped by Gap-Polymerase Chain Reaction or Multiplex Ligation-dependent Probe Amplification were analyzed. Among the cases, 160 individuals (52.6%) were α ⁺ thalassaemia silent carrier (-α/αα), comprising heterozygotes for -α ^3.7 , -α ^4.2 , –(α) ^20.5 and -α ^2.4 . Twelve individuals (3.9%) were α ⁺ thalassaemia carrier (-α/-α), including homozygous -α ^3.7 and compound heterozygous -α ^3.7 and -α ^4.2 . Meanwhile, 111 individuals (36.4%) were α ⁰ thalassaemia carrier (--/αα) consisting of heterozygotes for -- ^SEA , -- ^GB , -- ^FIL , -- ^AW , and – ^THAI deletions. Deletional Hb H disease (--/-α) was observed in individuals with compound genotypes such as -α ^3.7 /-- ^SEA , -α ^3.7 /-- ^GB , -α ^3.7 /-- ^AW , -α ^4.2 /-- ^SEA and -α ^2.4 /-- ^SEA . All parameters correlated with the number of α-globin gene deletions, with mean corpuscular volume (MCV) (r ² = 0.73) and MCH (r ² = 0.77) showing the strongest associations. Gender-specific MCH cut-offs of < 22.65 pg for males and < 22.25 pg for females demonstrated excellent diagnostic performance, with higher sensitivity and specificity in females (90.3% and 93.9%) than in males (89.5% and 88.9%). The findings indicate that the currently used MCH cut-off of 25 pg may be higher than optimal for identifying α⁰ thalassaemia carriers. A lower MCH threshold improves detection performance and may support refinement of the MTDC screening criteria to better prioritize high-risk individuals while reducing unnecessary DNA testing among α⁺ thalassaemia cases.

Thalassemia is a significant health burden in Malaysia. This report presents a retrospective analysis of nationwide diagnostic data compiled over seven years, investigating β-thalassemia spectrum in 731 referred family trios. Genomic analysis used gap-PCR, ARMS-PCR, MLPA, Sanger sequencing, and exome sequencing to validate rare CNVs. We identified 468 at-risk couples, with 440 children inherited biallelic mutations. Heterogeneous mutations were recorded among Peninsular Malaysian children with 43 β-, four δ-, 11 α-thalassemia and variant types, and five α-globin duplications. Among children in Sabah, 15 β-, one δ-, and two α-thalassemia and variants, and one α-globin duplications were recorded. Sarawak exhibited lowest heterogeneity. In Peninsular Malaysia, β ^E was most prevalent among Malays, followed by IVS 1–5 (G > C), β ^Malay , and IVS 1–1 (G > T). IVS 2–654 (C > T) and − 28 (A > G) were observed only among Chinese individuals in this referred cohort, while Cd 41/42 -TTCT was common to both ethnicities. β ^E /β-thalassemia was the most frequent disease with 188 cases. In Sabah, homozygous β ^Filipino was the most frequent genotype across multiple ethnicities, with 35.2% (31/88) of these cases exhibiting concomitant -α ^3.7 . Overall, 15.2% (111/731) of children with HBB mutations had concomitant α-thalassemia, and 10 had α-triplications. These findings highlight the necessity of HBA genotyping for accurate genetic counseling. We present two novel mutation, codon 122 (TTC > TGC) mutation (HBB:c.368 T > G; p.Phe123Cys) named β ^{Tanah Merah} , and α-globin cluster duplication, ααα ⁹¹ (NC_000016.10:g.87820_179080dup). This comprehensive dataset is essential for improving molecular diagnostics and optimizing DNA analysis workflows in highly heterogeneous populations.