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.

(1) Background: Alpha (α)-thalassaemia is a genetic disorder that affects 5% of the world population. Deletional or nondeletional mutations of one or both HBA1 and HBA2 on chromosome 16 will result in reduced production of α-globin chains, a component of haemoglobin (Hb) that is required for the formation of red blood cells (RBCs). This study aimed to determine the prevalence, haematological and molecular characterisations of α-thalassaemia. (2) Method: The parameters were based on full blood count, high-performance liquid chromatography and capillary electrophoresis. The molecular analysis involved gap-polymerase chain reaction (PCR), multiplex amplification refractory mutation system-PCR, multiplex ligation-dependent probe amplification and Sanger sequencing. (3) Results: With a total cohort of 131 patients, the prevalence of α-thalassaemia was 48.9%, leaving the remaining 51.1% with potentially undetected α gene mutations. The following genotypes were detected: -α^3.7/αα (15.4%), -α^4.2/αα (3.7%), --^SEA/αα (7.4%), α^CSα/αα (10.3%), α^Adanaα/αα (0.7%), α^{Quong Szeα}/αα (1.5%), -α^3.7/-α^3.7 (0.7%), α^CSα/α^CSα (0.7%), -α^4.2/α^CSα (0.7%), –^SEA/α^CSα (1.5%), –^SEA/α^{Quong Sze}α (0.7%), -α^3.7/α^Adanaα (0.7%), --^SEA/-α^3.7 (2.2%) and α^CSα/α^Adanaα (0.7%). Indicators such as Hb (p = 0.022), mean corpuscular volume (p = 0.009), mean corpuscular haemoglobin (p = 0.017), RBC (p = 0.038) and haematocrit (p = 0.058) showed significant changes among patients with deletional mutations, but not between patients with nondeletional mutations. (4) Conclusions: A wide range of haematological parameters was observed among patients, including those with the same genotype. Thus, a combination of molecular technologies and haematological parameters is necessary for the accurate detection of α-globin chain mutations.

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.

Rationale: Hemoglobin E beta-thalassemia (HbE-BT) is a prevalent and genetically diverse inherited hemoglobin disorder in Malaysia. While most cases are attributed to known genetic variants, discovering novel mutations is essential for understanding their wide-ranging clinical presentations and underlying genetic heterogeneity. Patient concerns: We present the case of a Malay male patient with an intermediate phenotype of HbE-BT whose clinical course was marked by a progression to a transfusion-dependent status, a change triggered by an acute febrile illness. The case highlights the complex challenges of long-term management, including a high risk of iron overload and the significant morbidity and mortality associated with postsplenectomy infections. Diagnoses: The patient was referred for a comprehensive DNA analysis of thalassemia syndromes. Full blood count showed hypochromic microcytic anemia (hemoglobin 7.90 g/dL, mean corpuscular volume 66.10 fL). Hemoglobin analysis via capillary electrophoresis revealed a characteristic pattern of HbA (49.1%), HbE (20.6%), and HbF (27.0%). Sanger sequencing subsequently confirmed a compound heterozygous state of HBB:c.79G>A (HbE) and a novel frameshift mutation at codon 124 of the beta-globin gene (HBB:c.375_376delAC). Predictive computational tools further supported the pathogenicity of this new variant, and its data have been submitted to the ITHANET database (IthaID: 4155). Interventions: Initially, the patient’s condition was managed with nutritional supplements and intermittent blood transfusions. Following an acute febrile episode at age 26, his clinical condition worsened, necessitating regular monthly blood transfusions to manage severe anemia. He also received subcutaneous chelation therapy to address severe hepatic iron overload. At age 36, he underwent a splenectomy to reduce his transfusion requirements. Outcomes: Despite these interventions, the patient passed away at the age of 37 from septic shock and multiorgan failure. This outcome occurred a year after his splenectomy and underscores the critical risk of overwhelming postsplenectomy infection, a significant complication in splenectomized thalassemia patients. Lessons: Identifying this novel compound heterozygosity contributes to our understanding of the genetic landscape of HbE-BT in Malaysia. This case reinforces the importance of comprehensive molecular analysis for accurate diagnosis, and it has important implications for improving long-term management and genetic counseling strategies for patients with thalassemia syndromes.

α-Globin triplication increases α/non-α-chain imbalance and may lead to a symptomatic β-thalassemia carrier. We describe a novel, de novo large segmental duplication, ααα¹⁰² in a patient who is a β-thalassemia IVS 1-1 (G > T) (HBB:c0.92 + 1G > T) carrier and presented with thalassemia intermedia. The duplication spans from intron II, position 203 of the N-methylpurine DNA glycosylase (MPG) gene, to the downstream region of the Hemoglobin Subunit Theta 1 (HBQ1) gene (GRCh38.p14) (HGVS NC_000016.10:g.79903_181947dup). The heterozygous β-thalassemia IVS 1-1 (G > T) was inherited from the father, while the mother was negative for thalassemia. α-MLPA analysis revealed multiple polymorphisms in the father; however, neither parent had the duplication. We concluded that the segmental duplication occurred de novo. In pre-marital screening programs, α-triplications and duplicated α-globin loci should be investigated when one partner has β-thalassemia or a β-thalassemia carrier.

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.