Publication: Analysis of thalassemia variant spectrum and developmental validation of exome sequencing for β-thalassemia in Malaysia
Loading...
Date
2025-12
Authors
Hassan, Syahzuwan
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
β-thalassemia poses a significant health problem in Malaysia, particularly among specific ethnicities. This study identified β-thalassemia mutations in 736 families participating in trio screening. Whole Exome Sequencing (WES) was used to validate the unknown Copy Number Variant (CNV), determine variants in the uninformative genotyping cases, and assemble a bioinformatics pipeline for simultaneous detections of thalassemia SNVs, indels, and CNVs. Hematological parameters were compared among β-thalassemia groups, and suitable combinations of predictors for heterozygous β-thalassemia were explored. Forty-six, 16, and 8 mutations were recorded in HBB, HBA, and HBD, respectively. More than half of the 571 Peninsular Malaysian children were patients, 163 (28.5%) were Hb E/β-thalassemia patients followed by 139 (24.3%) homozygous or compound heterozygous β-thalassemia. Hb E, IVS 1-5 (G>C), IVS 1-1 (G>T), and Hb Malay were the most common mutations among the Malays. IVS 2-654 (C>T), Cd 41/42 (-TTCT), and -28 (A>G) were common in Chinese. Sarawak had a low incidence of β-thalassemia. Of the 19 families, eight children were homozygous or compound heterozygous β-thalassemia, while four were Hb E/β-thalassemia. β-Filipino was prevalent in ethnicities of Sabah. Of 146 children, 90 (61.6%) were transfusion-dependent homozygous β-Filipino. The heterogeneity highlights the need for comprehensive genotyping, beyond population-specific variants. Compared to those without α-thalassemia, concomitant α-thalassemia mitigated microcytosis (p< .001)
and hypochromia (p< .001) in β-thalassemia carriers, exacerbated microcytosis (p= .004) and hypochromia (p< .001) in Hb E/β-thalassemia patients, and reduced Hb E carrier variant level (p< .001). However, co-inheritance of α-thalassemia did not significantly alter hematological parameters in homozygous or compound heterozygous β-thalassemia. A parsimonious combination of decreased MCH (OR=0.52, p<0.001) and increased HbA2 (OR=18.1, p<0.001) in Model 4 were sufficient for heterozygous β-thalassemia prediction. All models demonstrated good predictive performance (93.5-93.8%), however, Model 1 (MCV, RBC, MCH, and HbA2), Model 2 (RBC, MCH, and HbA2), and Model 3 (MCV, MCH, and HbA2) did not significantly improve heterozygous β-thalassemia prediction. To simplify the genotyping process, this study used The Genome Analysis Toolkit (GATK) HaplotypeCaller and CalculateGenotypePosteriors pipelines and demonstrated 100% concordance with conventional methods for variant detections. Three CNV callers were compared (CNVkit, cn.mops, and Control-Freec). Control-Freec exhibited superior performance, assisting in the identification of two novel CNVs of ααα91 and de novo ααα102. In conclusion, this multifaceted study provides a comprehensive understanding of the β-thalassemia spectrum in Malaysia. The assembled bioinformatics pipeline offers simpler and valuable strategies for a more efficient workflow and contributes to developing more effective molecular diagnostic strategies.