A next-generation sequencing-based universal target panel and algorithm for one-stop detection of copy number alterations and single-nucleotide variations in the HBB gene cluster for rapid diagnosis of β-thalassemia

Background: This study aimed to develop and validate a targeted next-generation sequencing (NGS) panel along with a data analysis algorithm capable of detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) within the beta-globin gene cluster. The aim was to reduce the turnaround time in conventional genotyping methods and provide a rapid and comprehensive solution for prenatal diagnosis, carrier screening, and genotyping of β-thalassemia patients.

Methods and results: We devised a targeted NGS panel spanning an 80.4 kb region on chromosome 11, encompassing the beta-globin gene cluster and 5' locus control region. We also developed an advanced data analysis algorithm consisting of variant calling and depth plot analysis that facilitates simultaneous detection of SNVs and CNVs in a single run. The test panel and algorithm were validated with 14 in-house β-thalassemia carrier/patient samples and cross-checked against the HbVar database. We identified seven pathogenic SNVs and five CNVs within the beta-globin gene cluster in various combinations, such as heterozygous, homozygous, and compound heterozygous conditions. Additionally, the coordinates of 169 rare deletions and 11 fusion mutations reported in the HbVar database were checked to verify the theoretical ability of our developed gene panel to detect all the CNVs within the target region.

Conclusion: The developed panel and NGS technology can detect both SNVs and CNVs in a single run and can also be utilized for prenatal diagnosis and carrier screening for hemoglobinopathies, underscoring its versatility and clinical utility.