A comparative study of SNPscan/CNVplex assay and routine PCR in genetic analysis of thalassemia

Accurate molecular diagnosis is essential for thalassemia prevention and carrier screening, particularly in high-prevalence regions. Conventional PCR-based methods are widely used in clinical practice but have limited ability to detect rare variants, homologous recombination events, and large structural rearrangements. This study evaluated the clinical performance of combined SNPscan/CNVplex assay for comprehensive thalassemia screening.

A total of 1,026 hematologic testing-positive individuals from Hunan Province, China, were analyzed using routine PCR and SNPscan/CNVplex assay in parallel. The SNPscan assay targeted 48 single-nucleotide variants and indels, while CNVplex targeted 28 deletional mutations and homologous recombination events in HBA1/HBA2 and HBB. All positive results and discordant results were confirmed by Sanger sequencing or specially designed PCR.

SNPscan/CNVplex assay identified thalassemia-associated variants in 302 individuals (29.43%), compared with 283 correctly detected by routine PCR, representing a 6.71% increase in diagnostic yield. Concordant results between the two methods were observed in 98.15% of cases, while 19 cases showed discordant findings. Additional variants detected by SNPscan/CNVplex assay included 14 α-globin gene triplications, one HKαα rearrangement, two rare SNVs/indels, and two large fragment copy number variants involving the α-globin gene cluster. The most common α-thalassemia mutation was--^SEA, whereas HBB:c.316-197C>T was the most frequent β-thalassemia variant. Three individuals carried both α-globin gene triplications and β-thalassemia mutations, indicating potential risk for aggravated globin chain imbalance. CNVplex assay also successfully identified large deletions and duplications encompassing the α-globin gene cluster that were missed by routine PCR.

Combined SNPscan/CNVplex assay demonstrated broader mutation coverage and higher diagnostic yield than routine PCR, particularly for detecting homologous recombination events and large structural variants. This integrated strategy may improve carrier detection and genetic risk assessment in thalassemia screening programs.