Misdiagnosis of α-Thalassemia Heterozygotes as Homozygotes Due to Base Mutations in the Primer Binding Region.

Abstract

Misdiagnosis of α-thalassemia genotypes due to primer site mutations presents diagnostic challenges in Chinese populations where -α^3.7 deletion and HBA2:c.427T>C (Hb Constant Spring) heterozygotes are prevalent. We describe two cases where conventional diagnostic approaches erroneously identified heterozygous carriers as homozygotes. Diagnostic algorithms employing gap-PCR and PCR-RDB for common thalassemia mutations initially suggested homozygous status in both probands. Proband 1 (28-year-old male) was misclassified as -α^3.7 homozygote by gap-PCR, but MLPA analysis revealed heterozygous status, subsequently confirmed by third-generation sequencing which identified concurrent NG_000006.1:g.32793 C > T mutation validated through Sanger sequencing. Proband 2 demonstrated discordant HBA2:c.427T > C results, with MLPA detecting atypical signal intensities in HBA2 and HBA1 loci. Comprehensive TGS analysis revealed trans configuration with HBA2:c.300+55T > G and HBA2:c.301-24delGinsCTCGGCCC variants. These cases highlight the important impact of mutations in the priming region on molecular diagnosis and emphasize the need for further characterization by other methods to avoid misdiagnosis when results from traditional methods are equivocal.