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

Misdiagnosis of α-thalassemia genotypes due to primer site mutations presents diagnostic challenges in Chinese populations where -α deletion and :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 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 :c.427T > C results, with MLPA detecting atypical signal intensities in and loci. Comprehensive TGS analysis revealed trans configuration with :c.300+55T > G and :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.