Hb Q-Thailand heterozygosity unlinked with the (-a4.2/) a+-thalassemia deletion allele identified by long-read SMRT sequencing: hematological and molecular analyses

Objective: In the present study, two unrelated cases of Hb Q-Thailand heterozygosity unlinked with the (-alpha(4.2)/) alpha(+)-thalassemia deletion allele were identified by long-read single molecule real-time (SMRT) sequencing in southern China. The aim of this study was to report the hematological and molecular features as well as diagnostic aspects of the rare manifestation.Methods: Hematological parameters and hemoglobin analysis results were recorded. A suspension array system for routine thalassemia genetic analysis and long-read SMRT sequencing were applied in parallel for thalassemia genotyping. Traditional methods, including Sanger sequencing, multiplex gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA), were used together to confirm the thalassemia variants.Results: Long-read SMRT sequencing was used to diagnose two Hb Q-Thailand heterozygous patients for whom the hemoglobin variant was unlinked to the (-alpha(4.2)/) allele for the first time. The hitherto undescribed genotypes were verified by traditional methods. Hematological parameters were compared with those of Hb Q-Thailand heterozygosity linked with the (-alpha(4.2)/) deletion allele in our study. For the positive control samples, long-read SMRT sequencing revealed a linkage relationship between the Hb Q-Thailand allele and the (-alpha(4.2)/) deletion allele.Conclusions: Identification of the two patients confirms that the linkage relationship between the Hb Q-Thailand allele and the (-alpha(4.2)/) deletion allele is a common possibility but not a certainty. Remarkably, as it is superior to traditional methods, SMRT technology may eventually serve as a more comprehensive and precise method that holds promising prospects in clinical practice, especially for rare variants.