Accurate genotype diagnosis of Hong Kongαα thalassemia based on third-generation sequencing

Background: The Hong Kongaa (HK alpha alpha) allele is a complex structural rearrangement of the alpha-globin gene containing -alpha(3.7) and alpha alpha alpha(anti 4.2) crossover junctions. Clinically, individuals carrying the HK alpha alpha allele are often misdiagnosed or missed using conventional thalassemia gene detection technology. This study aims to identify and validate different HK alpha alpha thalassemia subtypes using third-generation sequencing (TGS) technology. Methods: Between January 2015 and June 2021, 32 patients suspected of having HK alpha alpha thalassemia were included in this study. Genomic DNA was extracted, and gap-polymerase chain reaction (PCR), two-round nested PCR, multiplex ligation-dependent probe amplification (MLPA), and TGS were used for thalassemia gene detection. Results: The results of HK alpha alpha/alpha alpha and HK alpha alpha/-alpha(3.7) were similar to -alpha(3.7)/alpha alpha using the gap-PCR method. Tworound nested PCR could be used to verify the HK alpha alpha gene, but could not distinguish the subtypes of HK alpha alpha thalassemia. The MLPA assay was used to detect the change in the copy number of the alpha-globin gene, but it could not determine whether -alpha(3.7) and aaaanti 4.2 were in cis or in trans. Long-read TGS technology could accurately detect the HK alpha alpha allele and distinguish the genotypes of HK alpha alpha/alpha alpha, HK alpha alpha/-alpha(3.7), HK alpha alpha/-alpha(4.2), and HK alpha alpha/--S-EA without pedigree analysis. The contiguous sequence of the HKaa allele was detected using the TGS approach. This study also demonstrated that individuals with HK alpha alpha/alpha alpha and beta N/beta N genotypes tended to have normal hematological phenotypes. Conclusions: Long-read TGS is a reliable and efficient approach for accurate detection of HKaa thalassemia, which can be widely used in clinical practice. Accurate molecular diagnosis of HKaa thalassemia will benefit clinical genetic counseling and prenatal diagnosis.