Resolving diagnostic complexity: long-read sequencing unmasks a Hong Kongαα/‒α4.2 genotype mimicking a heterozygous ‒α3.7 deletion on multiplex ligation-dependent probe amplification

INTRODUCTION: Complex structural variants in thalassemia are rare and often undetectable or misdiagnosed by conventional genetic testing methods. We report a case initially misclassified by multiplex ligation-dependent probe amplification (MLPA) as a heterozygous ‒α3.7 deletion that was subsequently correctly identified by third-generation sequencing (TGS) as the Hong Kongαα (HKαα)/‒α4.2 genotype. METHODS: Gap polymerase chain reaction (PCR) was used to screen for 4 common deletional α-thalassemia variants (-SEA, -THAI, ‒α3.7, and ‒α4.2). A PCR-reverse dot blot detected 3 nondeletional mutations. We used MLPA to analyze copy number variations in the α-globin gene cluster; TGS characterized large deletions, cis/trans phasing, and complex structural rearrangements. RESULTS: Gap PCR identified the female patient as a heterozygous -SEA carrier. In the male patient, gap PCR showed 3 bands (‒α3.7, wild type, and ‒α4.2), making genotyping uncertain. The MLPA findings suggested a heterozygous ‒α3.7 deletion; TGS revealed coexisting deletion and duplication fragments, leading to the final diagnosis of HKαα/‒α4.2. DISCUSSION: For HKαα and ‒α4.2 coinheritance, gap PCR cannot provide an accurate genotype, and MLPA may erroneously report a heterozygous ‒α3.7 deletion. Third-generation sequencing accurately identified this complex variant, demonstrating its suitability for detecting such structural variants in clinical diagnostics.