Diagnosis of the accurate genotype of HKαα carriers in patients with thalassemia using multiplex ligation-dependent probe amplification combined with nested polymerase chain reaction

BACKGROUND: Patients carrying the HongKongαα (HKαα) allele and -α(3.7)/ααα(anti-4.2) could be misdiagnosed as -α(3.7)/αα by the current conventional thalassemia detection methods, leading to inaccurate genetic counseling and an incorrect prenatal diagnosis. This study was aimed to accurately analyze the genotypes of HKαα carriers and -α(3.7)/ααα(anti-4.2). METHODS: Samples were collected in our hospital from July 2017 to October 2019. Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction (Gap-PCR) and reverse dot blot (RDB). Anti-4.2 multiplex-PCR was used to confirm carriers of the ααα(anti-4.2) duplication with -α(3.7) deletion. Two-round nested PCR and multiplex ligation-dependent probe amplification (MLPA) were applied to accurately identify and confirm their genotypes. For data analysis, we used descriptive statistics and Fisher's exact tests. RESULTS: Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples. The results showed that α, β, and αβ compound thalassemia were identified in 1190 (46.78%), 1286 (50.55%), and 68 (2.67%) cases, respectively. A total of 227 samples from thalassemia patients were identified as -α(3.7)/αα by Gap-PCR, and the genotypes of two samples were uncertain. There was a difference between Gap-PCR and combined groups (Gap-PCR combined with nested PCR and MLPA) in detecting HKαα (P < 0.05). Among the 229 patients, 20 patients were identified as HKαα carriers and one was identified as -α(3.7)/ααα(anti-4.2) by two-round nested PCR and MLPA, including 15 patients with HKαα/αα, three with HKαα/αα and β-thalassemia coinheritance, one with HKαα/--(SEA), one with HKαα/-α(4.2) and β-thalassemia coinheritance, and one with -α(3.7)/ααα(anti-4.2) and β-thalassemia coinheritance. CONCLUSIONS: ααα(anti-4.2) and HKαα genotypes of patients carrying -α(3.7) need to be detected to reduce the misdiagnosis rate of patients carrying HKαα and -α3.7/ααα(anti-4.2) alleles. More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.