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

Abstract

Background:

Patients carrying the HongKong αα (HK αα) 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 HK αα 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 HK αα ( P < 0.05). Among the 229 patients, 20 patients were identified as HK αα carriers and one was identified as - α ^3.7/ ααα ^anti-4.2 by two-round nested PCR and MLPA, including 15 patients with HK αα/ αα, three with HK αα/ αα and β-thalassemia coinheritance, one with HK αα/-- ^SEA, one with HK αα/-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 HK αα genotypes of patients carrying - α ^3.7 need to be detected to reduce the misdiagnosis rate of patients carrying HK αα and - α3.7/ ααα ^anti-4.2 alleles. More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.