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

Abstract

Background: Patients carrying the HongKong alpha alpha (HK alpha alpha) allele and -alpha(3.7)/alpha alpha alpha(anti-4.2) could be misdiagnosed as -alpha(3.7)/alpha alpha 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 alpha alpha carriers and -alpha(3.7)/alpha alpha alpha(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 alpha alpha alpha(anti-4.2) duplication with -alpha(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 alpha, beta, and alpha beta 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 -alpha(3.7)/alpha alpha 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 alpha alpha (P < 0.05). Among the 229 patients, 20 patients were identified as HK alpha alpha carriers and one was identified as -alpha(3.7)/alpha alpha alpha(anti-4.2) by two-round nested PCR and MLPA, including 15 patients with HK alpha alpha/alpha alpha, three with HK alpha alpha/alpha alpha and beta-thalassemia coinheritance, one with HK alpha alpha/--(SEA), one with HK alpha alpha/-alpha(4.2) and beta-thalassemia coinheritance, and one with -alpha(3.7)/alpha alpha alpha(anti-4.2) and beta-thalassemia coinheritance. Conclusions: alpha alpha alpha(anti-4.2) and HK alpha alpha genotypes of patients carrying -alpha(3.7) need to be detected to reduce the misdiagnosis rate of patients carrying HK alpha alpha and -alpha 3.7/alpha alpha alpha(anti-4.2) alleles. More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.