Clinical validation of a single-tube PCR and reverse dot blot assay for detection of common α-thalassaemia and β-thalassaemia in Chinese

Abstract

Objective To evaluate a novel reverse dot blot assay for the simultaneous detection six types of common α-thalassaemia alleles (three deletional and three common non-deletional mutations) and 19 types of common β-thalassaemia alleles in a Chinese population. Methods Genomic DNA samples were collected from three hospitals in southern China. The novel thalassaemia gene assay involved one multiplex polymerase chain reaction amplification system and one round of hybridization. Each of the clinically validated DNA samples was re-tested using the new multiplex polymerase chain reaction/reverse dot blot assay II (M-PCR/RDB II) assay in a double-blind manner. Results A total of 1060 unrelated study participants, including 829 patients with thalassaemia and 231 healthy control subjects, were analysed. The whole PCR and RDB procedures were completed in 260 min. All the samples, including heterozygous thalassaemia, homozygous thalassaemia and compound heterozygous thalassaemia, were correctly genotyped, yielding 100% concordance with the reference assays. HKαα/--SEA and HKαα/−α4.2, which were not included in the detection panel, yielded a contradictory result with this new assay. Conclusion The novel M-PCR/RDB II assay was simple, rapid and accurate, suggesting that it could be used for the genetic screening and clinical diagnosis of common α-thalassaemia and β-thalassaemia variants in Chinese populations.

The gene spectrum of thalassemia in Yangjiang of western Guangdong Province

“…Frontiers in Genetics frontiersin.org -α 3.7 , -α 4.2 , α WS , and α CS , with allele frequencies of 19.54%, 7.47%, 3.56%, and 3.30%, respectively ( Frontiers in Genetics frontiersin.org more than 2% were −29 (A>G), CD27-28 (+C), CD26 (Hb E) (G>A), IVS-I-1 (G>T), CD43 (G>T), CD14-15 (+G), and Cap (Table 5). Furthermore, a small number of cases suspected with rare genotypes were referred to undergo gene sequencing or gap-PCR (Liang et al, 2022). Rare thalassemia genotypes were identified in 19 cases which were not included in our detection kit; they included four types of rare α-globin genotypes of αα/--THAI (two cases), HKαα/--SEA (four cases), α WS α CD74 GAC>CAC /-α 4.2 (two cases), and α CD31 AGG>AAG α/--SEA (one case).…”

“…Thalassemia is prevalent in Middle East, Mediterranean countries, Central Asia, India, and southern China, as well as countries along the north coast of Africa and in South America; about 5% of the world's population is a carrier of thalassemia (Weatherall, 1981;Weatherall, 1997;Lai et al, 2017). Previous studies indicated that there was a high frequency of thalassemia cases in southern China, especially in Guangxi, Guangdong, Jiangxi, and Hainan provinces (Xu et al, 2004;Xiong et al, 2010;Lin et al, 2014;Yao et al, 2014;Yin et al, 2014;Lai et al, 2017;Liang et al, 2022). Patients of each ethnic population carry their own specific types of mutations, including a few very common ones and a variable number of rare ones.…”

“…Genomic DNA was extracted from all peripheral blood leukocytes, umbilical cord blood, fetal villi, and amniotic fluid. PCR and reverse dot blot (RDB) were used to detect the three commonly known α-thal deletions (-- SEA , -α 3.7 , and -α 4.2 ) and three known point mutations (Hb CS (α CS α) HBA2 : c.427T>C; Hb QS (α QS α), HBA2 : c.377T>C; Hb WS (α WS α), and HBA2 : c.369C>G) responsible for α-thal, and 19 known β-thal mutations most commonly seen in the Chinese population [IVS-II-654 (C>T), HBB : c.316–197C>T; codons 41/42 (‒TCTT), HBB : c.126_129delCTTT; codon 17 (A>T), HBB : c.52A>T; −28 (A>G), HBB : c.‒78A>G; codon 26 (G>A), HBB : c.79G>A; codons 71/72 (+A), HBB : c.216_217insA; codons 27/28 (+C), HBB : c.84_85insC; −29 (A>G), HBB : c.‒79A>G; IVS-I-1 (G>A, G>T), HBB : c.92 + 1G>A, c.92 + 1G>T; codons 14/15 (+G), HBB : c.45_46insG; codon 43 (G>T), HBB : c.130G>T; Cap (-AAAC, A>C), HBB : c.-11_-8delAAAC; IVS-I-5 (G>C), HBB : c. 92 + 5G>C; codon 31 (‒C), HBB : c.94delC; initiation codon ATG>AGG, HBB : c.2T>G; −32 (C>A), HBB : c.‒82C>A; −30 (T>C), and HBB : c.‒80T>C] were also identified ( Liang et al, 2022 ). All genotyping was analyzed using a thalassemia gene chip kit (Guangdong Hybribio Limited Corporation, Chaozhou, China), as described in our previous study ( Liang et al, 2022 ).…”

“…Those cases with abnormal capillary electrophoresis behavior or thalassemia traits which could not be identified by this gene chip kit were further tested by gap-PCR and PCR sequencing based on their primary gene analysis result ( Liang et al, 2022 ), and anti-3.7 and anti-4.2 α-globin gene triplications were identified by single-tube multiplex PCR ( Wang et al, 2005 ). Only a small number of suspected cases with rare variants were further tested.…”

Clinical Performance Study of a New Fully Automated Red Blood Cell Permeability Fragility Analyzer

“…With the development of highly automated and informatized laboratories, most cumbersome methods have been gradually eliminated. At present, there are many fully automatic methods that are widely used in clinics, but the requirements for clinical laboratories are high, and the machines are expensive [ 18 ]. Moreover, these instruments are used for incidental detection of EFT, which has poor specificity and is not suitable for the preliminary screening of thalassemia in economically underdeveloped areas and grassroots hospitals.…”