A Single-Tube Multiplex Gap-Polymerase Chain Reaction for the Detection of Eight β-Globin Gene Cluster Deletions Common in Southeast Asia

Abstract

Up to now, more than 200 different β-thalassemia (β-thal) mutations have been characterized. The majority are point mutations causing expression defects. Only approximately 10.0% of the defects are caused by large deletions involving the β-globin gene cluster causing β(0)-thal, (δβ)(0)-thal, (G)γ((A)γδβ)(0)-thal and other conditions with or without persistence of fetal hemoglobin (Hb). For the prevention of severe forms of β-thal intermedia and β-thal major, it is important to identify carriers of point mutations as well as carriers of deletions. β-Thalassemia and related disorders are most commonly present among populations from all Mediterranean countries as well as Southeast Asia, India, Africa, Central America and the Middle East. Twelve relatively frequently occurring deletion types have been described involving the β-globin gene cluster. These include the 105 bp β(0)-thal deletion, the 619 bp deletion, the 3.5 kb deletion, the Southeast Asian (SEA) deletion, the Filipino deletion, Hb Lepore, the Thai (δβ)(0)-thal, the Siriraj J (G)γ((A)γδβ)(0)-thal, the Chinese (G)γ((A)γδβ)(0)-thal, the Asian Indian deletion-inversion (G)γ((A)γδβ)(0)-thal as well as the (hereditary persistence of fetal hemoglobin) HPFH-6 and HPFH-7 deletions. To improve the rapid detection of the eight common β-globin cluster deletions in Southeast Asian countries, a simple molecular technique based on a single-tube multiplex gap-polymerase chain reaction (PCR) has been developed in this study. This technique provides a fast, simple and cost effective diagnostic test for deletion types of β-thal that can be applied in every molecular diagnostic laboratory having standard PCR equipment.

Phenotypic expression of Hb F in common high Hb F determinants in Thailand: roles of α‐thalassemia, 5′ δ‐globin BCL11A binding region and 3′ β‐globin enhancer

“…Genomic DNA was prepared from peripheral blood leukocytes using a standard method. Screening for DNA deletions causing δβ 0 ‐thalassemia and HPFH found in Thailand and other Asian countries was carried out using multiplex PCR as previously described . Identifications of α o ‐thalassemia (SEA and THAI‐deletions), α + ‐thalassemia (3.7 and 4.2 kb deletions), Hb Constant Spring and Hb Pakse' mutations are routinely performed in our laboratory using PCR methodologies described elsewhere .…”

State of the art and new developments in molecular diagnostics for hemoglobinopathies in multiethnic societies

“…To detect the most common deletions causing alpha-and beta-thalassemia worldwide, a relatively simple, inexpensive multiplex gap-PCR is used [17][18][19][20][21]. A limitation to this otherwise excellent system is that primers can be included into the multiplex only when breakpoint sequences are known.…”

Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/Deletion (δβ)0 Mutation in Antalya

“…While only the β-globin gene is partially or completely removed in some of those deletions, the δ-globin gene or δ- and γ-globin genes are deleted in addition to the β-globin gene in some others [1,2]. It was also stated that 10% of the β-globin gene mutations are large deletions causing phenotypes associated with β-thal [3]. The phenotypes produced by deletions in the β-globin gene cluster are classified according to the gene(s) involved, such as β-thal, δβ-thal, εγδβ-thal, and hereditary persistence of fetal hemoglobin (HPFH) [4].…”

“…Despite general carrier frequency for β-globin gene mutations being reported at 2% for Turkey and at as high as 10% for Antalya Province, large deletional mutations in the β-globin gene cluster have rarely been reported so far and there is no systemic study on mutation profiles of large deletional mutations in the β-globin gene cluster in Turkey [4,5,6,7,8,9]. On the other hand, the number of studies on variety and allelic frequencies of large deletions in the β-globin gene cluster has been growing recently [2,3,10,11,12]. Previous studies revealed that HPFH-1, HPFH-2, HPFH-3, Sicilian (δβ)0-thal, Chinese Gγ(Aγδβ)0-thal, Hb Lepore, Asian-Indian inversion-deletion Gγ(Aγδβ)0-thal, and Turkish inversion-deletion (δβ)0-thal mutations are among the most recurrent large deletional mutations in the β-globin gene cluster [10,13].…”

“…Researchers have recently applied strategies like Southern blotting, FISH, quantitative polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA), and gap-PCR for molecular detection of large deletional mutations of the β-globin gene cluster [10,12,14,15,16]. Among them, gap-PCR is a fast and reliable method allowing us to detect the previously characterized mutations [3,13]. In this study, we screened patients in whom we were not able to find the underlying β-globin gene mutation(s) by direct DNA sequencing for the 8 different common deletional mutations of the β-globin gene cluster by gap-PCR.…”