Reliable detection of beta-thalassemia and G6PD mutations by a DNA microarray.

Abstract

β-Thalassemia is an autosomal recessive disorder caused by the absence or reduction of β-globin chain synthesis. There are >400 million β-thalassemia carriers worldwide, and >160 β-thalassemia mutations have been described (1). Different populations exhibit a specific subset of mutations, as in Sardinia, where carriers are ∼11% of the population and 95% of them present the β 39 mutation (1)(2)(3). In those populations, glucose 6-phosphate dehydrogenase (G6PD) deficiency is also common (4)(5)(6). For the G6PD gene, ∼130 mutations or combinations of mutations have been described (7), and early detection might reduce the risk of hemolytic crisis in childhood. A program of screening newborns would be desirable in those populations. The molecular diagnosis of β-globin and G6PD mutations currently involves a combination of classic methodologies such as restriction fragment length polymorphism analysis, allele-specific oligonucleotide (ASO) hybridization, reverse dot blots, amplification refractory mutation system (ARMS), and direct sequencing (2)(8)(9)(10)(11). These methods are laborious for large-scale screening. We set up a microarray-based assay for parallel one-shot detection of 17 mutations commonly found in the Mediterranean population: β+ −101(C→T); β+ −87(C→G); β codon 6 (-A); β codon 39 (C→T); β-IVSI-1 (G→A); β+-IVSI-6 (T→C); β+-IVSI-110 (G→A); β-IVSII-1 (G→A); β+-IVSII-745 (C→G); β+-IVSII-844 (C→G); G6PD A− variant (202G→A; 376A→G); Mediterranean variant (563C→T); Seattle variant (844G→C); Montalbano variant (854G→A); S. Antioco variant (1342A→G); and Maewo (1360C→T). We called this microarray “Thalassochip”. Thalassochip is based on the arrayed primer extension (APEX) technology (12) implemented with allele-specific primed extension (ASPEX) (13). APEX consists of a sequencing reaction primed by an oligonucleotide anchored to a glass slide (with its 5′ end) terminating just one nucleotide before the mutation site. DNA polymerase extends it by adding one fluorescently labeled …