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Reliable Detection of ß-Thalassemia and G6PD Mutations by a DNA Microarray

¹ IARC, International Agency for Research on Cancer, 150, Cours Albert Thomas, Lyon 69372, France

² University of Pisa, Dipartimento di Scienze dell’Uomo e dell’Ambiente, Via S. Giuseppe 22, 56100 Pisa, Italy

³ Dipartimento di Scienze Biomediche e Biotecnologie, Universita’ di Cagliari, Ospedale Regionale Microcitemie, Via Jenner, 09121 Cagliari, Italy

⁴ Institute of Molecular and Cell Biology, Estonian Biocentre, University of Tartu, 23 Riia Street, 51010 Tartu, Estonia

⁵ Dipartimento di Medicina Interna, Cardioangiologia ed Epatologia, Università di Bologna, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40125 Bologna, Italy

⁶ Asper Biotech, Ltd., 3 Oru St., 51014 Tartu, Estonia

^aaddress correspondence to this author at: Dipartimento di Medicina Interna, Cardioangiologia ed Epatologia, Università di Bologna, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40125 Bologna, Italy; fax 39-051-30-61-71, e-mail romeo{at}iarc.fr

ß-Thalassemia is an autosomal recessive disorder caused by the absence or reduction of ß-globin chain synthesis. There are >400 million ß-thalassemia carriers worldwide, and >160 ß-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 ß⁰ 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 ß-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: ß+ -101(CT); ß+ -87(CG); ß⁰ codon 6 (-A); ß⁰ codon 39 (CT); ß⁰-IVSI-1 (GA); ß+-IVSI-6 (TC); ß+-IVSI-110 (GA); ß⁰-IVSII-1 (GA); ß+-IVSII-745 (CG); ß+-IVSII-844 (CG); G6PD A^- variant (202GA; 376AG); Mediterranean variant (563CT); Seattle variant (844GC); Montalbano variant (854GA); S. Antioco variant (1342AG); and Maewo (1360CT). 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 dideoxynucleoside triphosphate complementary to the variant base. The reading of the incorporated fluorescence identifies the base in the target sequence. In ASPEX, the oligonucleotide ends with the variant base (ASO). Two pairs of oligonucleotides are needed for forward (F) and reverse (R) strands for both the mutant (M) and the wild-type (WT) alleles (ASO-M-F, ASO-M-R, ASO-WT-F, and ASO-WT-R, respectively). The extension occurs only when ASO completely matches to the target. Fig. 1 shows two examples. In this report we describe a validation study of Thalassochip.

View larger version (39K): [in this window] [in a new window]   Figure 1. Examples of ASO with complete matching. (A), WT homozygote for ß-IVS I-110. The WT homozygote produces two ASO-WT signals and two APEX signals. (B), heterozygote (carrier) for ß-IVS I-1. The heterozygote produces four signals from ASPEX and four signals from APEX. Boxed nucleotides indicate the variant position; underlined nucleotides indicate mismatched bases for weakening secondary structures of nucleotides; bolded nucleotides indicate the bases extended by Thermo Sequenase.

We studied 117 individuals from Sardinia who were referred to the hematologic service of the Ospedale Regionale Microcitemie, Cagliari, Italy. We used classic routine methods considered "gold standards" [restriction fragment length polymorphism analysis, ARMS, reverse dot blots, and direct sequencing, (3)(9)(14)]. All participants gave informed consent in accordance with the Helsinki Declaration. Participants were selected to cover the mutations of Thalassochip for validation purposes. Blood samples were also analyzed for G6PD activity (15).

In the present study, the ß-globin gene was amplified from human genomic DNA in two fragments and the G6PD gene in five fragments as described elsewhere (box I in the supplemental data, available with the online version of this Technical Brief at http://www.clinchem.org/content/vol48/issue11/). One unique protocol and thermocycling profile was used for all seven PCRs. 5' (C-12) aminolinker oligonucleotides were purchased from Sigma Genosys Ltd and spotted onto silanized slides as reported elsewhere [Refs. (16)(17)(18) and box III in the supplemental data]. A detailed list is given in Table 1 of the supplemental data. PCR products were pooled together, purified, and concentrated on Millipore Y30 columns, and 15 µL of eluate was collected. The PCR products were reduced in size by fragmentation to allow better hybridization with arrayed oligonucleotides. We treated 15 µL of purified PCR products (performed with 200 µM dATP, dCTP, and dGTP; 160 µM dTTP; and 40 µM dUTP) with 1 U of uracil N-glycosylase (Epicentre Technologies) and 1 U of shrimp alkaline phosphatase (Amersham Biosciences). The mixture was incubated at 37 °C for 3 h and then at 95 °C for 30 min to denature DNA with abasic sites and inactivate the uracil N-glycosylase and shrimp alkaline phosphatase. More details are provided in box III of the supplemental data. APEX and ASPEX work at the same time on the same slide with the reagent mixture and templates. Briefly, the 20-µL reaction mixture, containing fluorescently labeled dideoxynucleoside triphosphates (50 pmol of each), 10x buffer, fragmented PCRs (9 µL), and 4 U of Thermo Sequenase (Amersham Biosciences), was placed on the spotted slide and incubated for 25 min at 58 °C. Slides were washed, a droplet of SlowFade^® Light Antifade Reagent (Molecular Probes) was added to limit the bleaching of fluorescein, and the slide was imaged on a Genorama-003 four-color detector equipped with Genorama image analysis software (Asper Biotech). More details about the APEX/ASPEX reaction as well as the detection of signals are given in box II in the supplemental data.

Each mutation is specified by a pattern of six different oligonucleotides. Two positions are for APEX, and four positions are for ASPEX. A simple algorithm has been established for combining the six signals to give the final genotype. Two examples are shown in Fig. 1 . Shown in Fig. 1A is a WT homozygote for ß IVS I-110. The WT homozygote gives two ASO-WT signals and two APEX signals for WT genotype. The four signals correspond to the WT genotype. No signal corresponds to the mutant allele. This condition is scored as 4/0. A heterozygote (carrier) for ß IVS I-1 (shown in Fig. 1B ) produces four signals from ASPEX and four signals from APEX. The score is 4/4. A mutant homozygote is identified by four signals from the mutant allele and will show a score of 0/4. Intermediate scores such as 4/2, 2/4, 3/2, and 2/3 were considered "heterozygotes"; 4/1, 3/1, 3/0, and 2/0 were considered WT homozygotes; and 1/4, 1/3, 0/3, and 0/2 were considered mutant homozygotes. Scores of 1/0, 0/1, 2/1, and 1/2 were considered inadequate for diagnosis (marked as N/A). We calculated the 95% confidence intervals, comparison proportions, sensitivities, and specificities by binomial distribution, considering the gold standard as 100%. Statistical analyses were carried out with Statgraphics plus 2.1 for Windows (Manugistic Inc.).

In our sample set, we were not able to analyze the mutant alleles ß-IVSII-844 and G6PD854 because of their rarity in the Mediterranean area. In most cases, one or two of six oligonucleotides were enough to determine the genotype correctly. Twenty-seven oligonucleotides showed 100% correct extensions, and other 42 showed correct extensions in >90% of the samples. This means that, in the future, it will be possible to reduce the size of the microarray just picking the two to three best oligonucleotides within each mutation. Details about each specific oligonucleotide are reported in Table 2 of the supplemental data. Thalassochip and the gold standards are compared in Table 1 . Of 1989 genotypes, only 9 were called incorrectly, making the overall error rate 0.45%. All nine mistakes were samples misclassified as heterozygotes (six WT and three homozygotes) and would have been retested with different methods. ß-IVS II-745 demonstrated a lack of signal in most cases, which was reflected in a lower rate of detection (see Table 1 ). The region surrounding the ß-IVS II-745 mutation is AT rich, and 58 °C was probably too high for hybridization. Further trials with longer APEX and ASPEX oligonucleotides seemed to solve this problem (data not shown). Redesigning of oligonucleotides was also helpful in other situations (data not shown), leading to the idea that melting temperature could be one of the critical factors for the assay. Alternatively, longer oligonucleotides could allow a more stable interaction between the target, the oligonucleotide, and the Thermo Sequenase.

The specificity of Thalassochip was 100% for 13 mutations, with the lowest sensitivity being 98.5% for G6PD563. Sensitivity was 100% for 14 mutations and 90.9% for ß-codon 6. However, two carriers and two homozygotes for hemoglobin S (HbS) were correctly detected by APEX codon 6 oligonucleotides (data not shown), despite the fact that oligonucleotides for codon 6 were not specifically designed to detect HbS. More detailed information about the sensitivity and specificity is provided in Table 3 of the supplemental data.

Redundancy is important in microarray technology, both from a statistical point and for the correct interpretation of signals. Many different technologies require a redundancy of targets, which leads to more accurate responses with increasing numbers of signals. In our hands, six oligonucleotides per mutation were highly reliable for analysis, and we could have reached the same results with a lower number of oligonucleotides. The redundancy allowed us to circumvent artifacts such as lack of signals and nonspecific extensions.

Our results, along with the low reagent costs (approximately US $0.50/genotype) and short processing time (2 days for 30 samples testing for 17 mutations) for the microarray, indicate a potential use of this technology for screening programs. This approach may be expanded to other common diseases, such as -thalassemia, Wilson disease, or cystic fibrosis.

Acknowledgments

Dr. Landi is a recipient of a Marie Curie fellowship (IHP-MCIF-99-1) of the European Commission. Dr. Gemignani is recipient of a special training award (STA) by the International Agency for Research on Cancer. This work was supported in part by grants from the foundation "Fondazione Italiana Leonardo Giambrone per la Guarigione dalla Thalassemia". Andres Metspalu was supported by a core grant from the Estonian ministry of education (No. 0180518s98) and Estonian SF Grant 4479 and was the recipient of the IARC senior fellowship.

Footnotes

¹ these authors contributed equally to the project

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