Inexpensive DNA Microarray Application for Screening of Beta-Globin Mutations in Low-Resource Settings.

Abstract

Abstract 4067Poster Board III-1002Development of public health related products are, in general, driven by clinical usefulness in combination with economic feasibility. Not surprisingly, a high throughput, automated robotic system is not an option in a low-resource setting as exists in the majority of countries with high prevalence of hemoglobinopathies. Diagnostics developed for resource rich areas may not be feasible in such areas but certain parts of these technologies can be transformed into low-cost diagnostics suitable for resource poor settings. Hemoglobinopathies are traditionally presumptively diagnosed using a combination of methods including complete blood count, MCV or MCH, and hemoglobin electrophoresis or HPLC. Recently, sequence specific nucleic acid recognition methodologies have allowed the discovery of more than 200 mutations which cause thalassemia and an even higher number of mutations leading to hemoglobin variants. Importantly, such methods provide a more precise diagnosis and allow for prenatal analysis. Microarrays use specific nucleic acid sequences to detect up to thousands of mutations simultaneously but, due to their cost and requirements of equipment, are principally only suited to resource rich areas. However, pairing microarray technology with alkaline phosphatase colorimetric staining, an established visualization technology, significantly reduces assay cost and initial cost and complexity of required equipment and microarray-based diagnostics can thus become applicable to resource poor settings. This study focused on the development of a highly specific and low-cost method for genotyping beta-thalassemia mutations and hemoglobin variants using colorimetric staining of allele specific DNA microarrays. Target was easily prepared by in vitro incorporation of a small amount of biotin-labeled ribonucleotides after an initial PCR reaction and was immediately ready for subsequent visualization via the well characterized and highly specific biotin-streptavidin interaction. Streptavidin-conjugated alkaline phosphatase coupled to commercially available BCIP/NBT, well established in ELISA and western blots, allowed results visualization. Microarray assays that correctly and accurately identify mutations in multiple nucleic acid sequences (genechips) are difficult to produce as these are usually processed at a single condition. This single condition does not represent the optimum condition for all the individual and necessary reactions required to detect all mutations. This can be circumvented by changing reaction temperature during analysis. This requires multiple readouts during a genotyping cycle and precise thermal control, both of which are not yet compatible with a low-budget application. In a previous paper, we demonstrated a very low cost alternative solution that used buffers with different ionic strength ([Na+]) and a polymeric microfluidic assembly rather than temperature control to optimize microarray conditions. Arrays, including an internal batch quality control, were manufactured on ordinary glass microscope slides coated with a film of agarose. Detection is based on colorimetric grayscale readout from an ordinary flatbed desktop scanner at low resolution (1200 dpi). In this ongoing study, 30 of the most common mutations leading to beta-thalassemia and those responsible for HbC, HbS, HbD-Punjab, HbE and HbO-Arab hemoglobin variants were included. The microarray based technique is currently being compared to molecular diagnosis via sequencing. To date, a 100% concordance has been obtained. The study also continues to optimize probes for mutagenic sites in order to increase the number of analysis sites and confidence in genotype calls. In conclusion, combining a polymeric device easily manufacturable by high volume methods such as injection molding, microarrays printed on ordinary glass slides, and detection based on colorimetric staining a low-budget analysis system for genetic variations was obtained. Start-up and assay costs were roughly $2000 and $5 US, respectively. As such the method provides interesting and new diagnostic possibilities for those third world countries where beta-thalassemia and other hemoglobinopathies are highly prevalent and where the socioeconomic conditions exclude introduction of expensive methods for molecular diagnostics. Disclosures:No relevant conflicts of interest to declare.