Rapid and easy prenatal diagnosis of sickle cell anemia using double‐dye LNA probe technology

Abstract

Taking advantage of the high specificity of double-dye hydrolysis-locked nucleic acid (LNA) probes (Ugozzoli et al, 2004), we developed a qualitative three-colour real-time polymerase chain reaction (PCR) assay for the genotyping of sickle cell disease (SCD). The term SCD encompasses a group of disorders associated with mutations in the Haemoglobin Beta gene (HBB). The most common forms are defined by the presence, in codon 6, of an E6V mutation (GAG > GTG; Haemoglobin S; Hb S) on both alleles (sickle cell anaemia) and co-inheritance of Hb S and Hb C (E6K mutation; GAG > AAG; Hb SC disease). The highest prevalence of SCD is found in sub-Saharan Africa (sickle-cell gene carrier prevalence varying between 5% and 40% of the population), and most affected children die in the early years of life without being diagnosed or because of non-adapted treatments (Weatherall & Clegg, 2001). The methodological approach presented here combined the use of a primer pair designed to amplify the region of interest in the HBB gene and three double-dye probes containing LNA nucleotides. Each probe was labelled with a different fluorochrome to enable the different alleles to be discriminated (Fig 1). Primers were designed with the freeware meltcalc, ver. 2.0 (http://www.meltcalc.de). The Tm of each double-dye LNA probe was calculated by using the Exiqon Tm prediction tool (http://lna-tm.com). Specificity of the probes used in this approach. 20 ng of genomic DNA from three patients homozygous for the wild-type (WT/WT), S (S/S) and C (C/C) alleles respectively was tested in this experiment. The results given by the each probe are illustrated on different panels. The Cy5 channel was applied for the analysis of the WT probe, while FAM and Yakima Yellow were used for the S and C probes, respectively. The three probes showed absolute specificity because there was no signal detected when amplified genomic DNA did not contain the nucleotide targeted by the probe. The no template control did not give any signal for the three probes either. Samples from patients that were homozygous for each of the three aforementioned alleles were used to demonstrate the specificity of the probes (Fig 1). We used unique PCR conditions on the ABI 7500 Fast machine (Applied Biosystems, Foster City, CA, USA), which generated specific results for each probe in <40 min (see Table SI, Fig S1). A cohort of 37 genomic DNA representing the different possible genotypes was used to compare our new technique with either a PCR followed by restriction endonuclease cleavage to detect the A and S alleles (Saiki et al, 1988), or an allele-specific PCR designed to detect A and C alleles (Fischel-Ghodsian et al, 1990). A 100% concordance was observed between our technology and the two published methods. Moreover, the deduced genotypes matched perfectly with the available phenotypic analysis (Gulbis et al, 2006). To investigate the sensitivity of this analysis, we tested different amounts of genomic DNA per PCR reaction. The results were perfectly interpretable from 100 to 1 ng of starting material (data not shown), and enabled the testing of prenatal diagnosis samples even when there was scarce fetal tissue (demonstrated on 18 gDNA obtained from fetal samples; data not shown). Finally, to demonstrate the robustness of our approach, we tested 10 representative samples on currently available instruments that are widely used for multicolour real-time PCR analysis. The results obtained on the LC480 (Roche, Basel, Switzerland), the iCycler (Bio-Rad, Hercules, CA, USA) and the M × 3000 p (Stratagene, La Jolla, CA, USA) were perfectly comparable from one machine to another (see Fig S1). Although a fast PCR protocol was not applicable on all these machines, a longer PCR protocol did not affect the results. In summary, we have developed a very robust and rapid method that takes advantage of the high specificity of double-dye LNA probes, which enabled the identification of the two main mutations in SCD in <40 min. The protocol developed in our laboratory is easily transferable to other real-time PCR machines. The high sensitivity of our approach represents a great advantage for the analysis of samples containing small amounts of DNA. Moreover, this single closed tube method offers another obvious advantage for prenatal diagnosis as it prevents risk of cross contamination. We gratefully acknowledge the skillful technical assistance of Sandrine Delbauve. Table SI. Oligonucleotides used for the three-colour real-time polymerase chain reaction (PCR) assay in this study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.