Here, we discuss the usefulness of simultaneous genotyping and mutation scanning in the context of large-scale mutation screening projects. The search for new potentially deleterious genetic variants in candidate susceptibility genes requires the screening of coding sequences and splice junctions of entire genes in large sets of cases and controls. HR-melt analysis has repeatedly been reported as an efficient method for mutation scanning. Although it has been reported that different heterozygotes within the same amplicon could be distinguished from each other based on their curve shape differences (Graham, et al., 2005
) (Garritano et al.
personal communication), mutation screening performed on a large number of samples renders the analysis more complex. Moreover, screening of entire genes often requires the screening of genomic regions containing common SNPs that can interfere with the mutation scan and complicate the interpretation of the results (De Leeneer, et al., 2008
). Systematic resequencing of all variant samples is the most common approach to this issue. However, when applied on large series, this latter approach is expensive, laborious and time-consuming (Sevilla, et al., 2002
Simultaneous genotyping and mutation scanning by HR-melt analysis represents an attractive alternative for high-throughput analysis. The genotyping method was chosen because it could be easily integrated in our existing mutation scanning workflow. Other genotyping methods could have been chosen, but they would have added extra steps to our mutation screening protocol, and would also have required the use of another laboratory instrument. By performing genotyping and mutation scanning simultaneously using HR-melt, we avoided multiple manipulations, and waste of biological material and reagents. Lab contaminations issues were also reduced. For amplicons that contain a common SNP, we postulated that stratification of HR-melt data by common SNP genotype prior to mutation scanning analysis would increase the detection sensitivity for those rare variants, whose melting patterns may be either 1) essentially the same as, and consequently masked by, the melt curve of a common SNP heterozygote; 2) masked by the extra noise present in a large scale melt curve analysis that contains 2 common genotypes; or 3) buried within the melt curve data of an amplicon whose data complexity overcomes the standard software’s ability to group.
Here, we showed that simultaneous genotyping and mutation scanning is suitable to easily distinguish up to nine different genotype combinations, in the case of the 36th coding exon of ATM. Automatic clustering by the analysis software showed complete concordance with sequencing results. In addition, this approach offers the advantage of directly queuing asymmetric PCR products for sequencing. We validated on a series of 90 samples that sequencing reactions from asymmetric PCR products and standard sequencing reactions performed equally.
Study of the 59th
coding exon pointed out that the position of a variant within the amplicon and/or the nature of the sequence surrounding the variant are likely to play a critical role on the accuracy of mutation detection by standard HR-melt analysis. Our study provides evidence that in some sequence contexts, some sequence variants may be missed by the classical HR-melt approach, especially when mutation scanning is performed in a 384-well format. This issue has been discussed in a technical assessment of the HR-melt protocol by the UK National Genetics Reference Laboratory (http://www.ngrl.org.uk/Wessex/downloads.htm
), and the authors concluded to the existence of sequence variations “intrinsically difficult” to detect by HR-melt.
Simultaneous genotyping and mutation scanning represents therefore a valuable asset since it can easily be integrated in large-scale, high-throughput mutation scanning workflows. Although the risk of missing a rare variant might still remain, this method showed better sensitivity for the identification of novel rare variants, and better accuracy for distinguishing different genotype groups, than the standard HR-melt mutation scanning method. Having validated this approach to screen the 36th and 59th coding exons of ATM efficiently, 8 additional probes were designed to improve the mutation screening of the whole gene in our sample sets. Our general experimental strategy was to design an unlabeled probe for each variant reported to have a frequency >1% in dbSNP database in the regions of interest, in our sample series.
Thus, 10 out 66 ATM amplicons could have been predicted beforehand to require simultaneous genotyping and mutation scanning (15%). We also applied this approach to two additional amplicons during the course of the study to facilitate their mutation screening. The first one contained a SNP found to be common in our sample series (rs3092910:T>C), and the second one contained a novel SNP 54 bp upstream of the 43th coding exon of ATM, that we initially identified using the standard mutation scanning approach (). For all studied amplicons, cycling conditions (annealing temperature and number of cycles) were optimized in presence of LC Green for mutation screening. Our experience showed that after PCR optimization, none of the ATM amplicons failed to amplify in the presence of LCGreen. For amplicons requiring simultaneous genotyping and mutation scanning, we re-optimized the PCR conditions in presence of the probe. We also verified that the 1:5 primer concentrations ratios would not impair the HR-melt analysis. Initial protocols had to be modified in some cases, especially by adjusting MgCl2 concentration.
Unlabeled probes used to perform the simultaneous genotyping and mutation scanning of ATM, in the Breast Cancer Genetics Study.
Using our strategy, a higher level of confidence in mutation scanning results can be reached when simultaneously proceeding to genotyping using unlabeled probes. We have shown that this approach can dramatically reduce the amount of sequencing required, compared to sequencing all variants that have a melt curve indicative of the presence of a sequence variant, and recommend the method whenever one of three criteria is met 1) the cost of excess sequencing due to the presence of a known common variant in an amplicon will exceed the ~$50-$75 setup cost of the unlabeled probe assay; 2) there is great concern that the presence of a known common SNP will mask the presence of an unknown rare SNP; or 3) it is important, within the mutation screening context, to detect all of the minor allele homozygotes of a common SNP located with an amplicon of interest.
The potential of HR-melt for cost-effective and sensitive high-throughput genotyping and mutation scanning has been reported in numerous studies. For example, Takano et al
. and De Leeneer et al
. described HR-melt as an economical screening method to detect mutations in BRCA1
(De Leeneer, et al., 2008
; Takano, et al., 2008
). In their work, the authors emphasized the advantages, both in time and cost, offered by the use of HR-melt. Cost-effective and rapid methods for screening are indeed highly needed for mutation screening and testing, particularly for molecular diagnostic purposes in medium and low-resources countries. For mutation discovery studies, this technique would also be beneficial since it enables large-scale case-control or population studies at low-cost, but with a sensitivity and an accuracy higher than the current mutation scanning gold-standard, DHPLC (Chou, et al., 2005
In conclusion, simultaneous genotyping and mutation scanning is another methodology that confirms that HR-melt is a rapid, efficient and cost-effective tool that can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes.