BMC Cancer 2008, 8:59 http://www.biomedcentral.com/1471-2407/8/59 HRM is an in-well scanning method in which the melting analysis is performed immediately after the PCR amplifi- cation and is thus particularly suitable for high-through- put applications. HRM is predicted to have close to a 100% sensitivity as each heterozygous mutation should create a readily detected heteroduplex [17]. Heterozygotes are readily recognisable by HRM because they form a pro- portion of heteroduplexes which melt differently to per- fectly matched homoduplexes. The use of HRM to detect both known and novel germline mutations has been reported for several other genes including the EXT1, EXT2, RET, GJB1 and CFTR genes [18-21]. All germline muta- tions in the BRCA1 and BRCA2 genes are expected to occur in heterozygotes as mutant homozygotes are lethal [22] and consistent with this, none have been reported. A comparative study has recently shown that the sensitiv- ity and specificity of HRM is better than DHPLC which is the current gold standard of scanning methods [17]. In addition, HRM is more rapid as the melting analysis is performed in all wells simultaneously whereas DHPLC involves the removal of the PCR product (in itself prob- lematical because of the PCR contamination issue) and then the sequential analysis of each sample. The instru- mentation for DHPLC is more expensive and the analysis generates chemical waste which requires disposal. There are therefore significant time and cost advantages in adopting HRM. In our hands, all the different mutations in the 3 regions that we analysed from the BRCA1 and BRCA2 genes were detected, giving us a sensitivity of 100% in this pilot study. The true sensitivity remains to be determined in a larger study, but publications for other genes indicate that it is likely to be very high [14,17]. A larger study is cur- rently underway in our lab testing several hundred known mutations in historic samples across all BRCA1 and BRCA2 coding regions in combination with the screening of prospectively collected diagnostic samples. This will provide further insight into the true sensitivity of HRM and applicability of the method for mutation scanning of the entire coding regions of BRCA1 and BRCA2. Currently, the three founder mutations associated with Ashkenazi Jewish ancestry are generally identified using sequencing and, once a mutation is identified in a family, other family members can undergo predictive testing by sequencing for the exon in which the mutation was iden- tified. However, as the frequency of the three founder mutations is relatively high, it is possible that a second mutation might be segregating in the family and so ideally all founder mutations should be sought in other family members. HRM would allow testing for all three Ashkenazi mutations in family members at relatively low cost. The testing would also enable inexpensive popula- tion-based screening of individuals with Jewish ancestry for the founder mutations. We also detected the 5331G>A mutation in BRCA1 exon 20 that has recently been shown to be a Greek founder mutation [10]. Large scale screening for this founder mutation can now be undertaken in relevant populations. A similar approach can be applied to other founder muta- tions in regions not screened for in this study. The ability to detect mutations by HRM alone has implications for economical large scale population screening studies to understand the prevalence and characteristics of other known or suspected founder mutations. HRM can also be used as an inexpensive methodology for tracking mutations across pedigrees. Each mutation had a characteristic melting profile that would allow it to be identified by using a control for that mutation. In some cases, this would remove the necessity for sequencing. However, for diagnostic purposes, any apparently muta- tion positive individual should be confirmed by sequenc- ing as different mutations can also have similar profiles. Dufresne and colleagues [23] reported the use of melt curve analysis for the specific detection of the Ashkenazi mutations using SYBR Green 1 incorporation as moni- tored by a standard real time PCR machine. This approach is melting analysis rather then the high resolution melting (HRM) we have used here. Whereas their methodology is capable of detecting known mutations such as the Ashkenazi mutations by designing primers flanking those mutations, our methodology is an essentially different and more powerful methodology which is designed pri- marily as a screening methodology. We used an appropri- ate dye for high resolution melting, an HRM enabled real time PCR machine (only recently available) and software that allows HRM analysis to be performed. The greater sensitivity of HRM allowed longer amplicons to be used (534 base pairs was successfully used for the region sur- rounding the 6174delT mutation) which allows for the screening of unknown mutations as we have demon- strated. Thus by examining extra, non-founder mutations in close proximity to the founder mutations we show that this method easily detects missense mutations as well as small insertions and deletions. Interestingly, the missense mutation heterozygotes melt earlier than some of the deletion/insertion heterozygotes; the 6174delT initially being more stable than even the wildtype. Based on our current findings, we are confident that HRM for mutation detection will prove superior to the current germline mutation screening approaches in terms of speed, and cost and give at least equivalent sensitivity. HRM is the most rapid of the current screening methods as the only set-up involved is the PCR amplification, up to Page 5 of 7 (page number not for citation purposes)