A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes

Abstract

Background: Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous.

Methods: Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput.

Results: 169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays.

Conclusions: This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.

Figure 1

Detection of both BRCA2 c.26delC mutant and wild type alleles where deoxyinosine is used at the c.-26G>A SNP. The red and blue samples both contain the c.26delC deletion but differ according to their genotype for the BRCA2 c.-26G>A polymorphism. The red profile is homozygous wildtype for the polymorphism. The blue profile is heterozygous for the polymorphism. The left panel shows the difference curves where the baseline comprises both wildtype (grey) and heterozygous (green) genotypes. The right panel shows the corresponding melting peak curves. The difference curves are independent of the SNP meaning that both alleles are equally amplified using the deoxyinosine containing primers

Figure 2

Detection of different mutations within the same amplicon (BRCA1 11I region). The left panel shows the difference curves and the right panel shows the corresponding melting peak curves. All mutations produce obvious biphasic melting which is caused by the earlier melting of the heteroduplexes. The normalised and temperature-shifted difference plots in the left panel allow easy detection of the c.2863delTCATC (navy), c.2800C>T (red) and c.2885delA (green) relative to the wildtype HRM profile (grey). The melting peak curves in the right panel show that the c.2885delA is the most subtle mutation in that there is a minimal early melting heteroduplex component compared to the c.2863delTCATC and c.2800C>T mutations.

Figure 3

The influence of amplicon choice on mutation detection. The top panels show difference curves and melting peak curves for the original BRCA1 exon 7 amplicon which did not readily detect single base insertions or deletions. While the c.427G>T (red) and the c.314A>G (green) mutations are readily detectable in both visualisations, the pathogenic c.329insA (navy) and c.302-2delA (yellow) single base pair insertion and deletion mutations were difficult to detect as they melt like the wildtype controls (grey). The original amplicon was then divided into two overlapping amplicons. The bottom panels show difference curves and melting peak curves for BRCA1 amplicon exon 7A which detects the single base insertion and deletion. The c.329insA (navy) and c.302-2delA (yellow) clearly differ from the wildtype controls (grey) in the shorter amplicon. The BRCA1 c.314A>G (dark green) mutation is also readily detectable in both visualisations.

Figure 4

Detection of an insertion within a long nucleotide repeat by HRM (BRCA2 exon 23). The mutation c.9097insA (green) is an insertion of an adenine nucleotide into an 8 adenine repeat. Like the other mutations here; c.9117G>A (red) and c.9117+1G>A (navy); it is readily distinguishable from the wildtype (grey) in this 264 bp amplicon.

Figure 5

Two variations within the same sample result in greater HRM differences. (BRCA1 exon 13). The left panel shows the difference plot where 3 samples have been compared to the wildtype controls (grey). The green profile represents the a heterozygote for common polymorphism c.4308T>C while the blue represents the mutation c.4327C>T. The presence of both sequence variants (red) results in greater instability than each individual sequence variation due to the double mismatch in heteroduplexes. The right panel with melting peak curves show the complex melting nature of two co-existing sequence variants (red).

Figure 6

Detection of a sequence variation immediately adjacent to the 3' end of a primer. The class 1 SNP (BRCA2 c.1365A>G) (red) is readily detectable at the position immediately adjacent (1 base pair) to the 3' end of the reverse primer in this 232 bp amplicon (BRCA2 exon 10C).