With the recent media coverage of Angelina Jolie’s decision to have a preventative double mastectomy following genetic testing for BRCA mutations and with the price of this genetic test (approximately $3000AUD) from Myriad Genetics often being referred to, we were curious as to what is actually involved in the test.
Before we start, we should point out that the Australian and United States patents associated with this test have been the subject of recent high-profile decisions relating to the patentability of genetic materials, which we will not discuss in this post (but which we have previously discussed here and here). The Australian patent related to the BRACAnalysis test expires on 11 August 2015, and Reuters have reported the relevant United States patents all expire by 2015.
So, patents aside, what is actually involved in conducting a comprehensive BRACAnalysis test?
The BRACAnalysis Test:
The technical specifications of the BRACAnalysis test require (1) full sequencing of BRCA1 and BRCA2 genes and (2) an examination of BRCA1/BRCA2 gene rearrangements.
1 DNA Sequencing
The types of mutations in the BRCA1 and BRCA2 genes that are associated with risk of developing breast or ovarian cancer include single “letter” changes and small insertions or deletions in the DNA that can be detected by sequencing. Sequencing effectively determines the ‘spelling’ of the DNA of these genes.
For BRCA1, gene sequencing, approximately 5400 base pairs of DNA is sequenced in full in both the forward and reverse directions (the amplified products of 35 polymerase chain reactions (PCR) are sequenced).
For BRCA2, approximately 10200 base pairs of DNA is sequenced in full in both the forward and reverse directions (the amplified products of 47 PCR reactions are sequenced).
While sequencing (above) effectively determines the ‘spelling’ of the DNA of these genes, there are a number of large rearrangements of the genes (insertions or deletions of DNA) that cannot be detected by sequencing of the genes alone – and that is where more lab coat elbow grease is required.
Rearrangement testing is carried out by a technique such as recombination specific PCR, quantitative PCR or microarray comparative genomic hybridization (CGH).
Since rearrangements associated with breast or ovarian cancer are difficult to pick up by sequencing alone, 5 commonly known large genomic rearrangements are detected using PCR performed using primers specific for these 5 large genomic rearrangements (and for the normal gene).
But the work does not end there. When a single site mutation is a BRCA1/BRCA2 deletion or duplication mutation that is not one of the 5 commonly known BRCA1/BRCA2 large genomic rearrangements above (and this appears to be often the case – see here) – even more hard yakka is required.
At this point, multiplexed quantitative PCR or microarray-CGH analysis is used to determine copy number abnormalities indicative of deletion or duplication mutations across the BRCA1 and BRCA2 genes as outlined below:
2a Microarray-CGH analysis
So, let’s say we need to test for rearrangements, since a single site mutation is a BRCA1/BRCA2 deletion or duplication mutation that is not one of the 5 commonly known BRCA1/BRCA2 large genomic rearrangements above. At this point, a microarray-CGH analysis is required.
This technique involves the use of approximately 1700 probes which have been designed to interrogate all the coding DNA and various portions of non-coding DNA of BRCA1 and BRCA2, and are used to identify regions of altered copy number.
So the test can involve approximately 1700 probes for microarray-comparative genomic hybridization (CGH) analysis in addition to the sequencing other PCR methods.
It is fair to say, in researching this post, our science boffins at POF were surprised by the amount of work (and analysis) involved in the test.
Post Sequencing and Rearrangement
Having exhaustively carried out (1) sequencing and (2) testing for rearrangements above, any patient samples which test positive for any detected genetic variant/mutation/deletion are of course independently confirmed. For example:
Genetic variants/mutations are independently confirmed by repeated PCR and sequencing;
Deletions or duplications are confirmed by repeat multiplex quantitative PCR or microarray analysis of the BRCA1/BRCA2 genes;
Rearrangement positive samples detected by multiplex quantitative PCR are further assessed for sequence polymorphisms affecting the PCR primer binding sites, to minimize the possibility of false positive results; and
In some cases, long range PCR analysis and/or sequencing of the resulting PCR product is used to detect specific, previously reported insertions.
Given the above, and the fact that that the tests are all performed with relevant positive controls for all rearrangements/copy number in all of the approaches used, these tests are more of an undertaking than most people (including scientific types) realise.
 For those who love a bit of detail, the 5 rearrangements are:
a 3.8 kb deletion of exon 13
a 510 bp deletion of exon 22
a 6kb duplication of exon 13
a 7.1 duplication of exons 8 and 9 and
a 26 kb deletion of exons 14-20