Product update: BRCA1 and BRCA2

Both AmoyDx^® BRCA Panels are based on the HANDLE (Halo-shape Annealing and Defer-Ligation Enrichment) technology and allow the detection of point mutations (SNVs), insertions and deletions (InDels). Additionally, in DNA isolated from blood samples, large gene rearrangements (LRs) can be analyzed.

While until now this required parallel analysis of five blood specimen the new kit enables analysis of LRs in only one blood sample. With the new AmoyDx® BRCA Pro panel, the verification of large gene rearrangements with MLPA is no longer necessary. 

Functional principle

The AmoyDx® BRCA Pro Panel is based on the HANDLE (Halo-shape Annealing and Defer-Ligation Enrichment) technology (Fig. 1). It enables the preparation of NGS libraries in just five steps within approximately five hours, the hands-on time being only about one hour. The probes first hybridize at the target region of the genomic DNA with their terminal complementary ends. Using polymerase, the DNA is extended at the 3‘-end of the probes along the target DNA, and ligated to the 5‘-end of the probes by means of a ligase. Linear DNA molecules are removed by enzymatic digestion. The remaining ring-shaped DNA sequences are PCR amplified, in which process index sequences and UMIs are added. All reactions take place in only one tube per patient specimen.

The use of UMI (unique molecular identifier) sequences allows elimination of artefacts by amplification of the library and sequencing. The NGS libraries created with the AmoyDx® BRCA Pro Panel are suitable for sequencing on Illumina platforms.

The sequencing data is analyzed on the AmoyDx® NGS data analysis system (ANDAS) which consists of a local workstation with pre-installed analysis software. This allows reliable evaluation in just a few steps.

Background information on the AmoyDx^® BRCA Pro Panel (CE/IVD)

The BRCA1 and BRCA2 tumor suppressors are essential for repairing DNA double-strand breaks by means of homologous recombination. Deleterious mutations in the respective genes are frequently found in solid tumors. The enzyme PARP-1 (type I poly ADP-ribose polymerase) plays an important role in the repair of DNA single-strand breaks. PARP inhibitors approved for the treatment of patients with ovarian cancer or breast cancer with mutations in the BRCA1 and BRCA2 genes can inhibit tumor growth. In the process, the inhibition of PARP-1 causes DNA damage which cannot be repaired without functional BRCA1 and BRCA2 proteins [1-3].

The BRCA1 and BRCA2 genes are located on chromosomes 17 and 13, respectively. BRCA1 contains 22, BRCA2 26 coding exons. Meanwhile, more than 3,000 mutations are known that impair the function of the BRCA proteins. They include point mutations, insertions, deletions and large gene rearrangements (LRs) and are not concentrated in specific hot-spot regions on the BRCA genes. According to the guidelines of the IARC (International Agency for Research on Cancer) and the ACMG (American College of Medical Genetics), BRCA1 and BRCA2 mutations can be classified into five categories. PARP inhibitors can act effectively in patients with BRCA1/2 class 4 or class 5 mutations.

Purpose

The AmoyDx^® BRCA Pro Panel (CE/IVD) is an in vitro diagnostic assay based on Next Generation Sequencing (NGS) designed for qualitative detection of BRCA1 and BRCA2 variants in DNA extracts from peripheral whole blood samples and from FFPE tissues. The analysis covers all coding exons, the border regions between exons and introns, some introns, and UTR regions of the BRCA1 and BRCA2  genes. The kit can be used to detect point mutations (SNVs) as well as insertions and deletions (InDels) in DNA from tissue. In DNA from whole blood, large rearrangements (LRs) can be analyzed additionally. The assay is designed for identification of pathogenic and probably pathogenic variants (IARC class 4 and class 5) in patients with breast cancer or ovarian cancer. The kit is intended to be used by trained professionals in a laboratory environment only.