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Saturation Editing of BRCA1 Exons Reveals Functional, Non-Functional Variants

NEW YORK (GenomeWeb) – By systematically introducing mutations into the BRCA1 gene, researchers have sought to better classify which variants are likely to be pathogenic or benign.

BRCA1 is one of the best-studied genes, but the impact of its numerous variants on breast and ovarian cancer risk remains unclear. Such variants of uncertain significance (VUS) limit the clinical utility of a patient's data, said the authors of the new study.

"Frequently women are being tested for BRCA1 mutations because they have a family history of breast or ovarian cancer," paper co-author Lea Starita from the University of Washington said in a statement. "To be told that they have a genetic variant in this cancer-predisposing gene, but that the doctor doesn't know what it means, does not reduce their stress or their anxiety."

Using saturation genome editing, she and her colleagues tested how changes to 13 exons in the BRCA1 gene affected cells' abilities to grow in culture and see whether those alterations affect gene function. As they reported in Nature today, the variants determined by the researchers to be functional highly corresponded with what has been reported in the ClinVar database.

The researchers focused their saturation editing on the 13 BRCA1 exons that encode the RING and BRCT domains, which play a key role in the tumor suppressor function of BRCA1. They also used human haploid HAP1 cell line for their analysis, as BRCA1 is an essential gene for that line.

They designed a CRISPR/Cas9 approach to alter each nucleotide within those exons to all possible mutations — making a total of about 4,000 individual mutations — and let the cells to grow for 11 days. After that, they collected genomic DNA for analysis and used the frequency of the SNVs to gauge whether the induced variant was functional or not.

For each SNV, the researchers generated function scores based on that frequency. Those scores then indicated if the variant had no affect on BRCA1 function, if it did and led to cell death, or had an intermediate effect.

The SNVs largely fell in a bimodal distribution, with 72.5 percent being functional, 21.1 percent non-functional, and 6.4 percent intermediate.

These function scores largely agreed with how variants were interpreted in the ClinVar database. For 169 SNVs considered pathogenic by ClinVar, 162 were also considered non-functional by the researchers' analysis. Similarly, for 22 SNV considered benign by ClinVar, the researchers deemed 20 functional. This showed a sensitivity of 96.7 percent at 98.2 percent specificity.

These scores likewise agreed with allele frequencies in other databases and with computational metrics. They also corresponded with other assays of BRCA1 function, including a similar assay performed by the same team that relies on an homology-directed repair (HDR) reporter cell line that also came out today in the American Journal of Human Genetics. Functional BRCA1 is needed for HDR.

In that paper, the UW team made 1,056 amino acid substitutions in the first 192 residues of BRCA1 — a stretch that includes the RING domain. The multiplex reporter assay, based on one developed by the Jasin lab, uses green fluorescent protein to indicate whether HDR is still functional in the mutated HeLa cells. The two approaches, the researchers said, differed by 4 percent, which they said could reflect the biology of the two assays.

For their saturation editing study, the researchers also performed targeted RNA sequencing of transcripts obtained from edited cells. They generated RNA scores to indicate the expression consequences of the SNVs. They reported that 89 percent of non-functional missense SNVs didn't greatly reduce RNA levels, which they said indicated that their effects instead occur at the protein level.

Starita and her colleagues argued that these scores could help classify BRCA1 variants. "Our hope is that this database will continue to grow and will become a central point for guiding the interpretation of actionable variants as they are first observed in women," she said.

In an accompanying commentary in Nature, the National Cancer Institute's Stephen Chanock called the work "innovative."

The researchers' approach represents a potential "game-changer" for assessing VUS, he wrote, adding that additional clinical data is needed to validate the UW team's findings and that it must be determined how to best incorporate such data into variant classification.

The UW researchers plan to extend their analysis to encompass the full BRCA1 gene.