8. Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as human cancer susceptibility gene

Meindl A., et al., 2010

BRCA1, BRCA2, ATM, CHEK2, BRIP1 and PALB2 have been known to play crucial roles in genomic stability and homologous recombination in DNA repair, and disruption in any of these proteins can lead to breast cancer. Specifically, biallelic mutations in BRCA2 (FANCD1), BRIP1 and PALB2 (FANCN) lead to Fanconi anemia (FA). Recently, RAD51C R258H, a homologous missense mutation in RAD51C, was found to cause cells to be hypersensitive to DNA cross-linking agents and decrease RAD51 foci formation, which are typical features of FA. In this paper, they were interested in testing whether monoallelic germline variants in RAD51C can make cells more susceptible to breast and ovarian cancer, by screening more than 1000 patients who had gynecological cancers but negative for BRCA1 and BRCA2 mutations.

They found 14 RAD51C variants in 1100 affected, unrelated women who carry hereditary breast cancer and BC/OC. These 14 variants were catergorized into: 2 single-base pair insertions, 2 splice-site mutations, and 10 missense mutations. They also identified three missense mutations, A126T, G264S and T287A, which appeared to be more frequent in affect individuals from normal German controls.

A splice mutation 145+1G>T disrupted the canonical GT dinucleotide. It reduced the expression of functional RAD51C-001 and increased expression of nonfunctional RAD51C-008 while not affecting expression of nonfunctional RAD51C-009 compared to WT.

They tested 10 missense amino acids variants to see if they alter the function of RAD51C protein. They insert human RAD51C mutants into chicken DT40 cells where RAD51C ortholog was disrupted.
     - The survived cells expressed G125V and L138F at the highly conserved sequence, so they failed to complement the Rad51c mutant phenotype in DT40 cells.
     - RAD51C G3R, A126T, V169A and G264V complementary DNA corrected the mytomycin (MMC) hypersensitivity of Rad51c mutant in DT40 cells to the WT RAD51C cDNA levels.
     - D159N, G264S, T287A and R366Q partially restored the MMC sensitivity of Rad51c mutant in DT40 cells to WT RAD51C levels.
==> G125V and L138F did not restore normal RAD51 filament formation, while the other eight expressed the RAD51 foci formation just as seen in WT RAD51C.

Six germline variants were considered pathogenic, including two insertions, two splice site mutations, and two missense mutations G125V and L138F. However, two recurrent missense mutations, G264S and T287A were found to be not associated with cancer in 1100 tested women even though they resulted in "reduced cell survival and normal RAD51 foci formation,"

This paper established the first link between RAD51C and human cancers, breast and ovarian cancers, and confirmed RAD51C as a FA-gene, for the first time. It was suggested that women who who have strong history of breast and ovarian cancer, but are negative for BRCA1, BRCA2, and RAD51 mutations, should be screened using exomic sequencing.