7. Functionally null RAD51D missense mutation associates strongly with overian carcinoma

Rivera B., et al., June 23, 2017. Cancer Research.

RAD51S is an ATPase with the N terminal domian required for ssDNA binding. This domain includes a conserved ATP binding Walker A and Walker B motifs which are necessary for its role in HR. These motifs were also suggested to bind RAD51C and XCRR2 while residues 60-78 contribute to the  Linker domain responsible for interaction with RAD51C and XRCC2.

RAD51D c.620C>T; p.S207L (located in Walker B, highly conserved) has shown in this article for its role in disrupting the interaction of RAD51D and XRCC2 and increasing sensitive to PARPi. While the RAD51 paralogs, including RAD51B,C,D are known to involved in hereditary breast and ovarian cancer, but XRCC2 and XRCC3 are still not well understood. Truncated mutations of RAD51D in germ line cells have been known to predispose to ovarian cancer and cause cells to be susceptible to PARP inhibitors. Furthermore, high grade serous carcinoma (HGSC) is recently found to be associated with RAD51 missense mutations. Here, they found RAD51D c.620C>T; p.S207L mutation to be involved in ovarian cancer through studies of case-control, effect of this variant on the role of RAD51D in DNA repair activity and genomic profiling of known RAD51 tumors.

Control - RAD51D.3 cell line
Chinese Hamster Ovary (CHO) RAD51D deficient cell line - RAD51D.1
             stable expresses the c.620C>T; p.S207L - RAD51D.1-p.S207L.
 Wild-type RAD51D - RAD51D.1-wt

All RAD51D c.620C>T; p.S207L carriers do not contain BRCA1 and BRCA2 French Canadian founder mutations. However, they all have another missense RAD51D missense mutation, RAD51D c.698A>G; p.E233G. This result suggested that c.620C>T; p.S207L comes from chromosome carrying c.698A>G; p.E233G. [How? Why not c.620C>T; p.S207L causes the c.698A>G; p.E233G?]
The sequence data showed allelic imbalance increasing the ratio of RAD51D c.620T-mutant to c.620T-WT, ranging from 0.74 to 0.93, where 0.5 represents a balanced allele ratio. Loss of hereterozygousity at the RAD51D c.620C>T; p.S207L locus was found in all selected ovarian HGSC. TP53 somatic mutations were identified in all of them.  Since RAD51D c.620C>T; p.S207L located in Walker B, it disrupted the interaction between RAD51D and as seen in XRCC2  p.D206A mutation. RAD51D.1 and  RAD51D.1-p.S207L showed a significant decrease in RAD51 filament formation. Gene targeting frequency appeared to be significantly lower in RAD51D.p.S207L cells compared to RAD51D.1-wt, confirming HR defect.

 RAD51-p.E233G variant did not affect HR-dependent DSB repair; however, this mutation can rstore DNA repair activty in RAD51D deficient cells in the presence of p53, suggesting that pE233G does not negatively affect RAD51D HR activity by itself.


[Loss of heterozygosity (LOH) is a gross chromosomal event that results in loss of the entire gene and the surrounding chromosomal region.^[1]

Most diploid cells, for example human somatic cells, contain two copies of the genome, one from each parent (chromosome pair); each copy contains approximately 3 billion bases (adenine (A), guanine (G), cytosine (C) or thymine (T)). For the majority of positions in the genome the base present is consistent between individuals, however a small percentage may contain different bases (usually one of two; for instance, ‘A’ or ‘G’) and these positions are called ‘single nucleotide polymorphisms’ or ‘SNPs’. When the genomic copies derived from each parent have different bases for these polymorphic regions (SNPs) the region is said to be heterozygous. Most of the chromosomes within somatic cells of individuals are paired, allowing for SNP locations to be potentially heterozygous. However, one parental copy of a region can sometimes be lost, which results in the region having just one copy. The single copy cannot be heterozygous at SNP locations and therefore the region shows loss of heterozygosity (LOH). Loss of heterozygosity due to loss of one parental copy in a region is also called hemizygosity in that region.] - Wiki

PARPi causes RAD51D deficient cells by causing syntehtic lethality. PARPi (olaparib and talazoparib) or ciplatin impair the cell survival in cells expressing RAD51D-c.620C>T; p.S207L, similar to what was seenn in RAD51D.1 (RAD51-null) cells. This result helps confirms that PARPi can be a candidiate therapy for treating RAD51D c.620C>T; p.S207L