Functional analysis of the RAD51D gene conficting variant in breast cancer

Germline pathogenic variants in DNA repair genes (BRCA1/2, RAD50, RAD51D, PTEN and etc.) are responsible for the development of hereditary breast and ovarian cancers. The large number of variants detected by NGS technology have unknown or conficting clinical signifcance. Reclassifcation of these variants plays a crucial role in their application in routine laboratory practice.
The aim of the current study was to reclassify conficting RAD51D gene variant (rs145309168) in a young Buryat breast cancer patient using the translation-dependent nonsense-mediated mRNA decay (NMD) pathway.
Material and Methods. Wholeexome sequencing (WES) was performed in the germline DNA of 16 non-BRCA BC Buryat patients (data not shown). The diagnosis in all patients was confrmed morphologically (T1–3N0–2M0). All tested women were diagnosed with invasive (ductal) carcinoma of no special type. Rare variants (MAF<0.005) were analyzed to assess their impact on the RNA splicing using in silico tools like SpliceAI, ESEFinder, RESCUE-ESE, and EX-SKIP. A rare missense variant in the RAD51D gene (rs145309168) was identifed in a 39-year-old Buryat breast cancer patient. Frozen patient leukocytes were divided into experimental and control samples. The samples were cultured for 5–6 days and treated with puromycin (only experimental samples) for 4–6 hours prior to RNA isolation to avoid NMD followed by Sanger sequencing.
Results. In vitro experiments on live leukocytes from a breast cancer patient with the c.932T>A variant of the RAD51D gene were conducted. cDNA amplicons were obtained from RNA isolated from control and experimental leukocytes (treated with puromycin to avoid nonsense-mediated decay). For an accurate assessment of splicing aberrations, transcripts from the experimental leukocytes were compared to transcripts from control leukocytes by using Sanger sequencing. In both cases, the presence of the studied variant in the RNA signifed that the variant did not activate NMD and therefore did not affect splicing.
Conclusion. This study presents the frst in vitro functional analysis of the RAD51D variant (rs145309168) identifed in a young Buryat breast cancer patient. Our experimental data demonstrate that this variant does not disrupt normal splicing, providing evidence for its reclassifcation as «Likely Benign», which is consistent with published data and previous classifcations.

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