Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Bioinformatics Analysis
2.2. Functional Analysis
2.3. Transcript Analysis
2.4. ACMG/AMP-Like Classification of RAD51C Variants Based on PS3/BS3 Functional Evidence
3. Discussion
Clinical Interpretation of Variants
4. Materials and Methods
4.1. Ethics Approval
4.2. Variant and Transcript Annotations
4.3. Bioinformatics Analysis
4.4. Minigene Construction and Mutagenesis
4.5. Transfection of Eukaryotic Cells
4.6. Reverse Transcription Polymerase Chain Reaction and Fragment Analysis
4.7. ACMG/AMP-Like Classification of 20 RAD51C Variants Based on PS3/BS3 Functional Evidence
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Variant (HGVS) 1 | Bioinformatics 2 | Transcripts | |||
---|---|---|---|---|---|
Canonical | PTC 3 | In-Frame | Uncharacterized | ||
Wild type | 98.6% ± 0.2% | 1106-nt (1.4% ± 0.2%) | |||
c.146-3C > T | [↓]3′SS (9.5→8.7) | 100% | |||
c.404G > A | [−]5′SS (4.8→−3.5) | - | ▼(E2q27): 69.3% ± 2.9% Δ(E2q175): 19.9% ± 0.6% Δ(E2q22): 4.3% ± 0.5% Δ(E2): 2.4% ± 0.2% | 913-nt (4.1% ± 3.0%) | |
c.405-6T > A | [−]3′SS (7.7→2.2) [+] 3′SS (8.6) 4-nt upstream | - | ▼(E3p4):95.2%± 1.6% Δ(E3): 4.8% ± 1.6% | ||
c.571 + 4A > G | [↓]5′SS (10.5→8.1) [+] 5′SS (5.5) 4-nt downstream | 5.4% ± 0.1% | Δ(E3): 76.5% ± 0.3% ▼(E3q4): 11.6% ± 0.2% | Δ(E3q114): 4.0 ± 0.0% | 808-nt (1.4% ± 0.0%) 774-nt (1.1% ± 0.0%) |
c.571 + 5G > A | [↓] 5′SS (10.5→5.8) | - | Δ(E3): 91.5% ± 0.3% | Δ(E3q114): 4.8 ± 0.2% | 808-nt (1.6% ± 0.0%) 917-nt (1.1% ± 0.1%) 774-nt (1.0% ± 0.0%) |
c.572-1G > T | [−]3′SS (7.4→−1.2) | - | Δ(E4): 93.4% ± 0.2% | 1005-nt (3.3% ± 0.1%) 1058-nt (3.3% ± 0.1%) | |
c.705G > T | [−]5′SS (9.1→2.6) | - | Δ(E4): 100% | ||
c.705 + 5G > C | [↓]5′SS (9.1→7.2) | 51.6% ± 2.4% | Δ(E4): 48.4% ± 2.4% | ||
c.706-2A > C | [−]3′SS (11.1→3.1) [+]3′SS (3.3) 10-nt downstream | - | Δ(E5p10): 91.4% ± 1.5% Δ(E5p52): 1.8% ± 0.9% | Δ(E5): 4.0% ± 0.1% | 886-nt (2.8% ± 1.6%) |
c.706-2A > G | [−]3′SS (11.1→3.1) [+]3′SS (3.2) 10-nt downstream | - | Δ(E5p10): 33.5% ± 0.2% | Δ(E5): 65.4% ± 0.3% | 972-nt (1.1% ± 0.1%) |
c.837 + 2T > C | [−]5′SS (8.6→0.8) | - | Δ(E4_5): 2.2% ± 0.1% | Δ(E5): 89.3% ± 0.2% | 972-nt (8.5% ± 0.1%) |
c.905-3C > G | [−]3′SS (8.2→−4.9) | - | Δ(E7): 98.1% ± 1.0% Δ(E7_8): 1.9% ± 1.0% | ||
c.905-2A > C | [−]3′SS (8.2→0.1) | - | Δ(E7): 97.4% ± 0.4% | 660-nt (2.6% ± 0.4%) | |
c.905-2_905-1del | [−]3′SS(8.2→0.6) | - | Δ(E7): 100% | ||
c.965 + 5G > A | [↓]5′SS(8.7→3.8) | - | Δ(E7): 100% | ||
c.966-3C > A | [−]3′SS (7.3→4.4) | 2% ± 1.7% | Δ(E8): 86.8% ± 3.2% | ▼(E8p3)-a: 9.7% ± 0.4% | 881-nt (1.5% ± 1.4%) |
c.966-2A > G | [−]3′SS (7.3→−0.7) [+]3′SS(7) 3-nt upstream | - | Δ(E8): 86.7% ± 0.5% | ▼(E8p3)-b:11.0% ± 0.4% | 881-nt (1.2% ± 0.0%) 940-nt (1.1% ± 0.2%) |
c.966-2A > T | [−]3′SS (7.3→−1.1) [+]3′SS(7.6) 3-nt upstream | - | Δ(E8): 89.1% ± 0.3% | ▼(E8p3)-c:5.9% ± 0.1% | 881-nt (2.8% ± 0.3%) 940-nt (2.2% ± 0.0%) |
c.1026 + 5_1026 + 7del | [−]5′SS(2→?) (NNSplice: 0.8→ <0.1) | - | Δ(E8): 79.5% ± 1.4% ▼(E8q41): 3.3% ± 0.2% | Δ(E8q18):13.8% ± 0.7% | 881-nt (2% ± 0.6%) 778-nt (1.4% ± 1.6%) |
c.1026 + 5G > T | [−]5′SS(2→?) (NNSplice: 0.8→ <0.1) | - | Δ(E8): 78.0% ± 0.5% ▼(E8q44): 1.4% ± 0.2% | Δ(E8q18):18.7% ± 0.5% | 881-nt (1.9% ± 0.2%) |
c.HGVS 1 | Clinvar 2 | PVS1 3 | PP3/BP4 4 | PS3/BS3 5 | PS4 6 | PM2 7 | PM 8 | Proposed pSAD-Based ACMG/AMP-Like Variant Classification 9 |
---|---|---|---|---|---|---|---|---|
c.146-3C > T | Conflicting (*) LB (2), VUS, (2) | N/A | (−4%) N/A | BS3 | N/A | (4/303,851) N/A | N/A | (BS3 only) Uncertain Significance |
c.404G > A | LP (**) | N/A | (−99.5%) PP3 | PS3_VS | N/A | (1/300,225) PM2 | N/A | (PS3_VS + PM2) Likely Pathogenic |
c.405-6T > A | VUS (*) | N/A | (−79%) PP3 | PS3_VS | N/A | (0/304,932) PM2 | N/A | (PS3_VS + PM2) Likely Pathogenic |
c.571 + 4A > G | Conflicting (*) LB (1), LP(1), VUS (6) | N/A | (−30.5%) PP3 | (88%VS + 4%S + 5%N/A) PS3 | N/A | (1/84,873) PM2 | N/A | (PS3 + PM2) Likely Pathogenic 10 |
c.571 + 5G > A | VUS (**) | N/A | (−33.9%) PP3 | (95% vs. + 5%S) PS3_VS | PS4 | (8/336,321) N/A | PM3 11 | (PS3_VS + PS4 + PM3) Pathogenic |
c.572-1G > T | not reported | PVS1 | N/A | PS3_VS | N/A | (1/304,681) PM2 | N/A | (PS3_VS + PM2) Likely Pathogenic |
c.705G > T | VUS (**) | N/A | (−75.8%) PP3 | PS3_VS | N/A | (2/304,499) PM2 | N/A | (PS3_VS + PM2) Likely Pathogenic 10 |
c.705 + 5G > C | not reported | N/A | (−16.8%) PP3 | (48%VS + 52% N/A) N/A | N/A | (1/304,406) PM2 | N/A | (PM2 only) Uncertain Significance |
c.706-2A > C | LP (**) | PVS1 | N/A | (95%VS + 5% S) PS3_VS | N/A | (0/336,207) PM2 | N/A | (PS3_VS + PM2) Likely Pathogenic |
c.706-2A > G | P/LP (**) | PVS1 | N/A | (34%VS + 65% S) PS3 | PS4 12 | (10/336,207) N/A | N/A | (PS3 + PS4) Pathogenic |
c.837 + 2T > C | LP (**) | PVS1 | N/A | (90% S + 2% VS) PS3 | N/A | (0/304,832) PM2 | N/A | (PS3 + PM2) Likely Pathogenic |
c.905-3C > G | not reported | N/A | (−92.8%) PP3 | PS3 | N/A | (1/336,187) PM2 | N/A | (PS3 + PM2) Likely Pathogenic |
c.905-2A > C | P/LP (**) | PVS1 | N/A | PS3 | PS4 | (5/336,191) N/A | N/A | (PS3 + PS4) Pathogenic |
c.905-2_905-1del | P/LP (**) | PVS1 | N/A | PS3 | PS4 | (4/304,579) N/A | N/A | (PS3 + PS4) Pathogenic |
c.965 + 5G > A | LP(1); VUS(2) | N/A | (−59.9%) PP3 | PS3 | N/A | (2/304,579) PM2 | N/A | (PS3 + PM2) Likely Pathogenic |
c.966-3C > A | not reported | N/A | (−35.3%) PP3 | (90%S + 10%N/A) N/A | N/A | (1/304,818) PM2 | N/A | (PM2 only) Uncertain Significance |
c.966-2A > G | LP (*) | PVS1 | N/A | (90%S + 10%N/A) N/A | N/A | (0/304,818) PM2 | N/A | (PM2 only) Uncertain Significance |
c.966-2A > T | not reported | PVS1 | N/A | (90%S + 10%N/A) N/A | N/A | (0/304,818) PM2 | N/A | (PM2 only) Uncertain Significance |
c.1026 + 5_1026 + 7del | P/LP (**) | N/A | (−98.8%) PP3 | PS3 | PS4 | (6/304,853) N/A | N/A | (PS3 + PS4) Pathogenic |
c.1026 + 5G > T | not reported | N/A | (−98.8%) PP3 | PS3 | N/A | (0/304,840) PM2 | N/A | (PS3 + PM2) Likely Pathogenic |
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Sanoguera-Miralles, L.; Valenzuela-Palomo, A.; Bueno-Martínez, E.; Llovet, P.; Díez-Gómez, B.; Caloca, M.J.; Pérez-Segura, P.; Fraile-Bethencourt, E.; Colmena, M.; Carvalho, S.; et al. Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene. Cancers 2020, 12, 3771. https://doi.org/10.3390/cancers12123771
Sanoguera-Miralles L, Valenzuela-Palomo A, Bueno-Martínez E, Llovet P, Díez-Gómez B, Caloca MJ, Pérez-Segura P, Fraile-Bethencourt E, Colmena M, Carvalho S, et al. Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene. Cancers. 2020; 12(12):3771. https://doi.org/10.3390/cancers12123771
Chicago/Turabian StyleSanoguera-Miralles, Lara, Alberto Valenzuela-Palomo, Elena Bueno-Martínez, Patricia Llovet, Beatriz Díez-Gómez, María José Caloca, Pedro Pérez-Segura, Eugenia Fraile-Bethencourt, Marta Colmena, Sara Carvalho, and et al. 2020. "Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene" Cancers 12, no. 12: 3771. https://doi.org/10.3390/cancers12123771