Table 1.
Bioinformatics analysis and splicing outcomes of RAD51D canonical splice variants.
| Variant (HGVS) 1 | Bioinformatics (MaxEnt Scan) 2 | Transcripts 3 | |||
|---|---|---|---|---|---|
| Canonical | PTC | In-Frame | Uncharacterized | ||
| mgR51D_ex2-9 | Primers V1-ex9 | ||||
| Wild type | - | 57.8% ± 5.6 | ∆(E4_5) (13.8% ± 1.3); ∆(E3_7) (9.9% ± 2.6); ∆(E2_5) (5.2% ± 0.8) |
∆(E3_5) (13.3% ± 2.4) | - |
| c.83-2A>G | [−] 3′SS (8.52→0.56) | - | ∆(E2_5) (41.9% ± 1.1); ∆(E2) (39.6% ± 0.9); ∆(E2_3) (9.6% ± 0.1) |
- | 487-nt (9.0% ± 0.2) |
| c.83-4_83-3delinsAG | [−] 3′SS [+] 3′SS (5.42) 2-nt upstream |
- | ▼(E2p2) (54.2% ± 2.0); ∆(E2_5) (38.6% ± 2.1); ∆(E2) (7.2% ± 0.4) |
- | - |
| mgR51D_ex2-9 | Primers ex2-V2 | - | - | ||
| Wild type | 73.1% ± 5.6 | ∆(E4_5) (9.4% ± 3) | ∆(E3_5) (17.5% ± 5.2) | - | |
| c.145-2A>G | [−] 3′SS (2.43) | - | ∆(E3) (55.5% ± 0.6) | ∆(E3_5) (44.5% ± 0.6) | - |
| c.263+6T>C | 5′SS: 7.44→4.86 | 49.0% ± 1.2 | ∆(E3) (15.6% ± 0.3) | ∆(E3_5) (35.4% ± 1.2) | - |
| c.343C>T | 5′SS: 7.79→4.36 | 3.4% ± 0.7 | ∆(E4) (45.7% ± 1.0); ∆(E4_5) (28.1% ± 0.6) |
∆(E3_5) (22.7% ± 1.2) | - |
| c.345+2T>C | [−] 5′SS (7.79→0.04) | - | ∆(E4) (49.6% ± 1.4); ∆(E4_5) (26.4% ± 1.0) |
∆(E3_5) (24.0% ± 0.7) | - |
| c.480+1G>A | [−] 5′SS (11.08→2.9) | - | ∆(E4_5) (29.2% ± 0.4) | ∆(E3_5) (41.2% ± 0.7); ∆(E5) (29.6% ± 0.4) |
- |
| c.476_480+1dup | [−] 5′SS (11.08→0.5) | - | ∆(E4_5) (8.6% ± 0.4); (∆(E4) ▼(E5q6)) (6.3% ± 0.1); (∆(E2) ▼(E5q6)) (4.3% ± 0.1); (∆(E3) ▼(E5q6)) (3.6% ± 0.4) |
▼(E5q6) (60.4% ± 4.3); ∆(E3_5) (16.7% ± 5.0) |
- |
| c.481-8C>A | 3′SS: 8.21→1.75 [+] 3′SS (11.06) 6-nt upstream |
0.4% ± 0.1 | (∆(E4_5) ▼(E6p6)) (13.4% ± 0.2) | ▼(E6p6) (70.0% ± 0.8); (∆(E3_5) ▼(E6p6)) (8.8% ± 0.3); ∆(E3_5) (7.5% ± 0.3) |
- |
| c.577-2A>G | [−] 3′SS (10.36→2.41) | - | ▼(E7p41) (65.5% ± 0.5); ∆(E7) (14.3% ± 0.2) |
∆(E6_9) (20.2% ± 0.8) | - |
| c.738+1G>A 4 | [−] 5′SS (6.13→−2.05) | - | ▼(E8q43) (27.9% ± 0.1); ∆(E8) (51.1% ± 0.2); ∆(E4_7) (10.9% ± 0.1) |
- | 1363-nt (10.1% ± 0.0) |
1 Variants without full-length transcripts or residual amounts (<5%) are indicated in bold font. 2 [−]: site disruption; [+]: new site. 3 Transcripts are described with a combination of the following symbols: ▼ (incorporation of intronic sequences that were not present in the reference transcript), ∆ (skipping of exonic sequences that were present in the reference transcript), E (exon), p (new acceptor site), q (new donor site) and a number representing the exact number of nucleotides incorporated or skipped. For example, ▼(E2p2) denotes the use of an alternative acceptor site 2 nucleotides upstream of exon 2, causing the addition of 2-nt to the mature mRNA. 4 According to reference [33], c.738+1G>A causes only a 37-nucleotide intron retention ▼(E8q37). Yet, some methodological issues (location of PCR primers not reported, RT-PCR products analyzed only by low-sensitivity agarose electrophoresis, no agarose band isolation before Sanger sequencing and no allele-specific information) precluded, in our opinion, direct comparison with our minigene results.