Table 2. In silico evaluation of 39 GAA sequence variants that may affect the pre-mRNA splicing.
| MUT ss score (% of WT ss score) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Sroogle | |||||||||||
| Modification | Position | MES | NNSplice | HBond | ESEfinder | Rescue-ESE | PESX | DG | Senep | Observed consequence | Ref |
| Donor 5′ss | |||||||||||
| c.546G>A | Ex 2 | NR | NR | 5.8 (−38) | SRp40: 3.9 (−10) | None found | Loss 2 Pese | 2.2 (+61) | 57.60 (−17) | Normal spliced mRNA 6.3% of NC | 29 |
| c.546G>C | Ex 2 | NR | NR | 5.8 (−38) | SRp40: 3.5 (−19) | None found | Loss 2 Pese | −2.3 (+59) | 56.32 (−19) | 30 | |
| c.546G>T | Ex 2 | NR | NR | 5.8 (−38) | SF2/AF2 (Ig-BRCA): 2.3 (−23) SRp40: 3.7 (−14) | None found | None found | −2 (+64) | 57.04 (−18) | Normal spliced mRNA reduced with respect to NC | 31 |
| c.546+1G>T | In 2 | NR | NR | 5.8 (−38) | SF2/AF2 (Ig-BRCA): 3.7 (+23) | None found | Loss 1 Pese | NR | NR | 32 | |
| c.692+1G>C | In 3 | NR | NR | NR | None found | None found | None found | NR | NR | r.0 | 33 |
| c.692+5G>T | In 3 | NR | NR | 5.10 (−58) | SC35: 4.5(+32) SRp40: 4.7 (+88) | None found | None found | −0.3 (+92) | 58.22 (−17) | 34 | |
| c.955+2T>G | In 5 | 1.19 (−87) | NR | NR | SRp40: 2.2 (−19) | None found | 1 Pese creation | NR | NR | 34 | |
| c.1194+2T>A | In 7 | 1.91 (−81) | NR | NR | None found | None found | 4 Pese creation | NR | NR | 33 | |
| c.1194+2T>C | In 7 | 2.34 (−77) | NR | NR | None found | None found | 3 Pese creation | −3.5 (+51) | 69.19 (−20) | Retention 89 nt In 7 | 35 |
| c.1326+1G>A | In 8 | NR | NR | NR | None found | None found | None found | NR | NR | r.0 | 36 |
| c.1437+2T>C | In 9 | 1.00 (−89) | NR | NR | SF2/AF2: 3.2 (+88) | None found | None found | −4.4 (+48) | 67.39 (−20) | Skipping 111 nt Ex 9 | 37 |
| c.1437G>A | Ex 9 | 2.67 (−70) | NR | 9.3 (−34) | None found | None found | None found | −3.8 (+55) | 72.59 (−14) | Normal spliced mRNA 1.2% of NC | 34,35 |
| c.1551+1G>C | In 10 | NR | NR | NR | SC35: 4.0 (+14) | None found | 1 Pese creation | NR | NR | Skipping 114 nt Ex 10 | 37, 38 and 39 |
| c.1636+1G>C | In 11 | NR | NR (no change) | NR | SRp40: 2.0 (−31) | None found | 1 Pese creation | NR | NR | 34 | |
| c.1636+5G>C | In 11 | NR | NR (no change) | NR | None found | None found | 1 Pese creation | −1.0 (+68) | 62.99 (−16) | Retention 957 nt In 11 | 40 |
| c.1636+5G>T | In 11 | NR | NR (no change) | 8.30 (−27) | None found | None found | None found | −0.9 (+71) | 66.48 (−12) | Normal spliced mRNA 13.7% of NC | 34 |
| c.1888+1G>A | In 13 | 1.42 (−85) | NR | NR | SF2/AF2: 3.8 (+65) SF2/AF2 (Ig-BRCA): 3.2 (+33) | +2 ESE | None found | NR | NR | 34 | |
| c.2040G>A | Ex 14 | NR | NR | 8.90 (−26) | SC35: 3.7 (+12) | None found | Loss 2 Pese | −3.8 (+45) | 64.18 (−16) | Retention 190 nt In 14 | 41 |
| c.2189+1G>A | In 15 | NR | NR | NR | None found | +1 ESE | None found | NR | NR | 34 | |
| c.2331+2T>A | In 16 | 3.27 (−71) | NR | NR | None found | None found | Loss 1 Pese | NR | NR | 34 | |
| c.2331+2T>C | In 16 | 3.70 (−68) | NR new cryptic ss creation, | NR | None found | None found | Loss 1 Pese | −3.8 (+49) | 68.28 (−20) | Skipping 16 nt Ex 16 | 42 |
| c.2331+4A>G | In 16 | 8.27 (−28) | 0.28 (−75) | 12.5 (−22) | SRp40: 1.8 (+40) | None found | Loss 1 Pese | −7.00 (no change) | 72.42 (−15) | Normal spliced mRNA 3.6% of NC | 43 |
| c.2646_2646+1delTG | Ex 18–In 18 | NR | NR | NR | None found | None found | None found | NR | NR | 33 | |
| c.2646+2T>A | In 18 | NR | NR | NR | SRp40: 4.0 (+67) SRp55: 2.6 (+86) | +1 ESE | None found | NR | NR | 44 | |
| Acceptor 3′ ss | PSSM | ||||||||||
| c.-32-2A>G | In 1 | 5.75 (−58) | NR | NA | SF2/AF2: 2.3 (−70) SF2/AF2 (Ig-BRCA): 3.3 (−20) SRp55: 2.7 (+29) | None found | None found | 66.02 (−26) | 34 | ||
| c.-32-3C>A | In 1 | 9.35 (−32) | 0.94 (no change) | NA | SF2/AF2: 4.5 (+36) SRp55: 1.5 (−29) | None found | None found | 85.46 (no change) | Skipping 578 nt Ex 2 | 40 | |
| c.1076-1G>C | In 6 | NR | NR | NA | SF2/AF2: 3.2 (+33) SRp55: 1.5 (−50) | None found | Loss 1 Pese | 56.11 (−30) Branch site NRa | Retention 79 nt In 6 and 89 nt In 7 | 30,33 | |
| c.1195-2A>G | In 7 | NR | NR | NA | SF2/AF2: 4.0 (+21) | None found | None found | 58.39 (−29) | 45 | ||
| c.1327-2A>G | In 8 | NR | NR | NA | SF2/AF2: 1.3 (−70) SF2/AF2 (Ig-BRCA): 2.8 (−38) SRp40: 2.6 (−33) | None found | Loss 1 Pese | 64.57 (−21) | 46 | ||
| c.1438-1G>T | In 9 | NR | NR | NA | None found | None found | None found | 60.63 (−28) | 34 | ||
| c.1438-1G>C | In 9 | NR | NR | NA | SRp55: 4.3 (+34) | None found | None found | 60.63 (−28) | 5 | ||
| c.1552-3C>G | In 10 | NR | NR | NA | SRp40: 3.0 (−12) | +1 ESE | 3 Pese creation | 75.66 (−10) | -Normal spliced mRNA 8.3% of NC -SV1: retention 28 nt in 3′ In 10 (2.8% of NC) | 43 | |
| c.1755-1G>A | In 12 | NR | NR | NA | SF2/AF2: 3.0 (−14) SF2/AF2 (Ig-BRCA): 3.6 (−12) SC35: 2.6 (−19) SRp40: 3.1 (−14) SRp55: 1.7 (+113%) | None found | None found | 56.86 (−29) | 39 | ||
| c.2041-1G>A | In 14 | NR | NR new cryptic ss creation | NA | SC35: 3.3 (+11) SRp55: 2.9 (−38) | +1 ESE | 1 Pese creation | NR | Skipping 6 nt at 5′ Ex 15 | 34 | |
| c.2041-2A>C | In 14 | 1.00 (−89) | NR new cryptic ss creation | NA | SRp55: 3.0 (−36%) | None found | None found | NR | Skipping 6 nt at 5′ Ex 15 | 29 | |
| Others | DG | Senep | |||||||||
| c.1626C>G | Ex 11 | NR | NR (no change) new cryptic ss creation | 11.38 (no change) | None found | None found | None found | NR | NR | 33 | |
| PSSM | |||||||||||
| c.-32-13T>G | In 1 | 11.96 (−13) | 0.98 (no change) | NA | SRp55: 3.8 (+40) | None found | None found | 87.03 (no change) PolyPyrimidin sitea Algorithm by Kol et al47 0.88 (no change) Algorithm by Schwartz et al48 10.71 (−13%) | -Normal splice product -SV1 retention 36 nt 5′ In 1+ skipping 578 nt Ex 2 -SV2 skipping 578 nt Ex 2 -SV3 retention 60 nt 3′ Ex 2 | 38 | |
| c.1076-22T>G | In 6 | 3.73 (no change) | 0.38 (no change) New cryptic ss creation | NA | SF2/AF2: 4.9 (+69) SF2/AF2 (Ig-BRCA): 4.8 (+55) SRp40: 4.9 (+69) | +1 ESE | None found | 79.56 (no change) | -SV1 skipping 120 nt Ex 6. Retention 21 nt In 6. -SV2 retention 393 nt In 5. Skipping 45 nt Ex 6. Utilization of a cryptic donor site in Ex 6. Retention 21 nt In 6 | 49,50 | |
| c.1195-8G>T | In 7 | 9.13 (+16) | 0.73 (+62) | NA | SRp55: 3.0 (−21) | None found | 1 Pese creation | 84.32 (no change) | 34 | ||
Abbreviations: DG, delta free energy; Ex, exon; Esefinder, exonic splicing enhancer finder; HBond, hydrogen bond; In, intron; MES, MaxEntScan (maximum entropy scan); Mut ss, mutant splice site; NA, non-applicable; NC, normal control; NNSplice, neural network splice site prediction; NR, not recognizable because below detection threshold; Pese, putative exonic splicing enhancer; PESX, putative exonic splicing enhancer/silencer; PSSM, position-specific scoring matrix; Rescue-ESE, relative enhancer and silencer classification by unanimous enrichment; r.0, mRNA transcribed from the mutant allele non-detected in patient's fibroblasts; Senep, senepathy; Sroogle, splicing regulation online graphical engine; SV, splice variant; WT ss, wild-type splice site.
Threshold for NNSplice was set at 0.1. For the other programs, default thresholds were used.
Mutations barely predicted to affect the splicing process are indicated by underline.