Role of Splicing Regulatory Elements and In Silico Tools Usage in the Identification of Deep Intronic Splicing Variants in Hereditary Breast/Ovarian Cancer Genes
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Datasets
2.2. In Silico Variant Annotation and Analysis for All Datasets
2.3. Statistical Analysis
2.4. Experimental RNA Analysis in Patient’s Data Set
2.4.1. Reverse Transcription-PCR (RT-PCR) and Sanger Sequencing
2.4.2. Qualitative Analysis by Capillary Electrophoresis of Fluorescent Amplicons
2.5. Editorial Policies and Ethical Considerations
3. Results
3.1. SpliceAI Optimally Predicts Deep Intronic Splice-Altering Variants but with Less Sensitivity Those Affecting Splicing by Altering Regulatory Elements
3.2. Splicing Regulatory Elements Balance Is Similar between Pseudoexons and Canonical Exons
3.3. Inclusion of SRE Landscape in the In Silico Detection of Deep Intronic Splice-Altering Variants
3.4. Experimental Analysis of Hereditary Cancer Gene Variants
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Dataset | Splicing Altering Variants | No Splicing Altering Variants | Sensitivity | Specificity | Accuracy | MCC |
---|---|---|---|---|---|---|
All variants | 133 | 100 | 93.99 | 92.00 | 93.13 | 0.86 |
Cryptic splice | 117 | 100 | 95.73 | 92.00 | 94.01 | 0.88 |
SREs altering | 16 | 100 | 81.25 | 92.00 | 90.52 | 0.66 |
Pipeline | Sensitivity | Specificity | Accuracy | MCC |
---|---|---|---|---|
(1) ∆ SpliceAI ≥ 0.05 | 93.98 | 92.00 | 93.13 | 0.86 |
(2) ∆ SpliceAI ≥ 0.05 + SpliceAI < 0.05 and ∆ESRseq ≥ 0.63 | 96.24 | 69.00 | 84.55 | 0.69 |
(3) ∆ SpliceAI ≥ 0.05 + SpliceAI < 0.05 and ∆ESRseq ≥ 0.63 and Abs. Dif. 0.51 | 95.49 | 86.00 | 91.42 | 0.83 |
Gene * | cNomenclature ** | Intron | SpliceAI † (Position of Predicted Splice site) | ∆ESRseq ‡ | ABS dif. | Splicing Outcome § | Population Variant Frequencies (GnomAD) | ClinVar Review Status ¥ |
---|---|---|---|---|---|---|---|---|
ATM | c.1899-123A > G | 12 | AG 0.15 (−51 bp) and AG 0.74 (−90 bp)/DG 0.71 (−1 bp) | 0.633 | 1.217 | Three pseudoexons: ▼12A.1 (r.1899_1900ins1899-174_1899-124), ▼12A.2 (r.1899_1900ins1899-177_1899-124) ▼12A.3 (r.1899_1900ins1899-213_1899-124) | 0.000032 | NR |
c.2466+1552G > C | 16 | AG 0.93 (3 bp)/DG 0.69 (97 bp) | −0.144 | 1.816 | Pseudoexon ▼16A (r.2466_2467ins2466 + 1555_2466 + 1650) | NR | Likely pathogenic |1| | |
c.8850+2029A > G | 61 | AG 0.22(1 bp)/DG 0.16 (102 bp) | −0.187 | 0.765 | Pseudoexon▼61A (r.8850_8851ins8850 + 2030_8850 + 2131) | NR | NR | |
FAM175A | c.476+158G > T | 5 | AG 0.17 (2 bp)/DG 0.22 (−94 bp) | 0.559 | 0.591 | Pseudoexon ▼5A (r.476_477ins476 + 156_476 + 252 | 0.000446 | NR |
MUTYH | c.998-27G > A | 11 | AG 0.41 (−4 bp)/DG 0.05 (−215 bp) | 0.344 | 1.000 | Intron retention ▼12A (r.997_998ins998-23 + 998) | 0.001192 | Likely benign|Likely benign 1|1 |
Pipeline | Sensitivity | Specificity | Accuracy | MCC |
---|---|---|---|---|
Splice ≥ 0.05 | 100 | 71.42 | 75.75 | 0.62 |
Splice ≥ 0.05; ESRseq ≥ 0.63; Abs. Dif. 0.51 | 100 | 50.00 | 57.57 | 0.43 |
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Moles-Fernández, A.; Domènech-Vivó, J.; Tenés, A.; Balmaña, J.; Diez, O.; Gutiérrez-Enríquez, S. Role of Splicing Regulatory Elements and In Silico Tools Usage in the Identification of Deep Intronic Splicing Variants in Hereditary Breast/Ovarian Cancer Genes. Cancers 2021, 13, 3341. https://doi.org/10.3390/cancers13133341
Moles-Fernández A, Domènech-Vivó J, Tenés A, Balmaña J, Diez O, Gutiérrez-Enríquez S. Role of Splicing Regulatory Elements and In Silico Tools Usage in the Identification of Deep Intronic Splicing Variants in Hereditary Breast/Ovarian Cancer Genes. Cancers. 2021; 13(13):3341. https://doi.org/10.3390/cancers13133341
Chicago/Turabian StyleMoles-Fernández, Alejandro, Joanna Domènech-Vivó, Anna Tenés, Judith Balmaña, Orland Diez, and Sara Gutiérrez-Enríquez. 2021. "Role of Splicing Regulatory Elements and In Silico Tools Usage in the Identification of Deep Intronic Splicing Variants in Hereditary Breast/Ovarian Cancer Genes" Cancers 13, no. 13: 3341. https://doi.org/10.3390/cancers13133341