Klinische Genetica

M. Paola Lombardi

Clinical Molecular Geneticist

DNA-diagnostics Unit

Dept. Clinical Genetics, University of Amsterdam

Academic Medical Centre

Predictive value of in silico analysis of variants at intron-exon junctions: comparison of wet lab and

bioinformatics analysis.

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Introduction Splice prediction tools are commonly used by diagnostics laboratories to predict the

effect of a genetic variant on splicing.

A large number of prediction tools are available, either as standalone programs or as part of the Alamut splicing prediction module.

The performance of these algorithms have not been assessed and may give divergent results.

At this point, no comprehensive interpretation guidelines are available: the user must decide when a prediction is positive (i.e splice defect) or negative (i.e. no impact on splicing).

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“ Practice Guidelines for the Evaluation of Pathogenicity and the Reporting of Sequence Variants in Clinical Molecular Genetics (2013)”

In silico splice site predictions These are generally valid when used correctly and within the scope of their applicability. An NGRL study ( http://www.ngrl.org.uk/manchester/sites/default/files/publications/Informatics/NGRL_ Splice_ Site_ Tools_ Analysis_2009.pdf) showed that the better performing tools were capable of a good degree of accuracy, and that users can therefore be confident of the safe interpretation of results as part of the assessment of a variant. However they must be used with caution and should not be relied upon alone. Summary: it is acceptable to use in silico splice site prediction; however it is unacceptable to base an unequivocal clinical interpretation solely on this line of evidence. It is, however, acceptable to suggest further investigations based on the outcome. If this method of prediction is used it is recommended to arrive at an interpretation based upon a consensus of at least 3 splice site prediction programmes. It is not currently possible to set criteria for the change in prediction tools scores which should be considered significant (e.g. 10% deviation from the wild-type score). This remains a matter for local judgement and agreement. RNA studies Where possible, RNA studies are the best means of interpreting the consequences of a splicing mutation. Summary: Given the high predictive value of RNA studies they must be regarded as essential for the definitive interpretation of putative splicing mutations. However it is recognised that not all laboratories have the facilities to perform these analyses and that limited expression patterns may mean that the the required tissue is not available for analysis.

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From literature: Houdayer et al., 2011, In silico prediction of splice site affecting nucleotide variants. (In: In Silico Tools for Gene Discovery, Methods in Molecular Biology 760)

1. MES-Alamut and SSF-like provide the best predictions in that a decisional threshold can be defined. It is recommended to look at the score variations rather than the score themselves and to set a limit of significance for score variations.

2. Variations occurring at the AG-GT consensus canonical site can be considered as impacting splicing

3. Variations occurring at loosely defined consensus positions are also reliable. Using MES-Alamut we recommend that the mutant score should be at least 15% lower than the wild type score in order to consider the prediction as positive (deleterious). Using SSF-like, the threshold should be 5%.

4. Variations occurring outside the consensus positions are less reliable with specificity issue mainly and have to be interpreted in a context -dependent manner.

5. In silico tools cannot yet be used to define splicing outcome beacuse it is the result of a complex interplay between consensus sequences and other factors from the splicing machinery.

6. To use in silico predictions for diagnostic purposes a decision threshold must be selected. At this point there is no threshold allowing 100% sensitivity while keeping proper specificity.

7. The better the definition of the consensus (i.e. the higher the score) the better the reliability of the predictions. If the wild-type consensus site is not scored or poorly defined the tool should not be used.

8. Using MES-Alamut a cryptic site is considered as putative competitor if it reaches at least 80% of the score of the wild type one.

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● Blood was collected in PAX tubes and total RNA was isolated with the protocol provided by the manufacturer (PAX gene blood RNA kit- PreAnalytiX).

● cDNA was synthesized with Superscript III using the standard protocol (SuperscriptIII First-Strand Synthesis System for RT_PCR –Invitrogen).

● PCR amplification and sequencing primers were designed with Primer 3. PCR and sequence reactions were conducted with standard protocols and using the same primer pairs.

RNA analysis

Methods

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Mutation In silico prediction RNA analysis

MYBPC3: c.3815-1G>A P: acceptor ss abolished P:intron 33 retention

TTN: c.61121-1G>A P: acceptor ss abolished P: activation acceptor ss

MYBPC3: c.655-3C>G P: acceptor ss almost abolished P: activation de novo acceptor ss

PKP2: c.2578-3A>G P: acceptor ss scores strongly reduced P: activation de novo acceptor ss

MSH2: c.367-11T>G P: acceptor ss scores strongly reduced P: activation de novo acceptor ss

KCNH2: c.2146-3C>G P: acceptor ss almost abolished P: exon skipping + activation acceptor ss

TTN: c.28031-1G>A P?: acceptor ss poorly defined, 2 programs

P: activation acceptor ss

MYBPC3: c.1458-6G>A P?: acceptor ss abolished, 2 programs P: activation de novo acceptor ss

MYBPC3: c.2414-8T>A P?: acceptor ss not found P: activation de novo acceptor ss

SCN5A: c.393-5C>A P?: a-ss not found, new ss at 393-2 N-P: no effect on splicing

PRKAR1A: c.709-7_709-2del N-P: mildly reduced scores P?: skipping exon 7 (in a low % of mutant transcript )

Variants at the acceptor splice site with a potential effect on splicing

P: Pathogenic P? : Unknown Pathogenicity N-P: Non Pathogenic

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TTN: c.28031-1G>A

RNA analysis: activation splice site at 28036, 6 nt deletion

poorly defined ss

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MYBPC3: c.2414-8T>A

RNA analysis: activation of de novo splice site, insertion AGCCAG

ss not found

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SCN5A: c.393-5C>A

RNA analysis: no effect on splicing

Wild type ss signal not

found

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PPRvKAR1ARKAR1A PRKAR1A: c.709-7_709-2del

RNA analysis: in a low % of transcript skipping of exon 7 (Groussin L et al., J of Clin Endo & Metab 2006 91 1943–1949)

-1.1%

+0,1%

-0.18%

-65.2%

-4.2%

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Mutation In silico prediction RNA analysis

APC: c.531+3A>C P: donor ss scores reduced P: skipping exon 4

MYBPC3: c.624+4A>T P: donor ss scores reduced P: skipping of ex 17

MYBPC3: c.1457+5G>C

P: donor ss scores reduced P: skipping of ex 16

MYBPC3: c.3190+5G>A P: donor ss scores reduced P: activation donor ss

KCNQ1: c.683+5 G>A P: donor ss scores reduced P: skipping of exon 4

KCNQ1: c.477+5 G>A P: donor ss scores reduced P: skipping of exon 2

PAK3: c.1210+6C>T P?: donor ss scores poorly defined P: skipping of exon 14

SCN5A: c.4437+5G>A

P?: donor ss not found P: skipping of exon 24/24+25

SCN5A: c.3666+22G>T N-P?: donor ss scores unchanged P: activation de novo donor ss

MYBPC3: c.2994+33G>T N-P: donor ss scores unchanged N-P: no effect

MYBPC3: c.1790+7G>A N-P: donor ss scores unchanged N-P: no effect

KCNH2: c.2692+7C>T N-P: donor ss scores unchanged N-P: no effect

Variants at the donor splice site with a potential effect on splicing

P: Pathogenic P? : Unknown Pathogenicity N-P: Non Pathogenic

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MYBPC3: c.1457+5G>C

RNA analysis: skipping of exon 16

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MYBPC3: c.3190+5G>C

RNA analysis: activation cryptic splice site, del GTTGTTG

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SCN5A: c.4437+5G>A

ss not found: P?

RNA analysis: 2 transcripts skipping of exon 24 skipping and exon 24+25 skipping

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SCN5A: c.4437+5G>A

Exon 24 Exon 26

Exon 23 Exon 26

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SCN5A_c.3666+22G>T

RNA analysis: activation of de novo splice site, insertion 20 nt

N-P ? Scores unchanged

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Mutation In silico prediction RNA analysis

MYBPC3: c.1351G>A P: donor ss scores almost abolished P: activation cryptic donor ss

MYBPC3: c.2308G>A P: donor ss scores almost abolished P: NMD

KCNQ1: c.1685G>T P: donor ss scores almost abolished P: activation cryptic donor ss

SCN5A: c.1890G>A P: donor ss scores markedly reduced P: activation cryptic donor ss

MYBPC3: c.1790G>A P: donor ss scores reduced P: activation cryptic donor ss

MYBPC3: c.3814G>C P: donor ss scores reduced P: d-ss abolished, intron 33 included

KCNH2: c.1128G>A P: donor ss scores reduced P: skipping exon 5

DOCK8: c.3389A>G P: donor ss scores moderately reduced P: skipping exon 27

DES: c.1222C>G N-P: donor ss (c.1244) unchanged N-P: no effect

SCN5A: c.1008G>A N-P: acceptor ss (c.999) unchanged N-P: no effect

KCNQ1: c.502G>A N-P: acceptor ss (c.478) unchanged N-P: no effect

Variants within exons with a potential effect on splicing

P: Pathogenic N-P: Non Pathogenic

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MYBPC3: c.1790G>A

-14.5%

-29,5%

-46,9%

-55.8%

-11.5%

RNA analysis: activation cryptic splice site at 1696 (92 nt deletion)

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MYBPC3: c.3814G>C

-15.2%

-44.4%

-31.5%

-31.5%

-11.6%

RNA analysis: retention intron 33 (190 bp)

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KCNH2: c.1128G>A

RNA analysis: skipping exon 5

-14%

-46%

-38.7%

-20.5%

-11.8%

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DOCK8: c.3389G>A

-10.9%

-30.2%

- 4.3.%

100%

-5.7%

RNA analysis: skipping of exon 27, del 52 aa

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Conclusions Prediction tools appear to be reliable when the “UGG guidelines” can be applied: they

are able to discriminate between variants leading to splice defects or having no impact.

Prediction tools are also accurate in predicting the outcome of a splicing defect: they correctly detect de novo ss and cryptic splice site.

For variant prediction analyses in which the “UGG guidelines” cannot be applied, the prediction is not reliable. RNA studies or functional splicing assay remain mandatory.

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Discussion/Q&A

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MYBPC3: c.1458-6G>A

RNA analysis: activation de novo splice site, insertion CTAG

poorly defined ss

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PAK3: c.1210+6T>C

ss poorly defined

RNA analysis: exon 14 skipping

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APC: c.531+3A>C

-11.3%

-77.7%

100%

na

-8.7%

RNA analysis: exon 4 skipping

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KCNH2: c.2146-3C>G

RNA analysis: 2 transcripts, exon 9 skipping and activation of cryptic splice site at 2214

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MSH2: c.367-11T>G

100%

-78,1%

-8,9%

100%

-4.5%

RNA analysis: activation of de novo splice site at c.367-10 (ins10)

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MYBPC3: c.1351G>A

RNA analysis: activation cryptic splice site at 1310 (40 nt del)

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SCN5A: c.1890 G>A p.(Thr630Thr)

RNA analysis: activation cryptic splice site at 1890+34 (34 nt inserted)

-100%

-50.3%

-51.5%

-67.7%

-11.6%

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KCNQ1: c.502 G>A p.(Gly168Arg)

RNA analysis: no effect on splicing

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KCNQ1: c.1685G>T p.(Arg562Met)

RNA: activation crypptic splice site at 1660, 26 nt deleted

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MYBPC3: c.1624+4G>C

RNA analysis: skipping of exon 17

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KCNQ1: c.683+5G>T

RNA analysis: exon 4 skipping

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KCNQ1: c.477+5G>A

RNA analysis: exon 2 skipping

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KCNH2: c.2692+7C>T

RNA analysis: no effect on splicing

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MYBPC3: c.1790+7G>A

RNA analysis: no effect on splicing

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MYBPC3: c.655-3C>G

RNA analysis: activation of cryptic splice site, insertion AG

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PKP2: c.2578-3A>G

RNA analysis: new splice site at 2578-2, 2 nt insertion

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TTN: c. 61121-1G>A

RNA analysis: activation acceptor splice site at 61123, AG del