In silico analysis of variants at intron-exon junctions

40 Pages·2014·9.07 MB·English

by  T. Koopmann

Predictive value of in silico analysis of variants at
intron-exon junctions: comparison of wet lab and
bioinformatics analysis.
M. Paola Lombardi
Clinical Molecular Geneticist
DNA-diagnostics Unit
Dept. Clinical Genetics, University of Amsterdam
Academic Medical Centre

Klinische Genetica
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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RNA analysis
Methods
● 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.
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Variants at the acceptor splice site with a potential effect on splicing
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 )
P: Pathogenic P? : Unknown Pathogenicity N-P: Non Pathogenic
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TTN: c.28031-1G>A
poorly defined ss
RNA analysis: activation splice site at 28036, 6 nt deletion
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MYBPC3: c.2414-8T>A
ss not found
RNA analysis: activation of de novo splice site, insertion AGCCAG
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SCN5A: c.393-5C>A
Wild type ss signal not
found
RNA analysis: no effect on splicing
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PRKAR1A: c.709-7_709-2del
PPRvKAR1ARKAR1A
-1.1%
+0,1%
-0.18%
-65.2%
-4.2%
RNA analysis: in a low % of transcript skipping of exon 7 (Groussin L et al.,
J of Clin Endo & Metab 2006 91 1943–1949)
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