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Predicting effect of SNPs
and de novo variants on
splicing
presented by
Alexander Tchourbanov
Presentation structure
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Previous work on predicting
aberrant splicing events induced by
common and de novo genetic
variants
Proposed plan of action
Problem of aberrant splicing
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Splicing in vertebrate
genes is governed by
highly degenerate
motifs that include
donor, acceptor, branch
site and repertoire of
splicing enhancers and
silencers
Integrity of human
genes is constantly
compromised by de novo
mutations
~15% disease
associated mutations
cause aberrant splicing
Splicing components
Image credit: Understanding alternative splicing: towards a cellular code:
Arianne J. Matlin, Francis Clark and Christopher W. J. Smith, Nature Reviews Molecular Cell Biology 6, 386-398 (May 2005)
Importance of understanding
the aberrant splicing
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According to Human Gene Mutation Database
(HGMD) Professional 2010.4
(http://www.hgmd.cf.ac.uk)
60,489 mutations are missence/nonsense
10,210 mutations have consequences in mRNA
splicing
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Databases DBASS5 and DBASS3 currently
contain 900 well-annotated records of
disease causing aberrant splicing events
(Buratti et. al., Nucleic Acids Research, 2010).
Importance of understanding
the aberrant splicing
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Chen R, Davydov E, Sirota M, Butte A: NonSynonymous and Synonymous Coding SNPs
Show Similar Likelihood and Effect Size of
Human Disease Association. PLoS ONE 2010,
5(10):e13574.
Frequently it is difficult to get tissue samples
for RNA sequencing (brain samples, retina
samples)
We need to predict the effect of de novo
variants (which includes cancer mutations) and
common variants. No association study
possible.
Existing elements
Publication
Number of elements predicted
Fairbrother, W.G., et al.
(Science 2002)
238 hexamers as candidate ESEs
Zhang, X.H. and L.A. Chasin
(Genes Dev 2004)
Putative 2,069 octamers as exonic splicing enhancers and
974 octamers as exonic splicing silencers
Wang, Z., et al. (Cell 2004)
133 ESS-containg decanucleotides
Yeo, G.W., E.L. Van Nostrand,
and T.Y. Liang (PLoS Genet
2007)
133 5’SS ISEs and 299 3’SS ISEs pentamers
Goren, A., et al. (Mol Cell
2006)
285 hexamers putative exonic splicing regulatory
sequences
Zhang, C., et al. (Proc Natl
Acad Sci USA 2008)
Putative 1131 hexamers Exon-Identity Elements (EIEs) and
708 Intron-Identity Elements (IIEs)
Stadler, M.B., et al. (PLoS
Genet 2006)
380 hexamers as new candidate ESEs and 132 hexamers as
new candidate ESSs
Wang, E. T., et. al. (Nature
2008)
187 5’SS ISEs/ISSs and 175 3’SS ISEs/ISSs hexamers
supporting the tissue-specific splicing events
Orthologos blocks from UCSC GB
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2,333,379 extended exons from 23 Tetrapoda
organisms were obtained
A number of experimental reports showed that genes
from distantly related Tetrapoda organisms were
correctly expressed and post-transcriptionally
modified in transgenic animals (Capetanaki Y et al.:
Proc Natl Acad Sci USA 1989, Jacobs GH et al.:
Science 2007)
The genes encoding well-known RNA binding proteins
involved in splicing regulation are enriched with
ultraconserved elements (Bejerano G. et al.:Science
2004)
Counting oligos
Comparing oligo counts
Example of 5’SS ISEs found
Elements found
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Using the orthologous exons available for 23
Tetrapoda organisms we have identified
2,546 unique splicing regulatory elements.
Among these elements 203 (7.97%) 3’SS and
177 (6.95%) 5’SS supporting motifs are novel
and have not been previously reported in
systematic screens detecting such elements.
Among our predicted elements, 41.08% of
sequences were heptamers and 51.81% were
octamers and only 6.76% hexamers and
0.35% pentamers
Predicting donor splice site
Bayesian 5’ splice sites
sensor designed during
my PhD study has
performance better
than other sensors,
including maximum
entropy sensor from
MIT.
Exonic length distribution
Optimal exonic lengths substantially depend on the
flanking splicing signals strengths, considering
splice site (SS) strengths in the discrete range
from 1 (weakest) to 5 (strongest).
Example of LOD profiles (5’SS ISE)
Exon scoring method
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LOD scores associated with 5’SS,3’SS,
exonic length, competing SSs and
Enhancer/Silencer signals are combined
towards an exon strength
Existing splicing prediction
software
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http://www.umd.be/HSF
http://esrsearch.tau.ac.il/
http://genes.mit.edu/burgelab/rescue-ese/
http://genes.mit.edu/exonscan/
http://cryp-skip.img.cas.cz/
http://cubweb.biology.columbia.edu/pesx/
Strongest exonic silencers are the splice
sites themselves!!!
SpliceScan II performance on
mutations
Database
Prediction
method
DBASS 5
(Buratti E et al: Nucleic
Acids Res 2007)
Correct Wrong
Accuracy
DBASS 3
(Vorechovsky I: Nucleic
Acids Res 2006)
Correct Wrong
Accuracy
ExonScan
(Wang Z et al:
Cell 2004 )
42
320
11.6%
8
117
6.4%
GenScan
(Burge C:J Mol
Biol 1997)
52
310
14.36%
21
104
16.8%
SpliceScan II
100
262
27.62%
40
85
32%
Disturbing circadian pacemaker
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For example, the circadian pacemaker period homolog
1 (Per1) gene locus has intronic non-coding variant
rs885747 that has been previously associated with
Autism (Nicholas et. al., Molecular Psychiatry, 2007).
Haplotype analysis within per1 gave a single
significant result: a global P=0.027 for the markers
rs2253820-rs885747
We predicted creation of intronic splicing enhancer
GCGGGGT as one of the possible causative
mechanisms behind rs885747 that promotes aberrant
exonic isoform.
Disturbing circadian pacemaker
Disturbing circadian pacemaker
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Per1 is a member of the Period
family of genes and is expressed in
a circadian pattern in
the suprachiasmatic nucleus, the
primary circadian pacemaker in the
mammalian brain. Genes in this
family encode components of
the circadian rhythms of
locomotor activity, metabolism,
and behavior.
SNPs affect splicing
997 SNPs
Type of event
NEW!
NEW!
Alzheimer’s
associated
539 SNPs
Control
Ratio
Breast cancer
associated
Control
Ratio
Predicted exon corresponding
to an annotated exon
disappears (becomes too
weak)
0
2
0
0
0
-
Predicted exon corresponding
to an annotated exon
changes the score
43
12
3.58
11
2
5.5
Predicted exon sharing a SS
with an annotated exon
changes the score
242
78
3.10
59
29
2.03
Predicted exon sharing a SS
with an annotated exon
disappears
23
4
5.75
6
1
6.00
New predicted cryptic exon
appears sharing a SS with an
annotated exon
26
9
2.89
5
1
5.00
Predicted cryptic exon
disappears
50
49
1.02
30
17
1.76
New predicted cryptic exon
appears
50
46
1.08
24
25
0.96
rs849563 variant
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Am J Med Genet B Neuropsychiatr Genet. 2007 Jun
5;144B(4):492-5. Association of the neuropilin-2
(NRP2) gene polymorphisms with autism in Chinese
Han population. Wu S, Yue W, Jia M, Ruan Y, Lu T,
Gong X, Shuang M, Liu J, Yang X, Zhang D. Institute
of Mental Health, Peking University, Beijing, China.
Significant genetic association found between autism
and two of the SNPs of the NRP2 gene (rs849578:
P = 0.017, rs849563: P = 0.027), as well as specific
haplotypes, especially those formed by rs849563.
rs849563 is synonymous
rs849563 predicted mechanism
The neuropilin-2 (NRP2) gene is localized to 2q34, an autism susceptibility
locus. NRP2 has been demonstrated to both guide axons and to control
neuronal migration in the central nervous system. It has been reported
that NRP2 may be required in vivo for sorting migrating cortical and
striatal interneurons to their correct destination.
SpliceScan II tool
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SpliceScan II tool
http://www.wyomingbioinformatics.org/~achu
rban/docs/SpliceScanII.tar.gz
Is more sensitive than existing splicing
simulators (NetUTR, ExonScan)
Uses novel 5’ GC SS Bayesian sensor
Method allows predicting aberrant splicing
events associated with genomic variants
ACGMAP companion database
http://www.stritch.luc.edu/node/375
Proposed system architecture
Shotgun
Mate pairs
Transcriptome
Trios
Online submission
Phased reference
genomes of healthy
individuals
Haplotype trees
Variants calling (GMAP/gsNap)
Use PolyPhen (Ramensky et.al., NAR, 2002), SIFT (Kumar
et.al., Nature Protocols, 2009) or Panther
(Thomas et.al., Genomic research, 2003) to predict
destabilizing effects of non-synonymous genetic variants
Use SpliceScanII to predict effect of synonymous
mutations on splicing
Visualize information in the context of existing information
(HGMD, UCSC genome browser, dbSNP, PFAM, ASTD)
Variants analysis and visualization
Chromosome testing at BGI
DNA swap
Craven et al. Nature, 1-4 (2010)
Mito and Thacker
Tachibana et.al., Nature, 2009
Wellderly study
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The Wellderly Study is headed by Scripps Health Chief
Academic Officer Dr. Eric J. Topol, who has spent the
past four years recruiting healthy elderly individuals
 youngest participant is at least 80 years old, the median age of this
study group is 87 with oldest participant 108 years old
 free from major diseases and long-term medications
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This fall Complete Genomics announced that it will
sequence, at its own cost, the whole human genomes of
1,000 participants in the Wellderly Study
 Following announcement NASDAQ: GNOM stocks dropped 7%.
 The genomic sequences obtained in this study will be a private
property of Complete Genomics
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Archon Genomics X PRIZE
http://genomics.xprize.org/life-at-100-plus
Third generation platforms
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3rd generation
platforms (such as
Oxford nanopore
http://www.nanop
oretech.com/) will
revolutionize the
field soon.
Clarke, J. et al. “Continuous base identification for single-molecule nanopore DNA sequencing.” Nature Nanotech. 2009.
Thanks!