Alternative Splicing and Disease: an overview

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Alternative Splicing and Disease an overview

• Shoba Ranganathan
• Professor and Chair Bioinformatics
• Dept. of Chemistry and Biomolecular Sciences
  Adjunct Professor
• ARC CoE in Bioinformatics Dept. of Biochemistry
• Macquarie University Yong Loo Lin School of
  Medicine
• Sydney, Australia National University of
  Singapore, Singapore
• (shoba.ranganathan_at_mq.edu.au) (shoba_at_bic.nus.edu.s
  g)
• Visiting scientist _at_
• Institute for Infocomm Research (I2R), Singapore

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Outline of the talk

• Background
• Determining gene architecture
• Graph theory in AS
• Whole genome analysis results
• AS and disease

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Unexpectedly low number of genes in the human
genome
Drosophila 14,000 genes
C.elegans 19,000 genes
Human 22,000-25000 genes

• How can the genome of Drosophila contain fewer
  genes than the undoubtedly simpler organism C.
  elegans?
• This raises the possibility of expanded diversity
  leading to biological complexity

www.utexas.edu, www.sih.m.u-tokyo.ac.jp
http//pub.tv2.no/multimedia/na/archive/
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Sources of Biological complexity

• With a limited number of genes
• Enhanced regulation of genes and pathways
• Post-translational modifications
• Alternative splicing

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A Genomic View
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Spliceosomal splicing
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Maniatis Tasic, 2002
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Protein Diversity
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(No Transcript)
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Alternative splicing

• Splicing is a regulated process that removes the
  non-coding sequence from transcripts to produce
  mRNA (Bernot, 2004).
• Contradicts the central dogma of molecular
  biology
• One gene one protein

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Why AS?

• Protein diversity (Neverov et al., 2005).
• Form of spatial and temporal regulation (Lopez,
  1995)
• Errors in splicing lead to diseases (Orengo
  Cooper, 2007)
• Drug discovery (Levanon Sorek, 2003)

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Usual way of studying AS

• One gene at a time tedious for
  genomes
• Collect intron-exon structures for all isoforms
• Try to analyze them again one isoform at a time
  and then gene by gene.
• Unsuitable for genes with large numbers of
  transcripts.

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Usual way of studying AS
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Why use bioinformatics?

• Most research into alternative splicing is
  limited to a few genes (reductionist approach)
• Bioinformatics overcomes this by facilitating a
  systems biology approach
• Information can be obtained for all genes in a
  genome
• This can be done for many genomes allowing for
  comparative genomics

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Where is the splicing?

• Information on the intron-exon (coding/non-coding)
  arrangement of a gene is essential.
• Aligning mRNA/EST sequence to their co-ordinate
  genomic sequences will give the arrangement of
  exons in a gene. (MGAlign, Ranganathan et al
  2003 MGAlignIt, Lee et al 2003)

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Outline of the talk

• Background
• Determining gene architecture
• Graph theory in AS
• Whole genome analysis results
• AS and disease

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MGAlignIt (Lee et al., 2003)

• Fast heuristic approach and highly accurate
• Capitalizes on the fact that the mRNA sequence
  constitutes a very small percentage of the
  genomic sequence

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MGAligns biological alignment strategy
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MGAlignIt web service
http//origin.bic.nus.edu.sg/mgalign
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Benchmarking

• Dataset human Chr 22 from the Sanger Centre
  (Collins et al., 2003)
• 936 annotated mRNA (5176 exons)
• 48Mbp long human Chr 22 genomic sequence

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Some successes

• Short internal exons (exon 2 9 bp exon 9
  21bp)
• Short terminal exons (exon 1 15 bp)

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MGAlign performance

• More savings in computer time with longer gDNA
  sequences
• Based on 41 randomly chosen genomic fragments

sim4
spidey
mgalign
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Outline of the talk

• Background
• Determining gene architecture
• Graph theory in AS
• Whole genome analysis results
• AS and disease

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Problem Königsberg bridges (1700s)

• The residents of Königsberg, Germany, wondered if
  it was possible to take a walking tour of the
  town that crossed each of the seven bridges over
  the Presel river exactly once.
• Leonhard Euler, 1736 (father of graph theory)

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Graph theory for AS

• First used for AS by Heber et al. (2002).
• Each independent segment represented as a node,
  connected by arrows.
• Node here is not necessarily based on introns
  and exons simply a common contiguous segment of
  the gene.
• Human ADSL (adenylosuccinate lyase) gene

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Our splicing graph approach

• A biologists viewpoint each exon should be a
  node and each intron, an edge (connection).
• Automatic generation of AS clusters from gene
  structure.
• Identifying Reference distinct Exon and its
  associated variants.
• Simple rules for classifying alternative splicing
  events and visualization system for studying all
  variants from a single gene.
• Single-line diagram Experimentalist way of
  Alternative splicing analysis

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Making the splicing graph
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Usual classification of AS events(Leipzig et
al., 2004)
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Representing splice variants of the same gene as
a splicing graph
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Normal representation of transcripts human
hyalouronidase HYAL1 gene ENSG00000114378 (an
early version)
www.ebi.ac.uk/asd
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Splicing Graph representation of the same gene
Intron retention
Alternative Termination site
Exon skipping
Transcripts are shown as exon numbers 5239
639 1734 1834 124 134.
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Single-line Splice Diagram
Patterns using the above exon numbers are shown
as 5239 639 1734 1834 124
134.

• A Digraph or DAG (Directed Acyclic Graph)
• Graphs for which every unilateral orientation is
  traceable
• Experimentalists way of Alternative Splicing
  analysis (for a gene of interest with all
  transcripts) for validating splive junctions
• Intron retention is clearly visible

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Our extended classification
Automatic rule-based classification
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Our extended classification
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Where to make your splicing graphs
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Outline of the talk

• Background
• Determining gene architecture
• Graph theory in AS
• Whole genome analysis results
• AS and disease

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AS Databases (Of men and mice)
ASAP II (Kim et al., 2007) Comparative and evolutionary studies 17 genomes
EC Gene (Lee et al., 2007) Provides functional annotation for AS genes 9 genomes
ASTD (previously ASD) (Thanaraj et al., 2004) Genome wide analysis Human, mouse and rat
ASTALAVISTA (Foissac et al., 2007) Visual summary of the AS landscape Mainly for human genome

• Does not provide sufficient information for
  multi-gene comparison to understand the
  phenomenon of AS.

6
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Genome-wide AS analysisI said the fly
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Homology

• Similarity between biological sequences due to
  shared ancestry
• Orthology
• Homologous sequences are orthologous if separated
  by a speciation event
• The divergent copies of a singe gene in the
  resulting species are orthologous genes.
• At least 25 - 30 similarity at the protein level

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Gene Ontology

• Provides a controlled vocabulary to describe gene
  and gene product attributes in organisms.
• Three organizing principles
• Cellular component
• A component of a cell, e.g. nucleus
• Biological process
• Series of events accomplished by one or more
  ordered assemblies, e.g. signal transduction
• Molecular function
• Describes activities, e.g. catalytic activity

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AS genes in Bovine genome

• Part of bovine annotation project
• 16560 human genes, 15986 mouse genes, 4567 bovine
  genes
• Data extracted from ASTD and Ensembl (Hubbard et
  al., 2002)
• Orthologous genes found using Biomart from
  Ensembl
• Gene Ontology using Blast2GO (Conesa et al.,
  2005)
• 2458 (out of 4567) Ensembl AS genes have GO
  annotations
• 1716 AS genes can be further annotated

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Percentage of AS genes and orthologous spliced
genes in bovine, human and mouse

• Orthologous genes were analysed in order to
  reduce bias in the data.

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Gene Level AS Analysis of orthologous subset

• Percentage of bovine genes showing AS events are
  fewer compared to human.

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AS Event Analysis of the orthologous subset

• of AS events in bovine similar to human
• implies that more splice variants are obtained
  from fewer bovine genes.

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Gene Ontology analysis

• Gene Ontology using Blast2GO (Conesa et al.,
  2005)
• 2458 (out of 4567) AS genes has GO annotations in
  Ensembl
• 1716 AS genes can be further annotated

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Outline of the talk

• Background
• Determining gene architecture
• Graph theory in AS
• Whole genome analysis results
• AS and disease

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Implications for disease

• Diagnostics from early recognition of splice
  variants associated with disease, based on
  nucleotide detection
• Treatment options using siRNA
• Aberrant splicing in survival of motor neuron 1
  gene (SMN1) in spinal muscular atrophy (Cartegni
  and Krainer 2002)
• Suppressing anti-apoptotic AS variant of Bcl-x
  pre-mRNA in prostate and breast cancer cells
  (Mercatante et al. 2001)
• Correcting CFTR mis-splicing (Friedman et al.
  1999)

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Many diseases are caused by AS
Myotonic dystropy
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Why study farm animals?

• Provide valuable insights into gene function and
  genetic and environmental influences on animal
  production and human diseases. (Roberts et al.,
  2009 )
• The size and relatively long intervals between
  generations, domestic species are widely used to
  unravel the mechanisms involved in programming
  the development of an embryo and fetus, resulting
  in adult onset of diseases (King et. al., 2007 ,
  Padmanabhan et al., 2007)
• Mapping human disease genes to bovine orthologous
  genes is an excellent mode for carrying out
  analytical work and verifying the suitability of
  cow as a model organism.

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Mapping human disease genes to bovine genome

• 94 human disease genes were extracted from NCBI
  Genes and Disease database to analyse which of
  these genes were alternatively spliced in human
  and bovine genomes.
• AS analysis was conducted on 66 spliced genes.
• 17 orthologous spliced genes were observed in
  bovine.

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Human disease genes Conservation of cassette
exons in bovine orthologous genes

• Cassette exons occur in 38 of human disease genes
  and 14 orthologous bovine genes.

Number of cassette exons in 38 AS human disease genes 120
Exons present and constitutive in bovine orthologous gene 90
Exons present and regulated in bovine orthologous gene 7
Exons absent in bovine orthologous gene 23
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Human disease genes Cassette exons present and
regulated in bovine orthologous genes

• 3 genes with cassette exons in human were present
  and regulated in bovine.

Disease Gene name Cassette exon
Colon Cancer MLH1 Exon9
Exon10
Spinal muscular atrophy SMN1 Exon6
Exon5
Exon32
Tangier disease ABC1 Exon2
Exon10
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Human disease genes Intron retention present and
constitutive in bovine orthologues

• Intron retention in nine human genes out of
  which, IR in five genes was present and
  constitutive in bovine

Disease Gene name Intron retention
Glaucoma GLC1A Exon1
Spinocerebellar ataxia SCA1 Exon9
Polycystic kidney disease PKD1 Exon23
Exon15
Autoimmune polyglandular syndrome APS1 Exon10
Wilsons disease ATP7B Exon2
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Protein domain analysis of the orthologous
disease gene set

• Carried out Pfam domain search on 8 human disease
  genes to identify the effects of alternative
  splicing on the functional protein domains.
• Genes responsible for spinal muscular atrophy and
  colon cancer are spliced in bovine and resulted
  in probable structure and function disruption.
• 4 disease genes (glaucoma, Tangier, spinal
  muscular atrophy and colon cancer ) had all the
  domains from their human counterparts conserved
  in bovine.

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Conclusion
Our results provide a window of opportunity for
more in-depth analysis over a larger dataset,
where the cow can serve as a model organism for
many more human diseases.
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Acknowledgements

• PhD students at the
• National University of Singapore (Bernett T.K.
  Lee)
• Macquarie University (Durgaprasad Bollina and
  Elsa Chacko)
• Colleagues and A/Prof. Tin Wee Tan, NUS
• All of you

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Invitation to attend InCoB2009International
Conference in Bioinformatics(incob.apbionet.org)
Singapore, 7-11 Sept. at Matrix,
BiopolisKeynote Nobel Laureate Robert Huber,
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7 Sept Tutorials and Bioinformatics Education
workshop (WEBCB)8 Sept Clinical Bioinformatics
(CBAS) and SYMBIO (Students) Symposia9-11
Sept Scientific Meeting