http://. smoking habits there are over 1 billion people in the world that smoke tobacco there are...
TRANSCRIPT
Smoking Habits Smoking Habits
There are over 1 There are over 1 billion people in the billion people in the world that smoke world that smoke tobaccotobacco Of these 5-6 million Of these 5-6 million
will die on an annual will die on an annual basisbasis
This habit increases This habit increases the likelihood of the likelihood of developing lung developing lung cancer to 20 times cancer to 20 times that of a non-smokerthat of a non-smoker
Gail Butler, Chris Scodeller, Julie Ward, & Gail Butler, Chris Scodeller, Julie Ward, & Lori FosterLori Foster
OutlineOutline
Sequencing of SCLC cell lineSequencing of SCLC cell line
Somatic mutationSomatic mutation
Mutation signatures in NCI-H209Mutation signatures in NCI-H209
DNA repair pathwaysDNA repair pathways
Genomic Rearrangement-specifically CHD7Genomic Rearrangement-specifically CHD7
Sequencing of a SCLC Sequencing of a SCLC cell linecell line
Why use SCLC?Why use SCLC? Not surgically resectedNot surgically resected
Cell lineCell line NCI-H209NCI-H209 Immortal cell lineImmortal cell line 55-year-old male with SCLC55-year-old male with SCLC
Smoking history not recordedSmoking history not recorded Showed histologically typical small cellsShowed histologically typical small cells >97% of such tumors associated with tobacco >97% of such tumors associated with tobacco
smokingsmoking Taken before chemotherapyTaken before chemotherapy
Sequencing: The SOLiD Sequencing: The SOLiD PlatformPlatform
Massively parallel next-generation Massively parallel next-generation sequencingsequencing
Greater than 99.94% accuracyGreater than 99.94% accuracy
Relatively inexpensiveRelatively inexpensive
Allows for:Allows for: Whole genome sequencingWhole genome sequencing Targeted resequencingTargeted resequencing Gene expression dataGene expression data
Sample preparationSample preparation Fragment library or mate pair librariesFragment library or mate pair libraries
Libraries are sheared and adaptor Libraries are sheared and adaptor molecules are ligated to each unique molecules are ligated to each unique moleculemolecule
Each molecule attached to a beadEach molecule attached to a bead Amplified using emulsion PCRAmplified using emulsion PCR 3’ end modification3’ end modification
Beads are covalently attached to a glass Beads are covalently attached to a glass slideslide
A universal sequencing primer, ligase, and a A universal sequencing primer, ligase, and a set of fluorescently labeled di-base probes set of fluorescently labeled di-base probes are introducedare introduced
Multiple cycles of ligation, detection, and Multiple cycles of ligation, detection, and cleavage performedcleavage performed
After the template has been read, After the template has been read, synthesized strand removedsynthesized strand removed Primer attaches to template offset by 1 Primer attaches to template offset by 1
nucleotidenucleotide
CoverageCoverage
Figure 1AFigure 1A Minimum 30x Minimum 30x
coveragecoverage
Figure 1BFigure 1B 39x coverage for 39x coverage for
tumourtumour 31x coverage for 31x coverage for
normal cell linenormal cell line
BioinformaticsBioinformatics
Identify somatically Identify somatically acquired mutations acquired mutations from sequence datafrom sequence data
77 coding 77 coding substitutionssubstitutions
333 random 333 random variantsvariants
Indels difficult to Indels difficult to detectdetect
Supplementary Fig.1Supplementary Fig.1
Somatically acquired genomic Somatically acquired genomic variantsvariants
22,910 somatically 22,910 somatically acquired (not acquired (not inherited) inherited) mutationsmutations 70% intergenic70% intergenic 28% intronic28% intronic 0.8% non-coding 0.8% non-coding
translatedtranslated 0.6% coding0.6% coding
Figure 1CFigure 1C Somatic mutations Somatic mutations
of NCI-H209 of NCI-H209 genomegenome
Deletions, Deletions, insertions, insertions, heterozygous and heterozygous and homozygous homozygous substitutions, mis-substitutions, mis-sense, nonsense, sense, nonsense, and and rearrangements rearrangements
Point mutations in coding Point mutations in coding regionsregions
RB1 C706F Point MutationRB1 C706F Point Mutation Nonconservative amino acid Nonconservative amino acid
substitutionsubstitution Inhibits phosphorylation and Inhibits phosphorylation and
abolishes protein functionabolishes protein function
• TP53 Splice Site DisruptionTP53 Splice Site Disruption– TP53 encodes p53, a tumor suppressorTP53 encodes p53, a tumor suppressor
• Combination of RB1 and TP53 characteristic of Combination of RB1 and TP53 characteristic of SCLCSCLC
Non-synonymous vs. Non-synonymous vs. SynonymousSynonymous
Non-synonymousNon-synonymous Codes for different amino acidCodes for different amino acid
SynonymousSynonymous Amino acid produced not modifiedAmino acid produced not modified
Accumulation of mutations Accumulation of mutations increasing fitness will be shown as increasing fitness will be shown as an excess of non-synonymousan excess of non-synonymous
Observed ratio not different than Observed ratio not different than that expected by chancethat expected by chance Suggests that the majority of coding Suggests that the majority of coding
variants do not confer selective variants do not confer selective advantageadvantage
Mutations in regulatory Mutations in regulatory regionsregions
Little known about mutations occurring on Little known about mutations occurring on either side of transcription start siteseither side of transcription start sites
Supplementary Fig. 2ASupplementary Fig. 2A Find somatic substitutions within 2kb of Find somatic substitutions within 2kb of
known transcription start sitesknown transcription start sites
Apply hidden Markov modelsApply hidden Markov models AI program that can be trained to find AI program that can be trained to find
sequencessequences Predict which substitutions might affect Predict which substitutions might affect
transcription factor binding sitestranscription factor binding sites
Supplementary Fig. 2BSupplementary Fig. 2B
Distribution observed no different than that Distribution observed no different than that those mutations seen in random “simulated those mutations seen in random “simulated sets” of mutationssets” of mutations
May still be mutations that alter May still be mutations that alter transcription factor binding and affect gene transcription factor binding and affect gene regulationregulation
Example Supplementary Fig. 2CExample Supplementary Fig. 2C
T>G in RAS oncogene family gene, T>G in RAS oncogene family gene, RAB42RAB42Disrupts potential binding motifDisrupts potential binding motif
Big picture of somatic Big picture of somatic mutationsmutations
Data indicates that most of the Data indicates that most of the mutations in the coding and promoter mutations in the coding and promoter regions are passenger eventsregions are passenger eventsEvents that don’t contribute to the Events that don’t contribute to the
development of cancer, but have development of cancer, but have occurred during cancer growthoccurred during cancer growth
Mutations confer no selective Mutations confer no selective advantage to the cellsadvantage to the cells
Tobacco smoke Tobacco smoke contains more contains more than than 6060 carcinogens carcinogens which bind and which bind and chemically chemically modify DNA. modify DNA.
The carcinogen binds to the DNA The carcinogen binds to the DNA forming a bulky adducts at forming a bulky adducts at purinepurine bases (guanine and adenine).bases (guanine and adenine).
-Change the alpha -Change the alpha helixhelix
-Allow non-Watson–Allow non-Watson–Crick pairingCrick pairing
-Get in the wayGet in the way
Most Common Most Common TransversionsTransversions
G>T/C>A (34%)G>A/C>T (21%) A>G/T>C (19%)
Top 3 Top 3 transversions transversions are all are all purines…purines…
This distribution of transversions is This distribution of transversions is consistent with the literatureconsistent with the literatureShows there is consistenency with Shows there is consistenency with
mutational patterns.mutational patterns.Control for in vivo mutationControl for in vivo mutation
G>T transversions G>T transversions occur more occur more frequently at frequently at methylated methylated CpGCpG dinucleotidesdinucleotides
(34%) of total mutations(34%) of total mutations
CpG SitesCpG Sites
cytosine-phosphate- cytosine-phosphate- guanineguanine
G>T transversions occur G>T transversions occur more frequently at more frequently at methylatedmethylated CpG CpG dinucleotidesdinucleotidesIn mammals, 70% to In mammals, 70% to
80% of CpG are 80% of CpG are methylatedmethylated
(34%) of total (34%) of total mutationsmutations
5’3’
3’5’
CpG Island: High frequency of cytosine CpG Island: High frequency of cytosine connected to guanine.connected to guanine.
CpG islands are regions that contain a high CpG islands are regions that contain a high CpG content. CpG content. They are in and near approximately 40% of They are in and near approximately 40% of
promoters of mammalian genes.promoters of mammalian genes.
It’s getting complicated so lets recap:It’s getting complicated so lets recap:
Most transversion mutations (34% of total) Most transversion mutations (34% of total) are G>Tare G>T
The G >T mutations happen often at CpG sitesThe G >T mutations happen often at CpG sites
The G >T mutations which happen at CpG sites The G >T mutations which happen at CpG sites are often are often methylatedmethylated CpG sites CpG sites
When looking at guanines in the When looking at guanines in the genome, how often is the nucleotide genome, how often is the nucleotide preceding it a cytosine?preceding it a cytosine?
This often in the genome, a C is expected to precede a G This often in the genome, a C is expected to precede a G
When looking at guanines in the When looking at guanines in the genome, how often is the nucleotide genome, how often is the nucleotide preceding it a cytosine?preceding it a cytosine?
This often in a G>T mutations, a C This often in a G>T mutations, a C precedes the G precedes the G
Wait, what?Wait, what?
5’3’
3’5’
-N-N-N-N-?-G-N-N-N-N-N-N-N-C-G-N-N-N-N-?-G>T-N-N-N-N-N-?-G-N-N-N-
The expected fraction of CpG’s per Guanine in genomic DNAThe fraction of G>Ts mutations on CpG’s per guanine in CpG islands.If everything was random, we would expect the G>T mutations to have an equal make up of CpG/G, as genomic CpG/G…
…but that is not so!
Wait, what?Wait, what?
When looking at guanines in the When looking at guanines in the genome, how often is the nucleotide genome, how often is the nucleotide preceding it a cytosine?preceding it a cytosine?
This often in a G>T mutations, a C This often in a G>T mutations, a C precedes the G precedes the G
When looking at guanines in the When looking at guanines in the genome, how often is the nucleotide genome, how often is the nucleotide preceding it a cytosine?preceding it a cytosine?
This often in a G>A mutation, a C precedes the G This often in a G>A mutation, a C precedes the G •Often occur outside CpG islands.Often occur outside CpG islands.•Unusually high fraction likely due to spontaneous Unusually high fraction likely due to spontaneous deamination of methylated cytosine to thyminedeamination of methylated cytosine to thymine
When looking at guanines in the genome, When looking at guanines in the genome, how often is the nucleotide preceding it a how often is the nucleotide preceding it a cytosine?cytosine?
This often in a G>C mutation, a C precedes the G This often in a G>C mutation, a C precedes the G •similar to G>T but these were significantly more likely to similar to G>T but these were significantly more likely to occur within CpG islandsoccur within CpG islands
WHAT DOES THIS ALL WHAT DOES THIS ALL MEAN?MEAN?
““Thus, the sequence context of the 23,000 Thus, the sequence context of the 23,000 mutations in the NCI-H209 genome provides mutations in the NCI-H209 genome provides tremendous power to identify multiple tremendous power to identify multiple distinctive mutation signatures, not evident distinctive mutation signatures, not evident from targeted re-sequencing studies of from targeted re-sequencing studies of limited genomic regions.”limited genomic regions.”
It’s getting complicated (still) so lets recap:It’s getting complicated (still) so lets recap:
Most transversion mutations (34% of total) Most transversion mutations (34% of total) are G>Tare G>T
The G >T mutations happen often at CpG sitesThe G >T mutations happen often at CpG sites
The G >T mutations which happen at CpG sites The G >T mutations which happen at CpG sites are often are often methylatedmethylated CpG sites. CpG sites.
So how does the Methylation play So how does the Methylation play into all this?into all this?
Only 10–20% of CpG dinucleotides in CpG Only 10–20% of CpG dinucleotides in CpG islands are methylated while 60–70% CpG islands are methylated while 60–70% CpG sites are methylated outside the islands.sites are methylated outside the islands.
This provides a model to see how This provides a model to see how methylation of CpG sites affects C>T methylation of CpG sites affects C>T mutations.mutations.
5’3’
3’5’
CpG CpG IslandIsland
In other words, lets compare the In other words, lets compare the frequency of G>C mutations here and here frequency of G>C mutations here and here to see how methylation effects mutation.to see how methylation effects mutation.
Non CpG Non CpG IslandIsland
10-20 Percent 10-20 Percent MethylatedMethylated
60-70 Percent 60-70 Percent MethylatedMethylated
Non CpG Non CpG islandsislands
CpG CpG islandsislandsLess CpG mutations in CpG islands than CpGs in Less CpG mutations in CpG islands than CpGs in
non CpG islands. non CpG islands.
5’3’
3’5’
CpG IslandCpG Island10-20 Percent Methylated10-20 Percent Methylated
Less C>T MutationLess C>T Mutation
Non CpG IslandNon CpG Island60-70 Percent 60-70 Percent
MethylatedMethylatedMore C>T MutationMore C>T Mutation
Less G>C mutations in the islands…and there Less G>C mutations in the islands…and there is less methylation in the islands…..is less methylation in the islands…..
……suggesting that C>T mutations suggesting that C>T mutations preferentially occur at methylated CpGspreferentially occur at methylated CpGs
Can’t we fix this???Can’t we fix this???
Bulky adducts on purines are the most common Bulky adducts on purines are the most common source of DNA damage from tobacco carcinogens.source of DNA damage from tobacco carcinogens.
These bulky adducts get in the way of the RNA These bulky adducts get in the way of the RNA polymerase.polymerase.
When the RNA polymerase stops, it recruits When the RNA polymerase stops, it recruits nucleotide excision repair machinery, leading to nucleotide excision repair machinery, leading to excision of the altered nucleotide, preventing excision of the altered nucleotide, preventing mutation.mutation.
The more The more expression, the expression, the more the repair.more the repair.
Mutation repair in Mutation repair in non transcribed non transcribed regions occurred regions occurred less frequently less frequently than transcribed than transcribed regions (good!).regions (good!).
G>A mutations
•Mutations occurred about equally on transcribed and non-transcribed strands•Mutations on both strands were significantly reduced in more highly expressed genes.
A>G mutations
•Transcribed strand mutations decreased with higher gene expression.•Non Transcribed mutations were relatively level.
This suggests at least two separate DNA This suggests at least two separate DNA repair pathwaysrepair pathways
Which suggests “distinct physicochemical Which suggests “distinct physicochemical effects on DNA structure, with variable effects on DNA structure, with variable recognition and excision by the genome recognition and excision by the genome surveillance machinery.”surveillance machinery.”
Genomic Rearrangements Genomic Rearrangements & Copy Number& Copy Number
NCI-H209 genome has 58 somatic genome NCI-H209 genome has 58 somatic genome
rearrangementsrearrangements
• 18 deletions (31%)18 deletions (31%)• 9 tandem duplications (16%)9 tandem duplications (16%)• 15 Inverted intrachromosomal rearrangements (26%)15 Inverted intrachromosomal rearrangements (26%)• 9 non-inverted intrachromosomal rearrangements (16%)9 non-inverted intrachromosomal rearrangements (16%)• 7 interchromosomal rearrangements 7 interchromosomal rearrangements
Figure 3. Rearrangements Rearrangements between between
chromosomes 1 & 4chromosomes 1 & 4
Intrachromosomal inversionsIntrachromosomal inversions
Non-inverted Non-inverted intrachromosomal intrachromosomal rearrangementsrearrangements
Interchromosomal Interchromosomal rearrangementsrearrangements
Not classical inversions: Not classical inversions:
• Clear boundaries Clear boundaries separating changes in separating changes in copy number in genes on copy number in genes on both chromosomesboth chromosomes
• Breakpoints between Breakpoints between chromosomes aren't chromosomes aren't reciprocalreciprocal
• Unbalanced Unbalanced rearrangements rearrangements
Oncogenic Fusion GenesOncogenic Fusion GenesOncogenic Fusion Gene: A hybrid gene formed from two genes previously separated
Chromosomal rearrangements can result in an oncogenic fusion gene if:
2 genes side by side Intact ORF Genes in the same orientation
NCI-H209
Fusion gene: 240 bp deletion on chromosome 16:• 1st 2 exons of CREBBP• 3' portion of BTBD12
RT-PCR showed expression of fusion transcriptThis wasn't expressed in 55 other SCLS
Direct further studies here????
CHD7 significanceCHD7 significanceFigure 4.
CHD7 codes for a chromatin CHD7 codes for a chromatin helicase DNA binding helicase DNA binding proteinprotein
NCI-H209:NCI-H209:• 39.5kb tandem duplication of 39.5kb tandem duplication of
exons 3-8 of CHD7 exons 3-8 of CHD7 (Figure 4a &4c.)(Figure 4a &4c.) NCI-H2171:NCI-H2171:• Fusion gene of exons 1-3 of PVT1 Fusion gene of exons 1-3 of PVT1
(non-coding RNA gene immediately (non-coding RNA gene immediately downstream of MYC)downstream of MYC) & exons 4-38 of & exons 4-38 of CHD7CHD7 (Figure 4c.)-MYC amplification (Figure 4c.)-MYC amplification
LU-135:LU-135:• Fusion gene of exon 1 of PVT1 Fusion gene of exon 1 of PVT1 (non-(non-
coding RNA gene immediately downstream coding RNA gene immediately downstream of MYC) of MYC) & exons 14-38 of CHD7& exons 14-38 of CHD7 (Figure (Figure 4c.) -MYC amplification4c.) -MYC amplification
This suggests that CHD7 rearrangements This suggests that CHD7 rearrangements are a regular phenomenon in SCLC are a regular phenomenon in SCLC
Figure 4.
LU-135 studied by mate pair sequencing showed:LU-135 studied by mate pair sequencing showed: Fusion gene of exon 1 of PVT1 Fusion gene of exon 1 of PVT1 (non-coding RNA gene immediately (non-coding RNA gene immediately
downstream of MYC) downstream of MYC) & exons 14-38 of CHD7 & exons 14-38 of CHD7
CHD7 amplicon linked to MYC expression amplificationCHD7 amplicon linked to MYC expression amplification
• MYC codes for a transcription factor that regulates expression of multiple genesMYC codes for a transcription factor that regulates expression of multiple genes• Rearrangements resulted in increased expressivity in MYC & 3' end of CHD7Rearrangements resulted in increased expressivity in MYC & 3' end of CHD7
LU-135LU-135
FigureFigure 4. 4.
NCI-H2171 & LU-135 show elevated NCI-H2171 & LU-135 show elevated levels of expression levels of expression SCLC in general have a greater SCLC in general have a greater normalized expression of CHD7 than normalized expression of CHD7 than non-SCLC & other tumor typesnon-SCLC & other tumor types
CHD7 SummaryCHD7 Summary• CHD7 rearrangements found in 3 SCLC cell lines CHD7 rearrangements found in 3 SCLC cell lines
• LU-135 & NCI-H2171:LU-135 & NCI-H2171: have PVTI-CHD7 fusion genes + MYC amplification have PVTI-CHD7 fusion genes + MYC amplification • PVTI downstream of MYC & may be a transcriptional target of the MYC proteinPVTI downstream of MYC & may be a transcriptional target of the MYC protein• Insertion of CHD7 with subsequent amplification results in increased gene copy Insertion of CHD7 with subsequent amplification results in increased gene copy
number & regulatory elementsnumber & regulatory elements• OVEREXPRESSION OVEREXPRESSION
• NCI-H209:NCI-H209: duplication of parts of the CHD7 gene duplication of parts of the CHD7 gene• CHD7 is a chromatin remodeller that promotes enhancer-mediated transcription CHD7 is a chromatin remodeller that promotes enhancer-mediated transcription
through histone methylationthrough histone methylation• Histone modifiers have been implicated as cancer genes previouslyHistone modifiers have been implicated as cancer genes previously
Rearrangements of CHD7 would make for an Rearrangements of CHD7 would make for an
interesting extension of this paper interesting extension of this paper
SummarySummary
Each mutation due to the carcinogen affect Each mutation due to the carcinogen affect causes consequences in three processes:causes consequences in three processes:
Chemical modification of a purineChemical modification of a purine Failure to repair via surveillance pathwaysFailure to repair via surveillance pathways Incorrect nucleotide incorporation due to base Incorrect nucleotide incorporation due to base
distortion during DNA replicationdistortion during DNA replication
SummarySummary
Transcription-coupled repairTranscription-coupled repair Stall RNA polymerase observed with NCI-H209Stall RNA polymerase observed with NCI-H209 A>G mutationsA>G mutations
Expression-linked repair Expression-linked repair More effective in highly transcribed regionsMore effective in highly transcribed regions G>A mutationsG>A mutations
CombinedCombined G>T and A>T mutationsG>T and A>T mutations
After ThoughtAfter Thought
Lung cancer develops after 50 Lung cancer develops after 50 pack years of smokingpack years of smoking7,300 cigarettes a year7,300 cigarettes a year
On average you acquire one On average you acquire one mutation for every 15 cigarettes mutation for every 15 cigarettes smokedsmoked
Questions?Questions?