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Pr. Thierry Soussi
SESHAT: AN INNOVATIVE TOOL TO HANDLE SOMATIC AND GERMLINE TP53 VARIANTS
PITFALLS IN TP53 STATUS ANALYSIS
November 2017
SESHAT: AN INNOVATIVE TOOL TO HANDLE SOMATIC AND GERMLINE TP53 VARIANTS
PITFALLS IN TP53 STATUS ANALYSIS
November 2017
TP53 VARIANT PATHOGENICITY
HUNTING NOVEL TP53 SNP
SESHAT
Input amino acid substitution
and protein ID
Alignments
Sequence
Structure
Conservation score
Frequencies
Physicochemical properties
3D structureSecondary strcuture
Surface area properties
Output Predictions
TYPICAL PIPELINE FOR IDENTIFICATION OF DELETERIOUS VARIANTS IN CODING REGIONS
SIFTtakesaquerysequenceandusesmultiplealignmentinformationtopredicttoleratedanddeleterioussubstitutionsforeverypositionofthequerysequence.
DeleteriousNondeleterious
Borderline
TYPICAL PIPELINE FOR IDENTIFICATION OF DELETERIOUS VARIANTS IN CODING REGIONS
SIFTtakesaquerysequenceandusesmultiplealignmentinformationtopredicttoleratedanddeleterioussubstitutionsforeverypositionofthequerysequence.
PREDICTINGDELETERIOUS AMINOACIDSUBSTITUTIONSISNOT
PREDICTINGPATHOGENICITY.
DeleteriousNondeleterious
Borderline
pathogenic
TYPICAL PIPELINE FOR IDENTIFICATION OF DELETERIOUS VARIANTS IN CODING REGIONS
PREDICTING PATHOGENICITY
MUST BEWILL BE
GENE SPECIFIC
IDENTIFICATION OF PATHOGENIC VARIANTS IN CODING REGIONS
WHETHER IT WILL BE ALSO TUMOR TYPE SPECIFICIS AN OPEN QUESTION
IDENTIFICATION OF PATHOGENIC VARIANTS IN CODING REGIONS
UMD-TP53
83 004 TP53 records75 448 tumors*6 916 TP53 variants
Sequencing artfacts have been identified and tagged
Main problems: duplicates entries
http://p53.fr
* The difference between patients and records is due to the fact that some tumors have multiple TP53 mutations
0
200
400
600
800
1000
1200
1400
1600
1800
2000
1989
19
90 19
91 19
92 19
93 19
94 19
95 19
96 19
97 19
98 19
99 20
00 20
01 20
02 20
03 20
04 20
05 20
06 20
07 20
08 20
09 20
10 20
11 20
12 20
13 20
14 20
15 20
16 20
17
TCGA
Cumulated SNV 5-8
Cumulated Ins / Del 5-8
WE HAVE INDENTIFIED MOST POTENTIAL TP53 SNV
Cumulated TP53 novelty since 1989.Since 2008, the number of novel TP53 variants detected in human tumors has plateaued,
whereas the rate of detection of novel frameshift mutations has remained constant for more than 20 years.
IndelSNV
TCGA data
WE HAVE INDENTIFIED MOST POTENTIAL TP53 SNV
C G A
TGA
TCA T
CG
1 single codon can sustain 9 different single nucleotide substitutions
WE HAVE INDENTIFIED MOST POTENTIAL TP53 SNV
C G A
TGA
TCA T
CG
1 single codon can sustain 9 different single nucleotide substitutions
9
9
Codon 175
Codon 114
Codon 40
-9
-6
-3
0
3
6
9
Missensemuta+ononly Foundinhumancancer
NeverfoundinhumancancerMdm2 binding site
Hotspot170-175
183
114Regulatory domain (uncertain function)
WE HAVE INDENTIFIED MOST POTENTIAL TP53 SNV
1 393N
umbe
r of s
unst
itutu
in
0
500
1000
1500
2000
2500
3000
3500
4000
1 10 100 1000 10000
Freq
uenc
y in
the
data
base
Variant occurance in the database
p.R175H (3,500x)
FREQUENCY OF TP53 VARIANT IN THE UMD TP53 DATABASE
0
500
1000
1500
2000
2500
3000
3500
4000
1 10 100 1000 10000
Freq
uenc
y in
the
data
base
Variant occurance in the database
p.R175H (3,500x)
p.G302W (1x)
FREQUENCY OF TP53 VARIANT IN THE UMD TP53 DATABASE
0
500
1000
1500
2000
2500
3000
3500
4000
1 10 100 1000 10000
Freq
uenc
y in
the
data
base
Variant occurance in the database
p.R175H (3,500x)
p.G302W (1x)
507 variants have been found only once
FREQUENCY OF TP53 VARIANT IN THE UMD TP53 DATABASE
ASSESSING TP53 VARIANTS PATHOGENICITY
Missense mutations 73%
Nonsense mutations
11%
Synonymous mutations
2%
Splice mutations 3%
Indel 11%
UMD TP53: 82 000 MUTATIONS
ASSESSING TP53 VARIANTS PATHOGENICITY
Missense mutations 73%
Nonsense mutations
11%
Synonymous mutations
2%
Splice mutations 3%
Indel 11%
UMD TP53: 82 000 MUTATIONS
1% are pathogenic
ASSESSING TP53 VARIANTS PATHOGENICITY
chr17:g.7578552C>G
c.378C>G
p.Y126* 41
Evgeny M. Makarov et al. 2017 Plos one
ASSESSING TP53 VARIANTS PATHOGENICITY
chr17:g.7578552C>G
c.378C>G
p.Y126* 41
Evgeny M. Makarov et al. 2017 Plos one
ASSESSING TP53 VARIANTS PATHOGENICITY
chr17:g.7578552C>G
c.378C>G
p.Y126* 41
Evgeny M. Makarov et al. 2017 Plos one
ASSESSING TP53 VARIANTS PATHOGENICITY
chr17:g.7578552C>G
c.378C>G
p.Y126* 41
Evgeny M. Makarov et al. 2017 Plos one
p.Y126del
ASSESSING TP53 VARIANTS PATHOGENICITY
Is there a TP53 null genotype ?
All these cell lines have a TP53 non sense mutation
ASSESSING TP53 VARIANTS PATHOGENICITY
Is there a TP53 null genotype ?
Gain of functionIncrease metastatic potential
PREDICTING PATHOGENICITY
MUST BEWILL BE
GENE SPECIFIC
IDENTIFICATION OF PATHOGENIC VARIANTS IN CODING REGIONS
WHETHER IT WILL BE ALSO TUMOR TYPE SPECIFICIS AN OPEN QUESTION
SESHAT: AN INNOVATIVE TOOL TO HANDLE SOMATIC AND GERMLINE TP53 VARIANTS
PITFALLS IN TP53 STATUS ANALYSIS
November 2017
TP53 VARIANT PATHOGENICITY
HUNTING NOVEL TP53 SNP
SESHAT
R72P R213RP47S
TP53 AND RARE SNP
P36P
THE TP53 GENE HAS BEEN SEQUENCED IN MORE THAN 150 000 INDIVIDUALS
IN MOST CASES MATCHED DNA FROM THE SAME PATIENT IS NOT SEQUENCED
Gnomad(or previous Exac)
dbSNP
Include pathogenic variantsLow number of individuals
138,632 individualsOnly germline variant
TP53 AND RARE SNP
Gnomad(Exac)
138,632 individualsOnly germline variant
p.R72P 183,709p.P36P 3,520p.R213R 3,407p.P47S 433p.D21D 136
variantAllele count
p.R175H 1p.R248Q 5p.R273H 4p.R273C 3p.R248W 1p.Y220C 2….
variantAllele count
Gnomad database includes multiple pathogenic TP53 variants
What is the true frequency of TP53 pathogenic variant in the normal population ?
TP53 AND RARE SNP
National genome database(Finland, Sweden, Netherland, Japan,…)
Gnomad Flossie
TP53 AND RARE SNP
National genome database(Finland, Sweden, Netherland, Japan,…)
Gnomad Flossie
Exraction of all coding variants
TP53 AND RARE SNP
Remove well known non pathogenic SNP
48 remaining SNP
Remove pathogenic variants
UMD TP53 DATABASEGermline variants found at
frequency higher than expected
TP53 AND RARE SNP
48 remaining SNP
23 remaining SNP
Functional analysis
0
50000
100000
150000
200000
250000
300000
350000
T
Wt TP53 R175H
M1 M2
M3 M4
M5 M6
M7 M8
M9
Transactivation
annexin
Pi
Wt TP53
MutTP53
SNP M1
SNP M2
Apoptosis
ApoptosisNo Apoptosis
Apoptosis
Wt TP53 Mut1TP53
Mut2TP53SNP M1 SNP M2
Control
0
50000
100000
150000
200000
250000
300000
350000
T
Wt TP53 R175H
M1 M2
M3 M4
M5 M6
M7 M8
M9
Transactivation
Growth arrest
Apoptosis
TP53 AND RARE SNP
Sequencing primers and intronic SNP
Several intronic primers frequently used for sequencing overlapp SNP
SESHAT: AN INNOVATIVE TOOL TO HANDLE SOMATIC AND GERMLINE TP53 VARIANTS
PITFALLS IN TP53 STATUS ANALYSIS
November 2017
TP53 VARIANT PATHOGENICITY
HUNTING NOVEL TP53 SNP
SESHAT
SESHAT: A WEB PORTAL FOR TP53 MUTATION ANALYSIS
SESHAT
Annotation accuracy
The 3' rule:a misapplied guideline:
SESHAT
Annotation accuracy
Most TP53 insertions are duplications
SESHAT
Using a correct reference
p.R43HAnewmutationhotspot?
53 kDa
SESHAT
Using a correct reference
p.R43HAnewmutationhotspot?
35 kDa
53 kDa
BAM
USERS
Single mutantanalysis
Batch analysis
SESHAT: A WEB PORTAL FOR TP53 MUTATION ANALYSIS
BAM
USERS
Single mutantanalysis
Batch analysis
Seshat: transformation
SESHAT: A WEB PORTAL FOR TP53 MUTATION ANALYSIS
BAM
USERS
Single mutantanalysis
Batch analysis
Seshat: transformation
Mutalyzer:checking
SESHAT: A WEB PORTAL FOR TP53 MUTATION ANALYSIS
BAM
USERS
Single mutantanalysis
Batch analysis
Seshat: transformation
Mutalyzer:checking
Seshat: analysis
Batch analysisoutputs
Single mutant analysisoutputs
SESHAT: A WEB PORTAL FOR TP53 MUTATION ANALYSIS
SESHAT: A WEB PORTAL FOR TP53 MUTATION ANALYSIS
PatientID test1cDNA_variant c.743G>AHG19_Variant chr17:g.7577538G>ANG_017013.2_Variant NG_017013.2:g.18331G>ASNP_ID rs11540652Transcriptt1MN_000546.5 c.743G>ATranscriptt2NM_001126112.2 c.743G>ATranscriptt3NM_001126114.2 c.743G>ATranscriptt4NM_001126113.2 c.743G>ATranscriptt5NM_001126115.1 c.347G>ATranscriptt6NM_001126116.1 c.347G>ATranscriptt7NM_001126117.1 c.347G>ATranscriptt8NM_001126118.1 c.626G>AProteinP1TP53_alpha LRG_321p1:p.R248QProteinP3TP53_beta LRG_321p3:p.R248QProteinP4TP53_gamma LRG_321p4:p.R248QProteinP8Delta40_TP53_alpha LRG_321p8:p.R209QProteinP9Delta40_TP53_beta LRG_321p9:p.R209QProteinP10Delta40_TP53_gamma LRG_321p10:p.R209QProteinP5Delta133_TP53_alpha LRG_321p5:p.R116QProteinP6Delta133_TP53_beta LRG_321p6:p.R116QProteinP7Delta133_TP53_gamma LRG_321p7:p.R116QProteinP11Delta160_TP53_alpha LRG_321p11:p.R89QProteinP12Delta160_TP53_beta LRG_321p12:p.R89QProteinP13Delta160_TP53_gamma LRG_321p12:p.R89Q
PatientID test1Records_Number 2562Variant_Classification Missense_MutationVariant_Type SNP
Comment_1_Frequency ThissinglenucleotidevariantisveryfrequentComment_2_Activity Inactive
Comment_3_Isoforms Thissubstitutiontargets12TP53isoformsComment_4prediction DamagingComment_5_Outliers -Comment_6_Splicing NosplicedefectpredictedComment_7_Sequence -
Comment_9_SNPBothgermlineandsomaticvariantshavebeendescribed;seecomment10forpopulationdata
Comment_10_populationrs11540652;listedintheExAcdatabase(withouttheTCGAcohort)withallelecount=7
Pathogenicity Pathogenic
Final_commentPublishedresearchaswellasdatabaseanalysisprovidesufficientevidenceforclassificationofthisvariantaspathogenic.
mutalyzer_comment
PatientID test1Sift_Prediction DamagingSift_Score 0.006Polyphen-2_HumVar ProbablydamagingPolyphen-2_HumDiv ProbablydamagingMutassessor_prediction MediumMutassessor_score 2.970Provean_prediction DeleteriousProvean_Score -3.915Condel DeleteriousCondel_Score 0.905MutPred_Splice_General_Score 0.181MutPred_Splice_Prediction_Label SpliceNeutralVariant(SNV)MutPred_Splice_Confident_HypothesesNotrelevantSIFT_converted_rankscore 0.620Polyphen2_HDIV_rankscore 0.899Polyphen2_HVAR_rankscore 0.916LRT_converted_rankscore 0.856MutationTaster_converted_rankscore 0.708MutationAssessor_rankscore 0.886FATHMM_rankscore 0.998MetaSVM_rankscore 0.968MetaLR_rankscore 0.998VEST3_rankscore 0.860PROVEAN_converted_rankscore 0.728phyloP46way_primate_rankscore 0.782phyloP46way_placental_rankscore 0.950GERP_RS_rankscore 0.970SiPhy_29way_logOdds_rankscore 0.973Evol_Score_II 78.950RFscore_II 0.992phyloP100way_vertebrate_rankscore 0.996phastCons46way_primate_rankscore 0.973phastCons46way_placental_rankscore0.992phastCons100way_vertebrate_rankscore0.988CADD_raw_rankscore 0.999DANN_rank_score 0.999
SESHAT: A WEB PORTAL FOR TP53 MUTATION ANALYSIS
SESHAT: A WEB PORTAL FOR TP53 MUTATION ANALYSIS
What Future ?
Version 1
data
Analysisdata are not stored
SESHAT: A WEB PORTAL FOR TP53 MUTATION ANALYSIS
What Future ?
Version 1
Version 2.0
data
Analysisdata are not stored
data
AnalysisData can stored in private databaseAvailable to specific consortia
THANK YOU