panc4 results: learning lessons from a successful gwas

Panc4 Results: Learning Lessons from a Successful GWAS

Gloria M. Petersen, Ph.D. Mayo Clinic Cancer Center

Rochester, MN September 19, 2015 – Houston, TX

Pancreatic Cancer Case-Control Consortium (PanC4) Special Thanks to Alison Klein, Ph.D., Johns Hopkins University

Brief History of the PANC4

• February ‘06

• March ‘06 • April 4 ‘06

• May, Aug, Sept ‘06

• December ’06

• January ’08 and annually since

Paolo Boffetta contacts pancreatic cancer epidemiology investigators to gauge interest Survey of Investigators In-person meeting at AACR in Washington DC Follow-up, planning conference calls In-person meeting; share instruments; and establish pooled studies In-person meeting; pooled analyses, genotyping options

1. Organization and Oversight 2. Commitment 3. Communication

• Organizationally • Oversight • Feedback • Transparency

• Election Process • Authorship

4. Data Standardization/Harmonization

Lessons in Coordination

PanC4 Demographics

Study Cases Controls

Age at Diagnosis for Cases (SD)a

Age of Controls

(SD) Male (%)

NHW (%)b

IARC 448 456 63.84 (11.16) 61.88 (11.88) 57 100

Johns Hopkins 315 81 64.29 (11.53) 63.45 (14.73) 51 93

Mayo Clinic 1104 1027 65.92 (11.09) 63.34 (10.58) 56 93

MD Anderson 616 509 62.6 (9.69) 59.2 (10.6) 59 100

Memorial Sloan Kettering 317 139 64.03 (10.39) 61.68 (10.98) 64 87

Toronto 402 401 64.92 (11.03) 62.95 (11.73) 50 85

Queensland 559 604 66.64 (11.26) 67.51 (10.91) 60 96

UCSF 253 248 62.52 (10.35) 60.4 (10.96) 54 82

YALE 156 366 67.02 (10.43) 65.15 (10.6) 59 93

TOTAL 4164 3792 64.76 (10.92) 63.09 (11.33) 57 93

Childs, Mocci et al. Nat Gen, 2015

Quality Control • 8,052 subjects from PanC4 genotyped on the HumanOmni

ExpressExome-8v1 array

• QC • 20 Samples with unresolved identity issues • 45 Samples with relatedness issues • >2% missing call rate or missing genotype rate • HWE p<10-6

• 7956 individuals passed all QC and were retained for analysis


Primary Analysis Unconditional logistic regression adjusted for age and including the first seven principal components was conducted under a log-additive genetic model


Manhattan Plot of PanC4 Results


N e w l y G e n o t y p e d C a s e s P a n C 4

4 , 1 6 4 c a s e s 3 , 7 9 2 c o n t r o l s 6 5 4 , 4 7 0 S N P s

A s s o c i a t i o n R e s u l t s : F i g u r e 3

P r e v i o u s G W A S P a n S c a n 1 P a n S c a n 2 1 , 8 5 6 c a s e s 1 , 6 1 8 c a s e s a n d 1 , 8 9 0 c o n t r o l s 1 , 6 8 2 c o n t r o l s 5 2 8 , 1 7 9 S N P s 5 5 7 , 5 5 5 S N P s

1 0 0 0 G I m p u t a t i o n

C o m b i n e d S t a g e 1 A n a l y s i s

( P a n C 4 , P a n S c a n 1 , P a n S c a n 2 ) 7 6 3 8 c a s e s

7 3 6 4 c o n t r o l s 8 6 6 8 9 1 S N P s

A s s o c i a t i o n R e s u l t s F i g u r e 4

P A N D o R A R e p l i c a t i o n G e n o t y p i n g

2 4 9 7 c a s e s 4 6 1 1 c o n t r o l s

2 5 S N P s

C o m b i n e d A n a l y s i s

( P a n C 4 , P a n S c a n 1 , P a n S c a n 2 , P A N D o R A )

9 , 9 2 5 c a s e s 1 1 , 5 6 9 c o n t r o l s

2 5 S N P s

T o p S N P s

T o p S N P s


PanC4 Results

• Statistical Analysis of PanC4 samples identified 17q25.1 (LINC00673, rs7214041)

• OR=1.38 (1.26-1.51)

• P=1.95X10-10


PanC4 Results Chr Genea SNP Positionb Studyc

Reported OR

(CI)d

Reported P-valuee

PanC4 OR (CI)f

PanC4 P-value g

9q34 ABO rs505922 136149229 PanScan 1 1.2 (1.12 - 1.28) 5.37 X 10-8 1.27

(1.19 - 1.35) 1.72 X 10-13

13q22.1 KLF5 and KLF12 rs9543325 73916628 PanScan 2 1.26

(1.18 - 1.35) 3.27 X 10-11 1.24 (1.16 - 1.32) 2.26X 10-10

1q32.1 NR5A2 rs3790844 200007432 PanScan 2 0.77 (0.71 - 0.84) 2.45 X 10-10 0.83

(0.77 - 0.90) 3.05 X 10-6

5p15.33 CLPTM1L rs401681 1322087 PanScan 2 1.19 (1.11 - 1.27) 3.66 X 10-7 1.20

(1.13 -1.28) 2.70 X 10-8

5p15.33 TERT rs2736098 1294086 PanScan 3 0.80 (0.76 - 0.85) 9.78 X 10-14 0.85

(0.78 - 0.93) 2.31 X 10-5

7q32.3 LINC-PINT rs6971499 130680521 PanScan 3 0.79 (0.74 - 0.84) 2.98 X 10-12 0.81

(0.74 - 0.88) 7.10 X 10-6

16q23.1 BCAR1 rs7190458 75263661 PanScan 3 1.46 (1.3 - 1.65) 1.13 X 10-10 1.40

(1.22 - 1.60) 1.01 X 10-4

13q12.2 PDX1 rs9581943 28493997 PanScan 3 1.15 (1.1 - 1.2) 2.35 X 10-9 1.17

(1.10 - 1.24) 1.94 X 10-7

22q12.1 ZNRF3 rs16986825 29300306 PanScan 3 1.18 (1.12 - 1.25) 1.18 X 10-8 1.14

(1.04 - 1.24) 2.72 X 10-3



Combined Stage 1

Genotype imputation


Meta Analysis Results

We checked for population stratification considering only samples of European ancestry and the results did not vary significantly.



Stage 2: PANDoRA Replication

Stage 2 Analysis:

• Replication dataset: 2,497 cases and 4,611 controls from the PANDoRA consortium

• PANDoRa is a collection of European case-control studies inclulding Czech Republic, Germany, Greece, Italy, Lithuania, and Poland.

• 25 SNPs from 23 independent regions were selected to be genotyped


PANDoRA Statistical Analysis • Data from each country were analyzed

independently

• Logistic regression with additive effects of each allele were fit, including age as covariate (PCs not available)

• A test of HWE carried out on each SNP. Two SNPs (rs16867971 for Greece and rs10850078 for Lithuania) out of HWE (P<0.001). For those samples, analysis not completed for that SNP in that population


PANDoRA Results • Independent evidence of association at

17q25.1 (rs7214041) • OR=1.25 (1.11-1.41) • P=3.37X10-4

• rs9854771 on 3q29 is consistent, but not significant

• OR=0.93 (0.86-1.01) • P=0.101



Combined Analysis

17q25.1

Statistic Panc4 PanScan I PanScan II PANDoRA Combined Stage 1&2

rs11655237

maf cases;controls 0.146; 0.11 0.139; 0.129 0.149; 0.116 0.135; 0.114

OR (CI) 1.38 (1.26 - 1.52 )

1.09 (0.96 - 1.25 )

1.34 (1.16 - 1.55 )

1.24 (1.1 - 1.4)

1.26 (1.19 - 1.34)

p-value 1.38E-10 1.95E-01 2.95E-04 6.40E-04 1.42E-14

• Long intragentic non-coding protein RNA 673 (LINC00673)

• rs11655237 is a non-coding transcript variant and shows significant DNase hypersensitivity in multiple cancer cell lines

• rs11655237 binds transcription factors including P300, FOXA1, FOXA2, and the DNA repair protein RAD21

• Rs7214041 (intronic) alters regulatory motifs for HNF1 (HNF1A: hepatocyte nuclear factor-1)

3q29

TP63



OR (CI) 0.86 (0.81 - 0.92 )

0.88 (0.80 - 0.97 )

0.87 (0.79 - 0.97 )

0.93 (0.86 - 1.01)

0.89 (0.85 - 0.93)

p-value 3.10E-05 7.94E-03 1.55E-02 1.01E-01 2.35E-08

• rs9854771 and rs1515496 are intronic to p63, a transcription factor

• Homolog of p53

• Role in the prevention of cancer metastasis by playing a role in cell-cycle arrest and apoptosis

Tumor suppressive

Oncogenic

Melino, G. et al., PLoS (2011)

• The p63 family of proteins is highly conserved

• ΔNp63: predominant

isoform in pancreatic cancer cell lines and promoted pancreatic cancer growth, motility and invasion

Danilov, A.V. et al. PLoS One (2011)

• Variants in TP63 have been

previously found associated with lung and bladder cancer

Figueroa, J.D. et al , Hum Mol Genet (2014) , Lan, Q. et al , Nat Genet (2012) ,

Shiraishi, K. et al Nat Genet (2012) , Rothman, N. et al. Rothman, N. et al. Nat Genet (2010)

3q29

2p13.3



OR (CI) 1.14 (1.06 - 1.22 )

1.06 (0.96 - 1.18 )

1.15 (1.03 - 1.28 )

1.16 (1.06 - 1.27)

1.14 (1.09 - 1.19)

p-value 5.96E-05 1.57E-01 5.18E-03 9.42E-04 3.36E-09

rs1486134

ETAA1

• Prior GWAS in Han Chinese reported suggestive evidence for another SNP on 2p13.3 (rs2035565) Wu, C. et al , Nat Genet (2012)

• Strong LD between rs1486134 and rs2035565 in European and Asian populations based on 1000 Genomes samples (r2=0.91 and r2=0.90 respectively)

• ETAA1 alias (Ewing tumor-associated antigen 1) tumor-specific cell surface antigen in the Ewing's family of tumors Borowski, A. et al , Cancer Immunol Immunother (2006)

7p13



OR (CI) 0.89 (0.83 - 0.96 )

0.82 (0.73 - 0.91 )

0.85 (0.76 - 0.94 )

0.91 (0.83 - 1)

0.88 (0.84 - 0.92)

p-value 1.98E-03 1.66E-04 8.72E-03 3.93E-02 1.41E-08

rs17688601

SUGCT

• SUGCT (succinyl-CoA:glutarate-CoA transferase) is involved in glutarate metabolism.

• Mutations in this gene are associated with glutaric aciduria Marlaire, S. et al J Inherit Metab Dis (2014)

• Pathway of glutamine in human PDAC cells that is required for tumor growth Son, J. et al Nature (2013)

• rs17688601 alters binding of HNF1-4 (hepatocyte nuclear factor) and other DNA binding proteins (Haploreg)

Heritability • Proportion of phenotypic variance explained by

common SNPs

• PanC4: – Overall heritability: 16.4% (95%CI 10.4%-22.4%) – Significant and suggestive regions 3.0% (95%CI:

2.0%-3.9%)

• All GWAS – Overall heritability: 13.1% (95%CI 9.9%-16.3%) – Significant and suggestive regions: 2.1% (95%CI

1.7%-3.1%)

Childs, Mocci, et al Nat Gen, 2015

Manolio T et al. Nature. 2009 461: 747–753

“Missing Heritability?”

BRCA2 PALB2

PRSS1

HNPCC

CDKN2A

12 Confirmed GWAS HITS

Modified from Manolio T et al. Nature. 2009 461: 747–753

ATM

BRCA1

STK11 Genetic Susceptibility to Pancreatic Cancer

panc4 results: learning lessons from a successful gwas

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