pennsylvania geisinger medical center - chu-imaa

Geisinger Medical CenterDanville, PA USA

Pennsylvania(pop. 12,604,767119,283 km2)

Geisinger Health System

Weis Center for Research

Geisinger Clinic

Dept. of Vascular Surgery Primary Care ClinicsMyCode TM

Subject Enrollment for Research Studies

Confirmed AAA cases (N=925)•Blood (DNA)•Serum•Tissue (if surgical repair)•Authorization to access electronic medical records for research

Unselected “population” cohort (N=16,715)•Blood (DNA)•Serum•Authorization to access electronic medical records for research

•Population research•Epidemiology•Longitudinal outcome studies•Genetic and molecular studies

Males (78.2%)

74.0 yr

10.1%

18.4%

Females (21.8%)

72.9 yr

12.3%

20.8%

Mean age

Never smoked*

Family history

925 AAA cases

Characteristics of AAA Research Subjects

•surgical or endovascular repair 39%

•Mean age at repair 62.3 yr

•Mean diameter at repair 5.1 cm

•Repaired, with family history `18%

*most have history of secondary smoking exposure

0

10

20

30Females (191)Males (684)

Per

cent

Characteristics of AAA Research Subjects

40 45 50 55 60 65 70 75 80 85 90 95 100

105

0

Age (years)

AAA Size vs AgeFemales

40 60 80 1000

5

10

15

Age (years)

Dia

met

er (c

m)

AAA Size vs AgeMales

40 60 80 1000

5

10

15

RepairedUn-repaired

Age (years)

Dia

met

er (

cm)

Aortic Wall TissueAAA (N=4) and Normal (N=4)

��

ISOLATE TOTAL RNA��

EXPRESSION PROFILING USING AFFYMETRIX MICROARRAYS��

Genome-Wide Gene Expression Analysis of AAA Tissue

��

COMBINE RESULTS WITH LENK ET AL. 2007 Illumina Platform

7 cases, 7 controls ��

57 DIFFERENTIALLY EXPRESSED GENES IN COMMON��

VALIDATION & FOLLOW -UP:

Real-time Q-RT-PCR

Immunohistochemistry

Immunoblot analysis

Genome-Wide Gene Expression Analysis of AAA Tissue

Differentially Regulated Genes in AAA• Immune function

– CD4– CCL4– IL10RA– CD48– CXCR4– NCF4– NCF4– PTPRC

• Extracellular matrix – LAMA2– ITGA10– TMP2

• Oxidative Stress – GATM

Challenge: late-stage aneurysms; primary vs. secon dary changes

AAA Control

Genome Wide Association Using Pooled DNA Samples From Cases and Controls

DNA poolRep 1N=400

DNA poolRep 3N=400

DNA poolRep 2N=400

DNA poolRep 1N=400

DNA poolRep 3N=400

DNA poolRep 2N=400

Affymetrix SNP array

(500,000 SNPs)


(500,000 SNPs)


(500,000 SNPs)


(500,000 SNPs)


(500,000 SNPs)


(500,000 SNPs)

Calculate mean AF difference

Calculate AF for each SNP






(500,000 SNPs) (500,000 SNPs)(500,000 SNPs) (500,000 SNPs) (500,000 SNPs)(500,000 SNPs)

CV < 0.1 CV < 0.1

PDZRN3 CNTN3 ROBO2 ROBO1

74M 76M 78M 80M

GWAS Case-Control Allele Frequency Differences Chromosome 3p12.3

10

12 CNTN3

75M 76M

0

2

4

6

8

10

-log

10PΣ

5

Map position75,000,000 76,000,000

SNP ID Location P value

rs7635818 3p12.3 0.0003

rs11120508 1q41 0.0005

Top AAA -Associated SNPs

rs11120508 1q41 0.0005

rs1081244 9p21 0.008

rs11164404 1p21.1 0.01

Based on individual genotype analysis in 742 GVC cases and controls

74400k 74600k 74800k 75000k

NM_020872 CNTN3

NCBI SNP Reference

HapMap Position P-value

Mapping the Genetic Association with AAA of SNPs on Chromosome 3p12.3

Reference Position P-value

rs6549634 74762904 0.191

rs12714811 74841440 0.300

rs7642555 74852110 0.039

rs7635639 74866049 0.007

rs7635818 74866166 0.004

rs1569066 74881275 0.044

rs10222550 74902382 0.120

rs1527527 74919636 0.153

rs2178032 74943083 0.120

rs4417905 74998753 0.547

Association of SNP rs7635818 with AAA

SamplesNumber of subjects

Association P value

Geisinger Clinic

1,280 0.0083

Replication set 869 0.013

All samples 2,149 0.0014

4

213300k 213400k

NM_001017424KCNK2: potassium channel


AAA -Associated SNPs on Chromosome 1q41

213300 213400 2135000

1

2

3

Map position (kbp)

-Log

P v

alue

rs2841599 rs17024179 rs12039875 rs11120508 rs10494994 rs 1538547

0.37

0.41

0.44

0.32

0.38

0.57

0.54

0.60

0.96

0.67

0.48

0.66

0.48

0.26

0.42

LD correlation, R

0.330.67792 controls0.0085

0.290.71690 casesGeisinger Clinic

AC P value

Allele Frequency

NSamples

Association of SNP rs12039875 on Chromosome 1q41 with AAA

0.330.671,204 controls0.0072

0.290.711,142 casesAll

0.330.67412 controls0.029

0.300.70452 casesReplication

0.330.67792 controls0.0085Geisinger Clinic

A/CG/AA/GC/AT/CC/G P valuecontrolcase

haplotype frequency

SNPs

AAA is Associated With a Common Haplotype on Chromosome 1q41

0.00290.580.68A/CGACTC/G

0.000890.600.70A/CG/AACTC/G

0.000220.610.72A/CG/AA/GCTC/G

MMNPRAKRDFYLAAPDLLDPKSAAQNSKPRLSFSTKPTVLa:

MAAPDLLDPKSAAQNSKPRLSFSTKPTVLb:

MLPSASRERPGYRAGVAAPDLLDPKSAAQNSKPRLSFSTKPTVL c:

KCNK2 Expression in AAA Tissue

AAA AOD normal NTC

Tissue



bca

GAPDH

KCNK2

PCR amplification of reverse-transcribed total RNA

Are Effects of AAA -Associated Genetic Variants Additive?

SNP Chromosome Odds Ratio P value

rs7635818 3p12.3 1.3 0.0014

rs12039875 1q41 1.2 0.0072

rs10757278 9p21 1.3 0.0020

Does having multiple AAA -associated genetic variants increase the likelihood of AAA?

Are Effects of AAA -Associated Genetic Variants Additive?

2D

isea

se o

dds

)/(c

ontr

ols

n/c

ontr

ols

t) Non-zero slope, P<0.01R2 = 0.80

(Additive genetic model)

0 2 4 60

1

AAA Risk alleles

Dis

ease

odd

s(c

ases

n/c

ases

t)/(c

ontr

ols

5.0 ~0.6 cm/yr

AA

A D

iam

eter

(cm

)

Detailed Phenotyping – AAA Growth Rates

•AAA-growth rates from 3 patients based on serial imaging studies

•Data extracted from the patient’s electronic medical records (patients provided written research consent and authorization)

0 20 40 603.5

4.0

4.5

~0.2 cm/yr

Months

AA

A D

iam

eter

(cm

)

Acknowledgements

Alicia GoldenKathy MaskerGlenn GerhardDiane Smelser

James ElmoreDavid Franklin

Helena KuivaniemiDiane SmelserBill Bowen

Funding:American Heart Association, PA CURE Fund, Geisinger Clinical Research Fund, Ben Franklin Technology Development Fund, NIH

Helena KuivaniemiGerard Tromp

EHR

Research Data Broker and Clinical Decision Intelligence System (CDIS) Data Warehouse

Orders

Data

searchableexportable

DiagnosesLab values

MedsProcedures

Etc.

PHI FirewallTPO

Investigator

Central Biobank (MyCode)>30,000 consented participants

>72,000 samples

•Blood, serum, DNA, tissue

Governing Board

DataBroker

Research data

CDIS

FinanceClaims Ops

Quantitative RT -PCR Analysis of AAA Tissue

Geisinger Health System

Gray’s Woods

Geisinger Inpatient Facilities

Geisinger Medical Groups

Geisinger Health System Hub and Spoke Market Area

Geisinger Health Plan Service Area

Careworks Convenient Healthcare

Non-Geisinger Physicians With EHR

African -American

Native American

European

GC Pts

PC

2

European Ancestry of Geisinger Patient Population

Asian

African -American

Mexican

PC1

PC

2

Affymetrix 6.0 SNP genotype data

Pooled DNA GWAS Allele Frequency Data

Western Blot Analysis of AAA Tissue

0.0070.45MyCode controls

(N = 437)0.52

AAA(N = 650)

P valueG allele

frequencyControls

G allele frequency

Cases

Association of SNP rs10757278 on Chromosome 9p21 With AAA in the Geisinger Population


(N = 437)0.53All vascular

(N = 1,043)


(N = 437)0.54

Vascular disease(no AAA) (N = 353)

(N = 437)(N = 650)

3000

4000

Num

ber

10 Non-smokerSmoker

Females (N = 6,768) Males (N = 4,829)

Per

cent

with

AA

A

10 Non-smokerSmoker

Females (N = 6,768) Males (N = 4,829)

Per

cent

with

AA

A

Characteristics of Primary Care Research CohortGeisinger MyCode

18-29 30-39 40-49 50-59 60-69 70-79 80-89 >890

1000

2000

Age (years)

Num

ber

18-2

930

-39

40-4

950

-59

60-6

970

-79

80+

18-2

930

-39

40-4

950

-59

60-6

970

-79

80+

0

5

Age (years)

Per

cent

with

AA

A

18-2

930

-39

40-4

950

-59

60-6

970

-79

80+

18-2

930

-39

40-4

950

-59

60-6

970

-79

80+

0

5

Age (years)

Per

cent

with

AA

A

AAA diagnosis based on EMR query

pennsylvania geisinger medical center - chu-imaa

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