OUTLINE

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Genetics of Chronic Obstructive Pulmonary Disease
Edwin K. Silverman, M.D., Ph.D. Channing
Laboratory and Pulmonary and Critical Care
Division Brigham and Womens Hospital Harvard
Medical School Boston, Massachusetts USA
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Outline

• COPD Background
• Alpha 1-antitrypsin Deficiency
• Risk to Relatives for COPD-related Phenotypes
• Case-control Association Studies in COPD
• Genome Scan Linkage Analysis of Early-Onset COPD

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Chronic Obstructive Pulmonary Disease Definition

• Syndrome characterized by the presence of airflow
  obstruction due to chronic bronchitis or
  emphysema the airflow obstruction is generally
  progressive, may be accompanied by airway
  hyperreactivity, and may be partially reversible
  (ATS 1995).
• Includes
• Emphysema
• Chronic Bronchitis
• Small Airways Disease

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0 pack-yrs
0-20 pack-yrs
Percentage distribution of predicted FEV1 values
in subjects with varying pack-yrs of smoking.
Subjects with respiratory trouble before age 16
were excluded. Medians are shown for each group
on the x-axis.
21-40 pack-yrs
Population
41-60 pack-yrs
Burrows B, et al., Am Rev Respir Dis 1977
115195-205
61 pack-yrs
40
60
80
100
120
140
160
Predicted FEV1
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Alpha 1-Antitrypsin Background

• Alpha 1-antitrypsin, specified by the PI locus,
  is the major plasma protease inhibitor of
  leukocyte elastase.
• In Caucasian populations, the most common alleles
  are M (0.95), S (0.02 to 0.03), and Z (0.01 to
  0.02).
• Isoelectric focusing of serum can accurately
  determine PI type.

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Alpha 1-Antitrypsin Deficiency and COPD

• Individuals with two Z alleles, or one Z and one
  null allele, are referred to as PI Z.
• PI Z individuals have approximately 15 of normal
  plasma alpha 1-antitrypsin levels.
• PI Z individuals are at increased risk for
  severe, early-onset COPD.

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PULMONARY FUNCTION IN ALPHA 1-ANTITYRPSIN
DEFICIENCY EFFECTS OF CIGARETTE SMOKING
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Age, Smoking History, and Spirometry in Parents
of PI Z Subjects in St. Louis Alpha 1-antitrypsin
Study
n
FEV1 ( Pred.)
Age
Group
Pack-years
Parents of PI Zs with Preserved FEV1
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95.0 9.7
57.3 6.9
21.2 24.9
83
91.0 14.4
45.0 16.3
12.8 20.0
Parents of PI Zs with Reduced FEV1
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75.3 27.6
64.4 6.3
22.6 23.4
Note corresponds to p lt 0.05 by t test
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PI x Smoking Interaction in St. Louis AAT Study
PI M
PI MZ
PI Z
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FEV1 in Swedish PI ZZ Nonsmokers
(Piitulainen, Thorax 1997 53939)
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Risk to Relatives

• Definition The risk of a disease phenotype for
  relatives of affected individuals compared to the
  general population.

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Genetics of Pulmonary Function
Twins(Redline,S.,et al., 1987)

• Study of spirometry in 256 MZ and 158 DZ twin
  pairs not selected for respiratory problems
• For FEV1, correlation between MZ twins (0.72) was
  significantly higher than correlation between DZ
  twins (0.27)

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Genetics of Pulmonary Function COPD
Patients(Larson,R., et al., 1970)

• Study comparing spirometry in 156 first-degree
  relatives of COPD patients to 86 spouse controls
  with similar pack-years
• Airflow obstruction was found in 23 of
  first-degree relatives but only 9 of controls

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Association Studies

• Definition Methods which determine if a
  particular form of a DNA polymorphism occurs more
  frequently in subjects with a phenotype of
  interest case-control or family data may be used.

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Case-Control Association Study of COPD Heme
Oxygenase-1(Yamada et al., 2000)

• Distribution of HO-1 alleles at a dinucleotide
  repeat polymorphism in the 5 flanking region was
  compared in 101 Japanese emphysema patients and
  100 Japanese smoking controls. Alleles were
  grouped as S (lt 25 repeats), M (25-29 repeats),
  and L (gt 29 repeats).
• A higher frequency of class L alleles was noted
  in emphysema patients (21) than control subjects
  (10) with p lt 0.004. Transient-transfection
  assays demonstrated upregulation of 16 and 20
  repeat alleles, but not 29 or 38 repeat alleles,
  with exposure to hydrogen peroxide.
• Concerns
• How was classification scheme of alleles
  selected?
• Do functional differences correspond to this
  classification?

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Case-Control Genetic Association Studies in COPD
Do Not Support Association
Support Association
Candidate Gene
Category
Protease/ Antiprotease
PI MZ
Bruce (1984)
Lieberman (1986)
AlAT 3 Flanking Region
Sandford (1997)
Kalsheker (1990)
Alpha 1-antichymotrypsin
Sandford (1998)
Poller (1992)
Oxidant/ Antioxidant
Microsomal Epoxide Hydrolase
Yim (2000)
Smith (1997)
Heme Oxygenase-1
Yamada (2000)
Glutathione S1-Transferase P1
Ishii (1999)
CFTR
Artlich (1995)
Gervais (1993)
Other Candidates
ABO Blood Group
Vestbo (1993)
Cohen (1980)
ABH Secretor Status
Vestbo (1993)
Cohen (1980)
Vitamin D Binding Protein
Kauffmann (1983)
Schellenberg (1998)
Tumor Necrosis Factor a
Higham (2000)
Sakao (2001)
Note Selected references which support or do not
support an association to the specified locus are
presented
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Case-Control Association Studies in COPD
Potential Causes of Inconsistent Results

• Genetic heterogeneity Different genetic
  mechanisms in different populations
• Random error False positive/false negative
  results
• Population stratification Differences between
  cases and controls
• Study design/analysis problems
• Failure to correct for multiple comparisons
• Poor control group selection
• Small sample sizes

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Linkage Analysis

• Definition A group of methods which analyze the
  distribution of DNA markers within families to
  determine if a particular region of the genome
  contains a gene related to the phenotype of
  interest.
• Lod Score Result of a statistical test to
  determine if genetic loci are linked, expressed
  as log10 of the odds that the loci are linked.
  Lod score of 3-3.6 corresponds to highly
  significant linkage.

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Approaches to Circumventing the Complexity of
Early-Onset COPD

• Study severely affected young subjects
• Study intermediate phenotypes
• To approach the problem successfully requires
  multidisciplinary integration

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Plan to Identify Early-Onset COPD Susceptibility
Genes

• Step 1 Epidemiology Family Enrollment and
  Phenotype Measurement
• Step 2 Genetic Modeling Assessment for
  Familial Aggregation
• Step 3 Linkage Analysis Identification of a
  Linked DNA Marker
• Step 4 Susceptibility Gene Identification
  From Linkage to Gene

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Early-Onset COPD Study Population

• Proband Selection Subjects with severe COPD
  (FEV1 lt 40) at an early age (lt 52 years),
  without alpha 1-antitrypsin deficiency, were
  recruited.
• Recruitment of Relatives All available
  first-degree relatives and spouses were invited
  to participate. All available aunts/uncles and
  grandparents were also included.
• Linkage Data Set 72 early-onset COPD probands,
  320 first-degree relatives (48 parents, 147
  siblings, 125 children), 162 other relatives, and
  31 spouses.
• Control pedigrees 20 probands, 63 first-degree
  relatives and spouses

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Demographics and Spirometry in First-Degree
Relatives of Early-Onset COPD Probands Compared
to Control Subjects
FEV1 ( Pred.)
Age
Pack-years
n
Group
FEV1/FVC ( Pred.)
77.0 21.9
45.8 17.1
28.1 25.0
Smoking First Degree Relatives
207
84.0 15.5
89.2 14.4
48.6 13.9
22.1 22.1
Smoking Control Subjects
48
94.3 10.3
92.8 14.2
36.5 18.1
0.00
Nonsmoking First Degree Relatives
129
92.7 8.4
93.4 14.2
39.9 18.2
0.00
Nonsmoking Control Subjects
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95.5 7.2
Indicates p lt 0.05 compared to control
subjects Indicates p lt 0.01 compared to control
subjects
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Relative Odds of Reduced FEV1 and Chronic
Bronchitis (Adjusted for Age and Pack-years)
Among First-Degree Relatives of Early COPD
Probands
Relative Odds of Reduced FEV1 or Chronic
Bronchitis
FEV1lt80
FEV1lt60
GROUP
FEV1lt100
Chronic Bronchitis
All First-Degree Relatives
3.42
2.45
2.04
1.14
SMOKING First-Degree Relatives
4.50
3.53
1.77
3.63
NONSMOKING First-Degree Relatives
1.85
0.45
0.82
0.27
Note indicates odds ratio is significant at
p lt 0.05
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Phenotypes with Smoking-Related Effects in
First-Degree Relatives of Boston Early-Onset COPD
Probands

• FEV1
• FEV1/FVC
• Chronic Bronchitis
• Bronchodilator Responsiveness

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Genome Scan in Early-Onset COPD Families
Genotyping and Data Cleaning

• DNA samples from 607 early-onset COPD pedigree
  members
• 378 Short Tandem Repeat Markers genotyped by
  NHLBI Mammalian Genotyping Service (Mean
  spacing9.1 cM)
• Mendelian inconsistencies assessed with
  Relcheck/PedCheck
• Relcheck Assesses Pedigree Inconsistencies
  (removed 22 subjects)
• Pedcheck Assesses Individual Marker
  Inconsistencies

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Genome Scan in Early-Onset COPD Families
Phenotypes

• Qualitative Phenotypes
• Moderate Airflow Obstruction (FEV1 lt 60,
  FEV1/FVC lt 90)
• Mild Airflow Obstruction (FEV1 lt 80, FEV1/FVC lt
  90)
• Chronic Bronchitis (Assessed by Questionnaire)
• Quantitative Phenotypes
• FEV1
• FVC
• FEV1/FVC
• Future Analyses of Bronchodilator Responsiveness,
  IgE, etc.

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Genome Scan in Early-Onset COPD Families
Qualitative Phenotype Linkage Analysis

• Multipoint Nonparametric Analysis (ALLEGRO)
• 14 unaffected individuals removed
• ScoreAll option
• Affecteds only
• Smokers Only Analyses Missing phenotype data if
  lt 10 pack-years of smoking

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Genome Scan Multipoint Linkage Analysis with
ALLEGRO Chromosomal Regions with Lod Scores
Above 1.0
Moderate Airflow Obstruction
Mild Airflow Obstruction
Chronic Bronchitis
All Subjects Max Lod
Chr
Smokers Max Lod
cM
cM
All Subjects Max Lod
Smokers Max Lod
cM
cM
All Subjects Max Lod
Smokers Max Lod
cM
cM
3
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147
170
153
209
91
0.68
0.76
0.88
1.58
0.62
0.41
4
70
68
86
88
178
208
1.24
0.83
0.33
0.41
0.51
0.65
6
191
193
193
193
193
80
0.20
0.29
0.85
0.88
0.68
1.51
8
79
78
76
78
154
82
0.99
0.89
1.36
1.21
0.00
0.01
12
36
36
39
32
44
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1.70
1.28
0.63
1.54
0.43
1.14
15
38
45
20
72
5
5
1.07
1.18
0.56
0.36
0.06
0.31
18
13
13
13
64
126
80
1.01
0.50
0.59
0.40
0.08
0.36
19
42
40
42
42
42
42
1.54
1.65
1.09
1.64
1.21
1.73
22
46
46
14
14
36
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0.63
0.52
0.73
0.64
1.37
2.08
Note The highest allele-sharing lod score on
each chromosome for each phenotype is presented.
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Additional STR Markers on 12p

• Twelve additional STR markers between 18-49 cM on
  12p
• Linkage Analysis
• Multipoint Nonparametric Analysis (ALLEGRO)
• Multipoint Parametric Analysis with Heterogeneity
  (ALLEGRO)
• Dominant Model (58 of probands reported parental
  COPD)
• Penetrance 0.5
• Unaffected for airflow obstruction Agegt30,
  Packsgt10, FEV1gt80

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Candidate Genes in Regions of Suggestive Linkage
to COPD-related Phenotypes

• Chromosome 12p MGST1, MGP
• Chromosome 19p Leukotriene B4 omega-hydrolase
  (CYP4F2)
• Chromosome 22q Heme oxygenase 1, TIMP3
• Chromosome 2q IL8 Receptor

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COLLABORATORS

• New England Early-onset COPD Study Scott Weiss,
  Frank Speizer, Lyle Palmer, Jeffrey Drazen,
  Harold Chapman, Scott Weiss, John Reilly, Leo
  Ginns, Ed Campbell, D.C. Rao, Michael Province,
  Juan Celedon, Matthew Barth, Jody Senter,
  Jonathan Mosley