1. No category

“Cardiovascular disease genomics:
potential relevance to clinical medicine”
Dr. Bea Fowlow Lecture
University of Calgary
2007 May 17 1510-1610 h
Robert A. Hegele MD FRCPC FACP
Robarts Research Institute
London, Ontario, Canada
Financial disclosure: none
Atherosclerosis time course
Condition A
0
10
20
30
40
50
60
70
80
Atherosclerosis time course
Condition A
Condition B
0
10
20
30
40
50
60
70
80
Genetic determinants of atherosclerosis
Effect size
Mendelian
(monogenic)
common
complex
(polygenic)
Frequency
Genetic determinants of atherosclerosis
Effect size
Mendelian
(monogenic)
environment
common
complex
(polygenic)
Frequency
Single nucleotide polymorphisms (SNPs)
TAT CTG TGC CTC CCT GCC CCG CAG ATC AAC CCC CAC TCG
Y
L
C
L
P
A
P
Q
I
N
P
H
S
658 659
660
661
662
663
664 665 666 667 668 669 670
Single nucleotide polymorphisms (SNPs)
TAT CTG TGC CTC CCT GCC CCG CAG ATC AAC CCC CAC TCG
Y
L
C
L
P
A
P
Q
I
N
P
H
S
658 659
660
661
662
663
664 665 666 667 668 669 670
TAT CTG TGC CTC CCT GCC CTG CAG ATC AAC CCC CAC TCG
L
Q
I
N
P
H
S
Y
L
C
L
P
A
658 659
660
661
662
663
664 665 666 667 668 669 670
Single nucleotide polymorphisms (SNPs)
- swap of one nucleotide (“misprint”)
- common (>5%): 7.2 X 106 in human genome
Single nucleotide polymorphisms (SNPs)
- swap of one nucleotide (“misprint”)
- common (>5%): 7.2 X 106 in human genome
coding
silent
missense
non-coding
nonsense
silent
promoter
splicing
Single nucleotide polymorphisms (SNPs)
- swap of one nucleotide (“misprint”)
- common (>5%): 7.2 X 106 in human genome
coding
silent
missense
non-coding
nonsense
silent
promoter
splicing
Single nucleotide polymorphisms (SNPs)
- swap of one nucleotide (“misprint”)
- common (>5%): 7.2 X 106 in human genome
coding
silent
missense
non-coding
nonsense
silent
promoter
splicing
- SNP haplotypes
- groups of SNPs occurring together on a chromosome
APOA5
rare Mx
no Mx
APOA5
rare Mx
no Mx
APOA5
rare Mx
no Mx
APOA5
rare Mx
no Mx
Monogenic forms of common diseases
“caricatures”
-
may capture key elements of common phenotype
recapitulate key clinical and biochemical features
some features exaggerated
other features absent
temporal progression variable
may single out important pathways
Case report: HoFH
- 45 year-old Chinese woman
-
(G. Francis MD)
age 15: corneal arcus, AT thickening
age 30: angina, CABG
age 40: bilat carotid stenoses
chemosis X 3 years - extensive work-up
no focal neurological signs
HoFH response to Rx
20
TC
LDL-C
mmol/L
15
10
5
atorvastatin
100
colestipol
ezetimibe
80 mg
4g
10 mg
0
preRx
2002
2003
2004
LDLR exon 14: FH-Gujerat (P644L)
NORMA L
TAT CTG TGC CTC CCT GCC CCG CAG ATC AAC CCC CAC TCG
Y
L
C
L
P
A
P
Q
I
N
P
H
S
658 659
660
661
662
663
664 665 666 667 668 669 670
PATIENT
TAT CTG TGC CTC CCT GCC CTG CAG ATC AAC CCC CAC TCG
L
Q
I
N
P
H
S
Y
L
C
L
P
A
658 659
660
661
662
663
664 665 666 667 668 669 670
MRI:
- large calcified mass
- ? meningioma
MRI:
- large calcified mass
- ? meningioma
craniotomy:
- avascular mass with
necrotic yellowish core
microscopy:
-
cholesterol clefts
lipid-laden mφ
chronic inflammation
fibrosis
cholesterol shards
calcification
Dx:
- cholesterol granuloma
Heterozygous familial
hypercholesterolemia (HeFH)
-
autosomal dominant
1:500 (higher in Quebec)
corneal, skin and tendon deposits
early atherosclerosis
often undiagnosed
at least 3 genetic forms:
HCHOLAD1 (FH)
HCHOLAD2
HCHOLAD3
143890
144010
603776
LDLR (chr 19p)
APOB (chr 2p)
PCSK9 (chr 1p34)
Case report: HeFH
-
34 year-old truck driver, 2o CHD prevention
father & 2 uncles: fatal MIs <50 years
age 29: TC and LDL-C = 10 and 8 mmol/L
age 33:
- AMI
- diffuse CAD
- three stents placed
- Rx: atorvastatin 80 mg, ezetimibe 10 mg,
ramipril 5 mg, clopidogrel 75 mg,
bisoprolol 5 mg and ASA 81 mg
Case report: HeFH
Time
6 mo
Lipid Rx
TC
atorva 80 mg 5.36
eze 10 mg
LDL-C
3.76
9 mo
atorva 80 mg 4.23
eze 10 mg
2.90
12 mo
rosuva 40 mg 4.05
eze 10 mg
2.50
A
B
C
D
LDLR genomic DNA sequence:
exon 14:intron 14 splicing mutation
Ontario LDLR mutations
C152X
E119K
R103P-108X
E80K
C42R
∆36D
A29S
W23X
C6W
G314S
G197V-M243X
T413M* Y421C*
A370T* F381-391X
D203N
C163Y
L458P*
V408M*
∆198G
Q363P
E207K
R395W
K497L-528X*
L561P*
C660X
E760D*
N804K
1
2
4
3
5 6
9 10
11 12
1314
18
2g
*
16 17
t+
a
15
>a
*
15
5a
g+
c+
g
–10
8g
t–
2g
>c
–2a
1a
g+
t+
*
7 8
*
LDL-receptor
Ligand Binding
Domain
EGF Precursor Homology
Domain
O-linked Sugar
Domain
Cytoplasmic
Tail
Etiology of HeFH in Ontario
APOB
N=5
LDLR
N=44
other
N=21
OTHER …
- misdiagnosis
- other genes
- mutation types that are
missed by sequencing
Copy number variants (CNVs)
normolipidemic
Copy number variants (CNVs)
normolipidemic
unknown FH
Copy number variants (CNVs)
normolipidemic
unknown FH
Copy number variants (CNVs)
normolipidemic
unknown FH
Copy number variants (CNVs)
normolipidemic
unknown FH
Copy number variants (CNVs)
normolipidemic
unknown FH
Copy number variants (CNVs)
normolipidemic
unknown FH
Copy number variants (CNVs)
normlipidemic
unknown FH
Ontario LDLR mutations
C152X
E119K
R103P-108X
E80K
C42R
∆36D
A29S
W23X
C6W
G314S
G197V-M243X
F381-391X
T413M Y421C
A370T(3)
D203N
C163Y
V408M
L458P
∆198G
Q363P
R395W
E207K
K497L-528X
L561P(2)
C660X(3)
E760D
N804K
1
2
4
5 6
7 8
9 10
1314
1314
15
16 17
t>g
+2
3)
a(
g>
a
+5
g>
+1
∆exon 1-4
∆exon 1-6
11 12
a
5c>
+1
a
g>
–10 (3)
>g
–8t
>c
–2a (2)
a
g>
+1
t>g
+2
∆15 kb(5)
3
18
Ontario LDLR mutations
C152X
E119K
R103P-108X
E80K
C42R
∆36D
A29S
W23X
C6W
G314S
G197V-M243X
F381-391X
T413M Y421C
A370T(3)
D203N
C163Y
V408M
L458P
∆198G
Q363P
R395W
E207K
K497L-528X
L561P(2)
C660X(3)
E760D
N804K
1
2
4
5 6
7 8
9 10
∆exon 2-6
∆exon 6
16 17
18
t>g
+2
∆exon 3
15
3)
∆exon 2
1314
1314
a(
g>
a
+5
g>
+1
∆exon 1-4
∆exon 1-6
11 12
a
5c>
+1
a
g>
–10 (3)
>g
–8t
>c
–2a (2)
a
g>
+1
t>g
+2
∆15 kb(5)
3
27% more
∆exon 4-14
Etiology of HeFH in Ontario
APOB
N=5
e.g. CNPs
LDLR
N=44
OTHER …
- misdiagnosis
- other genes
- mutation types that are
missed by sequencing
other
N=21
Etiology of HeFH in Ontario
APOB
N=5
LDLR
N=56
other
N=9
OTHER GENES?
Between-mutation differences in LDL-C
*
*
*
LDL-C (mmol/L)
10
8
6
4
2
0
* P<0.05
Missense Splicing Nonsense CNVs
ABN
MLPA
SNPs
APOB
R3500Q
No
ABN
CNV types
C
deletion
5’
5’ regulatory region
B
duplication
A
•
promoter
•
enhancer elements
•
silencers
1
coding sequence
2
•
exons
•
introns (regulation)
3
3’ untranslated region
inversion
•
3’
mRNA stabilization
CNVs are common
and ubiquitous:
1447 across 360 MB
(12% of genome)
CASE REPORT
5 yr 3 mo old female
recurrent abdominal pain
acute respiratory distress, asystole
autopsy:
sacral xanthoma
aortic xanthomas
severe atheromata of R and L coronaries
post mortem total cholesterol: 12.2 mmol/L
2 affected sibs and 1st cousin
Circulation 2003; 107:791
Circulation 2003; 107:791
Mutation in sitosterolemia
ABCG5
ABCG8
S107X
Circulation 2003; 107:791.
The intestine and sterol metabolism
NPC1L1
Metabolic syndrome
-
multiplex CVD risk factor
deserves more clinical attention
common: 25-30% of North Americans
associated with:
aging
sedentary lifestyle
genetic predisposition
- fueled by:
obesity
- leads to:
type 2 diabetes
- core defect:
insulin resistance
Metabolic syndrome definitions
WHO (1998)
glycemia
IGT or T2DM or
top 25%ile clamp
NCEP (2001)
any 3 of:
FPG>6.1 mmol/L
plus any 2 of:
obesity
WHR >0.9/BMI >30
waist >102 cm in men
>88 cm in women
TG
>1.7 mmol/L
>1.7 mmol/L
HDL
<0.9 mmol/L
<1.0 mmol/L in men
<1.3 mmol/L in women
BP
other
>160/90
microalbuminuria
>130/85
Dunnigan-type FPLD
-
familial partial lipodystrophy
characteristic repartitioning of fat stores
insulin resistance
characteristic biochemical profile
hypertension
type 2 diabetes
early atherosclerosis
also: acanthosis nigricans, PCOS
at least 3 genetic subtypes: FPLD1, 2, 3
Case report: FPLD2
- 21 year old South Asian female
- age 6:
- age 10:
- age 12:
acanthosis nigricans
insulin resistance; diabetes
hypertension
TG >6600 mg/dL
pancreatitis
- OE: severe partial lipodystrophy; facial fat spared
- mother: diabetes, hypertension, high TG
MI at 23, pacemaker at 26, stroke at 32
- sister:
25 and similarly affected
LMNA splicing mutant: DNA analysis
A
GTG ACG g t g a g
5’
V
tgg
3’
T
normal gDNA
genomic DNA
proband gDNA
IVS8 +5G>C
2 cDNA species
B
M N P1 P2
1121 bp
1037 bp
C
exon 8 exon 9
5’
GTG ACG ATC TGG
3’
normal cDNA
intron 8
5’GTGACGgtgactggcagggcgcttgggac tctgggg aggccttggg tggcgatggg agcgct ggggTAAgtgtcc ttt tct cct ctccagATC TGG3’
retained
intron 8
IVS8 +5G>C
Stop
LMNA splicing mutant: western analysis
Lamin A/C
64KD
Mutant lamin A
50KD
N
Mx
N
Immunocytochemistry
A
97% of WT transfected
B
92% of mutant transfected
FPLD2 is a laminopathy
R336Q
R28W
Q6X
R62G
R60G
DK208
H222Y R249Q R321FS R343Q R377H R571S
L85R N195K E203G R298C
E145K
G465D R482L R527P R582H
R453W
I469T R482Q T528K R584H
R399C
R471C R482W
EDMD DCM
FPLD
LGMD CMTD
L530P G608G
K486N R527H G608S
R527C
OLS
MAD
HGPS
E536FS
J Clin Invest 2004; 113:349-351
Fat distribution in lipodystrophies
CONTROL
24 yrs
BMI 23.5
CONTROL
50 yrs
BMI 34.8
FPLD3
PPARG F388L
49 yrs
BMI 33.4
APL
FPLD2
HIV
LMNA R482Q LMNB2 R215Q 35 yrs
64 yrs
63 yrs
BMI 30.8
BMI 24.8
BMI 24.8
CGL
GNG3L1 fs
37 yrs
BMI 22.8
Hutchinson-Gilford progeria (MIM 176670)
-
accelerated aging
slowed growth
facial disproportion
micrognathia
alopecia
osteoporosis
thin parchment-like skin
depleted subQ fat
severe atherosclerosis
death before age 15
LMNA mutation effect on nuclei
5’ C GC GC C A C C C GC A GC
R
A
T
R
S
T10I
- 92% abnormal nuclei
- detachment from chromatin
- normal LMNA gene
- 10% abnormal nuclei
3’
Laminopathies
- >16 distinct rare human diseases
- >100 different mutations in LMNA
- cardiac involvement includes:
- dilated cardiomyopathy
- conduction disturbances
- atherosclerosis
Laminopathies listed according to mode of inheritance
MIM number
illustrative mutations*
Emery-Dreifuss muscular dystrophy (AD-EMD2)
181350
Q6X; R453W; R527P;
Dilated cardiomyopathy with CCA (CMD1A)
115200
R60G; L85R; N195K;
Early onset atrial fibrillation (EOAF)
607554
G161K
Familial partial lipodystrophy (FPLD2)
151660
R482Q; R482W; R482L
Hutchinson-Gilford progeria syndrome (HGPS)
176670
G608G; G608S
Autosomal dominant (AD) disorders
Atypical progeria syndromes
Atypical HGPS
T10I; G145K; R644C; E578V
Atypical Werner Syndrome
A57P; R133L; R133P; L140R;
Overlapping syndromes
LIRLLC
608056
R133L
CMD1A + QM
R377H
AD-EMD2 + LD + CCA
R527P
Autosomal recessive (AR) disorders
Emery-Dreifuss muscular dystrophy (AR-EMD2)
604929
H222Y
Charcot Marie Tooth disease (AR-CMT2B1)
605588
R298C
Limb-girdle muscular dystrophy (LGMD1B)
159001
R377H; delK208; IVS9 +5G>C
Mandibuloacral dysplasia (MAD)
248370
R527H; CoHtz R471C/R527C
Laminopathies listed according to mode of inheritance
MIM number
illustrative mutations*
Emery-Dreifuss muscular dystrophy (AD-EMD2)
181350
Q6X; R453W; R527P;
Dilated cardiomyopathy with CCA (CMD1A)
115200
R60G; L85R; N195K;
Early onset atrial fibrillation (EOAF)
607554
G161K
Familial partial lipodystrophy (FPLD2)
151660
R482Q; R482W; R482L
Hutchinson-Gilford progeria syndrome (HGPS)
176670
G608G; G608S
Autosomal dominant (AD) disorders
Atypical progeria syndromes
Atypical HGPS
T10I; G145K; R644C; E578V
Atypical Werner Syndrome
A57P; R133L; R133P; L140R;
Overlapping syndromes
LIRLLC
608056
R133L
CMD1A + QM
R377H
AD-EMD2 + LD + CCA
R527P
Autosomal recessive (AR) disorders
Emery-Dreifuss muscular dystrophy (AR-EMD2)
604929
H222Y
Charcot Marie Tooth disease (AR-CMT2B1)
605588
R298C
Limb-girdle muscular dystrophy (LGMD1B)
159001
R377H; delK208; IVS9 +5G>C
Mandibuloacral dysplasia (MAD)
248370
R527H; CoHtz R471C/R527C
Emery-Dreifuss muscular dystrophy (AD-EMD2)
(EMD2)
Dilated cardiomyopathy with CCA (CMD1A)
Early onset atrial fibrillation (EOAF)
Familial partial lipodystrophy - Dunnigan type (FPLD2)
Hutchinson-Gilford progeria syndrome (HGPS)
Atypical HGPS (AHGPS)
Atypical Werner Syndrome (AWRN)
OLS1 (LIRLLC)
OLS2 (LD + MW + DCM + CCA) *R28W
OLS3 (LD + DCM + CCA)
OLS4 (CMD1A + QM)
OLS5 (LD + MD + CCA)
Emery-Dreifuss muscular dystrophy (AR-EMD2) 604929
Charcot Marie Tooth disease (AR-CMT) 605588
Limb-girdle muscular dystrophy (LGMD1B) 159001
Mandibuloacral dysplasia (MAD) 248370
insulin resistance
hypogonadism
premature atherosclerosis
cardiac conduction defect
cardiomyopathy
peripheral neuropathy
short stature
bone disease
muscle weakness
muscle contractures
fat loss
voice changes
hair changes
skin changes
Class I laminopathy mutation 8.4 times more likely to occur 5’ of NLS
54
16
6
15
Genomic biomarkers in atherosclerosis
1. SNPs are workhorses, but new types are
on the horizon: e.g. genome-wide CNVs
2. Mutations in mendelian traits:
- specify pathways
- may aid diagnosis
- correlate with phenotypes
3. Common SNPs singly or in groups may:
- predict risk
- predict response to medications
- not yet “ready for prime time” use
It is not in the stars to hold
our destiny but in ourselves.
William Shakespeare
Hegele Lab Collaborators since 1999
Collaborator / Co-Author
Al-Shali K.
Anand S.S.
Anderson C.M.
Appell J.
Argmann C.A.
Balfe J.W.
Ban M.R.
Barrett P.H.
Behme M.T.
Bjerregaard P.
Boffa M.B.
Bowman K.A.
Brousseau M.E.
Brunt J.H.
Bull S.B.
Burnett J.R.
Cao H.
Carpentier A.
Carrington C.V.
Cattini P.A.
Chau L.A.
Chisholm R.J.
Clarson C.L.
Cohn J.S.
Cole E.H.
Connelly P.W.
Couture P.
Cummings E.
Cybulsky M.I.
De Angelis M.
Deshaies Y.
Devanesen S.
DeVries M.E.
Dewailly E.
Diffenderfer M.R.
Doering M.
Dolnikowski G.G.
Dolphin P.J.
Ebbesson S.E.
Edwards J.Y.
Eliasziw M.
Feldman R.D.
Fellows F.
Fisman M.
Institution
RRI
McMaster
Nippissing College
London Health Sciences
INSERM, Strassbourg
Sick Kids Hospital
RRI
U Western Australia
London Health Sciences
U Copenhagen
Queen's University
St. Michael's Hospital
Tufts University
U of Victoria
U of Toronto
U Western Australia
RRI
Laval
U of Trinidad & Tobago
U Manitoba
RRI
St. Michael's Hospital
London Health Sciences
McGill
UHN
U of Toronto
Laval
Dalhousie
U of Toronto
U di Milano, Italy
Laval
St. Michael's Hospital
U of Toronto
Laval
Tufts University
London Health Sciences
Tufts University
Dalhousie
U Copenhagen
UWO
U Calgary
RRI
UWO
London Health Sciences
Eliasziw M.
Feldman R.D.
Fellows F.
Fisman M.
Frankowski C.
Frohlich J.
Gagne C.
Gerstein H.
Giacca A.
Hahn A.
Hanley A.J.
Harris S.B.
Hramiak I.M.
Huff M.W.
Jack E.
Jones D.C.
Jones P.J.
Joubert G.I.
Kelemen L.
Kelly S.L.
Kelvin D.J.
Kennedy B.P.
Kirkpatrick R.D.
Klein G J
U Calgary
RRI
UWO
London Health Sciences Centre
Pfizer, Ann Arbor
UBC
Laval
McMaster
U of Toronto
London Health Sciences Centre
U of Toronto
UWO
London Health Sciences Centre
UWO
U of Toronto
RRI
McGill
London Health Sciences Centre
McMaster
U Manitoba
U of Toronto
Merck Research Labs, Dorval
U Manitoba
London Health Sciences Centre
Centre
Centre
Centre
Centre
Centre
Kirkpatrick R.D.
Klein G .J.
Koop B.F.
Koschinsky M .L.
Krahn A.D
Kwan J.
Lam arche B.
Lau H.K.
Leff T.
Lewis G .F.
Liede A.
Logan A.G .
Lonn E.
M adrenas J.
M ahon J.L.
M athews S.T.
M cKeown-Eyssen G .
M cKinney, J.
M cKnight C.J.
M cM anus R.
M etzger D.L.
M idgley J.
M iner S.E.
Montague P.A.
Morand O.H.
Mulvad G.
Mymin, D.
Narod S.A.
Nesheim M.E.
Norquay L.D.
O'Connor Jr. J.
Parkes R.K.
Pickering J.G.
Pinnaduwage D.
Prasad G.V.
Rabheru K.
Ramdath D.D.
Ran L.
Risica P.M.
Robinson J.
Rocnik E.F.
Rosen F.
Ruel I.L.
Sawyez C.G.
Schaefer E.J.
Sharma V.
Simard J.
Siu V.M.
Skanes A.C.
Spence J.D.
Spence J.D.
Stewart A.K.
Stewart L.
Strauss B.H.
Teitel M.
Teo K.K.
Tran K.
Triggs-Raine, B.L.
Tsui L.C.
Van Den Diepstraten C.H.
Vuksan V.
Wang J.
Welty F.K.
Wen X.Y.
Wolever T.M.
Wolfe B.M.
Xu L.
U M anitoba
London Health Sciences Centre
U Victoria
Q ueen's University
London Health Sciences Centre
U of Toronto
Laval
St. M ichael's Hospital
W ayne State, Detroit
U of Toronto
U of Toronto
UHN
M cM aster
RRI
London Health Sciences Centre
Pfizer, Ann Arbor
U of Toronto
RRI
U O ttawa
London Health Sciences Centre
UBC
U Calgary
St. M ichael's Hospital
McMaster
Roche, Basel
U Copenhagen
U Manitoba
U of Toronto
Queen's University
U Manitoba
Tufts University
St. Michael's Hospital
RRI
St. Michael's Hospital
St. Michael's Hospital
London Health Sciences Centre
U of Trinidad & Tobago
U of Toronto
U Copenhagen
RRI
Boston U
Scarborough General Hospital
Laval
UWO
Tufts University
London Health Sciences Centre
Laval
London Health Sciences Centre
London Health Sciences Centre
London Health Sciences Centre
London Health Sciences Centre
U of Toronto
Dalhousie
St. Michael's Hospital
St. Michael's Hospital
McMaster
U Ottawa
U Manitoba
Chinese University of Hong Kong
UWO
St. Michael's Hospital
RRI
Tufts University
U of Toronto
U of Toronto
London Health Sciences Centre
U of Toronto
Yamagata K.
Yao Z.
Yee R.
Yi C.
Young T.K.
Yusuf S.
Zinman B.
Tokyo University
U Ottawa
London Health Sciences Centre
McMaster
U of Toronto
McMaster
U of Toronto
Case report: FPLD3
-
45 year old Caucasian woman
age 13: muscular legs, lower arms
age 33: T2DM in 3rd pregnancy
age 40: insulin; TG >9000 mg/dL; pancreatitis
+ responded to pioglitazone
- OE:
BP 145/90, BMI 30 kg/m2, waist 90 cm
elbow xanthomata
extremities: subQ fat; musculature
- daughter age 12; TG 150 mg/dL; HDL 35 mg/dL
PPARG Y355X DNA sequence
PPARG Y355X activity
PPARG mutations in FPLD3