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Title: The Ischemic Stroke Genetics Study (ISGS) Protocol
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Title: The Ischemic Stroke Genetics Study (ISGS) Protocol
Physical Description: Book
Language: English
Creator: Meschia, James
Brott, Thomas
Brown, Robert
Crook, Richard
Frankel, Michael
Hardy,J ohn
Merino,Jose
Rich, Stephen
Silliman, Scott
Worrall, Bradford Burke
Publisher: BMC Neurology
Publication Date: 2003
 Notes
Abstract: BACKGROUND:The molecular basis for the genetic risk of ischemic stroke is likely to be multigenic and influenced by environmental factors. Several small case-control studies have suggested associations between ischemic stroke and polymorphisms of genes that code for coagulation cascade proteins and platelet receptors. Our aim is to investigate potential associations between hemostatic gene polymorphisms and ischemic stroke, with particular emphasis on detailed characterization of the phenotype.METHODS/DESIGN:The Ischemic Stroke Genetic Study is a prospective, multicenter genetic association study in adults with recent first-ever ischemic stroke confirmed with computed tomography or magnetic resonance imaging. Patients are evaluated at academic medical centers in the United States and compared with sex- and age-matched controls. Stroke subtypes are determined by central blinded adjudication using standardized, validated mechanistic and syndromic classification systems. The panel of genes to be tested for polymorphisms includes ß-fibrinogen and platelet glycoprotein Ia, Iba, and IIb/IIIa. Immortalized cell lines are created to allow for time- and cost-efficient testing of additional candidate genes in the future.DISCUSSION:The study is designed to minimize survival bias and to allow for exploring associations between specific polymorphisms and individual subtypes of ischemic stroke. The data set will also permit the study of genetic determinants of stroke outcome. Having cell lines will permit testing of future candidate risk factor genes.
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Study protocol

The Ischemic Stroke Genetics Study (ISGS) Protocol
James F Meschia* 1, Thomas G Brott', Robert D Brown Jr1, Richard JP Crook2,
Michael Frankel3, John Hardy4, Jose G Merino5, Stephen S Rich6,
Scott Silliman5 and Bradford Burke Worrall7


Address: 'Department of Neurology, Mayo Clinic, Jacksonville, Florida, U.S.A, 2Department of Neuroscience, Mayo Clinic, Jacksonville, Florida,
U.S.A, 3Emory University School of Medicine, Atlanta, Georgia, U.S.A, 4National Institute on Aging, Bethesda, Maryland, U.S.A, 5University of
Florida/Shands Hospital, Jacksonville, Florida, U.S.A, 6Department of Public Health Sciences and Neurology, Wake Forest University School of
Medicine, Winston-Salem, North Carolina, U.S.A and 7University of Virginia Health System, Charlottesville, Virginia, U.S.A
Email: James F Meschia* Meschia.James@mayo.edu; Thomas G Brott Brott.Thomas@mayo.edu; Robert D Brown Brown@mayo.edu;
Richard JP Crook Crook.Richard@mayo.edu; Michael Frankel mfranke@emory.edu; John Hardy- hardyj@mail.nih.gov;
Jose G Merino jose.merino@jax.ufl.edu; Stephen S Rich srich@wfubmc.edu; Scott Silliman scott.silliman@jax.ufl.edu; Bradford
Burke Worrall bbw9r@virginia.edu
* Corresponding author


Published: 08 July 2003
BMC Neurology 2003, 3:4


Received: 21 February 2003
Accepted: 08 July 2003


This article is available from: http://www.biomedcentral.com/1471-2377/3/4
2003 Meschia et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.



Abstract
Background: The molecular basis for the genetic risk of ischemic stroke is likely to be multigenic
and influenced by environmental factors. Several small case-control studies have suggested
associations between ischemic stroke and polymorphisms of genes that code for coagulation
cascade proteins and platelet receptors. Our aim is to investigate potential associations between
hemostatic gene polymorphisms and ischemic stroke, with particular emphasis on detailed
characterization of the phenotype.
Methods/Design: The Ischemic Stroke Genetic Study is a prospective, multicenter genetic
association study in adults with recent first-ever ischemic stroke confirmed with computed
tomography or magnetic resonance imaging. Patients are evaluated at academic medical centers in
the United States and compared with sex- and age-matched controls. Stroke subtypes are
determined by central blinded adjudication using standardized, validated mechanistic and syndromic
classification systems. The panel of genes to be tested for polymorphisms includes P-fibrinogen and
platelet glycoprotein la, Iba, and llb/Illa. Immortalized cell lines are created to allow for time- and
cost-efficient testing of additional candidate genes in the future.
Discussion: The study is designed to minimize survival bias and to allow for exploring associations
between specific polymorphisms and individual subtypes of ischemic stroke. The data set will also
permit the study of genetic determinants of stroke outcome. Having cell lines will permit testing of
future candidate risk factor genes.


Background
Cross-sectional, longitudinal, and twin studies strongly
support an inherited component to stroke risk, but except
for rare mendelian and mitochondrial stroke syndromes,


the molecular basis for inherited ischemic stroke risk
remains ill defined. The ability to identify high-risk
patients through genetic testing could make screening for
treatable intermediate phenotypes more cost-effective. For


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example, identification of patients with a high genetic risk
of cervical carotid atherosclerosis might enable the effi-
cient use of endarterectomy for primary stroke prevention
[ 1 ]. In addition, a clear and comprehensive understanding
of genetic risk may promote advances in gene therapy and
in the development of novel pharmaceutical agents.

Herein we describe the protocol of an ongoing prospec-
tive, multicenter study, the Ischemic Stroke Genetics
Study (ISGS). This study uses a candidate gene approach
in which rates of variant polymorphisms of the candidate
genes are compared between patients with ischemic
stroke and stroke-free control subjects.

Selection of Candidate Genes for ISGS
The candidate genes for this study include the gene encod-
ing P-fibrinogen and the genes encoding platelet glyco-
protein (GP) receptors Ia/IIa, Ib/IX/V, and IIb/IIIa. In
selecting candidate genes for an association study, effects
of polymorphisms on structure, function, or expression of
a gene product should be considered. Failure to consider
the underlying pathophysiologic mechanism when
searching for polymorphisms associated with stroke
might result in mistaking association for causation [2].
We studied genes related to thrombosis because the
importance of thrombosis in acute ischemic stroke has
been established conclusively in numerous clinical trials
of treatment and prevention [3-8]. We focused on genetic
variations in the fibrinogen gene cluster because of the
efficacy of fibrinolytic agents in the acute treatment of
ischemic stroke. In addition, we studied genes encoding
for platelet receptors because of the efficacy of platelet
anti-aggregant therapy in preventing first-time and recur-
rent ischemic strokes.

To restrict the choice of polymorphisms worthy of further
study, we constructed an evidence table from reports
appearing in MEDLINE-indexed English language jour-
nals describing cross-sectional or longitudinal studies of
at least one thrombosis gene polymorphism in at least
100 patients with stroke. Results were classified as positive
or negative according to whether a significant association
(P < 0.05) was found between stroke (or carotid athero-
sclerosis) and a polymorphism. Because it is biologically
plausible that a prothrombotic polymorphism may exert
a differential effect across different ages, sexes, and ethnic
groups, we classified studies as having positive results
even if they had only one positive subgroup. We consid-
ered a polymorphism worthy of further study if it was not
already a clearly established stroke risk factor and if at
least one association study was positive.

Regarding a possible relationship to stroke risk, most
studies of hemostasis genes have been inconclusive at best
and unconvincing at worst. On the basis of the evidence,


we concluded that the polymorphisms of factor VII
R353Q, factor XIII Val34Leu, plasminogen activator
inhibitor-1 4G/5G, and prothrombin G20210A were not
worthy of further investigation because large studies had
consistently yielded negative results (Table 1). For similar
reasons, we decided not to study factor V R506Q
(G1691A; i.e., the factor V Leiden mutation), despite its
apparent association with cerebral vein thrombosis [9].
Although unknown point mutations in the coding regions
of these genes may relate to stroke and relevant variations
in gene expression elements may exist, we decided to
focus on more immediately high-yield candidate genes.

The results of three large European studies listed in Table
1 led us to conclude that the P-fibrinogen gene might be a
promising candidate. Fibrinogen is a 340,000-Da GP con-
sisting of three polypeptide chains: a, P, and y. The genes
that encode these polypeptides reside on chromosome 4q
in a cluster. In a study of the P-fibrinogen G455A poly-
morphism, Kessler et al. [10] did not find an overall asso-
ciation between genotype and stroke, but heterozygosity
for the A allele was associated with large-vessel ischemic
stroke (P = 0.045). Schmidt et al. [11] observed an associ-
ation between carotid atherosclerosis and the C148T pol-
ymorphism in a population-based cross-sectional study of
persons with normal neurologic status. Carotid athero-
sclerosis was seen in 53.6% of persons with the C/C gen-
otype, 54.1% of those with the C/T genotype, and 88% of
those with the T/T genotype (P= 0.003). Abnormal results
on carotid ultrasonography were significantly more com-
mon in the T/T genotype group (OR, 6.29; 95% CI, 1.91
to 20.71). Data from the study by Carter et al. [12] on the
G448A polymorphism of the P-fibrinogen gene suggested
that mechanisms linking fibrinogen and the development
of cerebrovascular disease may be different in men and
women.

Several studies listed in Table 1 suggested that polymor-
phisms of genes controlling the three platelet glycoprotein
receptors la/IIa, Ib/IX/V, and IIb/IIIa, which play a role in
adhesion, might also be promising candidate risk factors
for stroke. GP Ia/IIa (integrin a21i) is involved in colla-
gen-induced platelet aggregation. It does not bind colla-
gen monomers, but it does bind collagen fibrils and
immobilized collagen. Binding of GPIa/IIa to collagen
induces a conformational change in receptor structure
that enhances affinity. Thus, one platelet GP of interest is
GPIa. Carlsson et al. [13] compared the GPIa (a2) C807T
genotype distribution in patients with ischemic stroke or
transient ischemic attacks with that in hospitalized
patients without cerebrovascular disease and in healthy
blood donors. An association between the polymorphism
and stroke was not seen overall. However, there was an
overrepresentation of the C807T polymorphism in
patients with stroke age 50 years or younger (n = 45)


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Table I: Association Studies of Thrombosis Genes in Ischemic and Carotid Atherosclerosis


Reference


Polymorphism Cases, n; controls, n


Factor V









Factor VII


Factor XIII
p-Fibrinogen


GPla/lla

GPIbCa

GPIIb/Illa



PAl I
Prothrombin


GP, glycoprotein; PAI, plasminogen activator inhibitor.


versus age-matched controls (OR, 3.02; 95% CI, 1.20 to
7.61). No such overrepresentation was detected in older
patients.

The second platelet GP of interest is GPIba, a transmem-
branous platelet GP (molecular weight, 143,000) that
forms noncovalent complexes with GPIbp, GPIX, and
GPV to form the GPIb/IX/V receptor, which is involved in
shear stress-induced platelet activation by binding to von
Willebrand factor (vWF). This receptor may be particu-
larly relevant in large-vessel atherosclerotic ischemic
stroke because high shear stresses like those seen in
atherosclerotic arteries increase ligand-receptor affinity.
The receptor may also have a role in so-called aspirin fail-
ure, in which patients suffer stroke despite taking daily
aspirin prophylaxis. Cyclooxygenase inhibition by aspirin
has little effect on initial aggregation in response to shear
forces. One GPIba polymorphism is referred to as "VNTR"
because it consists of a variable number of tandem repeats
of 39 base pairs, each repeat leading to a 13-amino acid
addition that pushes a vWF-binding domain further away
from the platelet membrane surface. Another is human


platelet antigen-2 (HPA-2), a mutation that codes for
either a thr (HPA-2a) or met (HPA-2b) at position 145.
The HPA-2 site resides next to the vWF and high-affinity
thrombin binding sites.

In a case-control study of these polymorphisms,
Gonzalez-Conejero et al. [14] found that cerebrovascular
disease was associated with both the C/B genotype of the
VNTR polymorphism (OR, 2.83; 95% CI, 1.16 to 7.07; P
= 0.0114) and the P allele of the HPA-2 polymorphism.
Of the 104 patients with cerebrovascular disease, 22.11%
carried at least one P allele compared with 10.58% of
controls (OR, 2.40; 95% CI, 1.04 to 5.63; P = 0.0244).
Neither polymorphism showed significant differences
related to age, sex, or type of cerebrovascular disease. Both
polymorphisms also correlated with coronary artery dis-
ease, but neither correlated with deep vein thrombosis.
This is the converse of what Ridker et al. [15] and others
found for factor V Leiden. Taken together, the studies sug-
gest that polymorphisms predisposing to arterial throm-
bosis may differ from polymorphisms predisposing to
deep vein thrombosis. This hypothesis supports the


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Gene


Catto et al., 1995 [56]; Catto et al., 1996 [57]
Kontula et al., 1995 [58]
Ridker et al., 1995 [15]
Martinelli et al., 1997 [59]
Press et al., 1996 [60]
van der Bom et al., 1996 [61]
Longstreth et al., 1998 [62]
Halbmayer et al., 1997 [63]
Markus et al., 1996 [64]
Nishiuma et al., 1997 [65]
Heywood et al., 1997 [66]
Corral et al., 1998 [67]
Catto et al., 1998 [68]
Schmidtetal., 1998 [I I]
Carter et al., 1997 [12]
Kessler et al., 1997 [10]
Carlsson et al., 1999 [13]
Carlsson et al., 1997 [69]
Gonzalez-Conejero et al., 1998 [14]

Carter et al., 1998 [16]
Ridker et al., 1997 [70]
Carlsson et al., 1997 [69]

Catto et al., 1997 [71]
Longstreth et al., 1998 [62]
Poort et al., 1996 [72]
Martinelli et al., 1997 [59]
Ridker et al., 1999 [73]


G 1691A
GI 1691A
G1691A
G 1691A
GI 1691A
G 1691A
G1691A
G 1691A
G 1691A
R353Q
R353Q
R353Q/323A2
Val34Leu
C148T
G448A
G455A
C807T
HPA-5
VNTR
HPA-2
PIA2
PIA2
HPA- I
HPA-3
4G/5G
G20210A
G20210A
G20210A
G20210A


348; 247
236; 137
209; 209
155; 155
116; 54/161/287
II 2; 222
106; 391
229; 71
180; 80
137; 83/87
286; 198
104; 104
529; 437
399
305; 197
227; 225
227; 170
218; 165/321
104; 104
104; 104
505; 402
209; 209
218; 165/321
218; 165/321
421; 172
106; 391
104; 104
155; 155
259; 1,744


Results


Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Negative
Positive
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative


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rationale for a hemostasis candidate-gene association
study such as ISGS, which investigates ischemic stroke
specifically and does not regard all acute thrombotic
events, whether arterial or venous, as a single clinical
entity.

The third candidate platelet GP gene controls GPIIb/IIIa
(integrin Ua11b3), a transmembranous heterodimer with
several ligands, including fibrinogen, fibrin, fibronectin,
and vWF. Many receptors are involved in platelet adhe-
sion and many agonists stimulate platelet aggregation,
but platelet aggregation requires GPIIb/IIIa. When plate-
lets aggregate, GPIIb/IIIa binds to fibrinogen and vWF.
Binding to vWF gains importance under conditions of
high shear stress. Carter et al. [16] found no overall asso-
ciation between the P1A2 polymorphism of the GPIIb/
Ilia gene and cerebral infarction confirmed by computed
tomography (CT). However, a subgroup analysis showed
significant genotype distribution differences in nonsmok-
ers. The risk of stroke was greater in nonsmokers hetero-
zygous for the P1A2 allele than in those homozygous for
P1A2 (OR, 2.37; 95% CI, 1.19 to 4.74; P = 0.01). Informa-
tion on young stroke patients was limited (n = 37), but in
a logistic regression model that included P1A genotype
status, smoking, hypertension, and diabetes, the OR for
stroke in those possessing the A2 allele was 1.68 (95% CI,
1.00 to 2.82; P = 0.05). This study highlights the need for
further studies of the interaction between genes and envi-
ronmental factors, in this case smoking, in attempts to
elucidate inherited stroke risk.

Aims
The primary aim of ISGS is to test the association between
ischemic stroke and the following putative risk factor pol-
ymorphisms: P-fibrinogen C148T, G448A, and G455A;
GPIa C807T; GPIba HPA-2 and VNTR; and GPIIb/IIIa
P1A. Exploratory aims are to investigate whether any asso-
ciation found between ischemic stroke and the panel of
tested polymorphisms is contingent on sex, age, ethnic
origin, smoking status, or stroke subtype and to investi-
gate whether hemostatic gene sequence variations are
associated with 90-day functional outcome after acute
ischemic stroke.

Methods
Design and Overview
The ISGS is a prospective multicenter study using a case-
control design (Fig. 1). Patients and control subjects are
screened at one of five clinical centers (Appendix 1 Addi-
tional file: 1), stroke status is verified, the index stroke for
each patient is subtyped, and baseline clinical and demo-
graphic data are collected. Blood samples are collected
from all enrolled patients and control subjects by means
of a one-time venipuncture. The samples are shipped to a
central DNA bank for processing and storage, and the


Multicenter
recruitment


Controls Patients


Blood Stroke 3 -month
samples Baseline data follow-up
cl dinical and data
DNA bank demographic
data Subtyping
S adjudicator
DNA

clinical
Genetics coordinating
laboratory centre


Statistical
Genotypic coordinating
data center

Figure I
Overview of flow of samples and data from study subjects to
genetic analyses.




processed DNA samples are sent to a central genetics lab-
oratory for genotyping. The genotype data are then
merged with the clinical, stroke, and follow-up data and
analyzed to ascertain potential associations between
stroke risk and genes for P-fibrinogen and platelet GPIa,
GPIba, or GPIIb/III.

Study Population
Patients With Stroke (Cases)
Each patient with suspected stroke admitted to a partici-
pating center is evaluated by a study neurologist according
to current standards for care [17,18]. The evaluation
includes patient history, physical examination, CT or
magnetic resonance imaging (MR) of the head, and labo-
ratory testing. Where clinically indicated, the evaluation
may also include carotid ultrasonography; MR, CT, or dig-
ital subtraction angiography; transthoracic or trans-
esophageal echocardiography; resting and ambulatory
electrocardiography; intracranial arterial imaging; and
additional blood testing.

Adult men and women who meet the following criteria
are entered into the study: 1) diagnosis of first-ever
ischemic stroke confirmed by the study neurologist on the
basis of history, physical examination, and head imaging
by CT or MR; 2) enrollment within 30 days after onset of


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stroke symptoms; 3) attained 18th birthday by the time of
enrollment; 4) complete blood cell count, casual or fast-
ing blood glucose, prothrombin time, and activated par-
tial thromboplastin time available; and 5) written
informed consent from the patient or surrogate.

Stroke is defined according to the World Health Organiza-
tion criteria [19] as rapidly developing signs of a focal or
global disturbance of cerebral function with symptoms
lasting 24 hours or longer or leading to death, with no
apparent cause other than vascular origin. A diagnosis of
ischemic stroke is made only if the patient has a clinical
diagnosis of stroke and if a CT scan or MR of the brain
done after onset of symptoms either is normal or shows
the relevant infarct. Patients with hemorrhagic transfor-
mation of an infarct remain eligible.

Time of stroke onset is defined as the time when the sub-
ject was last noted to be at baseline neurologic status. If
the patient awoke with stroke symptoms, the time of
onset is taken as the last time the patient was known to be
awake and without any symptoms of stroke. We restrict
enrollment to within 30 days after onset of symptoms
from the first-ever stroke to avoid potential survival bias
[20-23]. The date of enrollment is the date of obtaining
signed informed consent.

Exclusion criteria parallel those of the Siblings With
Ischemic Stroke Study (SWISS) [24]. Patients who are
already enrolled in SWISS are not eligible for participation
in ISGS.

To be able to assess the extent to which enrolled patients
represent all potential subjects, clinical study coordinators
at each site keep logs of every eligible stroke patient who
is offered participation in the study, whether or not they
are enrolled. The logs will contain initials and date of
birth of the eligible stroke patients, date of screening, sex,
and race/ethnicity.

Controls
Controls are adult men and women who have attained
their 18th birthday at the time of enrollment, have not
had a stroke, are unrelated by blood to patients enrolled
in the study, and who give written informed consent to
participate in the study. We confirm that controls have not
had a prior stroke by means of the Questionnaire for Ver-
ifying Stroke-free Status (QVSS), a structured interview
that was validated in an adult population (age, > 60 years)
using systematic review of electronic medical records as
the benchmark [25]. The QVSS was further validated in an
independent population using history and physical exam-
ination by a study neurologist as the benchmark [26].
Interviewers administering the QVSS may exclude a sub-
ject they judge to be an unreliable historian on the basis


of a global impression of moderate or severe impairment
of speech, language, hearing, or memory. Hospitalized
patients being treated for coronary or peripheral vascular
disease are not eligible for enrollment as controls, but
nonhospitalized subjects with a history of these condi-
tions are eligible.

Cases and controls are matched one-to-one. Matching cri-
teria are sex and age (within 3 years for patients who are
younger than 30 years and within 5 years for patients who
are 30 years or older). We recruit controls mainly from
among spouses and unrelated friends of the patients. Each
center has a backup plan for recruiting community volun-
teers should there be a lag in recruitment of properly
matched controls [27].

Data Collection
The schedule for data collection is shown in Table 2.

Structured Interview
A structured interview is conducted by the study coordina-
tor with each patient (case) or surrogate and each control
subject to explain the study, obtain informed consent, and
obtain standardized information on baseline medication
and demographic, medical, social, and behavioral varia-
bles. Information regarding race and ethnicity is recorded
according to self-report. A proband-derived family history
is taken for all living or deceased full siblings, all biologi-
cal children, and both biological parents [28]. Investiga-
tors do not independently verify stroke status of family
members as part of this protocol. Self-reported cerebrov-
ascular histories are obtained for all patients and control
subjects by administering the QVSS during the baseline
interview [25].

Medical Records
Study coordinators review the medical records of the ini-
tial evaluation of stroke cases to complete case report
forms for documenting eligibility and baseline data and
to construct the abstracted medical record used for stroke
subtyping. The following information is recorded on the
case report forms: patient history, physical examination,
CT or MR of the head, white blood cell count, platelet
count, and hemoglobin concentration, casual or fasting
blood glucose, prothrombin time, and activated partial
thromboplastin time, vital signs (height, weight, blood
pressure, and temperature), international normalized
ratio, lipid profile, plasma homocysteine concentration,
and size and location of the symptomatic cerebral infarct
as seen on head imaging.

The clinical coordinator also constructs the abstracted
medical record used for subtyping of the index stroke by
copying admission notes, physician progress notes, dis-
charge summaries, radiology reports, electrocardiograms,


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Table 2: Events Table for the Ischemic Stroke Genetics Study


Variables


90 14 days after stroke


Structured interview
Demographics
Medications
Family history
Past medical history
Social and behavioral history
QVSS
Blood sample
Medical records
Vital signs
Laboratory tests
Head imaging
Vascular imaging
Treatments
Stroke status
Subtyping
Time of stroke onset
NIH Stroke Scale
Barthel Index
Oxford Handicap Scale
Glasgow Outcome Scale


NIH, National Institutes of Health; QVSS, Questionnaire for Verifying Stroke-free Status; T, assessment by telephone; X, assessment by face-to-face
interview or examination.


echocardiography reports, laboratory reports, and rehabil-
itation notes.

Investigators and coordinators use standardized defini-
tions for major medical and surgical comorbid conditions
(Appendix 2 see Additional file: 2). Because blood
pressure typically falls during the first few days after an
acute ischemic stroke, we have adopted a modification of
the Northern Manhattan Stroke Study (NOMASS) tech-
nique [29], in which we use the systolic and diastolic
blood pressure values measured after admission to the
hospital floor (or intensive care unit) rather than meas-
urements taken by the emergency medical service or in the
emergency department. Blood pressure is measured from
the left brachial artery (if attainable) with a sphygmoma-
nometer, with the patient sitting upright. If the patient is
bedbound, the measurement is made with the head of the
bed elevated to at least a 45 angle.

Characterization of Ischemic Stroke
An on-site, study-appointed neurologist confirms the
diagnosis of ischemic stroke and the time of stroke onset
by interviewing patients or any available observers present
when the stroke was first noticed [30]. The examiner seeks
corroborating evidence (such as ambulance reports) and
carefully screens for the possibility of onset during sleep.


The severity of the neurologic deficit is assessed within 48
hours of the patient's enrollment in the study by means of
the National Institutes of Health Stroke Scale (NIHSS)
[31] administered by a certified examiner. The first CT or
MR obtained is used to measure infarct size by means of
standardized criteria (Appendix 3 see Additional file: 3).

Prestroke functional status is assessed retrospectively by
the study coordinator with the Oxford Handicap Scale
[32]. Acute poststroke functional status is assessed with
the Oxford Handicap Scale [32], Barthel Index [33,34],
and Glasgow Outcome Scale [35] within 48 hours of
enrollment.

Subtyping of the index ischemic stroke is done centrally
on the basis of the abstracted medical record by a neurol-
ogist adjudicator blinded to genotype data and personal
indentifiers. Because final subtype diagnosis has been
shown to vary from initial diagnosis in approximately
one-third of cases [36], the adjudicator uses all available
and relevant information obtained after completion of
the stroke work-up. The Trial of ORG10172 in Acute
Stroke Treatment (TOAST) [37], Oxfordshire Community
Stroke Project (OCSP) [38], and Baltimore-Washington
Young Stroke Study (BWYSS) [39] classification systems
are used for subtyping.


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Enrollment


Cases


Controls


Cases


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Follow-up of Stroke Patients
Patients with stroke are followed up by the study coordi-
nator at the center from which they were recruited. The
coordinator reassesses patients by using the Oxford Hand-
icap Scale, Barthel Index, and Glasgow Outcome Scale by
telephone interview 90 + 14 days after onset of stroke
symptoms. Coordinators preferentially interview the
patients themselves. However, if acquired deficits of
speech, language, or cognition prevent the patient from
participating in the telephone outcomes assessment, a
surrogate history is taken from a caregiver or live-in rela-
tive. Coordinators will also record mortality and history
of cause of death from collateral sources.

Genetic Data
The study coordinator at the local center obtains two
tubes of peripheral blood from each patient and each con-
trol subject, collected in a 10-mL (8.5-mL draw) acid-cit-
rate-dextrose solution A (ACD) tube. The blood samples
are shipped to a central DNA bank by overnight courier
and assigned a unique repository identifier. Lymphocytes
are isolated, and 0.5 mL of blood is retained in the origi-
nal tube as a quality control specimen for identity testing.
Isolated lymphocytes are cryopreserved using controlled-
rate freezing and stored at the liquid phase of nitrogen.

The DNA bank prepares high-quality, high-molecular-
weight DNA from the cell pellet using a modification of
the salting-out procedure of Miller et al. [40]. Quality con-
trol studies on DNA consist of estimation of quantity by
OD260/OD280 ratio, estimation of integrity by gel elec-
trophoresis and restriction digestion, and verification of
identity by microsatellite analysis and sex determination.

Transformation of lymphocytes is done using Epstein-
Barr virus and phytohemagglutinin. Lymphocyte cultures
are expanded to produce sufficient stock for 10 to 12
ampules. All cell cultures are done in the absence of anti-
biotics. Cryopreservation is again done by controlled-rate
freezing. Samples are stored at the liquid phase of nitro-
gen. In-house and remote fail-safe stocks are generated.
The following routine quality control studies for cell cul-
ture are performed: recovery of frozen stock and determi-
nation of viability, sterility testing for bacterial and fungal
contamination, testing for mycoplasma contamination by
polymerase chain reaction (PCR), and confirmation of
the identity of the culture by comparing the DNA finger-
print of the culture with that of the quality control speci-
men. If the first attempt fails, a second aliquot of
cryopreserved lymphocytes can be transformed.

Genotyping is done at a central genetics laboratory. Cur-
rently, the following sequencing methods are used, but
the laboratory will be responsive to technological
advances in the field.


PCR is carried out in a 75- tL volume on 40-ng genomic
DNA by use of plain primers under standard conditions
with a 57 to 52C (0.5C/cycle) "touch-down" anneal-
ing temperature. To remove excess unincorporated prim-
ers that would compete as sequencing primers in the cycle
sequencing reaction, the amplified product is filtered with
MultiScreen PCR filters (Millipore) and resuspended in 50
gL. The sequencing reaction is carried out using the
BigDye Terminator cycle sequencing kit (Applied Biosys-
tems) as per the manufacturer's conditions. To remove
excess dye terminators, the sequencing product is purified
by ethanol precipitation and resuspended in 10 gL of
HiDi formamide. The samples are then denatured and
electrophoresed on an ABI 3100 capillary analyzer. Data
analysis is carried out with a software suite (ABI) consist-
ing of Sequencing Analysis (base calling), Factura (heter-
ozygous base detection), and Sequence Navigator
(sequence comparison).

Adverse Events
All adverse events and serious adverse events will be
recorded by the study coordinators and forwarded to the
statistical center. Potential physical risks are minimal and
relate to the one-time phlebotomy. An independent med-
ical safety monitor will review summary reports of adverse
events and serious adverse events periodically and will
forward assessments to the study Principal Investigator.

Outcome Measures
The main end point of the study is whether any of the pol-
ymorphisms (the 3-fibrinogen polymorphisms C148T,
G448A, and G455A and the platelet GP polymorphisms
GPIa C807T, GPIba HPA-2 and VNTR, and GPIIb/IIIa
P1A) are associated with ischemic stroke. Thus, patients
with stroke will be compared with controls as to fre-
quency and distribution of these polymorphisms. Other
end points include potential associations among these
polymorphisms and individual subtypes of ischemic
stroke, ethnic origin, or 90-day poststroke functional out-
come and mortality. Additional analyses will include test-
ing for interactions between inherited susceptibility
(genotype) and environmental exposures (e.g., smoking).

Data Analyses
Several types of analyses will be performed to assess the
relationships between outcome (binary and continuous)
and risk factors (both genetic and environmental). The
statistical techniques we use to analyze the data depend
on the distribution of the independent (predictor) and
dependent (outcome) variables. When the outcome
variables are categorical (i.e., stroke [yes/no]) we will use
chi-square tests and logistic regression techniques; when
the outcome variables are continuous (with or without
inclusion of collected longitudinal data), we will use



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repeated measures analysis of covariance (ANCOVA)
techniques.

Cases will be compared with controls with respect to pre-
dictor variables of interest and with respect to genotypes
that may provide increased risk for stroke. Where needed,
adjustments for potential covariates (age, sex, hyperten-
sion, etc.) will also be included in these analyses. To per-
form the above comparisons, we will use logistic
regression, a statistical technique for modeling the rela-
tionship of a binary outcome and a set of independent (or
predictor) variables [41].

For our case-control comparisons, a second series of anal-
yses relates to the distribution of polymorphisms within
the selected candidate genes in the cases (stroke positive)
and controls (stroke negative). Each subject will be char-
acterized by the polymorphism (or mutation) found at
each candidate gene. Different analytical strategies need to
be employed for the different candidate loci under study.
For the fibrinogen gene cluster, it is proposed that the
genotype be considered as a binary variable, that is, as a
single nucleotide polymorphism (SNP) with two possible
alleles, since the outcome of polymorphisms in genes of
this cluster has resulted in apparent increased risk for
ischemic heart disease through increased circulating
fibrinogen levels. Thus, initial analyses using members of
the fibrinogen cluster (or with other candidate genes such
as GPIba) will focus on each case/control being classified
by the presence of a risk allele (yes/no), with comparison
of SNP allele frequencies between groups (stroke/con-
trol).

For other candidate genes (GPIba, etc.), distributions of
genotypes or strata determined by alleles or haplotypes
can be established for comparison. For example, GPIba
has a series of polymorphisms that may define an allele of
interest. Extending SNPs to haplotypes has the advantage
of increased information, yet decreased power in small
samples due to the increased number of possible haplo-
types to be tested. Restriction of haplotypes by inningn"
may provide some increase in power, yet it assumes previ-
ous knowledge of risk haplotypes. These few haplotypes
can be used to establish strata in the analyses proposed. In
separate analyses, the SNP/polymorphism status can be
considered to be an exposure variable. Thus, risk factor
distributions can be compared between "exposed" sub-
jects (with a polymorphism in a candidate gene) and
those "not exposed" (without a polymorphism). For lev-
els of continuous risk factors, this analysis will compare
means by analysis of variance methods. For dichotomous
risk factors, contingency table methods can be used.

In the case-control analyses, each candidate locus can be
analyzed individually as a potential modifier of disease


risk (stroke) and as a determinant of other intermediate
(continuous trait) end points. To evaluate the relation-
ships between gene interaction (gene-gene) and interac-
tion between inherited susceptibility (genotype) and
environmental exposures (e.g., hypertension or smoking),
several strategies can be employed. One approach is strat-
ification by genotype at each candidate locus, with analy-
sis of "case" status with the second genetic (or
environmental) exposure. For dichotomous exposures,
this reduces to a comparison of contingency tables within
genotype strata.

A second approach uses logistic regression. Logistic regres-
sion can be used for continuous risk factors within geno-
type strata. Similarly, multivariate logistic regression can
be used to predict group membership (stroke vs. control)
based on age, sex, environmental risk factors that appear
significant in univariate analyses, and genotype(s), with
first-order interaction terms of genotype with environ-
ment and gene 1 with gene 2.

Sample Size and Study Power
We intend to enroll a total of 900 participants, including
450 patients with ischemic stroke (cases) and 450 stroke-
free volunteers (controls), at five academic medical cent-
ers in the United States over 3 years. Estimates of potential
for recruitment were based on the assumption that the
rates of hospitalized stroke cases at the five participating
hospitals will remain at 1999 levels throughout the
patient recruitment phase of the study.

We anticipate that the study population will be approxi-
mately two-thirds white and one-third African American,
giving sample sizes of approximately 300 each for white
patients with stroke and control subjects and 100 each for
African American patients with stroke and control sub-
jects. Thus, for the above analyses, we are concerned pri-
marily with dichotomous outcomes with fixed sample
sizes (100, 300, or 450 stroke cases and 100, 300, or 450
controls). Power for any analyses involving continuous
measures can be approximated using an independent t
test comparison between the incident cases and controls.
In the case-control study, the power to address specific
issues relating to the genetic associations is dependent on
the sample size available, the frequency of the polymor-
phisms (in the fibrinogen cluster and other candidate
genes), and the size of the effect to be detected.

The power is determined on the basis of the frequency of
the SNP in the control group and the detectable difference
in SNP allele frequency in the control group. For example,
if an SNP has a frequency of 0.40 in the total group of con-
trols (n = 450), we can detect (with 75% power) a 20%
difference in cases [(0.20)(0.40) = 0.08] or frequencies
greater than 0.48 or less than 0.32 in cases. This is obvi-


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ously a small difference (or a relatively weak genetic rela-
tive risk). Using this same example, we would have 46%
power in whites and 22% power in African Americans. For
the African American group, we generally have power for
relatively high-frequency polymorphisms in controls
(approximately 45%-50%) and detectable differences of
40% in cases (0.20 difference). Thus, we could detect an
SNP allele frequency of 0.70 in cases and 0.45 in controls
with 80% power.

In general, the power to detect gene-environment interac-
tion effects is less than that for main effects and is depend-
ent on the type of interaction. For example, using
methods discussed by Goodman [42], the power to detect
interactions for multiple scenarios is possible. For these
scenarios, 2 x 2 tables can be used to describe the overall
main effects of genetic and environmental risks (i.e., both
the genetic and the environmental risk factors display a
main effect size [odds ratio] of about 4).

For large interactions we have > 99% statistical power.
Only in the smallest subgroup would power be apprecia-
bly affected. For example, for the African American group
(100 cases and 100 controls), with the same distribution
of individuals as above, the power to detect the large inter-
actions (OR of 0.33 in controls, OR of 5.33 in cases)
would still be -99%, while the power to detect the modest
interaction (OR of 0.80 in controls, OR of 1.50 in cases)
would be reduced to 13%.

Ethical Considerations
Local institutional review boards (IRBs) governing each
clinical center have approved the study protocol. Written
informed consent is obtained for every study subject. To
the extent permitted by local IRBs, surrogate consent is
permitted for patients rendered incompetent by stroke to
avoid biasing the study toward discovery of risk factors for
mild or moderate stroke but not severe stroke.

Genetic information has the potential to adversely affect
insurability and employability [43,44]. An individual's
genetic information could also lead to stigmatization
within a family and a community [45]. Because of the
highly sensitive nature of genetic information, we have
developed the following plan to prevent intentional or
unintentional misuse of genetic data, which is in keeping
with the Privacy Workshop Planning Subcommittee
Guidelines of the National Action Plan on Breast Cancer
[46].

Every ISGS investigator who obtains or has access to gen-
otypic data is blinded to individual personal identifiers,
and every investigator who obtains or has access to indi-
vidual personal identifiers is blinded to genotypic data.
Personal identifiers are defined as individual names,


addresses, phone numbers, fax numbers, and e-mail
addresses. Personal identifiers and linkage codes are kept
only at the clinical center where the study subject was
enrolled and will not be recorded on case report forms or
stored in the study electronic database. Experimental
research data are not placed in a participant's medical
record. There have been various opinions regarding
whether family members should be considered human
subjects in pedigree research and when it is permissible to
waive consent [28,47-49]. In ISGS, no personal identifi-
ers are collected on family members.

The protocol of ISGS calls for centrally banking DNA and
creating immortalized cell lines. Unlike the situation in
clinical trial research, no broad consensus on research eth-
ics exists among investigators and eligible participants in
the field of human molecular genetics research. Of recent
contention are procedures for ensuring ethical future use
of stored genetic material [50,51 ]. The potential for future
use of DNA should be anticipated, even if the specific
studies cannot be known. A future-use agreement should
respect genetic privacy rights and autonomy of human
subjects while encouraging scientific inquiry. Establishing
a transparent and ethically sound future-use agreement
for research in this field facilitates multicenter collabora-
tions and future research. After reviewing available policy
statements from genetics societies and governmental
agencies, we developed the following list of key princi-
ples, which are incorporated into a future-use agreement
governing DNA banked for this study: 1) Subjects must
provide informed consent to the original study at the time
of DNA collection. 2) The original study must have a rig-
orous procedure in place to protect privacy of study sub-
jects prior to DNA collection. 3) Patients should have the
option to consent to, or refrain from, participation in
future research at the time of the initial consent process.
4) Levels of consent must be clear, explicit, and exclusive
(e.g., original study only, any stroke study, any study). 5)
Applications for future use need to be reviewed formally.
6) Study investigators can release DNA for future use only
after determining whether the new study conforms to the
type of research permitted by the donor. 7) All specimens
are stripped of direct personal identifiers before future
use. 8) Anonymous data sets with genetic information
from different studies may be merged for hypothesis-gen-
erating analyses. We believe that this carefully developed,
publicly scrutinized future-use agreement is a unique
strength of this study and hope that this proactive
approach will avoid some of the rancorous
misunderstandings that other investigative groups have
encountered in genetic and pedigree research [51].

Discussion
Defining the molecular basis for the inherited component
to ischemic stroke risk will require converging lines ofevi-


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dence from various methodologies, including genom-
ewide screens [24] and candidate gene association studies.
Collection of DNA samples from a large cohort of
ischemic stroke pedigrees is logistically challenging. Col-
lection of DNA samples from patients with stroke and
unrelated stroke-free controls is more feasible.

Some stroke genetics research has been done in the con-
text of epidemiological studies; for example, a study by
Ridker and colleagues [15] tested for an association
between factor V Leiden and stroke. One limitation of this
approach is that there may be relatively few stroke end
points in an epidemiological study, particularly because
stroke, myocardial infarction, and vascular death are often
combined as the primary end point. Furthermore, for var-
ious reasons, epidemiological studies may include a
selected population, for example, only one sex or a lim-
ited socioeconomic stratum. The use of such highly
selected samples may compromise the ability to general-
ize the findings of a genetic association study.

Genetic association can be studied within the context of a
clinical trial of an intervention for primary prevention. In
a prevention study, DNA would be available both from
patients with stroke and from stroke-free controls, but the
intervention may alter the outcome (stroke) sufficiently to
confound interpretation of the results of a genetic associ-
ation study.

A genetic study in the context of a randomized trial of
treatment of stroke patients is a robust study design when
the goal is to discover genetic determinants of stroke out-
come or response to therapy (pharmacogenomics). How-
ever, such a study design may not achieve the goal of
discovering genetic risk factors for stroke because no
genetic material from stroke-free controls outside of the
study would be available. Furthermore, highly restrictive
criteria for eligibility into a clinical trial can drastically
limit the representativeness of a study population. For
example, in one study of intra-arterial thrombolysis for
treatment of acute ischemic stroke, 12,323 patients with
stroke were screened, and only 180 patients were selected
[4]. Other stroke studies include only patients with non-
disabling strokes [52], or include only patients with mod-
erate to severe strokes [4]. Because ISGS is a dedicated
stroke genetic association study, it has broad eligibility cri-
teria relative to clinical trials of drugs or devices. We
expect this to enhance the external validity of the results
of the study.

Additionally, we have given careful attention to appropri-
ate selection of controls [27]. The controls are concur-
rently enrolled at the same centers as are the patients, and
controls are also screened for a medical history of stroke
or transient ischemic attack and for the presence of symp-


toms of stroke or transient ischemic attack which may
have occurred in the absence of a corresponding medical
history.

A unique strength of ISGS is that the protocol was specifi-
cally developed to permit valid future pooled analyses
with the ongoing affected sibling pair linkage study SWISS
[24,53], which uses genome-wide screening within a
cohort of pedigrees to identify chromosomal regions -
rather than specific polymorphisms that are linked to
stroke. Conducting a candidate gene association study in
cases and controls has advantages compared with the
genome-wide linkage approach in siblings. Isolated cases
are more likely to be available than sibling pairs, increas-
ing the potential for statistical power. Furthermore, deter-
mination of phenotype is often retrospective in genome-
wide linkage studies because it is rarely possible to enroll
all affected members of a pedigree shortly after onset of
stroke. In contrast, the case-control approach allows pro-
spective phenotyping of all subjects at the time of onset of
stroke, which may be more reliable than retrospective
phenotyping. This is an advantage because ischemic
stroke has a heterogeneous phenotype, and distinctions
between specific clinical subtypes of ischemic stroke may
be relevant [54,55]. To address the heterogeneity of the
ischemic stroke phenotype, ischemic stroke is subtyped by
a genotype-blinded adjudicator in both ISGS and SWISS.

ISGS and SWISS also use the same definitions for ischemic
stroke and comorbidity and the same criteria for classify-
ing stroke mechanism. Both studies use the same key
exclusion criteria; for example, both studies exclude iatro-
genic and vasospastic ischemic stroke patients and
exclude the same mendelian and mitochondrial disor-
ders. In addition, the stroke-free status of controls in ISGS
and of discordant siblings in SWISS is verified using the
same structured interview instrument (i.e., the QVSS
[25]). We anticipate that the two studies will complement
each other in contributing to the broad, long-term objec-
tive of defining the molecular basis for inherited ischemic
stroke risk.

List of abbreviations
BWYSS, Baltimore-Washington Young Stroke Study

CT, Computed tomography

GP, Glycoprotein

ISGS, Ischemic Stroke Genetics Study

MR, Magnetic resonance imaging

NIHSS, National Institutes of Health Stroke Scale



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OCSP, Oxfordshire Community Stroke Project

OR, Odds ratio

PCR, Polymerase chain reaction

QVSS, Questionnaire for Verifying Stroke Status

SWISS, Siblings With Ischemic Stroke Study

TOAST, Trial of ORG10172 in Acute Stroke Treatment

vWF, von Willebrand factor

Competing interests
None declared.

Authors' contributions
Dr. Meschia is the principal investigator in ISGS, develop-
ing and writing the protocol from its inception to the final
version. Drs. Brott, Brown, Frankel, Merino, Silliman and
Worrall are site investigators who contributed to develop-
ing and writing the final protocol. Dr. Hardy and Mr.
Crook contributed to writing the portions of the manu-
script that refer specifically to DNA sequencing and anal-
ysis. Dr. Rich contributed to writing the portion of the
manuscript that refers to the statistical analytical plan.

Acknowledgements
This study is supported by NIH NINDS RO1 NS-42733
(J.F.M.)


Additional material


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