Keywords:
stroke - genetics - biobank
Palavras-chave:
acidente vascular cerebral - genética - biobancos
According to the Global Burden of Disease Study, in 2013, there were almost 25.7 million
stroke survivors (71% with ischemic stroke (IS)), 6.5 million deaths from stroke (51%
died from IS), 113 million disability-adjusted life years due to stroke (58% due to
IS) and 10.3 million new strokes (67% IS) worldwide[1]. The INTERSTROKE study defined the population-attributable risks for ischemic and
hemorrhagic strokes in 22 countries, concluding that 10 classical cardiovascular risk
factors are associated with 90% of the risk of stroke[2]. However, twins and familial aggregation studies suggest that the risk of stroke
has a substantial genetic component[3]. Although genetics, and more recently genomics, play an increasingly large role
in the practice of medicine, the daily clinical care of patients suffering from stroke
has not significantly been affected by the advances in these fields. Therefore, one
can assume that there is still much knowledge about stroke genetics and genomics needing
to be translated into clinical practice.
Currently, there are tremendous efforts to understand the genetic basis of both rare
and common cardiovascular and stroke disorders through strategies such as genome-wide
association studies and next-generation sequencing studies[4]. In this scenario, it has been pointed out that well-established stroke biobanks
with close collaboration between clinicians and geneticists are essential for supplying
the biological and clinical information required for these large-scale studies[4],[5],[6]. To address these challenges specifically, recent cooperative efforts have been
launched such as the Stroke Genetics Network and Bio-Repository of DNA in Stroke[7],[8].
As defined by Brazilian regulatory guidelines, a biobank represents an organized collection
of human biological material and associated data that are prospectively collected
and stored for research purposes, in accordance with predefined technical, ethical
and operational standards, under institutional responsibility and management. We present
the study protocol and current status of the Joinville Stroke Biobank (JSB), the first
Latin American DNA biobank of stroke.
METHODS
Participants and samples
The current biobank originated from two sequential initiatives to study epidemiological
aspects of stroke in recent years in Brazil[9]. The phenotypic data were extracted from the Joinville Stroke Registry, which is
a population-based registry that has been ongoing since 2005. In 2013, the registry
became supported by a municipal law[10]. The beginning of case and control data ascertainment, blood extraction and DNA
storage took place in 2010. These samples and data, which are still being collected
and processed in Joinville, constitute phase I of the JSB, which has no deadline for
completion.
Phase II was launched in 2015, when the JSB started to receive blood samples and phenotypic
data from four other Brazilian cities. This constitutes a task force of the Brazilian
Consortium of Stroke Research, which is sponsored by the Brazilian Ministry of Health
and the National Council for Scientific and Technological Development, to define stroke
incidence trends, case-fatality proportions and the prevalence of classic cardiovascular
risk factors according to the WHO Steps criteria[11] in different geopolitical scenarios than Joinville, which has a higher Human Development
Index than other cities. Data and samples will be collected until 2017.
Central site and contributing sites
In JSB phase I, inpatients from five hospitals and out-patient data (mild strokes)
from the town of Joinville (515,288 inhabitants, 2010 census) were included, whereas
JSB phase II covered the following cities: Sobral, northeast region (three hospitals;
147,135 inhabitants), Campo Grande, central region (eight hospitals; 774,202 inhabitants),
Sertãozinho, south-east region (two hospitals; 101,784 inhabitants) and Canoas, south
region (three hospitals; 323,827 inhabitants), whose geographic locations are shown
in the [Figure]. The central site, as defined by the International Stroke Genetics Consortium is
located in the facilities at the University of the Region of Joinville.
Figure Localization of cities, according to Brazilian mesoregions, corresponding to central
and contributing sites of the Joinville stroke biobank.
Inclusion and exclusion criteria
Since the beginning of both phases, data and blood samples have been collected from
each case and related controls. Patients included transient ischemic attacks, subarachnoid
hemorrhages, ischemic strokes and hemorrhagic strokes. We included first-ever or recurrent
patients, regardless of age and sex, residing in one of the five cities. All patients
had at least one cranial tomography. Stroke confirmation and diagnosis were performed
as previously reported[9]. In brief, the phenotype of each stroke patient includes demographic information,
social class (according to the Brazilian Criteria of Economic Classification based
on the National Household Sample Survey)[12], years of education, type of work (manual or not), family history of stroke, cardiovascular
risk factors, biochemical tests and functional status (modified Rankin scale)[13]. Furthermore, IS subtypes were classified according to the modified Trial of Org
10172 in Acute Stroke Treatment (TOAST) criteria and Causative Classification System
for Ischemic Stroke (CCS) criteria[14],[15]. Patients with IS of undetermined cause due to negative investigation or cryptogenic
stroke were classified according to the Embolic Strokes of Undetermined Source international
work group[16]. A neuroradiologist classified all hemorrhagic strokes as lobar and non-lobar subtypes.
Controls were matched by age and gender to patients, and individuals with a previous
history of stroke or blood ties to patients were excluded. In cases of a positive
family history of stroke, blood samples of all related patients and non-stroke relatives
were searched for and nominated as nuclear cases.
Phenotyping methods
[Table 1] shows the diagnostic examination and the categories of data retrieved by questionnaires.
After 2012, the CCS criteria for phenotyping IS patients were also employed. The neurologists
of all cities in the contributing sites were trained by stroke neurologists from the
Joinville Stroke Registry using the platform available on the CCS website[15]. After the TOAST diagnosis, the clinical history, physical examination findings
and the results of diagnostic work-up were reviewed for determining the CCS classification.
Ischemic stroke patients with incomplete diagnostic examination were classified as
a possible IS subtype or received an IS diagnosis as undetermined with incomplete
evaluation. All Joinville IS diagnoses were blinded for adjudication by two authors
(FIR and NLC).
Table 1
Demographic and diagnostic examination data available in the Joinville Stroke Biobank.
|
Category
|
Description
|
Cases
|
Controls
|
|
Date of birth/age
|
Y
|
Y
|
|
Gender
|
Y
|
Y
|
|
Skin color
|
Y
|
Y
|
|
Sociodemographic
|
City of residence
|
Y
|
Y
|
|
Address
|
Y
|
Y
|
|
Social status
|
Y
|
N
|
|
Education level
|
Y
|
N
|
|
First-ever event?
|
Y
|
N
|
|
Date of enrollment
|
Y
|
N
|
|
Hour of enrollment
|
Y
|
N
|
|
Creatinine
|
Y
|
N
|
|
Triglyceride
|
Y
|
N
|
|
Uric acid
|
Y
|
N
|
|
Clinical and biochemical data
|
Blood glucose
|
|
N
|
|
Cholesterol (hdl/ldl)
|
|
N
|
|
National Institutes of Health Stroke Scale (admission)
|
Y
|
N
|
|
Barthel Index
|
Y
|
N
|
|
Rankin Index
|
Y
|
N
|
|
Oxfordshire Community Stroke Project classifcation (Banford)
|
Y
|
N
|
|
TOAST classifcation
|
Y
|
N
|
|
CCS classifcation
|
Y
|
N
|
|
Transient ischemic attack
|
Y
|
Y
|
|
Hypertension
|
Y
|
Y
|
|
Diabetes mellitus
|
Y
|
Y
|
|
Dyslipidemia
|
Y
|
Y
|
|
Acute myocardial infarction
|
Y
|
Y
|
|
Premorbid risk factors
|
Congestive heart failure
|
Y
|
N
|
|
Atrial fbrillation
|
Y
|
N
|
|
Smoking
|
Y
|
Y
|
|
Alcohol consumption **
|
Y
|
N
|
|
Drug consumption
|
Y
|
N
|
|
Physical activity
|
Y
|
N
|
|
Brain CT
|
Y
|
N
|
|
Brain MRI
|
Y
|
N
|
|
Digital angiography
|
Y
|
N
|
|
Extracranial vessels evaluation
|
|
|
|
Ultrasound Doppler
|
Y
|
N
|
|
CT – angiography
|
Y
|
N
|
|
MR – angiography
|
Y
|
N
|
|
Diagnostic examination
|
Intracranial vessels evaluation
|
|
|
|
Transcranial Doppler
|
Y
|
N
|
|
CT – angiography
|
Y
|
N
|
|
MR – angiography
|
|
|
|
Embolic sources
|
Y
|
N
|
|
ECG
|
Y
|
N
|
|
Transthoracic echocardiography
|
Y
|
N
|
|
Transesophageal echocardiography
|
Y
|
N
|
|
24 h Holter
|
Y
|
N
|
|
Outcome measures
|
Rankin scale (30 days, 6 months, once a year up to 5 years)
|
Y
|
N
|
|
Death (cause, date) by death certificate
|
Y
|
N
|
TOAST: Trial of Org 10172 in Acute Stroke Treatment criteria16; CCS: Causative Classification
System criteria17. CT: computed tomography; MRI: magnetic resonance imaging. Y: yes
(available); N: no (not available).
*Self-declared skin color
**current moderate or heavy drinker was defined as 50 g/day, which is equivalent to
500 mL or two glasses of wine, 1000 mL of beer or 5 units of spirits, or being intoxicated
at least once a week.
Data and blood sample collection
Patient selection and blood collection were performed by neurologists and nurses,
respectively. Research nurses invited the controls to enroll from among the accompanying
persons willing to participate who fulfilled the inclusion criteria. After clarifying
the aims of the JSB and the corresponding roles of all parties involved, blood sampling
(phase I: 12 mL by venous puncture employing common EDTA-containing vacuum tubes;
phase II: finger puncture followed by drying and storage on an FTA Elute Card®, Whatman, Kent, United Kingdom) of patients and controls was performed by research
nurses. Baseline data included self-declared skin color, collected in strict accordance
with the mandatory procedures established by the Brazilian Institute of Geography
and Statistics for purposes of population census. Demographics, socio-economic data,
cardiovascular risk factors, National Institutes of Health Stroke Scale (clinical
stroke severity) and functional outcome can be extracted from the Joinville Stroke
Registry, which also includes causes and dates of death. The follow-up of patients
is being carried out by phone calls by previously-trained research nurses in one,
three, six, nine and 12 months in the first year and once a year for the following
four years after stroke diagnosis. Patients are asked about regular visits to a general
physician, blood pressure control, results of glycated hemoglobin and cholesterol
levels, and smoking habits.
An identification code, which comprises numbers that denote the date of recruitment,
contributing site and entry order, was assigned to each patient enrolled. Controls
were identified using numbers that keep their relationship with each matched patient.
These identification codes link clinical data and demographics with deposited DNA
samples and only the biobank managers or authorized personnel have access to the key
linking samples and phenotypic data.
Processing of samples, DNA extraction and quality control
Blood samples from phase I were maintained at 4°C until DNA extraction. Whole blood
samples were centrifuged for 10 minutes at 2,500 x g for separation of the buffy coat,
which contains most of the white blood cells. Thereafter, genomic DNA was obtained
by the classic “phenol-chloroform” procedure for total DNA extraction. The DNA was
resuspended in 200 μL of buffer TE (10 mM Tris-HCl; 1 mM EDTA; pH 8.0). For phase
II, we adopted the procedure for large-amount DNA extraction, starting with three
paper punches of 3 mm circles, as recommended by the manufacturer of the FTA Elute
Card®. The purity and yield of DNA extracted were evaluated by spectrophotometry at 260
and 280 nm (Epoch, Biotek Instruments, Winooski, USA). Long-term storage was considered
for A260/A280 ratios in the range 1.8–2.0. Finally, DNA was stored in aliquots at −80°C.
Ethical considerations
The JSB development plan and the informed consent forms were both approved by the
Institutional Review Board of the University of the Region of Joinville and by the
Brazilian National Commission for Ethics in Research (protocol 25000.142907/2013-07),
which holds the final approval prerogative for biobanks in Brazil. The adopted standards
and procedures strictly follow the ethical rules that came into force in 2011 and
are described in Resolution 441/11 of the National Health Council and in Ordinance
2.201 of the Brazil Ministry of Health, which correspond to the national guidelines
for biorepositories and biobanks of human biological material for research purposes.
The JSB also follows the international ethical regulations. All volunteers (patients
and controls) are enrolled after written informed consent has been given freely by
the participants or their legal representatives. The freedom to refuse participation
or withdraw at any time is guaranteed, and the confidentiality of personal data is
ensured in all circumstances. Free access to information and associated implications
is guaranteed to participants, thereby respecting the expression of individual will.
RESULTS
Since 2010, a total of 5,970 DNA samples have been registered belonging to patients
(2,688) and controls (3,282). The mean ages of patients and controls were 64.6 ± 19.3
and 56.9 ± 14.9 years, respectively. Men were more represented among the patients
(64%; 1,731/2,688), while women were more prevalent among controls (68%; 2,226/3,282).
[Table 2] shows the distribution of patients and controls per city, as well as their self-declared
skin color. White individuals predominated in the south (88%) and south-east (55%)
regions and brown in the north-east (53%) and central (41%) regions.
Table 2
Baseline characteristics of stroke patients and controls in the Joinville Stroke Biobank
database.
|
City
|
Campo Grande
|
Canoas
|
Joinville
|
Sertãozinho
|
Sobral
|
All
|
|
Demography
|
|
|
|
|
|
|
|
Cases (n)
|
507
|
187
|
1619
|
175
|
200
|
2688
|
|
Mean age (±SD) (years)
|
65.2 (14.9)
|
64.5 (14.3)
|
64.3 (14.1)
|
64.9 (15.1)
|
6 7.1 (16.1)
|
64.6 (19.3)
|
|
Male [n(%)]
|
290 (57.2)
|
100 (53.5)
|
1185 (73.2)
|
56 (32)
|
100 (50)
|
1731 (64.4)
|
|
Skin color [% (n/N)]
|
|
|
|
|
|
|
|
White
|
46.9
|
49.7
|
88.2
|
55.4
|
29.5
|
71.2
|
|
(238/507)
|
(93/187)
|
(1428/1619)
|
(97/175)
|
(59/200)
|
(1915 /2688)
|
|
Black
|
7.7
|
16.0
|
5.4
|
13.1
|
16.0
|
7.8
|
|
(39/507)
|
(30/187)
|
(87/1619)
|
(23/175)
|
(32/200)
|
(211/2688)
|
|
Brown
|
41.4
|
20.9
|
6.0
|
29.1
|
53.5
|
18.8
|
|
(210/507)
|
(39/187)
|
(97/1619)
|
(51/175)
|
(107/200)
|
(504/2688)
|
|
Native American
|
2.0
|
2.7
|
0.2
|
1 .1
|
0.5
|
0.8
|
|
(10/507)
|
(5/187)
|
(4/1619)
|
(2/175)
|
(1/200)
|
(22/2688)
|
|
Yellow
|
1. 4
|
0.5
|
0 .1
|
1 .1
|
0.5
|
0.5
|
|
(7/507)
|
(1/187)
|
(2/1619)
|
(2/175)
|
(1/200)
|
(13/2688)
|
|
Not-declared
|
0.6
|
10.2
|
0 .1
|
0.0
|
0.0
|
0.9
|
|
(3/507)
|
(19/187)
|
(1/1619)
|
0.0
|
0.0
|
(23/2688)
|
|
Controls (n)
|
180
|
24
|
2586)
|
276
|
216
|
3282
|
|
Mean age (±SD) years)
|
50.2 (14.6)
|
64.56 (13.1)
|
68.3 (14.7)
|
50.2 (13,9)
|
66.3 (15,5)
|
56.9 (14.9)
|
|
Male [n(%)]
|
61 (33.8)
|
14 (58.3)
|
865 (33.5)
|
65 (23.5)
|
51 (23.6)
|
1056 (32.2)
|
|
Skin color [% (n/N)]
|
|
|
|
|
|
|
|
White
|
53.9
|
62.5
|
90.4
|
46.7
|
32.9
|
80.7
|
|
(97/180)
|
(15/24)
|
(2338/2586)
|
(129/276)
|
(71/216)
|
(2650/3282)
|
|
Black
|
0.6
|
25.0
|
2.7
|
6.9
|
6.5
|
3.4
|
|
(1/180)
|
(6/24)
|
(71/2586)
|
(19/276)
|
(14/216)
|
(111/3282)
|
|
Brown
|
40.0
|
8.3
|
6.4
|
44.2
|
4 7.7
|
14.2
|
|
(72/180)
|
(2/24)
|
(166/2586)
|
(122/276)
|
(103/216)
|
(465/3282)
|
|
Native American
|
0.6
|
4.2
|
0.0
|
0.4
|
0.9
|
0.2
|
|
(1/180)
|
(1/24)
|
(1/2586)
|
(1/276)
|
(2/216)
|
(6/3282)
|
|
Yellow
|
0.6
|
0.0
|
0 .1
|
1. 4
|
0.0
|
0.2
|
|
(1/180)
|
0
|
(3/2586)
|
(4/276)
|
0
|
(8/3282)
|
|
Not-declared
|
4.4
|
0.0
|
0.3
|
0.4
|
12.0
|
1. 3
|
|
(8/180)
|
0
|
(7/2586)
|
(1/276)
|
(26/216)
|
(42/3282)
|
[Table 3] shows the distribution of stroke diagnoses of patients in the different cities.
Most of the patients (76%; 2,031/2,688) were IS, followed by 12% (319/2,668) transient
ischemic attacks, 9% (243/2,668) hemorrhagic stroke and 3% (95/2,688) subarachnoid
hemorrhage. A quarter of all stroke patients were aged ≤ 55 years.
Table 3
Major stroke diagnosis of patients in the Joinville Stroke Biobank database per city
[% (n/N)].
|
Variable
|
City
|
|
Campo Grande
|
Canoas
|
Joinville
|
Sertãozinho
|
Sobral
|
All
|
|
IS
|
16.5 (335/2031)
|
7. 5 (152/2031)
|
60.9 (1238/2031)
|
5.6 (112/2031)
|
9.5 (194/2031)
|
75.5 (2031/2688)
|
|
TIA
|
20.6 (66/319)
|
1.6 (5/319)
|
73.1 (233/319)
|
4.7 (15/319)
|
0
|
11.9 (319/2688)
|
|
HS lobar
|
-
|
-
|
11.7 (28/243)
|
0
|
-
|
-
|
|
HS non-lobar
|
-
|
-
|
7. 8 (19/243)
|
0
|
-
|
-
|
|
HS non-classified
|
34.6 (84/243)
|
11.1 (27/243)
|
20.1 (49/243)
|
13.9 (34/243)
|
0.8 (2/243)
|
9.1 (243/2688)
|
|
SAH
|
23.1 (22/95)
|
3.5 (3/95)
|
54.6 (52/95)
|
14.6 (14/95)
|
4.2 (4/95)
|
3.5 (95/2688)
|
|
Young adult (≤ 55 years old)
|
4.3 (118/2688)
|
0.4 (13/2668)
|
15.7 (424/2688)
|
1.9 (52/2688)
|
1.9 (53/2688)
|
24.5 (660/2688)
|
|
Nuclear cases
|
0
|
0
|
0.4 (7/1619)
|
0
|
0
|
0.4 (7/2688)
|
|
All cases
|
18.9 (507/2688)
|
6.9 (187/2688)
|
60.2 (1619/2688)
|
6.5 (175/2688)
|
7. 5 (200/2688)
|
2688
|
IS: ischemic stroke; TIA: transient ischemic attack; HS: hemorrhagic stroke; SAH:
subarachnoid hemorrhage.
The distribution of IS cases by TOAST criteria ([Table 4]) was: 20% (426/2,031) cardioembolic; 22% (440/2,031) small-artery occlusion; 16%
(317/2,031) large-artery atherosclerosis; 40% undetermined (807/2,031) and 2% (41/2,031)
other determined cause. After 2012, patients were also classified by CCS criteria
(56%; 1,503/2,688). As expected, among IS patients, the most common IS subtype was
undetermined, not only by TOAST criteria (40%; 807/2,031), but also by CCS criteria
(47%; 718/1,503). Out of those 807 undetermined IS patients classified by TOAST criteria,
55% (446/807) were patients with an incomplete evaluation, 42% (340/807) were cryptogenic
and 3% (21/807) were undetermined with two or more possible causes. Out of those 718
undetermined IS subtypes classified by CCS criteria, 25% (370/1,503) were IS patients
with incomplete evaluation, 17% (262/1,503) were unknown other cryptogenic, 8% (61/1,503)
were unknown cryptogenic embolism and 1% (25/1,503) were unclassified.
Table 4
Ischemic stroke subtypes diagnosis of patients in the Joinville Stroke Biobank database
[% (n/N)].
|
Variable
|
City
|
|
Campo Grande
|
Canoas
|
Joinville
|
Sertãozinho
|
Sobral
|
All
|
|
IS subtype by TOAST
|
|
|
|
|
|
|
|
Large-artery atherosclerosis
|
6.9 (22/317)
|
5 .1 (16/317)
|
81.8 (259/317)
|
2.8(9/317)
|
3.4 (11/317)
|
15.8 (317/2031)
|
|
Cardioembolic
|
12.2 (52/426)
|
3.2 (14/426)
|
78.3 (333/426)
|
2.8(12/426)
|
3.5 (15/426)
|
20.2 (426/2031)
|
|
Small-artery occlusion
|
17.1 (75/440)
|
4.3 (19/440)
|
6.9 (307/440)
|
0.9(4/440)
|
7. 9 (35/440)
|
21.7 (440/2031)
|
|
Other determined cause
|
17.1(7/41)
|
0
|
82.9 (34/41)
|
0
|
0
|
2.2(41/2031)
|
|
Undetermined
|
|
|
|
|
|
|
|
Cryptogenic/ESUS
|
5.6 (19/340)
|
16.2 (55/340)
|
51.8 (176/340)
|
2.6 (9/340)
|
23.8 (81/340)
|
16.8 (340/2031)
|
|
Incomplete investigation
|
35.6(159/446)
|
10.8 (48/446)
|
24.6 (110/446)
|
17.5 (78/446)
|
11.4 (51/446)
|
22.1 (446/2031)
|
|
Two or more determined causes
|
4.7 (1/21)
|
0
|
90.6 (19/21)
|
0
|
4.7 (1/21)
|
1.2 (21/2031)
|
|
IS subtype by CCS
|
|
|
|
|
|
|
|
Large-artery atherosclerosis
|
|
|
|
|
|
|
|
|
Evident
|
5.3 (8/150)
|
10.7 (16/150)
|
76.7 (115/150)
|
0
|
7. 3 (11/150)
|
9.9 (150/1503)
|
|
|
Probable
|
0
|
0
|
92.1(35/38)
|
7.9(3/38)
|
0
|
2.4 (38/1503)
|
|
|
Possible
|
12(3/25)
|
0
|
64 (16/25)
|
24 (6/25)
|
0
|
1.5 (25/1503)
|
|
Cardioembolic
|
|
|
|
|
|
|
|
Evident
|
11.7 (18/154)
|
7.1 (11/154)
|
75.3 (116/154)
|
3.2 (5/154)
|
2.6 (4/154)
|
10.2 (154/1503)
|
|
Probable
|
0
|
0
|
100 (40/40)
|
0
|
0
|
2.5 (40/1503)
|
|
Possible
|
15.7 (8/51)
|
0
|
49.1 (25/51)
|
13.6(7/51)
|
21.6 (11/51)
|
3.3 (51/1503)
|
|
Small-artery occlusion
|
|
|
|
|
|
|
|
|
Evident
|
6.8(12/175)
|
10.3 (18/175)
|
6.5 (113/175)
|
5.7(1/175)
|
17.7 (31/175)
|
11.5 (175/1503)
|
|
|
Probable
|
15.1 (10/66)
|
0
|
8.2(54/66)
|
3 .1 (2/66)
|
0
|
4.3 (66/1503)
|
|
|
Possible
|
2.1(1/48)
|
0
|
87.5 (42/48)
|
2.1(1/48)
|
8.3(4/48)
|
3.1 (48/1503)
|
|
Other determined cause
|
|
|
|
|
|
|
|
|
Evident
|
11.1 (4/36)
|
0
|
86.2(31/36)
|
0
|
2.7 (1/36)
|
2.3 (36/1503)
|
|
|
Probable
|
0
|
0
|
100(2/2)
|
0
|
0
|
1.2 (2/1503)
|
|
|
Possible
|
0
|
0
|
0
|
0
|
0
|
0
|
|
Undetermined
|
|
|
|
|
|
|
|
|
Unknown cryptogenic embolism
|
0
|
0
|
80.3(49/61)
|
0
|
19.7 (12/61)
|
4.1 (61/1503)
|
|
|
Unknown other cryptogenic
|
6.1 (16/262)
|
20.2(53/262)
|
44.6 (117/262)
|
26.7(7/262)
|
26.3 (69/262)
|
17.4 (262/1503)
|
|
|
Incomplete evaluation
|
27.6 (102/370)
|
12.9 (48/370)
|
24.3 (90/370)
|
21.3 (79/370)
|
13.8 (51/370)
|
24.6 (370/1503)
|
|
|
Unclassified
|
4(1/25)
|
24(6/25)
|
64 (16/25)
|
4 (1/25)
|
4 (1/25)
|
1.7 (25/1503)
|
|
Total IS by TOAST (since 2010)
|
16.5 (335/2031)
|
7. 5 (152/2031)
|
60.9 (1238/2031)
|
5.6 (112/2031)
|
9.5 (194/2031)
|
75.5 (2031/2688)
|
|
Total IS by CCS (since 2012)
|
12.2 (183/1503)
|
10.1 (152/1503)
|
57.3 (861/1503)
|
74.5 (112/1503)
|
12.9 (195/1503)
|
72. 0(1503/2100)
|
IS: ischemic stroke; TOAST: Trial of Org 10172 in Acute Stroke Treatment criteria16;
ESUS: Embolic Strokes of Undetermined Source; CCS: Causative Classification System
criteria17.
DISCUSSION
Up to October 2016, DNA from 5,970 blood samples distributed among patients (2,688)
and controls (3,282) had been extracted and stored. Since it is believed that most
of the genetic variants identified in stroke studies will have small, individual effects
on disease risk, efforts aimed at the successful discovery of the impact of genes
have required large sample sizes (usually involving tens or hundreds of thousands
of cases and controls) to achieve sufficient statistical power[10]. In addition, as with any case-control study, controls should be representative
of cases[17],[18].
With this in mind, the JSB comprises well-defined phenotypes and samples of high-quality
DNA. The priority recruitment of spouses or partners as case-matched controls represents
a suitable strategy for minimizing differences due to environmental effects to which
the participants (patients and controls) have been exposed. As controls are collected
in the same geographic region as patients, it is reasonable to expect a homogeneous
distribution of ethnic backgrounds between patients and controls. On the other hand,
despite having inflated influences due to shared variants and environmental effects
between patients and controls, the JSB may also provide relevant information on familial
aggregation analysis. It is also noteworthy to clarify that the JSB has a forecast
of unlimited continuity as, among other factors, the Joinville Stroke Registry is
supported by a municipal law. This gives rise to the fact that if there are cases
of stroke in individuals previously categorized as a control (since stroke events
may happen later in life), their biobank status changes, but maintains the traceability
of the change. This allows the possibility of evaluation of the impact on results
of any studies already performed with the data and samples of those individuals who
no longer belong to the control group. As far as we know, this is the first stroke
biobank in Latin America, a region with a massive race mixture.
Under TOAST, IS cases are assigned to one of three main subtypes (small-artery occlusion,
large-artery atherosclerosis, cardioembolic); to a rare cause (e.g., carotid or vertebral
artery dissection); or remain undetermined (because of more than one potential cause,
incomplete investigation or no apparent cause despite complete investigation/cryptogenic
stroke). The proportion in the undetermined category varies, but can be substantial
(up to 40% or more), and is usually excluded from ischemic subtype-specific analyses[19],[20]. In our cohort, between 22% (TOAST criteria) and 25% (CCS criteria) were undetermined
IS due to incomplete investigation. All patients with incomplete investigation were
assigned this diagnosis. In fact, the highly diverse sociodemographic scenarios at
sites contributing to the JSB, including difficulties related to health-care system
access and imaging diagnostic workup, might have influenced significantly. Nevertheless,
independent raters may perform blinded readjudications of all JSB IS diagnoses whenever
considered necessary.
Globally, large DNA biobanks have been contributing to the understanding of the different
genetic architectures and pathophysiological aspects of stroke. In this regard, the
UK Biobank (European and British Asian), Brains-SA (India and Sri Lanka), Bio-Repository
of DNA in Stroke – Middle East (Qatar)[21], China Kadoorie Biobank[22] and other collaborative initiatives, such as the International Stroke Genetics Consortium,
have contributed to the identification of several loci associated with IS subtypes.
Genome-wide association study approaches have been adopted for investigating many
complex diseases, such as stroke, and have been effective in identifying new genetic
variants associated with the risk of disease. However, for stroke studies, genome-wide
association studies have so far been applied principally in European, North American
and Japanese populations[3],[23].
In particular, four loci (PITX2, HDAC9, ZFHX3 and 12q24.2) were convincingly implicated by genome-wide association studies as associated
with IS in European-ancestry patients[24],[25]. Recently, a novel locus (rs12122341) was identified at 1p13.2 (near TSPAN2), associated with large-artery atherosclerosis stroke, and the locus 12q24 (near
ALDH2) was associated with small-artery occlusion[26]. In addition, the gene TRPV3 was associated with cardioembolic stroke after replication of exome sequencing analysis,
demonstrating that two polymorphisms were associated with cardioembolic stroke risk
in two cohorts, with rs151091899 being the most significant[27]. Therefore, replication studies in other populations are determinants in understanding
the significance of such polymorphisms as stroke risk factors. The Brazilian population
is one of the most heterogeneous populations in the world, comprising an admixture
of Native Americans, Europeans and Africans[28]. The admixture process occurred through different means in Brazilian geographic
regions. For instance, the Native American contribution is more pronounced in northern
Brazil, the African contribution is more elevated in the north-east, and the south
features a European predominance with few Native American and African influences[29]. Consequently, the JSB, unlike other biobanks[8],[22],[23], comprises an ideal admixed population with great ethnic variability linked to epidemiological
and clinical data, which may allow confirmation of polymorphisms found in specific
populations, contributing to global understanding of genetic mechanisms involved in
stroke.
In conclusion, the clinical, environmental and epidemiological issues related to the
different types of stroke, combined with the large genetic variability captured by
the JSB, may increase the chance of identifying new and relevant factors predisposing
individuals to stroke (and recovery) in Brazil. Our aim is to continue patient assessment
and blood collection, as well as begin genotyping samples in the near future. Some
initial studies are already being conducted in the Laboratory of Molecular Genetics
at the University of Campinas with the support of the Brazilian Institute of Neuroscience
and Neurotechnology and the use of a genome-wide association genotyping platform (Affymetrix™
6.0).
