Supplementary Methods
Patient selection
CVD was defined by the presence of at least one of the following features: (I)
myocardial infarction, proven by at least two of the following criteria: (a) classical
symptoms (chest pain that may radiate, oppressive pain, nausea, sweating and
absence of chest-wall tenderness on palpation), (b) specific electrocardiographic
abnormalities, (c) elevated cardiac enzymes (e. g. troponin and elevated creatinekinase (CK) and its myocardial band enzyme (CK-MB), levels); (II) percutaneous
coronary intervention or other invasive procedures; (III) coronary artery bypass grafting;
(IV) angina pectoris, diagnosed as classical symptoms (recurrent attacks of retrosternal
pain brought on by effort and emotion and relieved by rest and the administration of
nitroglycerin) in combination with at least one unequivocal result of one of the following:
(a) exercise test, (b) nuclear scintigram, (c) dobutamine stress ultrasound, (d) a more
than 70% stenosis on a coronary angiogram or (f) requiring treatment (V) ischemic
stroke, demonstrated by CT- or MRI scan; (VI) documented transient ischemic attack.
Blood pressure was measured using an oscillometric blood pressure device (Omron,
Hoofddorp, The Netherlands).
RNA isolation and RT-PCR analysis
Total cellular RNA from the buffy coat fraction was extracted using TriZol (Life
Technologies, The Netherlands) according to manufacturer’s protocol and reverse
transcribed using random primers and iScript reverse transcriptase (BioRad). The
conditions were 5 min 25°C, 30 in 42°C, 5 min 85°C. mRNA expression levels were
measured using SensiFast Sybr Green master mix (GE-Biotech) on CFX386 system
(BioRab, the Netherlands). Primers were designed using Primer3 software and were
exon-intron boundary crossing (Table S1). RT-PCR conditions were: 10 min 95°C
followed by 40 cycles 15’ 95°C 30’ 60°C and finally a melt reaction going from 65°-95°C
in 5’ per grade. Gene expression was calculated using the 2ΔΔCt method using 36B4
as reference gene.
Immunohistochemistry
Sections were fixed in 4% paraformaldehyde and subsequently embedded in paraffin.
For immunostaining, paraffin sections were deparaffinised before endogenous
peroxidase quenching. After blocking with Ultra V Block (Thermo Fischer Scientific,
Fremont, CA, USA) slides were incubated with anti-MCF2L 1:250 (rabbit polyclonal antiMCF2L, WH0023263M1, Sigma, Munich, Germany) overnight at 4ºC. Staining was
performed with anti-rabbit Horse Radish Peroxidase (HRP)-labelled IgG (ImmunoLogic,
Duiven, The Netherlands) followed by Bright DAB+ visualization (ImmunoLogic, Duiven,
The Netherlands). Counterstaining was performed using hematoxylin and slides were
cover-slipped with VectaMount (Vector Laboratories, Burlingname, CA, USA). Positive
controls consisted of samples of human tonsil. Negative controls consisted of
experimental tissues stained without the addition of primary antibodies following the
same protocol. Microscopy pictures were analyzed using Adobe Photoshop CS4.
To explore cellular localization of MCF2L in human atherosclerotic lesions the
sequential alkaline phosphatase (AP) double staining method was used as described
elsewhere [1]. MCF2L was stained in combination with either smooth muscle cell α-actin
(SMA) (1:500; mouse 1A4 antibody, DAKO, Glostrup, Denmark), macrophage CD68
(1:100; mouse monoclonal anti human CD68 PG-M1, DAKO, Heverlee, Belgium),
endothelial cell CD34 (1:1000; mouse anti human CD34Q, Bend10, ThermoFischer
Scientific, Waltham, MA, USA), antigen presenting cells (CD11c; 1:50; mouse
monoclonal 5D11, Monosan, Uden, The Netherlands), CD8 lymphocytes (1:50; mouse
monoclonal anti-human CD8, CD8/144B, DAKO, Heverlee, Belgium) and rabbit anti
human CD3 (1:5000;, IgG monoclonal SP7, ThermoFisher Scientific).
Visualization was performed with vector blue (Vector Laboratories) for MCF2L and
vector red (Vector Laboratories) for SMA, CD68, CD34, CD3, CDC11c and CD8 and
cover slipped.
Figure S1: The results of linkage analysis using Allegro software.
Figure S2: mRNA expression of MCF2L, RAC1 and RHOA in total circulating white
blood cells in 4 heterozygous carriers of the MCF2L c.2066A>G; p.(Asp689Gly) variant
and one non-carrier relative. RNA expression was analyzed in three white blood cell
RNA fractions per person. MCF2L expression was significantly decreased (P<0.05) in
carriers of the variant.
P<0.05
0.5
0.5
rel to 36B4
0.4
rel to 36B4
0.4
0.3
0.2
0.2
0.1
0.1
0.0
0.0
MCF2LAsp689
MCF2LGly689
MCF2LAsp689
MCF2LGly689
0.6
rel to 36B4
0.3
0.4
0.2
0.0
MCF2LAsp689
MCF2LGly689
Figure S3: Specifity of immunostaining of MCF2L.
A. Single staining of MCF2L (brown) in control tonsil tissue.
B. Negative control in tonsil tissue using only the secondary antibody.
A
B
Table S1: Clinical characteristics of the relatives.
Patient Sex Type of
CVD
Age
CVD
Medication
MCF2L
c.2066A>G;
p.(Asp689Gly)
Hyperte
nsion
(years)
Smoking BMI
(kg/m2)
I-1
F
None
N/A
None
No
No
Yes
23
II:3
F
None
N/A
None
No
No
No
22
II:1
F
PTCA
62
Lipitor 40
mg
Yes
No
No
22
II:2
M
AP
46
Inegy 40 mg Yes
No
Yes
25
II:4†
M
AMI
43
Lipitor 40
mg
Yes
No
No
23
II:5
M
CABG
40
Lipitor 20
mg
Yes
No
Yes
26
II:6
F
AMI/
PTCA
39
Zocor 20 mg Yes
No
No
24
PTCA = Percutaneous Transluminal Coronary Angioplasty; AP = Angina Pectoris; AMI = Acute Myocardial Infarction;
CABG = coronary artery bypass surgery; BMI = body mass index (kg/cm2). † = index case. Subjects were considered
smokers if they were current smokers or when they quitted smoking within the last 5 years. Hypertension was defined as
a systolic blood pressure > 140 mmHg and/or diastolic blood pressure > 80 mmHg or the use of anti-hypertensive
lowering drugs. Diabetes mellitus was defined as fasting plasma glucose ≥ 7.0 mmol/l or 2h plasma glucose ≥ 11.1 mmol/l
as defined by the World Health Organization (WHO).
Tabel S2: Linkage intervals with LODscore > -2 (NCBI137/hg19).
Chr#
Start
SNP
END
Chr7
64406438
rs10949950
65357776
Chr7
66689728
rs11761805
67026320
Chr7
117378355
rs739798
132945754
Chr9
136927656 rs1076148
141213431
Chr11
123289850 rs2156443
131697483
Chr13
112349868 rs9560166
113839747
Chr14
0
start
20709688
Chr17
64816464
rs4791032
65343997
Chr18
429354
rs17564131
4780620
Chr20
2340973
rs6082889
9378671
Chr21
43448796
rs220229
44715784
Chr22
0
start
17254399
SNP
rs35850374
rs4576304
rs10215367
End
rs11603321
rs515863
rs7156806
rs9904424
rs6506247
rs1997696
rs762391
rs2190742
SUM (bp)=
Interval
951338
336592
15567399
4285775
8407633
1489879
20709688
527533
4351266
7037698
1266988
17254399
82186188
Table S3: Clinical characteristics of participants in the PAS cohort (n = 935).
Male/Female Age (years)
BMI
LDL-c
HDL-c
TG
708/227
26.9 ± 1.2
3.11 ± 1.29
1.14 ± 0.32
2.06 ± 3.41
43 ± 5
Data are expressed as number (N) and presented as mean ± standard
deviation. BMI = body mass index in kg/m2; LDLc = low-density lipoprotein
cholesterol; HDL-c = high-density lipoprotein cholesterol; TG = plasma
triglycerides. All lipid data are expressed as mmol/l.
Table S4: Genotyping in replication cohort.
Cohort name
n
CAMSAP1
ZC3HC1
MCF2L
PAS
935
n/a*
6
1†
Sanquin
1440 13
3
0
CBR
8000 n/a*
5
0
PAS = Premature AtheroSclerosis; Sanquin = Sanquin Blood Bank; CBR =
Cambridge Bioresource and n/a = not applicable. * = Genotyping was not
performed due to a high frequency in the Sanquin cohort. † = Pedigree Index
case, other pedigree members were not included in the database.
Supplementary references
[1] van der Loos CM. Multiple immunoenzyme staining: methods and
visualizations for the observation with spectral imaging. J Histochem
Cytochem 2008;56;313-28.