Download Male-Specific Region of the Y Chromosome and Cardiovascular Risk

Male-Specific Region of the Y Chromosome and
Cardiovascular Risk
Phylogenetic Analysis and Gene Expression Studies
Lisa D.S. Bloomer, Christopher P. Nelson, James Eales, Matthew Denniff, Paraskevi Christofidou,
Radoslaw Debiec, Jasbir Moore, Cardiogenics Consortium, Ewa Zukowska-Szczechowska,
Alison H. Goodall, John Thompson, Nilesh J. Samani, Fadi J. Charchar, Maciej Tomaszewski
Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017
Objective—Haplogroup I of male-specific region of the human Y chromosome is associated with 50% increased risk of
coronary artery disease. It is not clear to what extent conventional cardiovascular risk factors and genes of the malespecific region may explain this association.
Approach and Results—A total of 1988 biologically unrelated men from 4 white European populations were genotyped
using 11 Y chromosome single nucleotide polymorphisms and classified into 13 most common European haplogroups.
Approximately 75% to 93% of the haplotypic variation of the Y chromosome in all cohorts was attributable to I, R1a,
and R1b1b2 lineages. None of traditional cardiovascular risk factors, including body mass index, blood pressures, lipids,
glucose, C-reactive protein, creatinine, and insulin resistance, was associated with haplogroup I of the Y chromosome in
the joint inverse variance meta-analysis. Fourteen of 15 ubiquitous single-copy genes of the male-specific region were
expressed in human macrophages. When compared with men with other haplogroups, carriers of haplogroup I had ≈0.61and 0.64-fold lower expression of ubiquitously transcribed tetratricopeptide repeat, Y-linked gene (UTY) and protein
kinase, Y-linked, pseudogene (PRKY) in macrophages (P=0.0001 and P=0.002, respectively).
Conclusions—Coronary artery disease predisposing haplogroup I of the Y chromosome is associated with
downregulation of UTY and PRKY genes in macrophages but not with conventional cardiovascular risk factors. (Arterioscler Thromb Vasc Biol. 2013;33:1722-1727.)
Key Words: association ◼ cardiovascular risk ◼ DNA ◼ gene ◼ genetics ◼ mRNA
T
he human Y chromosome is one of the smallest in the
genome. Its major part, the male-specific region (MSY),
constitutes ≈95% of its length, does not recombine with the X
chromosome during meiosis, and is inherited as an indivisible
unit from fathers to sons.1 Because of its haploid nature, the
MSY has been routinely excluded from large-scale genomewide association studies and is one of the most underexplored
portions of the human DNA.
A growing body of evidence suggests that the human MSY
may contribute to cardiovascular risk in men. Several studies conducted in the general population revealed associations
between single nucleotide polymorphisms of the MSY and
blood pressure,2–4 low-density lipoprotein-cholesterol,5 a surrogate of proatherogenic B phenotype of low-density lipoprotein-cholesterol particles, and paternal history of myocardial
infarction.5 Our recent phylogenetic analysis showed that one
of the most common European lineages of the Y chromosome
(haplogroup I) was associated with increased risk of coronary
artery disease (CAD) among British men.6 Indeed, the analysis of ≈3000 men from 2 British cohorts revealed that compared with other lineages of the Y chromosome, haplogroup I
increased risk of CAD by ≈50%.6 This makes haplogroup I of
the Y chromosome one of the strongest common genetic risk
factors of CAD known to date.6
The biological mechanisms underlying the association
between CAD and human Y chromosome are not fully
understood. Our previous transcriptome-wide analysis of
human macrophages revealed that compared with other Y
chromosome lineages, haplogroup I was associated with
suppression of adaptive immunity and increased response to
inflammation.6 Specifically, 19 Kyoto Encyclopedia of Genes
and Genomes (KEGG) pathways connected by genes of
immunity and inflammation showed differential regulation in
macrophages between men with haplogroup I and carriers of
the other MSY lineages.6 However, it is not clear which genes
of the MSY may drive the association between haplogroup I
Received on: October 27, 2012; final version accepted on: April 11, 2013.
From the Department of Cardiovascular Sciences (L.D.S.B., C.P.N., J.E., M.D., P.C., R.D., J.M., A.H.G., N.J.S., F.J.C., M.T.) and Department of Health
Sciences (J.T.), University of Leicester, Leicester, United Kingdom; Leicester National Institute for Health Research Biomedical Research Unit in Cardio­
vascular Disease, Glenfield Hospital, Leicester, United Kingdom (C.P.N., A.H.G., J.T., N.J.S., M.T.); Department of Internal Medicine, Diabetology, and
Nephrology, Medical University of Silesia, Zabrze, Poland (E.Z.-S.); and School of Science and Engineering, University of Ballarat, Ballarat, Australia (F.J.C.).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.301608/-/DC1.
Correspondence to Maciej Tomaszewski, MD, FAHA, FRCP, Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing,
Glenfield Hospital, Leicester LE3 9QP, United Kingdom. E-mail [email protected]
© 2013 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1722
DOI: 10.1161/ATVBAHA.113.301608
Bloomer et al Y Chromosome and Cardiovascular Risk 1723
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and predisposition to CAD. Of the 27 distinct protein-coding
genes of the MSY, 15 single-copy genes with homologs on
the X chromosome (so-called X-degenerate genes) show
mostly ubiquitous tissue expression (including cells and
organs of cardiovascular system) and thus are the strongest
biological candidates for further functional studies on MSY
and cardiovascular disorders. So far little is known about their
function and specific roles in male health and disease.
It is also not clear whether the altered immune response may
entirely explain the increased susceptibility to CAD in men
with haplogroup I. The effect of the MSY on CAD risk could
be mediated, at least to some extent, by traditional cardiovascular and metabolic risk factors, such as adiposity, blood pressure,
lipids, glucose, or insulin resistance. Taken together these risk
factors explain <50% individual variation in susceptibility to
CAD. Although in our previous study the association between
haplogroup I and CAD in the West of Scotland Coronary
Prevention Study was independent of some sociodemographic
and conventional cardiovascular risk factors, this analysis was
limited only to 1 cohort of middle-age Scottish men, many of
whom were on pharmacological treatment with a potential to
affect measurements of several key phenotypes.6 Thus, it is difficult to extrapolate these data into apparently healthy general
population of European men.
Here, we examined whether CAD-related haplogroup I of
the Y chromosome is associated with either previously investigated or new traditional cardiovascular risk factors in young
men of white European ethnicity recruited from the general
population. This was followed by a systematic comparative
expression analysis of 15 ubiquitous X-degenerate genes of
the MSY in macrophages from carriers of haplogroup I and
men from other MSY lineages.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
In brief, 1940 biologically unrelated, apparently healthy men from 3
populations of white European ethnicity (Young Men Cardiovascular
Association Study 1-YMCA1,5 Young Men Cardiovascular
Association Study 2-YMCA27 and Genetic Regulation of Arterial
Pressure in Humans in the Community-GRAPHIC8) were included
in the genetic association analysis. Macrophage RNA from 48
white British men with a history of premature myocardial infarction (recruited into Cardiogenics Transcriptome Project)6 was used
in MSY genes expression studies. Reconstruction of the MSY phylogenetic tree was based on information provided by 11 bi-allelic
polymorphisms (M9, M35, M45, M89, M170, M173, M201, M207,
M269, M304, and SRY10831) genotyped in all cohorts, as reported
before (Table I in the online-only Data Supplement).6 This set of
polymorphic variants allows classification of >95% of European Y
chromosomes into major haplogroups (Y[xBR], BR[xE1b1b1,F],
E1b1b1, F*, G, I, J, K*, P*, R*, R1*, R1a, R1b1b2).9–11 The presence
of population stratification/population admixture was examined using
information provided by autosomal SNPs from previous array-based
experiments (GRAPHIC8, Cardiogenics6) or a panel of 34 ancestrally
informative SNPs (YMCA1 and YMCA2).12–15 Gene expression analysis of all ubiquitous single-copy genes of MSY was conducted using
quantitative real-time PCR.
Results
Clinical and Demographic Characteristics
A total of 1068 and 509 unrelated men in Young Men
Cardiovascular Association Study 1 (YMCA1) and YMCA2,
respectively, were informative in the Y chromosome phylogenetic analysis. In the Genetic Regulation of Arterial Pressure
in Humans in the Community (GRAPHIC) Study, 363 biologically unrelated men from the offspring generation provided full genetic information for haplogroup resolution. In
the Cardiogenics cohort, 48 men had full DNA and mRNA
information necessary for further haplogroup I–stratified gene
expression analysis. The clinical characteristics of YMCA1,
YMCA2, and GRAPHIC sons are shown in Table 1 and
are largely consistent with the phenotypic characteristics of
young, apparently healthy males from general population. The
basic phenotypic information on men from the Cardiogenics
Study included in this project is shown in Table II in the
online-only Data Supplement.
Table 1. Demographic and Clinical Characteristics of Men From YMCA1, YMCA2, and the GRAPHIC Study
Phenotype
YMCA1
YMCA2
GRAPHIC Sons
1068
509
363
Age, y
19.3 (3.6)
18.9 (3.3)
BMI, kg/m2
22.9 (3.0)
22.6 (3.0)
25.1 (4.1)
Clinic SBP, mm Hg
118.1 (13.2)
118.8 (13.2)
128.4 (13.0)
Clinic DBP, mm Hg
n
25.7 (5.0)
74.2 (7.9)
74.1 (7.9)
76.4 (9.6)
TC, mmol/L
4.3 (0.9)
3.7 (1.0)
4.6 (0.9)
HDL-C, mmol/L
1.2 (0.3)
1.1 (0.3)
1.3 (0.3)
Triglycerides, mmol/L
1.1 (1.0–1.1)
0.9 (0.8–0.9)
LDL-C, mmol/L
2.6 (0.9)
2.2 (0.8)
Glucose, mmol/L
Creatinine, µmol/L
CRP, mg/L
HOMA-IR
…
2.5 (0.7)
4.8 (0.7)
4.4 (0.8)
4.9 (0.8)
82.8 (11.1)
74.9 (9.8)
90.8 (9.1)
…
…
1.6 (1.6–1.7)
1.6 (1.6–1.7)
0.08 (0.07–0.08)
…
Data are mean and SD or geometric mean and 95% confidence intervals (triglycerides, CRP, and HOMA-IR). BMI indicates body mass index; CRP, C-reactive protein;
DBP, diastolic blood pressure; GRAPHIC, Genetic Regulation of Arterial Pressure in Humans in the Community; HDL-C, high-density lipoprotein-cholesterol; HOMAIR, homeostatic model assessment insulin resistance index; LDL-C, low-density lipoprotein-cholesterol; n, No. of subjects; SBP, systolic blood pressure; TC, total
cholesterol; and YMCA, Young Men Cardiovascular Association Study.
1724 Arterioscler Thromb Vasc Biol July 2013
Figure 1. Frequency of Y chromosome haplogroups in each
cohort. Colored bars and letters
represent each haplogroup
running from the evolutionarily oldest on the left to the
youngest of the right (youngest
haplogroup is shown in pink
[R1b1b2]); M and SRY markers
indicate the terminal marker of
each haplogroup; data are percentages of individuals in each
haplogroup. GRAPHIC sons
indicates Genetic Regulation
of Arterial Pressure in Humans
in the Community; and YMCA,
Young Men Cardiovascular
Association Study.
Phylogenetic Analysis of the Y Chromosome
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A total of 1988 individuals from YMCA1, YMCA2,
GRAPHIC, and Cardiogenics with sufficient genetic/phenotypic information were classified into 1 of 13 major European
MSY haplogroups. Approximately 75% to 93% of the haplotypic variation in these cohorts was attributable to 3 lineages
(I, R1a, and R1b1b2; Figure 1). As expected, haplogroup R1a
was the most common in Polish populations, whereas R1b1b2
predominated in the British men (Figure 1). Haplogroup I
(previously associated with increased risk of CAD) was the
second most common haplogroup in each cohort.
There was no evidence of population stratification/admixture in any of the cohorts (Figures I–III in the online-only Data
Supplement).
Association of Haplogroup I and
Major Cardiovascular Risk Factors:
Individual and Joint Analysis
The cohort-specific associations between haplogroup I and
conventional risk factors are shown in Tables III–V in the
online-only Data Supplement. None of traditional cardiovascular risk factors was associated with haplogroup I in the
meta-analysis of 1944 men from 3 populations (Table 2).
There was no evidence of heterogeneity in the joint analysis
(Table 2). Sensitivity analyses showed that adjustment for
blood pressure–lowering effect of antihypertensive treatment did not affect the association findings from analysis
of systolic blood pressure and diastolic blood pressure (data
not shown).
Association Between Haplogroup I and Expression
of Single-Copy MSY Genes in Human Macrophages
Of 15 MSY genes, 14 were expressed in human macrophages.
Two genes (ubiquitously transcribed tetratricopeptide repeat,
Y-linked gene [UTY] and protein kinase, Y-linked, pseudogene [PRKY]) showed statistically significant difference in
expression between men with haplogroup I and those with
other lineages of the Y chromosome after correction for multiple testing (Table VI in the online-only Data Supplement).
Specifically, carriers of haplogroup I had ≈0.61- and 0.64-fold
Table 2. The Association Between Haplogroup I and Cardiovascular Risk Factors in YMCA1, YMCA2, and GRPAHIC Sons:
Fixed-Effect Age-Adjusted Inverse Variance Meta-Analysis
Phenotype
β-Coefficient (SE)
BMI, kg/m
−0.07 (0.19)
0.709
0.590
0.14 (0.81)
0.865
0.555
2
Clinic SBP, mm Hg
P Value
Heterogeneity
Clinic DBP, mm Hg
−0.32 (0.49)
0.524
0.356
TC, mmol/L
−0.01 (0.06)
0.862
0.131
HDL-C, mmol/L
0.00 (0.02)
0.798
0.113
Triglycerides, mmol/L*
0.04 (0.03)
0.161
0.823
LDL-C, mmol/L
−0.01 (0.05)
0.775
0.017
Glucose, mmol/L
−0.01 (0.05)
0.820
0.445
Creatinine, µmol/L
−0.02 (0.62)
0.972
0.937
0.18 (0.17)
0.290
—
−0.11 (0.10)
0.263
0.624
CRP, mg/dL**
HOMA-IR*
Data are expressed as β-coefficients, SE, and levels of statistical significance from meta-analysis; heterogeneity, level of statistical significance from χ2 test for
heterogeneity. BMI indicates body mass index; CRP, C-reactive protein; DBP, diastolic blood pressure; GRAPHIC, Genetic Regulation of Arterial Pressure in Humans in the
Community; HDL-C, high-density lipoprotein-cholesterol; HOMA-IR, homeostatic model assessment insulin resistance index; LDL-C, low-density lipoprotein-cholesterol;
SBP, systolic blood pressure; TC, total cholesterol; and YMCA, Young Men Cardiovascular Association Study.
*YMCA1 and YMCA2 only; and **GRPAHIC sons only.
Bloomer et al Y Chromosome and Cardiovascular Risk 1725
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Figure 2. Macrophage expression of UTY and PRKY mRNA in
British men from Cardiogenics cohort. A, Differences in relative (β-actin normalized) expression of PRKY between men with
haplogroup I and carriers of other haplogroups. B, Differences
in relative (β-actin normalized) expression of UTY between men
with haplogroup I and carriers of other haplogroups. C, Linear
correlation in macrophage expression of UTY and PRKY.
lower age-adjusted levels of UTY and PRKY in macrophages
than men with non-I haplogroups (P=0.0001 and P=0.002,
respectively; Figure 2). There was a significant linear correlation in mRNA levels of both genes in human macrophages
(r=0.486; P=0.0005; Figure 2).
Discussion
Our data revealed that CAD-predisposing haplogroup I
of the Y chromosome6 was not associated with any major
conventional cardiovascular risk factors in apparently healthy
men from the general population of white European ancestry.
The systematic analysis of all ubiquitous single-copy MSY
X-degenerate genes revealed that a vast majority of them are
indeed expressed in human macrophages. We also uncovered a
significant downregulation of 2 MSY genes (UTY and PRKY)
in macrophages from men with haplogroup I when compared
with carriers of other lineages.
Our data from YMCA studies and the GRPAHIC cohort
provide important support for previously proposed notion that
the association between haplogroup I of the Y chromosome
and CAD is independent of traditional cardiovascular risk factors.6 Indeed, using continuous measures of cardiovascular
risk (essentially un-confounded by medications), we showed
that neither blood pressures nor metabolic phenotypes were
associated with haplogroup I of the Y chromosome among
apparently healthy men. The absence of association between
the Y chromosome and C-reactive protein in this analysis is
particularly important given that CAD-related haplogroup I
was linked to immunity and inflammation in the recent transcriptome-wide analysis of human macrophages.6 The data
collected here show that C-reactive protein, a simple measure
of low-grade inflammation16,17 and increasingly recognized
surrogate of cardiovascular risk,16 is unlikely to explain the
reported associations between haplogroup I and increased susceptibility to CAD6 or possibly higher risk of AIDS progression or failure of retroviral therapy.18
Our data also reveal that other correlates of CAD not
included in the previous observation,6 such as creatinine levels (a measure of renal function) and insulin resistance, do not
track with haplogroup I of the Y chromosome among young
apparently healthy men. Insulin resistance is a recognized contributor to metabolic syndrome, type 2 diabetes mellitus, as
well as CAD19 and data from experimental models suggested
a linkage between the Y chromosome and measures of insulin
sensitivity.20 Estimated glomerular filtration rate, a measure
of renal function, is also associated with the risk of CAD and
cardiovascular mortality in the general population.21,22 In this
study, instead of estimated glomerular filtration rate we used
creatinine levels because a significant proportion of YMCA
men was aged <18 years, and The Modification of Diet in
Renal Disease-estimated glomerular filtration rate equation is
simply not validated as a measure of renal function in this age
category. The inclusion of these additional cardiovascular risk
factors in our analysis extended the range of conventional correlates of CAD that are unlikely to account for its association
with the haplogroup I of the Y chromosome.
Our gene expression analysis provided the first insights
into MSY transcriptome in human macrophages. This cell
type is a recognized player in the development of atherosclerosis.23 Perhaps more importantly, macrophages from men
with haplogroup I showed suppression of adaptive immunity and upregulation of proinflammatory response pathways when compared with other haplogroups.6 Indeed, we
hypothesized that changes in activity/expression of these
pathways in human macrophages could possibly mediate the
effect of MSY on CAD.6 However, it was not known which
MSY genes and transcripts may be the biological drivers of
the identified findings. In this context, association between
1726 Arterioscler Thromb Vasc Biol July 2013
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CAD-predisposing haplogroup I and 2 mRNAs of MSY
(PRKY and UTY) reported here is an important step forward
to elucidate the role of the Y chromosome in atherosclerosis.
PRKY encodes one of cAMP-dependent serine/threonine protein kinases. These enzymes are key messengers in the cellular
responses to cAMP and act through phosphorylation of proteins to activate (or deactivate) receptors in different cells and
tissues. PRKY is classified as a transcribed pseudogene; it lost
its exon 6 and a part of exon 5 (encoding functionally active
domains of the kinase) and is prone to nonsense-mediated
decay. The biological role of PRKY is not clear. Its fully functional homolog on the X chromosome (PRKX) is involved in
macrophage maturation and kidney development.24,25 The protein product of UTY belongs to one of the histocompatibility
antigens recognized by T cells.26 Expressed in a wide variety
of tissues,26 UTY elicits T-cell response in allogenic transplantation and is hypothesized to play a major role in graft-versushost disease and male grafts rejections.27 Interestingly, both
allograft rejection and graft-versus-host-disease KEGG pathways were associated with haplogroup I in macrophage transcriptome profiling in our previous study.6 The results shown
here together with previously published data suggest that of 2
genes, UTY is a biologically more likely mediator of the association between haplogroup I and immune system, and possibly CAD. However, further studies are warranted to confirm
the direct association between the UTY (PRKY) and CAD and
elucidate the biological mechanisms of these findings.
In summary, our results show that haplogroup I of the Y
chromosome previously linked to increased risk of CAD does
not track with conventional cardiovascular and metabolic risk
factors in young men from the general white European population. We also show for the first time that haplogroup I is
associated with expression of ≥2 MSY genes, of which 1 is a
strong biological candidate linked to immune system. Further
studies should focus on functional characterization of biological underpinnings of the association between haplogroup I and
UTY/PRKY expression to fully elucidate the mechanism of
increased susceptibility to CAD among men with haplogroup
I of the Y chromosome.‍
Disclosures
This study was funded by the British Heart Foundation project grants
(PG/06/097/21331 and PG/12/9/29376) to Maciej Tomaszewski;
Departmental PhD scholarship to Lisa D.S. Bloomer and University
Hospitals of Leicester National Health Service (NHS) Charitable
Funds (M61RT31) to Maciej Tomaszewski and Lisa D.S. Bloomer.
Christopher P. Nelson is supported by National Institute for Health
Research (NIHR) Leicester Cardiovascular Biomedical Research
Unit. James Eales is supported by British Heart Foundation grant
(PG/12/9/29376) to Maciej Tomaszewski. Fadi J. Charchar is supported by research grants from the LEW Carty Charitable Fund and
National Health and Medical Research Council of Australia. Nilesh
J. Samani holds a personal chair supported by the British Heart
Foundation and is a UK NIHR senior investigator. The Cardiogenics
project was supported by the European Union 6th Framework Program
(LSHM-CT-2006–037593). The remaining authors report no conflicts.
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Significance
Compared with carriers of other haplogroups, men with haplogroup I of the Y chromosome exhibit increased susceptibility to coronary artery
disease, possibly because of downregulation of adaptive immunity and upregulation of response to inflammation pathways in macrophages.
Traditional cardiovascular risk factors, including body weight, blood pressure, lipids, glucose, C-reactive protein, measures of renal function,
and insulin resistance, are unlikely mediators of the association between haplogroup I and coronary artery disease. Fourteen of 15 ubiquitous
single-copy genes of the male-specific region of the Y chromosome are expressed in human macrophages. Two of these genes (UTY and
PRKY) are downregulated in macrophages from carriers of haplogroup I when compared with men with other haplogroups of the Y chromosome. Future functional studies will clarify contributions of these genes to haplogroup I–driven risk of coronary artery disease.
Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017
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Male-Specific Region of the Y Chromosome and Cardiovascular Risk: Phylogenetic
Analysis and Gene Expression Studies
Lisa D.S. Bloomer, Christopher P. Nelson, James Eales, Matthew Denniff, Paraskevi
Christofidou, Radoslaw Debiec, Jasbir Moore, Cardiogenics Consortium, Ewa
Zukowska-Szczechowska, Alison H. Goodall, John Thompson, Nilesh J. Samani, Fadi J.
Charchar and Maciej Tomaszewski
Arterioscler Thromb Vasc Biol. 2013;33:1722-1727; originally published online May 2, 2013;
doi: 10.1161/ATVBAHA.113.301608
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Male-specific region of the Y chromosome and cardiovascular risk – phylogenetic analysis and
gene expression studies
Lisa D. S. Bloomer (1), Christopher P. Nelson (1,2), James Eales (1), Matthew Denniff (1), Paraskevi
Christofidou (1), Radoslaw Debiec (1), Jasbir Moore (1), CARDIOGENICS, Ewa ZukowskaSzczechowska (3), Alison H Goodall (1,2), John Thompson (2,4), Nilesh J. Samani (1,2), Fadi J.
Charchar (1,5) and Maciej Tomaszewski (1,2)
1. Department of Cardiovascular Sciences, University of Leicester, Leicester, UK
2. Leicester National Institute for Health Research Biomedical Research Unit in Cardiovascular
Disease, Glenfield Hospital, Leicester, UK
3. Department of Internal Medicine, Diabetology and Nephrology, Silesian School of Medicine,
Zabrze, Poland
4. Department of Health Sciences, University of Leicester, Leicester, UK
5. School of Science and Engineering, University of Ballarat, Ballarat, Australia
Corresponding author:
Maciej Tomaszewski, MD, FAHA, FRCP
Department of Cardiovascular Sciences,
University of Leicester,
Clinical Sciences Wing,
Glenfield Hospital,
LE3 9QP, UK,
Tel: +44 (0)116 2044752
e-mail: [email protected]
1
Supplementary Table I. SNPs defining the major European Y chromosome haplogroups.
Polymorphism symbol
db SNP "rs" number
M201
rs2032636
M304
rs13447352
M269
rs9786153
M207
rs2032658
M45
rs2032631
SRY10831
rs2534636
M173
rs2032624
M35
N/A
M89
rs2032652
M9
rs3900
M170
rs2032597
SRY10831 is a recurrent mutation
Alleles
G->T
A->C
C->T
A->G
G->A
A->G
A->C
G->C
C->T
C->G
A->C
2
Haplogroup
G
J
R1b1b2
R1b1b2
P
B-R; R1a
R1b1b2
E1b1b1
F-R
K-R
I
Supplementary Table II. Clinical characteristics of men from Cardiogenics with available RNA.
Phenotype
Values
n
48
Age (years)
54.6 (6.0)
BMI (kg/m2)
28.7 (4.5)
SBP (mmHg)
133.8 (20.1)
DBP (mmHg)
82.5 (10.8)
Data are means and standard deviations, n – number of subjects, BMI – body mass index, SBP –
systolic blood pressure, DBP – diastolic blood pressure
3
Supplementary Table III. Cardiovascular risk factors stratified by Y chromosome haplogroups (I
versus others) in Young Men Cardiovascular Association Study 1 (YMCA1).
Phenotype
I
Other
P-value
n
190
878
BMI (kg/m2)
22.8 (3.1)
22.9 (3.0)
0.607
Clinic SBP (mmHg)
117.9 (12.8)
118.2 (13.3)
0.589
Clinic DBP (mmHg)
73.5 (7.4)
74.3 (8.0)
0.258
TC (mmol/L)
4.1 (0.9)
4.3 (0.9)
0.147
HDL-C (mmol/L)
1.2 (0.4)
1.1 (0.3)
0.213
Triglycerides (mmol/L)
1.1 (1.0 – 1.2)
1.1 (1.0 – 1.1)
0.190
LDL-C (mmol/L)
2.4 (0.9)
2.6 (0.9)
0.034
Glucose (mmol/L)
4.8 (0.8)
4.8 (0.7)
0.669
Creatinine (µmol/L)
82.6 (11.0)
82.9 (11.2)
0.978
HOMA-IR
1.6 (1.5 – 1.8)
1.6 (1.5 – 1.7)
0.548
Data are means and standard deviations or geometric means and 95% confidence intervals
(triglycerides and HOMA-IR), n – number of subjects, BMI – body mass index, SBP – systolic blood
pressure, DBP – diastolic blood pressure, TC – total cholesterol, HDL-C – high-density lipoprotein
cholesterol, LDL-C – low-density lipoprotein cholesterol, HOMA-IR – homeostatic model assessment
insulin resistance index, P-value – level of statistical significance after age-adjustment
4
Supplementary Table IV. Cardiovascular risk factors stratified by Y chromosome haplogroups (I
versus others) in Young Men Cardiovascular Association Study 2 (YMCA2).
Phenotype
I
Other
P-value
n
85
424
BMI (kg/m2)
22.7 (2.9)
22.5 (3.1)
0.650
Clinic SBP (mmHg)
120.3 (11.9)
118.6 (13.4)
0.354
Clinic DBP (mmHg)
73.9 (7.2)
74.1 (8.0)
0.786
TC (mmol/L)
3.9 (1.2)
3.7 (1.0)
0.204
HDL-C (mmol/L)
1.0 (0.3)
1.1 (0.3)
0.124
Triglycerides (mmol/L)
0.9 (0.8 – 1.0)
0.9 (0.8 – 0.9)
0.584
LDL-C (mmol/L)
2.4 (0.9)
2.2 (0.8)
0.054
Glucose (mmol/L)
4.4 (0.6)
4.4 (0.8)
0.560
Creatinine (µmol/L)
75.9 (9.7)
74.7 (9.8)
0.874
HOMA-IR
1.6 (1.5 – 1.7)
1.7 (1.6 – 1.7)
0.282
Data are means and standard deviations or geometric means and 95% confidence intervals
(triglycerides and HOMA-IR), n – number of subjects, BMI – body mass index, SBP – systolic blood
pressure, DBP – diastolic blood pressure, TC – total cholesterol, HDL-C – high-density lipoprotein
cholesterol, LDL-C – low-density lipoprotein cholesterol, HOMA-IR – homeostatic model
assessment insulin resistance index, P-value – age-adjusted level of statistical significance
5
Supplementary Table V. Cardiovascular risk factors stratified by Y chromosome haplogroups (I
versus others) in sons recruited into Genetic Regulation of Arterial Pressure in Humans in the
Community (GRAPHIC).
Phenotype
I
Other
P-value
n
51
312
BMI (kg/m2)
25.5 (4.5)
25.1 (4.0)
0.395
Clinic SBP (mmHg)
128.7 (12.3)
128.4 (13.1)
0.814
Clinic DBP (mmHg)
77.5 (9.9)
76.3 (9.6)
0.292
TC (mmol/L)
4.6 (1.0)
4.6 (0.9)
0.544
HDL-C (mmol/L)
1.3 (0.3)
1.3 (0.3)
0.483
LDL-C (mmol/L)
2.5 (0.8)
2.5 (0.7)
0.884
Glucose (mmol/L)
5.0 (0.9)
4.9 (0.8)
0.285
Creatinine (µmol/L)
90.4 (9.7)
90.8 (9.1)
0.746
C-reactive protein
0.09 (0.06 – 1.12)
0.10 (0.06 – 0.08)
0.291
Data are means and standard deviations or geometric means and 95% confidence intervals, n –number
of subjects, BMI – body mass index, SBP – systolic blood pressure, DBP – diastolic blood pressure,
TC – total cholesterol, HDL-C – high-density lipoprotein cholesterol, LDL-C – low-density
lipoprotein cholesterol, CRP – C-reactive protein, P-value – age-adjusted level of statistical
significance
6
Supplementary Table VI. Association between haplogroup I of the Y chromosome and expression of
15 ubiquitous single-copy MSY genes in human macrophages from Cardiogenics.
MSY gene
Haplogroup I
Other haplogroups
P-value
n
14
34
USP9Y
8.4 (1.0)
8.2 (0.8)
0.464
DDX3Y
6.1 (0.7)
5.8 (0.6)
0.087
NLGN4Y
13.9 (1.7)
13.3 (3.9)
0.637
TBL1Y
3.6 (1.0)
3.3 (0.9)
0.358
SRY
NOT EXPRESSED
TMSB4Y
10.0 (2.3)
11.1 (0.9)
0.011
EIF1AY
4.2 (0.5)
4.2 (0.5)
0.942
CYorf15A
9.4 (1.0)
9.4 (0.9)
0.693
CYorf15B
8.6 (1.5)
8.3 (1.6)
0.933
RPS4Y1
3.5 (0.5)
3.2 (0.6)
0.202
PRKY
8.2 (0.8)
7.6 (0.6)
0.002
UTY
8.4 (0.6)
7.8 (0.6)
0.0001
RPS4Y2
3.1 (0.4)
3.3 (0.8)
0.391
KDM5D
10.2 (1.9)
9.3 (2.1)
0.212
ZFY
7.0 (0.7)
7.0 (0.6)
0.881
MSY – male specific region of the Y chromosome, data are dCTs (means and standard errors) from
real-time quantitative PCR, P-value – level of statistical significance after age-adjustment
7
Supplementary Figure I. Analysis of population admixture in Young Men Cardiovascular Association Study 1 (YMCA1) and 2 (YMCA2). The method
predicts the likelihood of ancestry to a predefined set of subpopulations (Asian [blue], African [green] and European [red]). YMCA1 is shown in orange,
YMCA2 is shown in purple.
8
Supplementary Figure II. A Analysis of population stratification in the GRAPHIC sons based on autosomal SNPs from previous large scale genetic
association analysis. The X and Y axes represent the first two dimensions from multidimensional scaling (MDS) analysis on the matrix of genome-wide
identity-by-state pair-wise distances. There is a clear proximity between men from GRPAHIC (red) and HapMap CEU population (yellow). HapMap YRI and
JPT+CHB populations are shown in green and blue, respectively. B. individuals with haplogroup I (green) versus carriers of all other haplogroups (red).
A
B
9
Supplementary Figure III. A. Analysis of population stratification in men with available macrophage RNA from the Cardiogenics based on autosomal SNPs
from previous genome-wide association scan. The X and Y axes represent the first two dimensions from multidimensional scaling (MDS) analysis on the
matrix of genome-wide identity-by-state pair-wise distances. There is a clear proximity between men from Cardiogenics (red) and HapMap CEU population
(yellow). HapMap YRI and JPT+CHB populations are shown in green and blue, respectively. B. individuals with haplogroup I (green) versus carriers of all
other haplogroups (red).
A
B
11
10
Methods
Populations
Young Men Cardiovascular Association Study 1 and 2 (YMCA1 and YMCA2)
The Young Men Cardiovascular Association Study 1 (YMCA1) consists of 1157 biologically
unrelated apparently healthy males (mean age: 19.1 years) recruited in Silesia (Southern Poland)
(1,2). The Young Men Cardiovascular Association Study 2 (YMCA2) - an extension of YMCA1,
recruited an additional sample of unrelated 597 young (mean age: 19.0 years) men in Southern Poland
(2). Clinical and biochemical phenotyping protocols of each study were described in detail in previous
publications (1,2). In brief, clinical history was collected using anonomysed coded questionnaires.
Recorded anthropometric measurement included height and weight as well as three consecutive blood
pressure measurements (1,2). Each subject underwent thorough biochemical phenotyping (under
fasting conditions) including analysis of fasting lipids (total cholesterol, HDL-C, triglycerides)
glucose and creatinine. Circulating levels of insulin were measured by radioimmunoassay on 1470
Wizard automatic gamma counter and were used to calculate homeostatic model assessment insulin
resistance index (HOMA-IR) [(insulin
glucose)/22.5] (2). LDL-C was calculated using the
Friedewald formula. Only 1.6% and 0.3 % men in YMCA1 and YMCA2 were on antihypertensive
medication (1,2) and none were prescribed lipid-lowering medications. Blood pressure values from
those on antihypertensive treatment were adjusted for blood pressure-lowering effect of therapy using
previously reported method (1,2).
Genetic Regulation of Arterial Pressure in Humans in the Community (GRAPHIC)
The Genetic Regulation of Arterial Pressure in Humans in the Community (GRAPHIC) cohort
consists of 2037 healthy individuals from 520 European British families (two parents, two offspring)
recruited from the general population of Leicester, UK (3). A total of 1028 men of white European
ethnicity were recruited (516 fathers [average age: 53.8 years] and 512 sons [average age 25.0 years]).
Of 512 male offspring, 391 biologically unrelated individuals were selected for this analysis – in
families with two male offspring only the elder sibling was included in this study. Clinical and
biochemical phenotyping protocols of the GRAPHIC were described in detail elsewhere (3). In brief,
clinical history was collected through standardised questionnaires alongside anthropometric
measurements (height, weight and waist circumference). Each individual had their blood pressure
measured using both clinic and 24-hour ambulatory monitoring (3). A blood sample was secured to
measure total cholesterol, HDL-cholesterol, glucose, and creatinine. LDL-cholesterol was measured
enzymatically. 0.8% of GRAPHIC sons were on antihypertensive medication. Blood pressure values
from those on treatment were adjusted to correct for blood pressure-lowering effect of
antihypertensive therapy as reported before (3).
Cardiogenics Transcriptome Project
Of 918 individuals recruited into this international multi-centre collaborative project, 849 and 684
underwent transcriptomic analysis on Illumina Human Ref-8 array, as reported before (4). Of 255
men included in the previous transcriptome-wide analysis, 48 had sufficient amount of RNA for
measurement of MSY genes expression in macrophages. Only these individuals (all white British men
with a history of premature myocardial infarction) were included in this study.
Written, informed consent was obtained from each individual from all cohorts in accordance with the
Declaration of Helsinki and all the studies had the approval of relevant institutional ethics committees.
DNA and RNA analysis
Genotyping and Y chromosome phylogenetic analysis
DNA was extracted from peripheral leukocytes using standardised procedures. Genotyping was
conducted using the iPLEX genotyping method ([Sequenom (San Diego, USA)] in the GRAPHIC
Study or TaqMan [on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems,
Cambridge, UK)] in YMCA1,YMCA2 and Cardiogenics Study.
Eleven bi-allelic MSY polymorphisms (M9, M35, M45, M89, M170, M173, M201, M207, M269,
M304, and SRY10831) (Supplementary Table I) were genotyped in all cohorts. The genotypes of
1
these polymorphisms were then used to map each Y chromosome into one of 13 major European
branches (haplogroups) of the Y chromosome phylogenetic tree, as previously described (4). This set
of polymorphic variants allows classification of >95% of European Y chromosomes into major
haplogroups [Y(xBR), BR(xE1b1b1,F), E1b1b1, F*, G, I, J, K*, P*, R*, R1*, R1a, R1b1b2] (5). The
haplogroups were named in accordance with the Y chromosome Consortium nomenclature (6-7); all
males in one haplogroup are monophyletic for investigated SNPs.
Analysis of population stratification/admixture
The presence of population admixture in YMCA1 and YMCA2 was assessed using the extensively
validated Structure program (http://pritch.bsd.uchicago.edu/software.html) (8-11). This method uses
information from 34 ancestrally informative SNPs (rs10141763, rs1024116, rs1084344, rs12913832,
rs1321333, rs1335873, rs142665, rs149844, rs1573020, rs16891982, rs182549, rs1886510,
rs1978806, rs2026721, rs2040411, rs2065160, rs2065982, rs2303798, rs2304925, rs239031,
rs2572307, rs2814778, rs3785181, rs4540055, rs5033240, rs5997008, rs722098, rs727811, rs730570,
rs773658, rs7897550, rs881929, rs896788, rs917118) and predicts the likelihood of ancestry to a
predefined set of subpopulations (Asian, African and European). Reference population genotypes
were downloaded from http://spsmart.cesga.es/snpforid.php. The genotyping of the 34 ancestrally
informative markers was conducted using KASP genotyping and SNPline instrumentation
(KBiosciences).
The analysis of population stratification in the GRAPHIC was conducted using SNPs from previously
conducted 50 IBC gene-centric array-based experiment (3). In brief, ~1700 ancestrally informative
SNPs present on the array were included in the principal component analysis. Non-metric, multidimensional scaling was then applied to visualise the genetic similarity between the major Y
chromosome lineages. To ensure all individuals were of European descent, GRPAHIC men were
visualised in the context of Hap Map populations of different ethnicities (CEU, JAP+CHB, Yoruba).
Exclusion of population stratification in men from the Cardiogenics Study included in this project was
conducted using principal component method and non-metric, multi-dimensional scaling, as reported
before (4).
Monocyte and macrophage isolation and RNA extraction
Monocytes were isolated from whole blood using positive selection with CD14 magnetic beads on
AutoMACS system (Miltenyi Biotech, Bergisch Gladblach, Germany) (12,13). Macrophages were
derived from monocytes after culturing for 7 days in the presence of recombinant human M-CSF, as
described previously (12-13).
MSY gene expression analysis
Only 50% of ubiquitous single-copy MSY genes were present on the microarray platform used in
Cardiogenics and survived all quality control filters. Therefore, full expression analysis of all
ubiquitous single-copy genes of MSY in this study was conducted using quantitative real-time PCR
[on ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Cambridge, UK)] in all
Cardiogenics men with available macrophage RNA. Relative expression of each MSY mRNA was
calculated after normalisation to a housekeeping control (β-actin).
Statistical and bioinformatics analysis
Due to skewed distributions, triglycerides, C-reactive protein (CRP) and HOMA-IR were logtransformed prior to statistical analysis.
The cohort-specific association analysis was based on comparison of cardiovascular risk factors
between men with CAD-predisposing haplogroup I and all carriers of other lineages using multiple
linear regression. Inverse-variance fixed-effect meta-analysis was then used to examine the effect of
haplogroup I of the Y chromosome on clinical phenotypes across the three cohorts (YMCA1,
YMCA2, GRAPHIC). All analyses were conducted after adjustment for age.
2
Our calculations showed that the study had from good to excellent (79-100%) power to detect
associations between main cardiovascular risk factors and haplogroup I assuming that its effect on
CAD was mediated solely by each of these risk factors (Table below). Using the formula
ln(a/b)=ln(a)-ln(b) [whereby a – odds ratio CAD in men with haplogroup I: 1.56 (1.24-1.97) (4), b –
the effect size for each individual cardiovascular risk factor from previously published studies (14-20,
Table below)] we estimated the magnitude of an effect that the we would expect from haplogroup I to
have on each risk factor if it was the major driver of the association between haplogroup I and CAD.
The corresponding power estimates were then calculated using sampsi in Stata 12.1.
Analysis of association between haplogroup I and expression of MSY genes was conducted using
linear regression in men from Cardiogenics. Specifically; age-adjusted residuals of dCt from
quantitative real-time PCR were compared between haplogroup I and other lineages. The fold
difference in expression between these groups was calculated by previously used formula: fold
difference=2−difference in dCt . The threshold of statistical significance after correction for multiple testing
was calculated at P≈0.004 given that 14 out of 15 genes were expressed in macrophages (P=0.05/14).
Table. Risk factors of coronary artery disease and haplogroup I of the Y chromosome – power
calculations.
Phenotype
Effect size
–
CAD
L95
U95
Reference
Effect size
– haplo I
Power (%)
BMI
1.14
1.09
1.2
14
0.31
99.0
(kg/m2)
SBP
1.24
1.15
1.35
15
0.23
82.7
(mmHg)
DBP
1.13
1.04
1.22
15
0.32
99.3
(mmHg)
TC
1.08
1.02
1.15
16
0.37
99.9
(mmol/L)
HDL-C
0.78
0.73
0.83
16
0.69
100.0
(mmol/L)
LDL-C
1.25
1.18
1.33
17
0.22
79.1
(mmol/L)
Triglycerides*
1.19
1.08
1.32
18
0.27
99.9
(mmol/L)
Glucose*
1.06
1.00
1.12
19
0.39
99.9
(mmol/L)
CRP
1.23
1.07
1.42
20
0.24
85.9
(mg/L)
Effect size – CAD – estimated magnitude of effect of each individual phenotype on the risk of
coronary artery disease [per 1 SD unit increase/decrease or 1 mmol/L increase (triglycerides and
glucose)*], L95 – lower 95% confidence interval of the estimate, U95 – upper 95% confidence
interval of the estimate, Reference – a literature source of the estimates, Effect size – haplo I – the
expected magnitude of effect of haplogroup I on each individual risk factor, Power – power of the
study to detect the expected effect of haplogroup I on each individual cardiovascular risk factor, BMI
– body mass index, SBP – systolic blood pressure, DBP – diastolic blood pressure, TC – total
cholesterol, HDL-C – high-density lipoprotein cholesterol, LDL-C – low-density lipoprotein
cholesterol, CRP – C-reactive protein
3
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