Palomino Doza et al. BMC Genetics 2013, 14:57
http://www.biomedcentral.com/1471-2156/14/57
RESEARCH ARTICLE
Open Access
Low-frequency intermediate penetrance variants
in the ROCK1 gene predispose to Tetralogy
of Fallot
Julian Palomino Doza1*, Ana Topf1, Jamie Bentham2, Shoumo Bhattacharya2, Catherine Cosgrove3, J David Brook3,
Javier Granados-Riveron3, Frances A Bu’Lock4, John O’Sullivan5, A Graham Stuart6, Jonathan Parsons7,
Caroline Relton1, Judith Goodship1, Deborah J Henderson1 and Bernard Keavney1
Abstract
Background: Epidemiological studies indicate a substantial excess familial recurrence of non-syndromic Tetralogy
of Fallot (TOF), implicating genetic factors that remain largely unknown. The Rho induced kinase 1 gene (ROCK1) is
a key component of the planar cell polarity signalling pathway, which plays an important role in normal cardiac
development. The aim of this study was to investigate the role of genetic variation in ROCK1 on the risk of TOF.
Results: ROCK1 was sequenced in a discovery cohort of 93 non-syndromic TOF probands to identify rare variants.
TagSNPs were selected to capture commoner variation in ROCK1. Novel variants and TagSNPs were genotyped in a
discovery cohort of 458 TOF cases and 1331 healthy controls, and positive findings were replicated in a further 209
TOF cases and 1290 healthy controls. Association between genotypes and TOF was assessed using LAMP.
A rare SNP (c.807C > T; rs56085230) discovered by sequencing was associated with TOF risk (p = 0.006) in the
discovery cohort. The variant was also significantly associated with the risk of TOF in the replication cohort (p =
0.018). In the combined cohorts the odds ratio for TOF was 2.61 (95% CI 1.58-4.30); p < 0.0001. The minor allele
frequency of rs56085230 in the cases was 0.02, and in the controls it was 0.007. The variant accounted for 1% of the
population attributable risk (PAR) of TOF. We also found significant association with TOF for an uncommon TagSNP
in ROCK1, rs288979 (OR 1.64 [95% CI 1.15-2.30]; p = 1.5x10-5). The minor allele frequency of rs288979 in the controls
was 0.043, and the variant accounted for 11% of the PAR of TOF. These association signals were independent of
each other, providing additional internal validation of our result.
Conclusions: Low frequency intermediate penetrance (LFIP) variants in the ROCK1 gene predispose to the risk
of TOF.
Keywords: Congenital heart disease, Tetralogy of fallot, Genetics, Planar cell polarity pathway
Background
Congenital heart disease affects approximately 1% of live
births and is a major source of morbidity and mortality in childhood. Tetralogy of Fallot (TOF) is the
commonest cyanotic CHD, affecting approximately 3 per
10,000 newborns [1]. TOF is characterized by right ventricular outflow tract obstruction, a ventricular septal defect between the anterior and posterior limbs of the
* Correspondence: julianpalomino@gmail.com
1
Institute of Genetic Medicine, Newcastle University, Newcastle, UK
Full list of author information is available at the end of the article
trabecular septal band, over-riding of the aorta and right
ventricular hypertrophy.
Approximately 20% of TOF among live born children
occurs in the setting of chromosomal conditions
(notably 22q11 deletion syndrome), other multi-system
malformation syndromes, or maternal factors such as
teratogen exposure and diabetes. Recurrence risk studies
in the remaining ~80% of “sporadic” cases indicate a
significant familial predisposition, implicating genetic
factors [2,3]. Very rare or private variants in candidate
genes such as Nkx2.5 and Tbx1 have been shown in previous studies to account for small proportions of the
© 2013 Palomino Doza et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Palomino Doza et al. BMC Genetics 2013, 14:57
http://www.biomedcentral.com/1471-2156/14/57
Page 2 of 7
population attributable risk of TOF [4,5]. Recently, association between rare copy number variants [6,7] and between commoner genetic variation and TOF has been
demonstrated [8] but the associations discovered so far
account for only a small proportion of the estimated
heritability of the condition.
The non-canonical Wnt signalling pathway, also
known as the planar cell polarity (PCP) pathway, plays a
key role in cardio genesis [9,10]. The PCP pathway is
principally involved in the determination of cellular polarity in the orientation perpendicular to the apical/basal
plane. PCP pathway signalling is triggered by the interaction between a Wnt protein and a Frizzled (Fz)
receptor, which via the protein Dishevelled, activates
downstream signalling. Rho induced kinases (ROCKs)
are a family of serine/threonine kinases that act as
downstream effectors of PCP signalling and other signalling cascades. They phosphorylate a variety of cellular
substrates and thereby influence cell polarity, adhesion
and motility. A simplified schema of the principal proteins involved in PCP signalling is presented in Figure 1.
ROCK1 is expressed in critical structures during the
process of cardio genesis in both mouse and Xenopus
embryos [11]. Inhibition of ROCKs in murine embryos
induces defects in cardiac looping and septation,
possibly due to effects on mesodermal cell migration
[11,12]. Although the ROCK1 knockout mouse does not
show a cardiac phenotype, possibly due to functional redundancy between ROCK family members in the mouse
[13], phenotypic correspondence between mouse models
of heart development and human disease is known to be
imperfect. For example, cardiovascular malformation is
not seen in EVC −/− mice which are deficient in the
gene causing human Ellis van Creveld syndrome, a
multisystem malformation disorder in which cardiovascular malformation is a prominent feature [14].
ROCK1 is therefore a strong candidate gene for involvement in human CHD susceptibility. We explored
the involvement of both low frequency and common
genetic variation in ROCK1 on the risk of TOF in a
case/control study.
Methods
Study populations
British Caucasian patients with TOF (adults or children)
were recruited from seven participating UK congenital
heart disease units. Northern and Yorkshire Multicentre
Ethics Committee and Central Oxford Research Ethics
Committee approved the study, which was carried out
following the principles of the Helsinki declaration. All
patients (or their parents, if the patient was a child too
young to consent him/herself ) gave informed consent.
The presence of a recognized genetic syndrome associated with congenital heart disease (for example
DiGeorge, Noonan’s or Williams’ syndromes), or of developmental delay, were exclusion criteria. In addition to
review of the clinical records, patient samples underwent
screening for 22q11 deletion using a commercially
available Multiplex Ligation-dependent Probe Amplification (MLPA) kit (MRC-Holland) and the sample was excluded from analysis if a deletion was confirmed.
Following exclusions, 667 TOF proband samples were
available for study.
Controls were British Caucasians free of self-reported
CHD recruited to two previously described populationbased studies [15,16]. Although controls did not undergo
echocardiographic examinations or clinical assessment for
CHD, misclassification due to undiagnosed TOF in any
controls would have been extremely unlikely to have occurred. A total of 2715 control samples were available for
study. Cases and controls were arbitrarily subdivided into
test and replication cohorts.
Wnt
Fz
Cell membrane
Dvl
Cytosol
Rho
Rac
Jnk
ROCKs
AP1
Nucleus
Cytoskeleton
Figure 1 Simplified schema of the PCP pathway.
Rare variant discovery and replication
Screening for low frequency variants in ROCK1 was
performed using Sanger sequencing in 93 TOF probands. Primers were designed to amplify exonic sequence and 100 base pairs into the intron at either end.
Standard methodology was used. Optimized primer sequences, annealing temperatures and product lengths
are listed in Additional file 1: Table S1. All previously
undescribed variants were genotyped in the remainder
of the cases and controls in the test population using
SEQUENOM iPLEX assays. Assays were designed using
the RealSNP web based resource. The replication cohort
was genotyped for rs56085230, which achieved statistical
significance at p < 0.05 in the test cohort, using the same
SEQUENOM iPLEX assay. Additional file 1: Table S2
shows PCR primers, extension primers, mass and base
Palomino Doza et al. BMC Genetics 2013, 14:57
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call for the three variants in ROCK1 that were
undescribed at the time of the sequencing study.
TagSNP study of ROCK1
For the assessment of common variation in ROCK1, allele frequencies for SNPs in the first 28 exons of ROCK1
and 15Kb upstream were downloaded from the HapMap
phase 2 CEU data (www.hapmap.org). SNPs from exons
28–31 of ROCK1 were not included since during the
mutational screening a duplicated region of 19,413 bp
(chr 18: 16774338..16793806) incorporating those exons
was discovered to be present in all probands and control
individuals. A tagging strategy with an r2 threshold of
0.8 and mAF of 0.05 was adopted. Using these criteria a
total of 34 SNPs could be captured using 12 tagSNPs.
We forced the inclusion of the previously described nonsynonymous SNPs rs2271255 (Lys222Glu), rs45449301
(Ile432Val) and rs2292296 (Leu1097Phe). SNPs were also
genotyped using SEQUENOM iPLEX assays. Further detail
of SNPs, primers and conditions are presented in
Additional file 1: Table S3.
The conformation of genotypes to Hardy-Weinberg
equilibrium was checked and allele frequencies determined using PEDSTATS software [17]. Association tests
were carried out using LAMP version 0.0.9 (−−fastAssoc
switch) [18]. To make some allowance for the risk of
false-positive findings due to multiple comparisons, the
results were interpreted based on the false discovery rate
(FDR) using the program QVALUE [19]. We adopted an
FDR of 0.05, that is, one in twenty of the associations
detected at this level is anticipated to be false. Where
statistically significant association was observed, we calculated the population attributable fraction for the risk
genotype using Levin’s formula.
Results
Novel low frequency variants
Three variants undescribed at the time of the sequencing
study were found in screening of the first 26 exons of
ROCK1 in 93 TOF probands. The first variant was a C
to T substitution in exon 7 at position 807 of the mRNA
transcript (c.807C > T). The c.807C > T variant has been
recently identified by the 1000 Genomes Project and
assigned the identifier rs56085230. This variant causes
no change to the sequence of the ROCK1 protein where
a leucine is encoded at residue 268. Sequence traces for
the variant are shown in Additional file 2: Figure S1.
The second variant was a T to G substitution in exon
16, at position 1785 of the mRNA transcript (c.1785 T >
G), which causes no change to the sequence of the
ROCK1 protein where a serine is encoded at residue
595. The third variant was a C to G substitution at position 2318 of the mRNA transcript (c.2318C > G) which
results in a change in amino acid at residue 773 from
Page 3 of 7
Threonine to Serine (p.Thr773Ser). Each of the three
variants was present in one patient in the screening cohort, and no patient carried more than one variant.
The three novel variants were genotyped initially in a
test cohort of 458 TOF cases and 1331 controls. We did
not find significant differences in allele and genotype frequencies for c.1785 T > G and c.2318C > G. However,
the minor allele of rs56085230 was significantly associated with TOF risk (p = 0.006). Genotype frequencies in
the test cohort for the novel variants are shown in
Table 1. In the replication cohort (consisting of 209 TOF
cases and 1290 controls), rs56085230 was also significantly associated with TOF (p = 0.018). In the total
population of cases and controls the odds ratio for TOF
was 2.61 (95% CI 1.58-4.30; p < 0.0001) and the calculated population attributable risk was 1%. Genotype
counts are shown in Table 2. The association between
rs56085230 and TOF risk remained significant (p < 0.05)
after correction for multiple testing using QVALUE.
Analysis using ESEfinder [20] and Spliceview [21]
showed that rs56085230 decreases the strength of the
splicing acceptor site at the end of the exon and produces disappearance of a SrP40 binding site. Analysis of
the variant using the PhastCons algorithm at the UCSC
genome browser [22] showed that this nucleotide is
highly conserved among mammals and vertebrates
(Score = 1).
During the period this work was conducted, genome
wide data on common SNPs typed on the Illumina
660 W platform became available in a proportion of
cases and controls in this study. Principal components
analysis of these data indicated that all carriers of the
rare allele at rs56085230 clustered with the HapMap
CEU population, ruling out population stratification as a
cause of the association we observed. The minor allele
frequency at rs56085230 was 0.02 in the cases and 0.007
in the controls; the allele frequency in controls closely
agrees with that reported in the NCBI Exome Variant
Server in healthy people of White European origin
(0.008; http://evs.gs.washington.edu).
Haplotype-tagging SNP genotyping
Genotyping was successful for at least 98% of the samples for all SNPs. The estimated genotype miscall rate
was <1%. All markers were in Hardy-Weinberg equilibrium at the 5% significance level, and observed allele frequencies agreed well with HapMap data from the CEU
population (Additional file 1: Table S4). Seven common
haplotypes with frequencies above 1% accounted for
more than 95% of common variation within the population (Additional file 1: Table S5). Genotype at the
rs288979 SNP in intron 16 was significantly associated
with risk of TOF (OR: 1.64 [95% CI 1.15-2.30]; p =
1.5x10-5; Table 3). The association remained significant
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Table 1 Genotypes at the three previously undescribed rare variants in ROCK1 in case and control populations
Cases
Variant
Homozygote
major allele
Controls
Heterozygote
Homozygote
minor allele
Homozygote
major allele
Trend p-value
Heterozygote
Homozygote
minor allele
rs56085230
445
12
0
1283
5
0
0.006
c.1785 T > G
452
5
0
1309
10
0
0.49
p.Thr773Ser
441
17
0
1285
46
0
0.77
at the 5% FDR level after correction for multiple testing.
The minor allele frequency at rs288979 in the total
population was 0.043, and the PAR for genotype at
rs288979 was 11%. In silico splicing analysis of the
variant showed no hypothetical effect over splicing.
Analysis of the variant using the PhastCons algorithm at
the UCSC genome browser showed that this nucleotide
is not conserved among mammals and vertebrates
(Score = 0).
There was no significant LD between rs288979 and
rs56085230 (D′=0, r2 = 0; Figure 2), indicating that the
association signals from these two SNPs are independent
of, and therefore confirm, each other. In silico analysis
of rs288979 showed no evidence of splicing changes
associated with it. No other common ROCK1 SNP was
associated with TOF at p < 0.05.
Discussion
We present evidence for association between two low
frequency variants in the ROCK1 gene and TOF risk.
Sequencing studies detected a novel rare synonymous
variant in exon 7 of ROCK1 (c.807C > T) in a highly
evolutionary conserved nucleotide within the kinase
motif of the protein which, according to in silico analysis, might influence splicing. During the course of this
study, the variant was identified in the 1000 Genomes
Project and assigned the identifier rs56085230. The variant was found to significantly increase the risk of TOF
in a test cohort including 458 cases and 1331 controls.
The association was replicated in 209 cases and 1290
controls, and association in the whole population
remained significant after correction for multiple comparisons. The minor allele frequency of rs56085230 was
approximately 0.007 in our healthy population. Since
false positive results due to population stratification are
a particular concern in studying rare variants, we used
genome wide common SNP data to confirm that carriers
of the rare allele at rs56085230 and controls were ethnically homogeneous. Additionally, TagSNP studies of
ROCK1 showed association between TOF and a second,
intronic variant, rs288979, whose minor allele frequency
in the healthy population was 0.037. The evolutionary
conservation of rs288979 was low, suggesting that it is
in linkage disequilibrium with a causative variant, rather
than having a direct effect on TOF risk. There was no significant linkage disequilibrium between the rs56085230
and rs288979 SNPs, thus they may be considered as providing independent replication of the contribution of the
ROCK1 gene to TOF risk. To our knowledge this is the
first study to systematically examine genetic variation at
ROCK1 and TOF. Also, our sample is among the largest
thus far reported for the genetic study of CHD; we were
therefore able to carry out internal replication of the association we observed.
The finding that low frequency intermediate penetrance variants are associated with TOF, and confer
moderate odds ratios, is in keeping with what might be
expected from consideration of the natural history of
TOF in an evolutionary context. Prior to the modern
cardiac surgical era, some 80% of children born with
TOF died prior to the age of ten years, suggesting that
any variant that significantly increased the risk of TOF
would be subject to purifying selection in the population
and thus (in the absence of balancing positive selection
for some other character) uncommon [23,24]. We and
others have recently demonstrated association between
rare genic copy number variants, which are known to be
subject to strong purifying selection, and CHD risk.
Some associated CNVs have similar frequencies in cases,
and associated odds ratios, to the variants associated in
the present study, for example 1q21.1 duplication is
present in just under 1% of subjects with TOF and confers an odds ratio of around thirty [7]. The odds ratios
and allele frequencies of our two associated variants are
Table 2 Genotype counts for rs56085230 in the replication cohort
Cases
Variant
Homozygote
major allele
rs56085230
195
Controls
Trend p-value
Heterozygote
Homozygote
minor allele
Homozygote
major allele
Heterozygote
Homozygote
minor allele
14
0
1258
30
2
0.018
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Table 3 rs288979 Genotype counts and frequencies for
probands and controls
A/A
A/G
G/G
MAF
mAF
Probands
387
41
9
0.93249
0.06751
Controls
1658
126
4
0.96253
0.03747
inversely related, consistent with the operation of selective pressure: whereas rs288979 has a higher mAF it confers a modest OR, and the rarer variant rs56085230
confers a correspondingly higher risk of TOF [25].
The calculated population attributable risk (PAR) for
ROCK1 rs56085230 was 1% in our sample, while that
for rs288979 was 11%. Notwithstanding these figures,
neither variant can be considered as having an optimal
combination of moderate allele frequency and high risk
to carriers to be of potential use in population screening
[26]. Rather, the principal utility of our result lies in the
implication of ROCK1 in human outflow tract malformation and as a basis for further mechanistic studies
in man. The rs56085230 variant is synonymous, and
minigene splicing studies showed no evidence that it
acts to affect splicing (data not shown); the rs288979
variant is intronic and of unknown function. The mechanisms of action of these variants therefore require
further study.
“Low frequency intermediate penetrance” (LFIP) variants such as rs56085230 and rs288979 are presently the
Figure 2 SNPs genotyped (mAF > 0.001) and relationships
between them. The linkage disequilibrium relationships between
SNPs are represented by Haploview triangle plots; darker small
squares denote higher linkage disequilibrium (represented by r2),
and the numbers in the small squares show the significant pairwise
D-prime values derived from the present study.
subject of intensive investigation as the potential source
for the substantial “missing heritability” not detected by
GWAS approaches in many complex diseases. However,
there is relatively little evidence thus far that such variants do indeed make significant contributions to complex disease risk. The present study provides, to our
knowledge, the first evidence that LFIP variants contribute to the risk of sporadic, non-syndromic congenital
heart disease. Our study highlights some issues particular to the study of such LFIP variants that merit comment. First, despite our study being among the largest of
CHD genetics thus reported, the p-values for association
that we obtained were modest. This is an inevitable
consequence of the low frequency of the associated variants. In order to achieve significance at a level typically
considered acceptable in GWAS studies of commoner
variants (p < 5x10-8), hundreds of thousands of CHD patients and controls would have been required. While this
was unfeasible, our study importantly demonstrated internal replication, since two ROCK1 variants independently showed association with TOF; it is likely that such
internal replication by independent SNPs in the same
gene, in the same study population, will be an important
feature of future LFIP studies. Second, studies of LFIP
variants are inherently more susceptible to confounding
by population stratification than are studies of commoner variants. We used genome-wide SNP chip data in
our cases and controls to confirm that rare variant
carriers clustered with the HapMap CEU population;
however, only family-based studies would have the capacity to entirely remove concerns about small degrees of
population stratification not detected by this approach.
Further studies will be required to determine how both
rs56085230 and the intronic rs288979 SNP, which has
no known function, influence TOF risk; study in the
appropriate human cell type in an environment corresponding to early organogenesis will likely be challenging. The hypothetical influence of rs56085230 over
splicing should be explored in the future. It remains possible that the association observed at these SNPs is due
to LD with other LFIP variants that were untyped in this
study; further sequencing and genotyping studies will be
required to resolve this. Confirmation of the result we
have obtained for TOF in a similarly large cohort of patients with other congenital heart disease diagnoses
would be of interest to establish whether the influence
of ROCK1 is restricted to outflow tract defects. Notwithstanding these limitations, our study provides the first
evidence in man that disturbances of PCP pathway
signalling plays a role in the aetiology of cardiac
malformations. Study of other genes in the PCP pathway, and mouse modelling studies to further elucidate
the role of ROCK1 in cardiac development would be of
significant interest.
Palomino Doza et al. BMC Genetics 2013, 14:57
http://www.biomedcentral.com/1471-2156/14/57
Conclusions
We found evidence of two significant associations between low frequency variants in ROCK1, a plausible candidate gene for human cardiac malformations, and the
risk of TOF. This is the first large cohort study exploring
the relationship between genetic variation at the ROCK1
gene and human cardiac malformations. These results
also provide among the first evidence that low frequency
intermediate penetrance variants explain a significant
proportion of genetic predisposition to certain complex
diseases.
Page 6 of 7
3.
4.
5.
6.
7.
Additional files
Additional file 1: Table S1. ROCK 1 ROCK 1 primers, optimal
annealing temperature and PCR product length. Table S2. PCR primers,
extension primer s, masses and base call for the MassExtend genotyping
experiment for the previously undescribed variants. Table S3. PCR primer
extension primer and mass for the ROCK1 tagged SNPs Sequenom assay.
Table S4. Counts and allele frequencies for our population and Hapmap
CEU data, Hom WT: Homozygotes wild type; Hom NWT: Homozygotes
not wild type; mAF: minor allele frequencies; MAF: major allele
frequencies; Het Heterozygotes. Table S5. Common Haplotypes for
ROCK1 within our research population. Genotypes are specified for the
following SNPs: rs7227454, rs288989, 807 C > T, rs288979, rs17202368,
rs17202375, rs1481280, rs8085504 and rs398528.
Additional file 2: Figure S1. ROCK1 807C > T variant trace display.
Upper and medium panel show patient sequence, bottom shows a
normal trace.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JP participated in the design, performed the sequencing and the Sequenom
assays, participated in the statistical analysis and drafted the manuscript; AT
participated in the sequencing and the Sequenom assays and also in the
statistical analysis; CC, DB, JG, FB, JO, GS, JP, JB and CR recruited patients,
collected the samples and participated in the design and coordination of
the study; JG and DH participated in the study design, data analysis and
manuscript draft; BK participated in the design, coordination and supervision
of the study, data analysis and manuscript draft. All authors read and
approved the final manuscript.
Acknowledgements
This research was funded with grants from the British Heart Foundation and
Heart Research UK.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Author details
1
Institute of Genetic Medicine, Newcastle University, Newcastle, UK.
2
Department of Cardiovascular Medicine, Oxford University, Oxford, UK.
3
Institute of Genetics, Nottingham University, Nottingham, UK. 4University
Hospitals of Leicester NHS Trust, Leicester, UK. 5Newcastle upon Tyne
Hospitals NHS Foundation Trust, Newcastle, UK. 6Bristol Royal Hospital for
Children, Bristol, UK. 7Leeds Teaching Hospitals NHS Trust, Leeds, UK.
19.
20.
21.
Received: 13 June 2012 Accepted: 5 June 2013
Published: 19 June 2013
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doi:10.1186/1471-2156-14-57
Cite this article as: Palomino Doza et al.: Low-frequency intermediate
penetrance variants in the ROCK1 gene predispose to Tetralogy
of Fallot. BMC Genetics 2013 14:57.
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