Meta Gene 12 (2017) 47–49

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Meta Gene

journal homepage: www.elsevier.com/locate/mgene

Top SNPs from the phenome-wide association study catalog and the risk
of varicose veins of lower extremities: A replication study
Alexandra Shadrina a,b,⁎, Mariya Smetanina a, Ekaterina Sokolova a,b, Kseniya Sevost'ianova a, Andrey Shevela a,
Evgenii Seliverstov c, Elena Zakharova c, Evgeny Ilyukhin d, Elena Voronina a,b, Dmitry Makarov e,
Alexander Kirienko c, Igor Zolotukhin c, Maxim Filipenko a,b
a
Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentjev Avenue, Novosibirsk 630090, Russia
b
Novosibirsk State University, 2 Pirogova street, Novosibirsk 630090, Russia
c
Pirogov Russian National Research Medical University, 1 Ostrovitianova street, Moscow 117997, Russia
d
Private Surgery Center “Medalp”, 54 Leningradskaya street, Saint Petersburg 197758, Russia
e
Republican Clinical Hospital, 138 Orenburgskiy tract, 138, Kazan 420064, Republic of Tatarstan, Russia

a r t i c l e i n f o a b s t r a c t

Article history: Varicose veins are the most common venous pathology of lower limbs. A strong body of evidence indicates a
Received 10 November 2016 prominent role of heredity in the development of this condition, but genetic factors influencing its risk are still
Revised 10 January 2017 poorly investigated. Phenome-wide association study (PheWAS) is a novel approach to discovering associations
Accepted 18 January 2017
between genetic polymorphism and multiple phenotypes. The results of the largest PheWAS today are included
Available online 19 January 2017
in the PheWAS catalog, which contains the associations with a variety of phenotypes including varicose veins of
Keywords:
lower extremities. In our study we aimed to replicate top 5 associations from the PheWAS catalog in the popula-
Varicose veins tion of ethnic Russians. We determined the genotypes of polymorphisms rs735854, rs7023329, rs2857595,
Polymorphism rs6420094, and rs16856202 in the sample of 460 patients with primary varicose veins of lower extremities
Phenome-wide association study and 646 control subjects without a history of chronic venous disease. Though we had enough statistical power
to validate the reported associations, no evidence for statistical significant association was revealed in our
study. We can speculate that either these polymorphisms have no effect on the risk of varicose veins, or their ef-
fects can be modulated by currently uninvestigated ethnic, environmental or lifestyle factors.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction between 3144 single-nucleotide polymorphisms (SNPs) from the

A novel approach has recently been proposed to study genetic asso-
ciations with multiple phenotypes. It was called a phenome-wide asso-
ciation study (PheWAS) by analogy with its complementary approach –
a genome-wide association study (GWAS). The PheWAS algorithm was
developed by Denny et al (2010), who used International Classification
of Disease (ICD9) billing codes to categorize phenotypes of the study
population. The source of DNA samples were biobanks linked to elec-
tronic medical record (EMR) system. The largest PheWAS today was
conducted by Denny et al (2013). They scanned for associations
Abbreviations: CEAP, clinical severity, etiology, anatomy and pathophysiology; GWAS,
genome-wide association study; EMR, electronic medical record; PheWAS, phenome-
wide association study; SNP, single-nucleotide polymorphism.
⁎ Corresponding author at: Laboratory of Pharmacogenomics, Institute of Chemical
Biology and Fundamental Medicine, 8 Lavrentjev Avenue, Novosibirsk 630090, Russia.
E-mail address: weiner.alexserg@gmail.com (A. Shadrina).
GWAS catalog and 1358 EMR-derived phenotypes in 13,835 individuals
of European ancestry. The results of this study have been included in the
PheWAS catalog that contains the revealed associations with P b 0.05
(https://phewas.mc.vanderbilt.edu/). Salnikova et al (2016) aimed to
investigate genetic susceptibility to connective tissue disorders and
searched the PheWAS Catalog for SNPs associated with several condi-
tions including varicose veins of lower extremities. They found 188
SNPs associated with this pathology. Top 5 polymorphisms with the
most significant associations were rs735854, rs7023329, rs2857595,
rs6420094, and rs16856202, though none of them reached a false dis-
covery rate-adjusted level of statistical significance. These results
could therefore be considered as tentative and needed further valida-
tion. Although a strong body of evidence indicates that etiology of vari-
cose veins includes a moderate-to-strong genetic component (Krysa
et al., 2012), genetic factors influencing the risk of this condition remain
poorly explored, and validation of the results obtained from the
PheWAS would contribute to a better understanding of the genetic

http://dx.doi.org/10.1016/j.mgene.2017.01.005
2214-5400/© 2017 Elsevier B.V. All rights reserved.
48 A. Shadrina et al. / Meta Gene 12 (2017) 47–49

basis of this disease. In our study we aimed to replicate the findings re- Table 1
ported by Salnikova et al (2016) in the population of ethnic Russians. Sequences of the primers and probes.

Polymorphism Direction/label Sequence

2. Material and methods Primers

rs735854 Forward 5′-TGCCTGAGCTGTTTTTGTGCTA-3′
Reverse 5′-CTTCCCGCAAAATGTTTAGGTG-3′
2.1. Patients rs7023329 Forward 5′-CTAAATGGTACCTACACAGGTGC-3′
Reverse 5′-GAATCTGAAGAGTCTGAGGATACAAC-3′
We genotyped 460 patients with primary varicose veins of lower ex- rs2857595 Forward 5′-TAGTTGAAAAATATTGATAAGACTTGG-3′
tremities (145 (31.5%) men and 315 (68.5%) women; median age Reverse 5′-CCCACCAGGGTTCCTCTACG-3′
rs6420094 Forward 5′-GTGGCGAAAAGGAGAACTTC-3′
49 years, lower quartile 38 years, upper quartile 57 years) and 646 con-
Reverse 5′-CCACCAGGTGACCATAGCC-3′
trols without a history of chronic venous disease (236 (36.5%) men and rs16856202 Forward 5′-TTGAATTGCTGAAAGGACCG-3′
410 (63.5%) women; median age 50 years, lower quartile 37 years, Reverse 5′-GCTCGGCATTTCAAAGTGTC-3′
upper quartile 59 years). Blood samples were collected in Novosibirsk
Probes
State Regional Clinical Hospital (Novosibirsk, Russia), Center of New rs735854 R6G 5′-R6G-CGGACATCTTCACACACAAC-BHQ-3′
Medical Technologies (Novosibirsk, Russia), Pirogov City Clinical Hospi- FAM 5′-FAM-CGGACATCTCCACACACAAC-BHQ-3′
tal No. 1 (Moscow, Russia), Private Surgery Center “Medalp” (Saint Pe- rs7023329 R6G 5′-R6G-TCCTGTGAATCTATGCCTGCA-BHQ-3′
tersburg, Russia), and Republican Clinical Hospital (Kazan, Republic of FAM 5′-FAM-TCCTGTGAATCTGTGCCTGCA-BHQ-3′
rs2857595 R6G 5′-R6G-CACTCAATCTCACCCCTGCC-BHQ-3′
Tatarstan, Russia). All the patients in the case group underwent clinical FAM 5′-FAM-CACCCAATCTCACCCCTGCC-BHQ-3′
examination with a subsequent duplex ultrasound of a venous system rs6420094 R6G 5′-R6G-CCAGAGATAGCGGGTGTTGC-ВHQ-3′
of both legs and had objectively confirmed indications to varicose FAM 5′-FAM-CCAGAGGTAGCGGGTGTTGC-BHQ-3′
veins surgery. All the patients had visible varicose veins and reflux in rs16856202 R6G 5′-R6G-CAAGACAAATTACTGGAGAGTCT-BHQ-3′
FAM 5′-FAM-CAAGACAAATGACTGGAGAGTCT-BHQ-3′
the great saphenous vein or the small saphenous vein according to du-
plex ultrasound. Duplex ultrasound scanning was performed with the
patient in standing position. The condition of the deep veins was thor-
oughly examined to exclude post-thrombotic changes. The reflux of su-
nlm.nih.gov/), UGENE software (version 1.14, http://ugene.unipro.ru/),
perficial venous system was evaluated using the distal compression
and Oligo Analyzer software (version 1.0.3).
manoeuvre. The reflux duration of N0.5 s was considered pathologic.
Distribution of clinical severity, etiology, anatomy and pathophysiology
2.3. Statistical analysis
classes (Eklöf et al., 2004) among patients in the case group was as fol-
lows: 138 (30.0%) patients had C2 class; 232 (50.4%) had C3 class; 75
Statistical analysis was performed using R statistical software (ver-
(16.3%) had C4 class; 15 (3.2%) had C5 and C6 classes. Patients with
sion 2.15.1, http://www.r-project.org; glm function). Odds ratios and
post-thrombotic syndrome or congenital varicose veins were excluded
95% confidence intervals were calculated by logistic regression analysis
from the study. 115 (25.0%) individuals developed varicose veins before
under additive model of inheritance. All data were adjusted for sex and
the age of 20; 187 (40.7%) patients had the age of disease onset between
age. The expected frequencies of genotypes in the control group were
20 and 30 years; 97 (21.0%) – between 30 and 40 years; 57 (12.4%) – be-
tested for accordance with Hardy-Weinberg equilibrium using exact
tween 40 and 50 years; and 4 (0.9%) – over 50 years. 319 (69.3%) pa-
test. Differences were considered statistically significant at P b 0.05. To
tients had positive family history of varicose veins; 120 (26.1%) had
estimate the statistical power of study, Genetic power calculator
negative family history; and 21 (4.6%) case individuals were not able
(http://pngu.mgh.harvard.edu/~purcell/gpc/cc2.html) was used.
to provide such information.
All the individuals enrolled in this study signed the informed con-
3. Results
sent. All clinical investigations were conducted according to the princi-
ples expressed in the Declaration of Helsinki. The study was approved
Distribution of genotypes in the case and the control groups is pre-
by the Ethics Committee of Institute of Chemical Biology and Funda-
sented in Table 2. Genotype frequencies were in accordance with
mental Medicine (protocol No. 15, 13 September 2013) and the Ethics
Hardy-Weinberg equilibrium in both groups. Minor allele frequencies
Committee of Pirogov Russian National Research Medical University
were close to those reported by 1000 Genomes Project for European
(protocol No.123, 21 January 2013). All cases and controls were ethnic
populations (http://www.1000genomes.org). None of the studied poly-
Russians. Ethnical background was defined as the declaration of a sub-
morphisms showed statistical significant association with the risk of
ject (only those subjects were selected who defined themselves as
varicose veins of lower extremities (Table 2).
well as their parents as ethnic Russians).
4. Discussion
2.2. Genotyping
Varicose veins of lower extremities is a common multifactorial disor-
Genomic DNA was isolated from leukocytes in venous blood by pro- der with a prominent but yet poorly explored genetic component. The
teinase K digestion followed by phenol/chloroform extraction and etha- PheWAS catalog contains 188 associations with this pathology, with
nol precipitation. Genotyping was carried out by real-time PCR allelic the most pronounced signals obtained for SNPs in the MYH9
discrimination with TaqMan probes. PCR was performed in 20 μL reac- (rs735854), MTAP (rs7023329), SLC34A1 (rs6420094), and the DISC1/
tion volume containing 20–100 ng of genomic DNA, 65 mM Tris-HCl TSNAX-DISC1 (rs16856202) genes, and for polymorphism rs2857595 lo-
(рН 8.9), 24 mM ammonium sulphate, 3.0 mM magnesium chloride, cated between the NCR3 and the UQCRHP1 genes (Salnikova et al.,
0.05% Tween 20, 0.2 mM dNTP, 0.3 μM primers, 0.1 μM probes 2016). These results needed to be validated by independent studies. In
(Table 1), and 1.0 U of Taq polymerase. PCR thermal cycling conditions our study we found no evidence for association with varicose veins of
were as follows: denaturation for 3 min at 96 °С followed by 48 cycles of lower extremities for any of the polymorphisms examined. It is worth
8 s at 96 °С and 40 s at 60 °С. Amplification procedure was conducted mentioning that we had enough statistical power (85–94%) to reveal as-
using CFX96 Thermal Cycler (Bio-Rad, USA). sociations with the same effect size as it was reported in the PheWAS
Primers and probes were designed using sequences obtained from Catalog. We can speculate that associations identified by PheWAS
the National Center for Biotechnology Information (http://www.ncbi. could actually be false positive. Notably, varicose veins are poorly
 A. Shadrina et al. / Meta Gene 12 (2017) 47–49 49

Table 2
Association of top 5 SNPs from the PheWAS catalog with the risk of varicose veins of the lower extremities.

SNP, genotypes Cases: genotype distribution, Controls: genotype distribution, OR (95% CI), P OR, P reported in the PheWAS Statistical power (our
MAFa, HWEb MAFa, HWEb catalog study)

rs735854 CC:CT:TT 178:181:61, 36%, 0.20 257:298:86, 37%, 1.00 0.98 (0.82–1.18), 0.85 0.81, 1.4e–04 94%
rs7023329 AA:AG:GG 107:200:95, 49%, 0.92 160:307:168, 51%, 0.43 0.93 (0.78–1.11), 0.40 0.82, 1.9e–04 89%
rs2857595 GG:GA:AA 302:105:9, 15%, 1.00 448:181:12, 16%, 0.24 0.90 (0.70–1.15), 0.41 1.28, 2.2e–04 85%
rs6420094 AA:AG:GG 204:196:60, 34%, 0.26 314:260:72, 31%, 0.12 1.13 (0.95–1.34), 0.18 1.21, 6.0e–04 87%
rs16856202 TT:TG:GG 430:25:1, 3%, 0.33 605:29:0, 2%, 1.00 1.31 (0.77–2.23), 0.32 1.65, 7.1e–04 87%
a
Minor allele frequency.
b
P-value for deviation of genotype distribution from the Hardy–Weinberg equilibrium (exact test).

coded in administrative databases, therefore using ICD9 billing codes to
define this phenotype in the phenome-wide association study could be
the reason for false positive findings due to possible misclassification.
Alternatively, the effects of the analyzed SNPs could be modulated by
currently unexplored environmental/lifestyle factors making it difficult
to validate the revealed associations. Either way, these polymorphisms
do not seem to play a prominent role in genetic susceptibility to varicose
veins.
5. Conclusions
Our study failed to replicate top results of the PheWAS regarding
varicose veins. Nevertheless, to date the PheWAS catalog comprises
only a limited number of SNPs that have shown associations with differ-
ent traits in the previous studies. Expanding the PheWAS catalog with
additional SNPs and combination of the PheWAS and GWAS approaches
can enable new insights into the genetics of varicose veins as well as
other human diseases.
Funding
This work was supported by Russian Science Foundation (grant
number 14-15-00734, “Searching of genes involved in varicose vein dis-
ease pathology”).
Declaration of conflicting interests
The authors declare that there is no conflict of interest.
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