Autoimmunity

ISSN: 0891-6934 (Print) 1607-842X (Online) Journal homepage: http://www.tandfonline.com/loi/iaut20

Plasma VEGF-related polymorphisms are implied

in autoimmune thyroid diseases

Ines Zaaber, Marc Rancier, Maria G. Stathopoulou, Abdelsalam Saleh, Héla

Marmouch, Christine Masson, Helena Murray, Mary Jo Kurth, John Lamont,
Peter Fitzgerald, Selvia Mahjoub, Khaled Said, Besma Bel Hadj Jrad Tensaout,
Souhir Mestiri & Sophie Visvikis-Siest

To cite this article: Ines Zaaber, Marc Rancier, Maria G. Stathopoulou, Abdelsalam Saleh, Héla
Marmouch, Christine Masson, Helena Murray, Mary Jo Kurth, John Lamont, Peter Fitzgerald,
Selvia Mahjoub, Khaled Said, Besma Bel Hadj Jrad Tensaout, Souhir Mestiri & Sophie Visvikis-
Siest (2016): Plasma VEGF-related polymorphisms are implied in autoimmune thyroid diseases,
Autoimmunity

To link to this article: http://dx.doi.org/10.3109/08916934.2016.1151005

Published online: 08 Mar 2016.

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 http://informahealthcare.com/aut
ISSN: 0891-6934 (print), 1607-842X (electronic)

Autoimmunity, Early Online: 1–7

! 2016 Taylor & Francis. DOI: 10.3109/08916934.2016.1151005

RESEARCH ARTICLE

Plasma VEGF-related polymorphisms are implied in autoimmune

thyroid diseases
Ines Zaaber1,2*, Marc Rancier1,3*, Maria G. Stathopoulou1, Abdelsalam Saleh1, Héla Marmouch4, Christine Masson1,
Helena Murray5, Mary Jo Kurth5, John Lamont5, Peter Fitzgerald5, Selvia Mahjoub4, Khaled Said2, Besma Bel Hadj
Jrad Tensaout2, Souhir Mestiri2**, and Sophie Visvikis-Siest1,6**
1
UMR INSERM U1122; Interactions Gène-Environnement en Physiopathologie Cardio-Vasculaire (IGE-PCV), Université de Lorraine, Nancy, France,
2
Laboratory of Genetics, Biodiversity and Bioresource Valorization, Superior Institute of Biotechnology of Monastir, University of Monastir, Monastir,
Downloaded by [University of California, San Diego] at 03:42 09 March 2016

Tunisia, 3Department of Endocrinology, Centre Hospitalier Robert Schuman, Luxembourg, Luxembourg, 4Department of Internal Medicine-
Endocrinology, Hospital FattoumaBourguiba in Monastir, Tunisia, 5Randox Laboratories Ltd, Crumlin, UK, and 6Department of Internal Medicine
and Geriatrics, CHU Nancy-Brabois, France

Abstract Keywords
Autoimmune thyroid diseases (AITD), including Graves’ disease (GD) and Hashimoto thyroiditis Genetics, Graves’ disease, Hashimoto
(HT), are complex multifactorial diseases. Vascular endothelial growth factor (VEGF) is thyroiditis, VEGF, polymorphisms
implicated in various inflammatory diseases, especially autoimmune diseases. Our aim was to
elucidate the relationships between plasma VEGF levels and four genome-wide association History
study-identified single nucleotide polymorphisms (SNPs) related to VEGF with AITD in Tunisian
patients. A total of 364 healthy controls and 389 patients with AITD were genotyped for the Received 30 September 2015
SNPs rs6921438, rs4416670, rs6993770 and rs10738760. Levels of thyroid hormones and Revised 19 January 2016
antibodies were quantified simultaneously with plasma VEGF after a period of six months of Accepted 31 January 2016
treatment. We found that the minor alleles of rs10738760 and rs6921438 are associated with Published online 26 February 2016
the presence of GD. A allele of rs10738760 polymorphism is associated with increased plasma
levels of free tri-iodothyronin (FT3) while no relationship was found with circulating VEGF
plasma levels after six months of treatment. We also showed that the T allele of rs4416670
polymorphism was associated with increased risk of hyperthyroidism in patients treated for six
months, independently of their initial diagnosis. There was no significant association between
the SNPs and the risk for HT compared with controls. This study shows that AITD are influenced
by 3 SNPs linked to VEGF circulating levels. Whereas rs10738760 appeared specific to GD and
FT3 production after six months of treatment, rs6921438 and rs4416670 were implicated in the
risk for GD. This study opens new ways to test pharmacogenomics concepts in the future
especially in GD in which recurrence prognosis is still challenging.

Introduction apoptosis increased probability of hypothyroidism. However,

both HT and GD exhibit destructive consequences on thyroid
Autoimmune thyroid diseases (AITD) comprise essentially
parenchyma [2].
two major diseases in clinical practice: Hashimoto thyroiditis
AITD are complex autoimmune diseases involving mul-
(HT) and Graves’ disease (GD). Genetic and/or environmental
tiple genes, including both immune regulatory genes and
factors can elicit the immune response leading to infiltration
thyroid-specific genes such as thyroid-stimulating hormone
of the thyroid by lymphocytes and production of autoanti-
receptor (TSHR), thyroglobulin, human leukocyte antigen
bodies directed against constituents of the thyroid [1]. In GD,
(HLA), cytotoxic T-lymphocyte-associated protein 4
thyroid presents as hyperplasia, inflammation and neovascu-
(CTLA4), protein tyrosine phosphatase, non-receptor type
larization. Anti-TSHR auto-antibodies bind to TSH-receptor
22 (PTPN22), CD40, Fc receptor-like protein 3 (FCRL3),
and stimulate the hormone secretion, resulting in hyperthy-
interleukin-2 receptor alpha (IL2RA) and forkhead box P3
roidism. This also leads to goitre genesis. HT is associated
(FOXP3) [3,4].
with a lymphocyte infiltration leading to necrosis and
AITD can be accompanied by goitre. Angiogenesis plays
an important role in the development of goitre in the prolif-
*Equally first authors. eration of endothelial cells occurring before the increased
**Equally last authors. proliferation of the thyroid follicular cells [5]. Angiogenesis is
Correspondence: Dr Sophie Visvikis-Siest, UMR INSERM U1122; IGE- regulated by proteins, such as proinflammatory cytokines and
PCV, Université de Lorraine, 30 Rue Lionnois, 54000 Nancy, France. growth factors, which promote and regulate endothelial cell
Tel: + 33 (0)3 83 68 21 63. Fax: + 33 (0)3 83 32 13 22. E-mail:
[email protected] proliferation and stabilization of neovascularization. Whereas
 2 I. Zaaber et al. Autoimmunity, Early Online: 1–7

HT exhibits a Th1 profile (including elevated interleukin 1 were healthy blood donors with no declared autoimmune
beta (IL-1b), interleukin 2 (IL-2), interleukin 12 (IL-12), disease. In both patients and controls, there were some family
interferon gamma (IFNg), tumour necrosis factor alpha TNFa structures that were recorded and taken into account for
and CD40 ligand (CD40L)), GD follows a Th2 profile appropriate statistical analyses.
(interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL- GD and HT were diagnosed by standard criteria set by the
6), interleukin 10 (IL-10), interleukin 13 (IL-13) and CD40 American Association of Clinical Endocrinologists and the
ligand (CD40L)) [4,6–9]. American Thyroid Association (AACE/ATA) [24,25].
Vascular endothelial growth factor (VEGF or VEGF-A) is The patients were followed for more than six months after
the most potent angiogenic factor and is implicated in various their initial diagnosis and beginning of treatment. After the
inflammatory diseases, especially autoimmune diseases starting point of the discovery of the disease, GD patients
[10,11]. An increase in serum VEGF and intrathyroid were treated with synthetic antithyroid according to their
neovascularization has also been observed in patients with status with doses specific to the severity of their disease. After
GD and HT [12]. Among angiogenic proteins, VEGF has six months of treatment, all patients were expected to be
been studied extensively [13,14]. Several in vivo and in vitro euthyroid. However, in real-life conditions, the response to
studies have demonstrated the expression of VEGF in normal treatment is not the same for all patients, thus the hormonal
and diseased tissue of the thyroid [8,9,15,16]. status can be hypothyroid, hyperthyroid or euthyroid
A number of single nucleotide polymorphisms (SNPs) (TSH  6.82mUI/l, TSH  0.28mUI/l and TSH between
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have been described in VEGF gene, including A-2578C and 0.28 and 6.82 mUI/l, respectively).
T-460C in the promoter region, and G + 405C in the Written informed consent was obtained from all patients
50 -untranslated region, reported to be related with protein and controls. The study protocol was approved by the ethics
production [17–19]. Relationships between these polymorph- committee of the hospital-university Fattouma Bourguiba
isms and some autoimmune diseases such as psoriasis [20], Monastir, Tunisia.
and type 1 diabetes mellitus [21] have been demonstrated.
Only one study has examined the association between these Genotyping of VEGF gene polymorphisms
polymorphisms and GD showing that some of them may be Genomic DNA was obtained from peripheral blood leukocytes
risk factors for AITD [22]. by the salting out technique [26]. Genotyping was performed
Recently, in a genome-wide association study (GWAS), by Randox Laboratories Ltd (Crumlin, UK) (Evidence
we identified four SNPs mapping to three chromosomal InvestigatorÕ ) using an assay based on a combination of
regions explaining up to 50% of the inter-individual multiplex PCR and biochip array hybridization. Two SNPs
variability of circulating VEGF levels: rs6921438 and were in disagreement with the Hardy–Weinberg equilibrium
rs4416670 (6p21.1), rs6993770 (8q23.1), and rs10738760 (Table 1) in the controls sample, which may be explained by
(9p24.2) [23]. the pedigree structure of this sample. As detailed below, this
To our knowledge, no study has been published regarding structure has been taken into account in further analyses.
the relationship of these polymorphisms and AITD. Similarly,
whether these polymorphisms could have an effect on the Laboratory measurements
response to standard treatment of patients with AITD and the
status of thyroid hormones after treatment, especially in GD Levels of TSH, FT4 and FT3 after six months treatment
remains unknown. Indeed, GD still lacks accurate prognostic Plasma TSH, FT4 and FT3 quantification was performed by
factors for recurrence after non-surgical therapies. Monobind Inc. (Lake Forest, CA) using VAST (Versatile
Therefore, our aim was to investigate the role of VEGF in Analyse System Technology) FT3, FT4 and TSH ELISA Kit.
the development of AITD. First, we investigated the relation- The reference range of plasma TSH is 0.28–6.82 mIU/L, of
ship between the 4 VEGF-related SNPs with the presence of FT4 is 0.8–2 ng/dl and of FT3 is 1.4–4.2 pg/ml.
AITD together with the thyroid status after six months of
treatment in a Tunisian population. Furthermore, we aimed to Levels of anti-TPO antibody and anti-Tg antibody after six
assess the possible associations of VEGF circulatory levels months treatment
with thyroid status after treatment.
Plasma anti-TPO antibody quantification was performed by
EURO DIAGNOSTICAÕ (Malmö, Sweden) using the
Materials and methods
DIASTAT ANAÕ anti-TPO ELISA kit. Plasma anti-Tg
Patients and controls antibody quantification was performed by EURO
DIAGNOSTICAÕ (Malmö, Sweden) using DIASTAT
This study was conducted on 364 healthy controls and 389
ANAÕ anti-Tg ELISA kit. For anti-Tg and anti-TPO a
patients with AITD (297 with HT and 92 with GD). Controls
and patients were randomly selected from the same popula- reciprocal titer of 41:100 was considered positive.
tion living in the middle coast of Tunisia.
Levels of anti-thyroid-stimulating hormone receptor
Clinical data were obtained from medical records of
(anti-TSHR) antibody after six months treatment
patients recruited in the Department of Endocrinology,
University Hospital Fattouma Bourguiba Monastir. Patients Plasma anti-TSHR antibody quantification was performed by
with HT (279 women and 18 men) and with GD (74 women ALPHADIAGNOSTIC INTERNATIONAL INC (San
and 18 men) had an average age of 46 ± 11 years. Controls Antonio, TX) using the Human anti-TSHR antibody ELISA
(23 men and 341 women) having a mean age of 46 ± 12 years kit.
 DOI: 10.3109/08916934.2016.1151005 VEGF polymorphisms in autoimmune thyroid disease 3

Plasma levels of VEGF after six months treatment The A allele of polymorphism rs10738760 was signifi-
cantly associated with an increased risk of GD compared with
Plasma VEGF protein quantification was performed by
HT ( ¼ 0.54, p ¼ 0.003) and controls ( ¼ 0.46, p ¼ 0.006).
Randox Laboratories, Ltd. (Crumlin, UK) using a biochip
A significant association between rs6921438 and risk of
array analyser (EvidenceÕ ) [23].
GD compared with controls was also observed. The allele A
Statistical analysis of the SNP was associated with increased GD risk ( ¼ 0.324,
p ¼ 0.05). There was no significant association between the
The Hardy–Weinberg equilibrium was tested using a 2 test. SNPs and the risk for HT compared with controls.
The after treatment values of VEGF were not normally By studying the association between the SNPs and the
distributed and they were log-transformed. All analyses were current hormonal status (patients currently having hypothy-
performed using the GWAF package developed in R that roidism, hyperthyroidism or patients currently in normal
allows the adjustment of the analyses in the familial structure thyroid condition), we showed that the T allele of rs4416670
[27]. The associations between the genotyped SNPs and the polymorphism was associated with an increased risk of
AITD, the after treatment hormonal status and the presence of hyperthyroidism in six months treated patients ( ¼ 1.82,
thyroid antibodies were tested using a Generalized Estimation p ¼ 0.049), independently of their initial diagnosis.
Equation adjusted for age and gender to account for within Levels (after treatment) of TSH, FT4 and FT3 among the
pedigree correlation. The associations of the SNPs with the rs10738760, rs4416670, rs6921438 and rs6993770 genotypes
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continuous traits (VEGF, TSH, FT4 and FT3) were tested are shown in Table 6 and the distribution of anti-TPO, anti-Tg
through Linear Mixed Effects models also adjusted for age, and anti-TSHR antibodies is presented in Table 7.
gender and familial structure. The level of significance was By studying the association between polymorphisms and
set at p50.05. clinical parameters measured after six months of treatment
(FT3, FT4, TSH, anti-TPO antibody, anti-Tg antibody and
Results anti-TSHR antibody), we observed a significant positive
Genotype and allele frequencies distributions of rs10738760, association between the A allele of rs10738760 polymorph-
rs6921438, rs4416670 and rs6993770 polymorphisms are ism and plasma levels of FT3 ( ¼ 0.716, p ¼ 0.024).
presented in Tables 2–5. Our analyses gave no significant results concerning the
associations between plasma VEGF after treatment and initial
diagnosis and between different thyroid statuses after
Table 1. The Hardy–Weinberg equilibrium for the 4 SNPs.
treatment.
Control AITD HT GD
p p p p Discussion and conclusion
rs6921438 0.001 0.120 0.161 0.499 The change from traditional medicine to molecular persona-
rs4416670 0.087 0.465 0.119 0.205 lized medicine can only be performed by integrating large-
rs6993770 0.140 0.500 0.523 0.817 scale personalized molecular data in risk stratification tools.
rs10738760 0.023 0.407 0.472 0.525
The development of ‘‘-omics’’ technologies is already

Table 2. Distribution of rs10738760 genotypes and alleles in patients with AITD (n ¼ 378), HT (n ¼ 286), GD (n ¼ 92), healthy control subjects
(n ¼ 354), hypothyroidism, hyperthyroidism and euthyroidism.

Control AITD1 HT2 GD3 Hypothyroidism Hyperthyroidism Euthyroidism

n (%) n (%) n (%) n (%) n (%) n (%) n (%)
Genotype
GG 118 (33.33) 98 (25.92) 82 (28.67) 16 (17.39) 9 (33.33) 1 (20) 29 (33.33)
GA 154 (43.5) 197 (52.12) 149 (52.1) 48 (52.17) 15 (55.56) 3 (60) 44 (50.57
AA 82 (23.17) 83 (21.96) 55 (19.23) 28 (30.44) 3 (11.11) 1 (20) 14 (16)
Allele
G 390 (55.08) 393 (51.98) 313 (54.72) 80 (43.48) 33 (61.11) 5 (50) 102 (58.62)
A 318 (44.92) 363 (48.02) 259 (45.28) 104 (56.52) 21 (38.89) 5 (50) 72 (41.38)

Table 3. Distribution of rs6921438 genotypes and alleles in patients with AITD (n ¼ 377), HT (n ¼ 286), GD (n ¼ 91), healthy control subjects
(n ¼ 348), hypothyroidism, hyperthyroidism and euthyroidism.

Control AITD1 HT2 GD3 Hypothyroidism Hyperthyroidism Euthyroidism

n (%) n (%) n (%) n (%) n (%) n (%) n (%)
Genotype
GG 190 (54.6) 182 (48.27) 144 (50.35) 38 (41.76) 15 (55.56) 4 (80) 39 (44.32)
GA 117 (33.62) 150 (39.79) 110 (38.46) 40 (43.96) 10 (37.04) 0 37 (42.32)
AA 41 (11.78) 45 (11.94) 32 (11.19) 13 (14.28) 2 (7.4) 1 (20) 12 (13.63)
Allele
G 497 (71.41) 514 (68.17) 398 (69.58) 116 (63.74) 40 (74.07) 8 (80) 113 (64.94)
A 199 (28.59) 240 (31.83) 174 (30.42) 66 (36.26) 14 (25.93) 2 (20) 61 (35.06)
 4 I. Zaaber et al. Autoimmunity, Early Online: 1–7

Table 4. Distribution of rs6993770 genotypes and alleles in patients with AITD (n ¼ 372), HT (n ¼ 282), GD (n ¼ 90), healthy control subjects
(n ¼ 349), hypothyroidism, hyperthyroidism and euthyroidism.

Control AITD1 HT2 GD3 Hypothyroidism Hyperthyroidism Euthyroidism

n (%) n (%) n (%) n (%) n (%) n (%) n (%)
Genotype
AA 139 (39.83) 145 (38.98) 109 (38.65) 36 (40) 11 (40.74) 2 (40) 33 (37.93)
TA 152 (43.55) 181 (48.66) 137 (48.58) 44 (48.89) 12 (44.44) 3 (60) 43 (49.42)
TT 58 (16.62) 46 (12.36) 36 (12.77) 10 (11.11) 4 (14.82) 0 11 (12.65)
Allele
A 430 (61.61) 471 (63.31) 355 (62.94) 116 (64.44) 34 (62.96) 7 (70) 109 (62.64)
T 268 (38.39) 273 (36.69) 209 (37.06) 64 (35.56) 20 (37.04) 3 (30) 65 (37.36)

Table 5. Distribution of rs4416670 genotypes and alleles in patients with AITD (n ¼ 374), HT (n ¼ 282), GD (n ¼ 92), healthy control subjects
(n ¼ 352), hypothyroidism, hyperthyroidism and euthyroidism.

Control AITD1 HT2 GD3 Hypothyroidism Hyperthyroidism Euthyroidism

n (%) n (%) n (%) n (%) n (%) n (%) n (%)
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Genotype
CC 99 (28.13) 102 (27.27) 85 (30.14) 17 (18.48) 9 (33.33) 0 19 (21.19)
TC 160 (45.45) 180 (48.13) 126 (44.68) 54 (58.69) 13 (48.15) 2 (40) 52 (59.77)
TT 93 (26.42) 92 (24.6) 71 (25.18) 21 (22.83) 5 (18.52) 3 (60) 16 (18.39)
Allele
C 358 (50.85) 384 (51.34) 296 (52.48) 88 (47.83) 31 (57.41) 2 (20) 90 (51.72)
T 346 (49.15) 364 (48.66) 268 (47.52) 96 (52.17) 23 (42.59) 8 (80) 84 (48.28)

Table 6. Hormone levels among the rs10738760, rs6921438, rs4416670 and rs6993770 genotypes.

FT4 after six months FT3 after six months

of treatment of treatment TSH after six months VEGF after six months
Mean ± standard Mean ± standard of treatment of treatment
deviation deviation Mean ± standard deviation Mean ± standard deviation
rs10738760
GG 23.276 ± 6.912 4.075 ± 2.321 2.168 ± 2.688 119.408 ± 143.79
GA 24.661 ± 7.518 4.894 ± 2.714 1.835 ± 1.974 101.28 ± 114.04
AA 26.478 ± 9.793 5.532 ± 3.699 1.817 ± 2.041 136.73 ± 167.40
rs6921438
GG 24.165 ± 7.496 4.924 ± 2.475 2.249 ± 2.408 132.79 ± 134.57
GA 25.608 ± 8.432 4.62 ± 3.344 1.394 ± 1.318 110.38 ± 146.68
AA 22.817 ± 7.535 5.055 ± 1.803 1.965 ± 2.828 41.071 ± 51.844
rs4416670
CC 23.533 ± 6.756 5.064 ± 2.061 1.935 ± 1.845 104.65 ± 138.67
TC 24.264 ± 8.249 4.415 ± 2.629 1.777 ± 2.247 111.56 ± 120.58
TT 26.669 ± 8.384 5.229 ± 3.804 2.103 ± 2.324 136.22 ± 166.28
rs6993770
AA 25.21 ± 7.502 4.449 ± 2.631 1.784 ± 1.787 137.56 ± 155.93
TA 24.16 ± 8.736 5.055 ± 3.223 1.835 ± 2.227 94.84 ± 107.45
TT 24.956 ± 5.953 4.755 ± 1.953 2.427 ± 2.745 124.31 ± 167.08

facilitating this change by increasing our knowledge on the unique finding among biomarkers of chronic diseases, ensures
biological and molecular processes implicated in diseases, that the study of VEGF ‘‘-omics’’ determinants can lead to
and the translation of this knowledge into clinical practice. predictive biomarkers.
However, a major obstacle in the integration of molecular This hypothesis led us to a GWAS identification of
information in current risk prediction and prevention is that VEGF-related variants (four SNPs, in three chromosomes)
the identified ‘‘-omics’’ markers explain a low percentage of that explain up to 50% of VEGF circulating levels
the disease/trait variability. variability [23]. These findings represent an additional
This investigation is based on strong epidemiological exception in the field of complex traits, where typically the
evidence that demonstrates VEGF’s high heritable genetic identified genetic markers explain less than 20% of the
component [28,29]. In fact, we have previously shown that the trait’s heritability, consequently overcoming the above-
additive genetic heritability of VEGF plasma levels in mentioned major obstacle of the majority of GWAS for
families was 60.5% of the total variance of the trait [28]. integration of molecular information in current risk
The extremely high heritable component of VEGF, which is a prediction.
 DOI: 10.3109/08916934.2016.1151005 VEGF polymorphisms in autoimmune thyroid disease 5
Table 7. Distribution of anti-TPO, anti-Tg and anti-TSHR antibody among the rs10738760, rs6921438, rs4416670 and
rs6993770 genotypes.

Anti-TPO antibody Anti-TG antibody Anti-TSHR antibody

+n (%) n (%) +n (%) n (%) +n (%) n (%)
rs10738760
GG 25 (32.05) 25 (25) 7 (36.84) 43 (27.05) 1 (10) 3 (30)
GA 37 (47.44) 52 (52) 7 (36.84) 82 (51.57) 7 (70) 3 (30)
AA 16 (20.51) 23 (23) 5 (26.32) 34 (21.38) 2 (20) 4 (40)
rs6921438
GG 35 (44.3) 56 (54.91) 8 (42.11) 83 (51.23) 5 (50) 4 (40)
GA 34 (43.04) 36 (35.29) 7 (36.84) 63 (38.89) 3 (30) 4 (40)
AA 10 (12.66) 10 (9.8) 4 (21.05) 16 (9.88) 2 (20) 2 (20)
rs4416670
CC 19 (24.36) 24 (24) 9 (47.37) 34 (21.38) 2 (20) 2 (20)
TC 46 (58.97) 47 (47) 7 (36.84) 86 (54.09) 5 (50) 6 (60)
TT 13 (16.67) 29 (29) 3 (15.79) 39 (24.53) 3 (30) 2 (20)
rs6993770
AA 30 (38.46) 38 (37.62) 6 (31.58) 62 (38.75) 4 (40) 2 (20)
TA 38 (48.72) 51 (50.5) 12 (63.16) 77 (48.13) 6 (60) 7 (70)
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TT 10 (12.82) 12 (11.88) 1 (5.26) 21 (13.12) 0 1 (10)

In this study, we demonstrate for the first time that the revealed that VEGF +405 polymorphism might be a risk
above-mentioned polymorphisms associated with VEGF factor for GD, while the 2578 SNP is related with increased
levels are also associated with an increased risk of GD auto-antibody production.
(versus HT and versus controls) and with a risk of hyperthy- The conflicting results of the previous genetic studies
roidism even after treatment. Moreover, we have shown that could be due to the low pertinence of the studied polymorph-
one of these SNPs is also associated with increased levels of isms in terms of their implication in the VEGF circulating
FT3 after treatment. Finally, no association with VEGF levels levels inter-individual variability. No SNP among the previ-
was observed between GD and HT patients, neither between ously reported has been found associated with VEGF levels in
each group of AITD patients and controls nor between all our GWAS [28].
AITD patients and controls. Given this last result, a higher The four GWAS-identified SNPs are located close to the
diagnostic value to the genetic component of VEGF could be VEGF and MRPL14 genes (chromosome 6p21.1), within the
attributed. ZFPM2 gene (chromosome 8q23.1), and between the VLDLR
The proangiogenic effects of thyroid hormones (T4 and and KCNV2 genes (chromosome 9p24.2). To date a link
T3) have been first described by R. J. Tomanek et al. in 1985 between these loci and AITD pathophysiology has not been
in a heart model [24]. Later, the association of thyroid established.
hormone analog diiodothyropropionic acid (DITPA) with The A allele of polymorphism rs10738760 was more
VEGF protein expression was reported in non-ischemic heart frequent in the GD group and was associated with an
experiments [25]. Both T4 and DITPA therapies increase increased risk compared with HT and control groups. The A
brain vascularity after thyroidectomy-induced hypothyroidism allele of rs6921438 polymorphism was significantly asso-
in rats [26]. A first explanation could be that thyroid ciated with a high risk of GD compared with controls. These
hormones induce HIF1a production but not its stabilization results underline the specificity of VEGF-linked polymorph-
[27]. A second explanation stands in a non-genomic action isms in GD, inside the AITD family. Even if GD requires
through integrin ávâ3 action on VEGFR-2 pathway. This constant VEGF-dependent neovascularization, this cannot
integrin contains a site for thyroid hormone and induces the be the only explanation for these results. Hyperthyroidism
IP3K and MAPK pathways [28–30]. Therefore, thyroid is in itself associated with increased VEGF production [39].
hormone induces angiogenesis both through membrane, Therefore, it is difficult to determine if VEGF plays
cytosolic and nuclear actions [31]. The inhibition of thyroid its major role through inflammation and goitre genesis
hormone-activated ávâ3 leads to a decrease in VEGF or as a consequence of thyroid hormone-induced
synthesis [32]. vascularization.
This has also been confirmed by nanoTetrac While rs10738760 is associated with FT3 levels, the SNP
(Tetraiodothyroacetic Acid) prevention of thyroid hormones rs4416670 is associated with hyperthyroidism after six
effects, especially through ávâ3 integrin blockage [33]. months of treatment. This could lead to the separation of
Several studies have shown a positive association with patients with common hyperthyroidism and patients with
some polymorphisms such as rs699947, rs1570360, rs833061, gravity signs. Since higher FT3 levels are considered as a sign
rs2010963 and rs3025039 [34–37] while other studies have of severity and since the highest levels of FT3 are often found
found no association for different diseases [19,38]. Few data in GD, rs10738760 could be implicated in the genesis
in the literature describe the relationship between GD or HT of hyperthyroidism through inflammation (Figure 1).
and polymorphisms of VEGF gene. Only Vural et al. [22] Considering the apparent lack of specificity of rs4416670, it
have investigated the relationship between GD and A-2578C, could be hypothesized that the latter polymorphism acts in the
T-460C and G + 405C SNPs of VEGF in GD. This study downstream effects of inflammatory lesions.
 6 I. Zaaber et al. Autoimmunity, Early Online: 1–7

Figure 1. Plasma VEGF-related polymorph-

isms and their relationships with GD and
Hyperthyroidism.
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VEGF regulates many inflammatory and autoimmune way forward for pharmacogenomics studies is wide open in
mechanisms by promoting the migration and proliferation of the field of thyroiditis (especially in GD), which lacks a
endothelial cells. Also, it stimulates leukocyte adhesion to the reliable prognostic marker and as a result presents a challenge
endothelium, and initiates monocyte/macrophage chemotaxis to the physician in everyday practice.
as well as angiogenesis [40,41]. In patients with autoimmune
thyroid disease such as GD and HT circulating levels of
Declaration of interest
VEGF were increased and neovascularisation intrathyroid
observed [12]. A recent study in a Chinese population found This work was supported by the ‘‘Région Lorraine’’. No
decreased plasma levels of VEGF in hyperthyroid patients in competing financial interests exist.
comparison with healthy controls [42]. Another study found
the opposite results, in association with other angiogenic References
factor such as Ang-1, Ang-2 and STie-2 [43]. In the current
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