March 2013
Volume 54, Issue 3
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Retina  |   March 2013
A Genetic Case-Control Study Confirms the Implication of SMAD7 and TNF Locus in the Development of Proliferative Vitreoretinopathy
Author Affiliations & Notes
  • Jimena Rojas
    From the Institute of Applied Ophthalmolbiology (IOBA-Eye Institute), University of Valladolid, Spain; the
  • Itziar Fernandez
    Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (Ciber BBN), Valladolid, Spain; the
  • Jose C. Pastor
    From the Institute of Applied Ophthalmolbiology (IOBA-Eye Institute), University of Valladolid, Spain; the
  • Robert E. MacLaren
    Moorfields Eye Hospital and University College London Institute of Ophthalmology National Institute for Health Research Biomedical Research Centre, London, United Kingdom;
  • Yashim Ramkissoon
    Moorfields Eye Hospital and University College London Institute of Ophthalmology National Institute for Health Research Biomedical Research Centre, London, United Kingdom;
  • Steven Harsum
    Moorfields Eye Hospital and University College London Institute of Ophthalmology National Institute for Health Research Biomedical Research Centre, London, United Kingdom;
  • David G. Charteris
    Moorfields Eye Hospital and University College London Institute of Ophthalmology National Institute for Health Research Biomedical Research Centre, London, United Kingdom;
  • Jan C. Van Meurs
    The Rotterdam Eye Hospital and Erasmus University Medical Center, Rotterdam, the Netherlands; the
  • Sankha Amarakoon
    The Rotterdam Eye Hospital and Erasmus University Medical Center, Rotterdam, the Netherlands; the
  • Jose M. Ruiz-Moreno
    VISSUM, Alicante, University of Castile-La Mancha, Albacete, Spain; the
  • Amandio Rocha-Sousa
    Department of Senses Organs, Faculty of Medicine, University of Porto, São João Hospital, Porto, Portugal; the
  • Maria Brion
    Medicina Xenómica, Complexo Hospitalario Universitario de Santiago, Institute for Development and Integration of Health (IDIS), Santiago de Compostela, Spain; and the
    University of Santiago de Compostela, Galician Public Foundation for Genomic Medicine, CIBERER, Santiago de Compostela, Spain.
  • Angel Carracedo
    Medicina Xenómica, Complexo Hospitalario Universitario de Santiago, Institute for Development and Integration of Health (IDIS), Santiago de Compostela, Spain; and the
    University of Santiago de Compostela, Galician Public Foundation for Genomic Medicine, CIBERER, Santiago de Compostela, Spain.
  • Corresponding author: Jimena Rojas, Calle de Belen 17, Campus Universitario Miguel Delibes, Edificio IOBA, CP 47007, Valladolid, Spain; jimena@ioba.med.uva.es
Investigative Ophthalmology & Visual Science March 2013, Vol.54, 1665-1678. doi:10.1167/iovs.12-10931
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      Jimena Rojas, Itziar Fernandez, Jose C. Pastor, Robert E. MacLaren, Yashim Ramkissoon, Steven Harsum, David G. Charteris, Jan C. Van Meurs, Sankha Amarakoon, Jose M. Ruiz-Moreno, Amandio Rocha-Sousa, Maria Brion, Angel Carracedo; A Genetic Case-Control Study Confirms the Implication of SMAD7 and TNF Locus in the Development of Proliferative Vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 2013;54(3):1665-1678. doi: 10.1167/iovs.12-10931.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Proliferative vitreoretinopathy (PVR) is still the major cause of failure of retinal detachment (RD) surgery and although the risk for developing this complication is associated with some clinical characteristics, the correlation is far from absolute, raising the possibility of genetic susceptibility. The objective of this study was to analyze the genetic contribution to PVR in patients undergoing RD surgery, the Retina 4 Project.

Methods.: A candidate gene association study was conducted in 2006 in a Spanish population of 450 patients suffering from primary rhegmatogenous RD. Replication was carried out in a larger population undergoing RD surgery at several European centers among 546 new patients. Single nucleotide polymorphism (SNP) of 30 genes known to be involved with inflammation were analyzed. For replication stage, those genes previously detected as significantly associated with PVR were genotyped. Distribution of allelic and haplotypic frequencies in case and control group were analyzed. Single and haplotypic analysis were assessed. The Rosenberg two-stage method was used to correct for single and multiple analyses.

Results.: After correction for multiple comparisons, four genes were significantly associated with PVR: SMAD7 (P = 0.004), PIK3CG (P = 0.009), TNF locus (P = 0.0005), and TNFR2 (P = 0.019) In the European sample, replication was observed in SMAD7 (P = 0.047) and the TNF locus (P = 0.044).

Conclusions.: These results confirm the genetic contribution to PVR and the implication of SMAD7 and TNF locus in the development of PVR. This finding may have implications for understanding the mechanisms of PVR and could provide a potential new therapeutic target for PVR prophylaxis.

Introduction
Proliferative vitreoretinopathy (PVR) is still in this era the major cause of failure of retinal detachment (RD) surgery with an incidence of 5% to 10%. 1 It is believed to represent an abnormal wound healing process induced by a retinal break, which allows egress of RPE cells into the vitreous cavity and there is strong evidence that inflammation plays an important role. 2,3 Following RD, the blood–ocular barrier breaks down, possibly due to disruption of the photoreceptor-RPE cell interface, and inflammatory cells are recruited together and increase in inflammatory mediators. 46 Growth factors and cytokines present in the vitreous cavity may be responsible for cell migration, metaplasia, and proliferation, 79 which can result in the development of glial scar tissue and retinal contraction. 1013  
Most researches in this field have orientated their efforts to identify clinical factors responsible of this pathologic repairing process (such as, the method of retinopexy or tamponade used). However, it seems likely as a cell-based inflammatory response that genetic susceptibility may have a role, particularly as PVR can occur in patients with prompt and initially successful surgery following a primary rhegmatogenous RD. 14,15 Hence, we are still unable to predict the risk of PVR reliably and apart from surgery, there is currently no prevention or cure. 1618  
We have learned that many diseases are the consequence of interaction between environmental factors (clinical variables) and the genetic profile of each subject. 19,20 Considering this concept of complex disease, we decided to investigate the genetic component of PVR. One preliminary study conducted by our group in a small sample had showed in 2005 an association between the TGF-β1 and PVR. 21 We also developed three predictive models of PVR based on the analysis of genetic variables. 22  
The Retina 4 project, a candidate gene and replication study strategy, overcomes one of the major problems with association studies in generating false positives. 23,24 Associations need to be replicated in order to confirm their veracity. For that reason, this study comprises two stages: the discovery stage, where only samples coming from Spanish centers were analyzed; and the replication stage, where new samples coming from centers in Spain, Portugal, the Netherlands, and the United Kingdom were studied. 
Methods
Setting and Design
A case-controlled, candidate gene association study was conducted in 2006 among patients from eight centers in Spain, the discovery stage (Table 1). For the replication stage, a European multicenter case-controlled association study in those genes found significantly associated to PVR in the discovery stage (TNF locus, mothers against decapentaplegic (SMAD7), phosphatidyl inositol 3-kinase, catalytic, gamma (PIK3CG), and TNFR2) was conducted between 2009 and 2010. All patients provided written informed consent, and the study was approved by the institutional research committees of each center and followed the tenets of the Declaration of Helsinki. 
Table 1. 
 
Centers Involved in the Study
Table 1. 
 
Centers Involved in the Study
Country Center Discovery Stage Replication Stage
Cases PVR Controls RD Total Cases PVR Controls RD Total
England
 Moorfields Eye Hospital, London N - - - 32 118 150
 % Total - - - 5.9 21.6 27.5
Holland
 Rotterdam Eye Hospital, Rotterdam N - - - 39 89 128
 % Total - - - 7.1 16.3 23.4
Portugal
 Coimbra University Hospital, Coimbra  N - - - 6 1 7
 % Total - - - 1.1 0.2 1.3
 Sao Joao Hospital, Oporto N - - - 6 52 58
 % Total - - - 1.1 9.5 10.6
Spain
 Germans Trias i Pujol University Hospital, N - - - 11 11 22
  Badalona  % Total - - - 2.0 2.0 4.0
 Jiménez Díaz Foundation, Madrid N - - - 2 2 4
 % Total - - - 0.4 0.4 0.7
 Ophthalmological Foundation of Mediterranean, N - - 5 2 7
  Valencia  % Total - - 0.9 0.4 1.3
 San Millan San Pedro Hospital, Logroño N - - - 9 1 10
 % Total - - - 1.6 0.2 1.8
 Universitary Hospital La Fe, Valencia  N - - - 10 25 35
 % Total - - - 1.8 4.6 6.4
 University Hospital of Navarra  N - - - 11 19 30
 % Total - - - 2.0 3.5 5.5
 Universitay Hospital Ramón y Cajal, Madrid N - - - 2 3 5
 % Total - - - 0.4 0.5 0.9
 Barraquer Ophthalmology Centre  N 8 35 43 2 6 8
 % Total 1.8 7.8 9.5 0.4 1.1 1.5
 Donostia Hospital, San Sebastian N 7 35 42 2 0 2
 % Total 1.5 7.8 9.3 0.4 0 0.4
 IOBA (University Eye Institute), Valladolid N 28 22 50 10 15 25
 % Total 6.2 4.9 11.1 1.8 2.7 4.6
 Reina Sofia Universitary Hospital, Cordoba N 5 9 14 - - -
 % Total 1.1 2 3.1 - - -
 Universitary Hospital Pio del Rio Hortega N 7 10 17 - - -
 % Total 1.5 2.2 3.8 - - -
 University Hospital Vall d'Hebrón N 20 75 95 1 25 26
 % Total 4.4 16.7 21.1 0.2 4.6 4.8
 University Hospital of Valladolid N 32 28 60 2 8 10
 % Total 7.1 6.2 13.3 0.4 1.5 1.8
 Vissum, Alicante N 31 98 129 1 18 19
 % Total 6.9 21.8 28.7 0.2 3.3 3.5
TOTAL N 138 312 450 151 395 546
% Center 30.7 69.3 100.0 27.7 72.3 100.0
Study Population
Both samples were composed of patients who had undergone primary rhegmatogenous RD surgery. Careful ophthalmoscope examination by slit lamp and indirect ophthalmoscope were performed postoperatively to classify the patient as case or control. 
Those who developed PVR grade C1 or higher 25 after surgery were included as a case. Those who did not develop PVR after 3 months of follow up were included in the control group. To achieve a stringent phenotype classification, other causes than a primary rhegmatogenous RD, such as traumatic, tractional, exudative, or iatrogenic RD were excluded. Exclusion criteria were also RD secondary to macular hole, giant retinal tears defined by more than 3 hours, patients with preoperative PVR grade C1 or higher, 25 and patients with RD in the affected eye and RD with PVR in the fellow eye. In addition, clinical characteristics that could affect the stringent phenotypification were investigated like race, family history of RD, status of the lens, and so on (Table 2). Surgeon experience was also valorated. Experts were considered to be those who had already performed greater than or equal to 100 pars plana vitrectomy (PPV) and no experts those with less than 100 PPV. 
Table 2. 
 
Clinical Characteristics of the Replication Sample
Table 2. 
 
Clinical Characteristics of the Replication Sample
Replication Stage
Characteristic Case—PVR Control—RD Total P Value
% Type % Type
Sex
 Male 100 28.74 248 71.26 348 0.362
 Female 45 25.00 135 75.00 180
 Total 145 (6)° 27.46 383 (12)° 72.54 528
Race
 North-African 0 0 6 100 6 0.0644
 Asian 2 40.00 3 60.00 5
 European 133 26.44 370 73.56 503
 Hispanic 7 53.85 6 46.15 13
 Indian 5 41.67 7 58.33 12
 Subsaharian 2 50.00 2 50.00 4
 Total 149 (2)° 27.44 394 (1)° 72.56 543
RD family history
 No 142 27.52 374 72.48 516 0.932
 Yes 9 30 21 70 30
 Total 151 27.66 395 72.34 546
PVR family history
 No 149 27.49 393 72.51 542 0.4758*
 Yes 1 50 1 50 2
 Total 150 (1)° 27.57 394 (1)° 72.43 544
RD in fellow eye
 Unknown 5 38.46 8 61.54 13 0.5289*
 No 136 27.7 355 72.3 491
 Yes 10 23.81 32 76.19 42
 Total 151 27.66 395 72.34 546
PVR in fellow eye
 Unknown 5 38.46 8 61.54 13 0.0057*
 No 141 26.70 387 73.30 528
 Yes 5 100 0 0 5
 Total 151 27.66 395 72.34 546
Phakia/aphakia
 Unknown 7 33.33 14 66.67 21 0.2333
 Aphakia 19 38.78 30 61.22 49
 Pseudophakia 40 28.37 101 71.63 141
 Phakia 85 25.37 250 74.63 335
 Total 151 27.66 395 72.34 546
Retinopexy
 No 147 27.63 385 72.37 532 0.8268*
 Yes 4 28.57 10 71.43 14
 Total 151 27.66 395 72.34 546
Scleral surgery (SS)
 No 116 25.55 338 74.45 454 0.0281*
 Yes 34 38.2 55 61.8 89
 Total 150 (1)° 27.62 393 (2)° 72.38 543
SS + drainage
 No 131 29.18 318 70.82 449 0.0383*
 Yes 20 20.62 77 79.38 97
 Total 151 27.66 395 72.34 546
PPV
 No 15 10.87 123 89.13 138 <0.0001
 Yes 136 33.33 272 66.67 408
 Total 151 27.66 395 72.34 546
Air
 No 138 26.59 381 73.41 519 0.0153*
 Yes 13 48.15 14 51.85 27
 Total 151 27.66 395 72.34 546
SF6
 No 87 30.21 201 69.79 288 0.0077*
 Yes 64 24.81 194 75.19 258
 Total 151 27.66 395 72.34 546
C3F8
 No 116 26.54 321 73.46 437 0.3041*
 Yes 35 32.11 74 67.89 109
 Total 151 27.66 395 72.34 546
Silicone
 No 55 13.03 367 86.97 422 <0.0001*
 Yes 96 77.42 28 22.58 124
 Total 151 27.66 395 72.34 546
Laser
 No 35 13.41 226 86.59 261 <0.0001*
 Yes 116 40.7 169 59.3 285
 Total 151 27.66 395 72.34 546
Cryotherapy
 No 99 31.03 220 68.97 319 0.0481*
 Yes 52 22.91 175 77.09 227
 Total 151 27.66 395 72.34 546
In the discovery stage only patients coming from Spanish centers were included, while in the replication stage participants were completely new patients coming from centers in Holland, Portugal, Spain, and the United Kingdom (Table 1). Inclusion of patients during the replication stage coming from centers that participated in the former stage was consecutive and prospective. Simulation-based power analysis by bootstrap was used for sample size determination of replication stage. 
Measures
Blood Collection and DNA Extraction.
A peripheral blood sample was used for DNA extraction, which was performed using the commercial REALpure Kit protocol SSS (DURVIZ SL, Valencia, Spain), or similar. Genotyping was performed by the Genotyping Center (CeGen-ISCIII, Santiago de Compostela, Spain). For the discovery stage, the SNPlex Genotyping System (Applied Biosystem, Foster City, CA) was used. For the replication sample the MassARRAY SNP Genotyping System (Sequenom Inc., San Diego, CA) was used following the manufacturer's instructions. The principles of this method are detailed in Buetow et al. 26  
Genes and Single Nucleotide Polymorphism Selection.
According the inflammatory nature of PVR, 2,27 the following 30 candidate genes were investigated in the discovery stage: connective tissue growth factor (CTGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), interferon gamma (IFNG), insulin-like growth factor 2 (IGF-2), insulin-like growth factor I receptor (IGF1R), interleukin 1 alpha (IL1A), interleukin 1 beta (IL1B), interleukin 1 receptor antagonist (IL1RN), interleukin 6 (IL6), interleukin 8 (IL8), interleukin 10 (IL10), monocyte chemotactic protein 1 (MCP1), macrophage migration inhibitory factor (MIF), matrix metalloproteinase (MMP)-2, matrix metalloproteinase 7 (MMP7), nuclear factor kappa-b subunit 1 (NFKB1), nuclear factor of kappa light chain gene enhacer in B cells inhibitor alpha (NFKBIA), nuclear factor of kappa light chain gene enhacer in B cells inhibitor beta (NFKBIB), platelet-derived growth factor (PDGF), platelet-derived growth factor receptor alpha (PDGFRα), PI3K, SMAD3, SMAD7, transforming growth fator beta 1 (TGF-β1), transforming growth fatcor beta 2 (TGF-β2), tumor necrosis factor alpha (TNF-α), and tumor necrosis factor receptor 2 (TNFR2). Common tag SNPs with correlation coefficients greater than or equal to 0.85 and a minor allelic frequency greater than or equal to 10% were studied to explain as much as possible the known genetic variation for each gene. The Tagger method implemented in the Haploview program (provided in the public domain by Broad Institute; http://www.broad.mit.edu/mpg/haploview/) was used for this purpose. 28 According to the linkage disequilibrium observed in the TNF-α region, the following four genes were necessary to be investigated in the TNF locus: lymphotoxin alpha (LTα), TNF-α, leucocyte specific transcript 1 (LST1), and the activating natural killer receptor p30 (NCR3). Genic and extragenic regions 10 kb upstream and downstream were considered for each gene. Functional SNPs or ones previously described in association with other inflammatory diseases were added for analysis. 2933 In the replication stage same SNPs previously studied in PIK3CG, SMAD7, TNF locus, and TNFR2 (30 SNPs) were investigated. 
Statistical Analysis
Preliminary and Descriptive Analysis.
The data quality were evaluated by Hardy-Weinberg equilibrium using a Pearson goodness-of-fit test. When there was a low genotype count, a Fisher exact test was used. Data quality were also evaluated by differential missingness between cases and controls. Allelic and genotypic frequencies were estimated. 
Test of Association: Single SNPs.
Single associations were established using the χ2 and Fisher tests. Five inheritance models were defined: (1) In the codominant model, every genotype gave a different and nonadditive risk, (2) in the dominant model, a single copy of the variant allele was enough to modify the risk, (3) in the recessive model, two copies of the variant allele were necessary to change the risk, (4) in the over dominant model, heterozygosity was compared with a pool of each allele homozygosity, and (5) in the additive model, each copy of the variant allele modified the risk in an additive form. The Akaike Information Criteria (AIC) was used to choose the inheritance model that best fit the data. 
Test of Association: Haplotypic SNPs.
Haplotypes consistent with the sample were analyzed taking into consideration blocks from as few as two consecutive markers (subhaplotypes) to as much as all of the markers included in each gene (haplotypes). Haplotypic frequencies were estimated using the expectation-maximization algorithm. 34 To evaluate the association between haplotypes and disease, generalized linear models were used. The effects of haplotypes were modeled as additive, dominant, and recessive. The log-likelihood criterion was used to select the best model. 
Multiple Testing and False Discovery Rate Analysis.
To correct for multiple analyses, the Rosenberg two-stage method was used. 35 For the first stage, single-SNP association tests and haplotype-SNP association tests within one gene were considered. An omnibus test that combined SNP and haplotype analysis was then constructed. This omnibus test used the test statistic  where pSNP was the Simes-adjusted P value 36 for the most significant SNP, and pHAP was the Simes-adjusted P value for the most significant haplotype. The distribution of the omnibus test statistic under the independence hypothesis was computed from the permutation distribution obtained by shuffling case and control indicators. In the second stage, summary gene P values were adjusted for multiplicity by controlling the expected false discovery rate with the Benjamini-Hochberg procedure (q-value). 37  
All statistical analyses were conducted using R software including SNPassoc and haplo.stat packages. 3840  
Results
Discovery Stage
Four hundred fifty patients were analyzed, including 138 cases and 312 controls. A total of 230 SNPs distributed along the 30 candidate genes were investigated. Six SNPs did not pass the design pipeline, and 27 failed during the genotyping process. All informative SNPs verified the Hardy-Weinberg equilibrium except one. It was located in the PDGFα gene and was eliminated because it exhibited poor performance during the genotyping process. Thus, a total of 196 SNPs were analyzed, yielding a conversion rate of 85.2%. 
Single and Haplotypic Associations.
Twenty two single significant associations were observed in 16 genes: EGF, IGF1, IL1RN, MIF, MMP-2, NFKB1, NFKBIA, NFKBIB, PDGFA, PDGFRA, SMAD3, SMAD7, TGFB1, TGFB2, TNF locus, and TNFR2 (Table 3). Following permutation, only three single significant associations in three genes were detected: the rs243845 in MMP-2 (P = 0.050), the rs7226855 in SMAD7 (P = 0.0015), and the rs2229094 in TNF locus (P = 0.0283) (Table 3). 
Table 3. 
 
Single Associations in the Discovery and Replication Stages
Table 3. 
 
Single Associations in the Discovery and Replication Stages
Gene SNP Genotype Discovery Stage Replication Stage
Frequency, % Inheritance Model* OR CI 95% OR P Value Corrected P Value Frequency, % Inheritance Model* OR CI 95% OR P Value Corrected P Value
Control Case Lower Upper Control Case Lower Upper
EGF rs1024600 CC/CG 92.7 85.7 Recessive 0.02846 0.6870 - - - - - - - -
C→G GG 7.3 14.3 2.12 1.09 4.11 - - - - - - - -
IGF1 rs2195240 TT/CT 93.4 98.4 Recessive 0.23 0.05 0.98 0.017 0.1816 - - - - - - - -
T→C CC 6.6 1.6 - - - - - - - -
rs5742629 AA/AG 90.4 97.5 Recessive 0.25 0.07 0.84 0.0087 0.1816 - - - - - - - -
A→G GG 9.4 2.5 - - - - - - - -
IL1RN rs3087270 AA 47.6 56.8 Additive 0.72 0.53 0.99 0.0420 0.9132 - - - - - - - -
A→G AG 38.8 35.2 - - - - - - - -
GG 13.6 8 - - - - - - - -
MIF rs1007888 AA 30 19 Dominant 0.0201 0.2306 - - - - - - - -
A→G AG/GG 70 81 1.82 1.08 3.07 - - - - - - - -
rs243845 CC 45.4 29.6 Dominant 0.0023 0.0500 - - - - - - - -
C→T CT/TT 54.6 70.4 1.98 1.26 3.09 - - - - - - - -
rs243864 TT/GT 92.2 97.5 Recessive 0.30 0.09 1.01 0.026 0.3446 - - - - - - - -
T→G GG 7.8 2.5 - - - - - - - -
rs243866 GG/AG 92.4 97.5 Recessive 0.31 0.09 1.05 0.0317 0.3446 - - - - - - - -
G→A AA 7.6 2.5 - - - - - - - -
NFKB1 rs4698858 CC 45.1 32.3 Dominant 1.73 1.11 2.69 0.01435 0.2614 - - - - - - - -
C→G CG/GG 54.9 67.7 - - - - - - - -
NFKBIA rs17103274 TT 84.9 72.1 Dominant 0.0034 0.0515 - - - - - - - -
T→C CT/CC 15.1 27.9 2.17 1.3 3.61 - - - - - - - -
NFKBIB rs3136640 GG/CC 51.3 65.1 Overdominant 0.0087 0.0993 - - - - - - - -
G→C CG 48.7 34.9 0.57 0.37 0.87 - - - - - - - -
PDGFA rs7806249 TT/CC 52.4 62.9 Overdominant 0.04696 0.3917 - - - - - - - -
T→C CT 47.6 37.1 0.65 0.42 1.00 - - - - - - - -
PDGFRA rs7656613 TT/CC 65.1 51.4 Overdominant 0.1654 0.1884 - - - - - - - -
T→C CT 34.9 48.6 1.76 1.11 2.8 - - - - - - - -
SMAD3 rs6494634 CC 40.8 50.8 Additive 0.71 0.50 0.99 0.04016 0.8741 - - - - - - - -
C→T CT 49.3 43 - - - - - - - -
TT 9.9 6.2 - - - - - - - -
PIK3CG rs4460309 CC/CT - - - - - - - - 95.1 90.2 Recessive 0.04566 0.047
C→T TT - - - - - - - - 4.9 9.8 2.12 1.03 4.35
rs7226855 AA 56.3 36.2 Overdominant 2.27 1.48 3.48 0.00014 0.0015 34.8 22.8 Dominant 0.006382 0.0070
A→G 65.2 77.2
43.7 63.8 1.8 1.17 2.79
rs6507877 GG/AA 57.2 46.9 Overdominant 0.03446 0.0070
A→G AG 1.51 1.03 2.22
TGFB1 rs2241713 GG/CG 79.2 87.6 Recessive - - - - - - - -
G→C CC 20.8 12.4 0.54 0.29 0.99 0.03810 0.3175 - - - - -
TGFB2 rs1891467 AA 53.5 45 Additive 1.44 1.03 2.02 0.03447 0.25 - - - - - - - -
A→G AG 42.2 45.7 - - - - -
GG 4.3 9.3 - - - - -
rs2000220 AA 48.1 39.1 - - - - - - - - -
A→G AG 41.8 43.8 Addictive - - - - -
GG 10.1 17.2 1.41 1.04 1.90 0.02595 0.25 - - - - -
rs947712 GG 58.6 47.6 - - - - - - - -
G→A AG 35.4 38.9 Addictive - - - - -
AA 6.1 13.5 1.54 1.12 2.11 0.00787 0.1718 - - - - -
TNF locus rs2857706 AA/GG 75.2 62.2 Overdominant 0.0077 0.0837 - - - - - - - -
G→A AG 24.83 37.80 1.84 1.18 2.87 - - - - -
rs2229094 CT/CC 6.33 8.94 Dominant 2.02 1.31 3.11 0.0013 0.0283 - - - - - - - -
T→C TT 52.00 34.96 - - - - -
rs2256974 GG 64.55 74.59 Additive 0.64 0.42 0.97 0.0315 0.2783 64.7 74.7 Dominant 0.02615 0.0060
G→T GT 31.34 23.77 35.3 25.3
TT 4.10 1.64 0.62 0.40 0.95
rs909253 TT/CT - - - - - - - - 87.4 95.1 Recessive 0.0059 0.0060
T→C CC - - - - - - - 12.6 4.9 0.36 0.16 0.81
rs199964 TT/CT - - - - - - - - 93.6 97.9 Recessive 0.03152 0.0060
T→C CC - - - - - - - 6.4 2.1 0.31 0.09 1.06
rs1800629 GG/AA - - - - - - - - 76.3 67.8 Overdominant 0.04563 0.0060
G→A GA - - - - - - - 23.7 32.2 1.53 1.01 2.32
In multiple analyses, significant haplotypic and subhaplotypic associations were detected in 13 genes: IGF-IR, IL-10, IL6, MIF, MMP-2, NFKB1, NFKBIA, NFKBIB, PIK3CG, SMAD3, TGF-β2, TNF locus, and TNFR2 (Table 4). Following permutation, five genes maintained their significant association: NFKB1 (P = 0.0460), NFKBIA (P = 0.0460), PIK3CG (P = 0.0010), TNF locus (P = 0.0050), and TNFR2 (P = 0.0130) (Table 4). 
Table 4. 
 
Haplotypic Associations in the Discovery and Replication Stages
Table 4. 
 
Haplotypic Associations in the Discovery and Replication Stages
Gene N SNP Initial SNP Inheritance Model* Discovery Stage Replication Stage
Frequency, % OR CI 95% OR Multiple Association P Value Corrected by Gene Inheritance Model* Frequency, % OR CI 95% OR Multiple Association P Value Corrected by Gene
Control Case Inferior Superior P Value Corrected P Value Control Case Inferior Superior P Value Corrected P Value
IGF-IR 2 rs10794486 Additive 9.72 16.98 1.78 1.12 2.85 0.0125 0.0150 0.1760 - - - - - - - - -
3 rs7166287 Additive 6.69 14.42 2.49 1.37 4.52 0.0031 0.0050 - - - - - - - - -
4 rs7166287 Additive 2.74 7.78 3.19 1.36 7.49 0.0054 0.0110 - - - - - - - - -
rs10794486 Additive 2.10 7.94 4.15 1.61 10.70 0.0032 0.0043 - - - - - - - - -
5 rs7166287 Additive 2.54 6.90 4.19 1.37 12.83 0.0168 0.0180 - - - - - - - - -
rs10794486 Additive 2.01 7.82 4.18 1.59 10.98 0.0010 0.0020 - - - - - - - - -
6 rs7166287 Additive 2.51 6.72 3.88 1.27 11.86 0.0072 0.0080 - - - - - - - - -
rs10794486 Additive 2.0 7.27 3.36 1.30 8.66 0.0013 0.0030 - - - - - - - - -
IL-10 3 rs1800890 Additive 4.6 3.09 9.17 1.87 45.05 0.0015 0.0023 0.1700 - - - - - - - - -
IL-6 6 rs4719713 Additive 1.48 3.80 3.43 1.16 10.14 0.0533 0.0529 0.1100 - - - - - - - - -
8 rs22725336 Additive 1.06 3.29 3.75 1.02 13.75 0.0127 0.0112 - - - - - - - - -
0.46 3.18 9.98 1.99 49.95 0.0025 0.0010 - - - - - - - - -
1.42 3.81 3.49 1.17 10.42 0.0468 0.0360 - - - - - - - - -
MIF 2 rs1007888 Additive 7.07 11.60 1.90 1.12 3.24 0.0391 0.0430 0.1850 - - - - - - - - -
MMP2 2 rs9928731 Additive 33.88 42.54 1.43 1.02 1.99 0.0185 0.0180 0.2050 - - - - - - - - -
rs243845 Dominant 34.27 43.36 1.95 1.20 3.14 0.0127 0.0092 - - - - - - - - -
3 rs9928731 Additive 0.15 2.07 14.13 12.59 15.86 0.0267 0.0170 - - - - - - - - -
NFKB1 3 rs3774932 Dominant 2.36 5.81 2.69 1.19 6.07 0.0173 0.0230 0.0460 - - - - - - - - -
rs4698858 Dominant 18.68 10.01 0.49 0.28 0.87 0.0033 0.0030 - - - - - - - - -
rs11722146 Dominant 1.80 5.90 3.87 1.61 9.30 0.0026 0.0023 - - - - - - - - -
4 rs4698858 Dominant 18.46 10.82 0.55 0.32 0.95 0.0097 0.0063 - - - - - - - - -
0.84 3.27 4.89 1.32 18.11 0.0021 0.0047 - - - - - - - - -
rs11722146 Dominant 1.80 5.93 3.83 1.59 9.21 0.0025 <0.0001 - - - - - - - - -
5 rs2300540 Dominant 1.05 3.08 3.43 1.07 10.93 0.0326 0.0230 - - - - - - - - -
0.67 2.80 4.76 1.25 18.17 0.0081 0.0100 - - - - - - - - -
rs4698858 Dominant 0.82 3.45 5.25 1.48 18.67 0.0019 0.0022 - - - - - - - - -
6 rs3774932 Dominant 0.17 2.10 12.85 1.42 116.34 0.0020 0.0050 - - - - - - - - -
rs2300540 Dominant 1.05 3.17 3.37 1.06 10.67 0.0293 0.0320 - - - - - - - - -
0.67 2.71 4.46 1.16 17.16 0.0090 0.0070 - - - - - - - - -
NFKBIA 2 rs2007960 Dominant 8.04 14.48 2.47 1.44 4.24 0.0054 0.0037 0.0460 - - - - - - - - -
3 rs3138045 Dominant 8.02 13.64 2.59 1.49 4.52 0.0054 0.0020 - - - - - - - - -
6 rs7152826 Dominant 5.75 8.24 0.27 0.21 0.35 0.0493 0.0500 - - - - - - - - -
NFKBIB 2 rs11879872 Dominant 0.32 2.49 11.88 1.28 110.62 0.0333 0.0360 0.0830 - - - - - - - - -
PIK3CG 3 rs4460309 Dominant 0.01 1.14 15.8 × 104 15.8 × 104 15.8 × 104 0.0010 0.0410 0.0010 - - - - - - - - -
SMAD7 2 rs4939826 - - - - - - - - - Additive 41.85 49.69 1.40 1.06 1.86 0.0207 0.0330 0.3110
TGFB2 2 rs947712 Dominant 13.41 19.20 1.99 1.22 3.24 0.0205 0.0230 0.3180 - - - - - - - - -
rs2796821 Dominant 14.04 20.49 1.83 1.15 2.89 0.0189 0.0120 - - - - - - - - -
TNF locus 2 rs909253 Dominant 27.14 37.18 1.98 1.27 3.08 0.0024 0.0046 0.0050 - - - - - - - - -
rs2229094 Dominant 2.98 9.49 3.65 1.82 7.31 0.0001 <0.0001 - - - - - - - - -
3 rs2857706 Dominant 15.16 20.52 1.87 1.18 2.96 0.0484 0.0430 - - - - - - - - -
rs909253 Dominant 2.97 9.57 3.64 1.80 7.37 0.0001 0.0001 - - - - - - - - -
rs2229094 Dominant 2.97 9.53 4.08 2.01 8.28 0.0001 0.0001 - - - - - - - - -
4 rs2857706 Dominant 3.07 9.54 3.36 1.66 6.79 0.0002 0.0005 - - - - - - - - -
rs909253 Dominant 2.97 9.57 3.91 1.92 7.99 0.0001 0.0001 - - - - - - - - -
rs2229094 Dominant 2.98 9.53 4.15 2.04 8.45 0.0001 0.0002 - - - - - - - - -
5 rs2857602 Dominant 3.06 9.50 3.35 1.66 6.77 0.0002 0.0004 - - - - - - - - -
rs2857706 Additive 3.07 9.54 3.47 1.76 6.85 0.0002 0.0001 - - - - - - - - -
rs909253 Dominant - - - - - - - - -
- - - - - - - - -
6 - - - - - - - - -
- - - - - - - - -
- - - - - - - - -
- - - - - - - - -
7 - - - - - - - - -
rs2857706 Dominant - - - - - - - - -
- - - - - - - - -
- - - - - - - - -
8 - - - - - - - - -
rs2857706 Dominant - - - - - - - - -
- - - - - - - - -
9 - - - - - - - - -
TNFR2 6 rs12141399 Additive NA 1.04 NA NA NA 0.0093 <0.0001 0.0130 - - - - - - - - -
Taking into consideration now the total amount of genes, following the second stage of the Rosenberg method, four genes maintained significant association with PVR (Table 5): PIK3CG (P = 0.009), SMAD7 (P = 0.004), TNF locus (P = 0.005), and TNFR2 (P = 0.019). 
Table 5. 
 
Associations following the Correction for Single and Multiple Comparisons
Table 5. 
 
Associations following the Correction for Single and Multiple Comparisons
Gene Chr Extension Discovery Stage Replication Stage
Single Contrasts Haplotypic Contrasts Omnibus Test Single Contrasts Haplotypic Contrasts Omnibus Test
MinP Test, P Value, MinP Test, Value, MinP Test, P Value, MinP Test, Value, MinP Test, Value, MinP Test, P Value,
WYZ* P WYZ HFT P HFT § OMNI P OMNI # q-Value WYZ* P WYZ HFT P HFT § OMNI P OMNI # q-Value
CTGF 6q23.1 3.13 kpb (132311017–132314146) 6 0.0484 0.2096 0.1374 0.5432 0.2096 0.53 0.2409 - - - - - - -
EGF 4q25 99.37 kbp (111053499–111152860) 9 0.0214 0.1476 0.1686 0.749 0.328 0.47 0.2409 - - - - - - -
FGF2 4q26 71.53 kbp (123967313–124038840) 10 0.0674 0.68 0.1218 0.599 0.599 0.8 0.2667 - - - - - - -
HGF 7q21.1 68.01 kbp (81166258–81237388) 5 0.1326 0.605 0.1444 0.372 0.372 0.519 0.2409 - - - - - - -
IFNγ 12q14 4.972 kbp (66834816–66839787) 6 0.221 0.868 0.23 0.551 0.551 0.682 0.2526 - - - - - - -
IGF1 12q22-q23 84.65 kbp (101313809–101398471) 10 0.0087 0.154 0.1759 0.716 0.154 0.236 0.1854 - - - - - - -
IGF2 11p15.5 6.047 kbp (2106918–2125616) 5 0.0739 0.536 0.2921 0.735 0.536 0.734 0.2576 - - - - - - -
IGF-IR 15q26.3 308.7 kbp (97010302–97319034) 12 0.0574 0.706 0.0004 0.176 0.176 0.329 0.2056 - - - - - - -
IL10 1q31-q32 4.892 kbp (205007570–205012462) 9 0.0735 0.665 0.0233 0.17 0.17 0.238 0.1854 - - - - - - -
IL1α 2q12-q21 11.48 kbp (113247966–113259442) 7 0.2414 0.912 0.2356 0.48 0.48 0.619 0.2526 - - - - - - -
IL1β 2q13-q21 7.02 kbp (113303567–113310586) 5 0.1298 0.735 0.1649 0.494 0.494 0.641 0.2526 - - - - - - -
IL1RN 2q14.2 16.12 kbp (113591941–113608063) 9 0.042 0.47 0.1095 0.447 0.447 0.59 0.2526 - - - - - - -
IL-6 7p21 4.797 kbp (22732028–22738091) 8 0.1806 0.891 0.0151 0.11 0.11 0.162 0.1800 - - - - - - -
IL-8 4q13-q21 3.157 kbp (74825139–74828295) 5 0.054 0.25 0.5179 0.8 0.25 0.363 0.2135 - - - - - - -
MCP1 17q11.2-q21.1  1.923 kbp (29606409–29608329) 6 0.1538 0.805 0.2292 0.507 0.507 0.666 0.2526 - - - - - - -
MIF 22q11.2 845 bp (22369647–22567417) 7 0.0202 0.223 0.0434 0.185 0.185 0.284 0.2029 - - - - - - -
MMP2 16q13-q21 27.52 kbp (54070589–54098101) 8 0.0023 0.048 0.0185 0.205 0.048 0.113 0.1425 - - - - - - -
MMP9 20q12-q13 7.653 kbp (44070954–44078607) 7 0.0549 0.421 0.2193 0.579 0.421 0.528 0.2409 - - - - - - -
NFκB1 4q24 116 kbp (103641518–10357506) 9 0.0144 0.162 0.0088 0.046 0.046 0.079 0.1425 - - - - - - -
NFκBIA 14q13 3.228 kbp (34940467–34943694) 6 0.0035 0.059 0.0036 0.046 0.046 0.097 0.1425 - - - - - - -
NFκBIB 19q13.1 8.919 kbp (44082454–44091372) 7 0.0087 0.13 0.023 0.083 0.083 0.114 0.1425 - - - - - - -
PDGFα 7p22 21 bp (179285–179305) 5 0.047 0.367 0.0637 0.148 0.148 0.241 0.1854 - - - - - - -
PDGFRα 4q11-q13 68.97 kbp (54790204–54859171) 8 0.0165 0.197 0.5423 0.913 0.197 0.313 0.2056 - - - - - - -
PIK3 7q 41.67 kbp (106292977–106334801) 10 0.0551 0.611 <0.0001 0.001 0.001 0.009 0.0300 0.0457 0.0470 0.5491 0.982 0.0456 0.331 0.1866
SMAD3 15q21-q22 129.3 kbp (65145249–65274586) 10 0.0402 0.551 0.0188 0.371 0.371 0.747 0.2576 - - - - - - -
SMAD7 18q21.1 30.86 kbp (44700222–44731079) 7 0.0001 0.002 0.0603 0.313 0.002 0.004 0.0250 0.0064 0.0070 0.0554 0.311 0.0064 0.047 0.0397
TGFβ1 19q13.1 23.17 kbp (46528490–46551655) 6 0.0381 0.3 0.1671 0.397 0.3 0.434 0.2409 - - - - - - -
TGFβ2 1q41 95.1 kbp (216586200–216684584) 9 0.0079 0.151 0.035 0.318 0.151 0.237 0.1854 - - - - - - -
TNF 6p21.3 2.763 kbp (31651328–31654090) 11 0.0013 0.034 0.0004 0.005 0.005 0.005 0.0250 0.0059 0.0060 0.0870 0.416 0.0059 0.044 0.0397
TNFR2 1p36.22 42.22 kbp (12149647–12191872) 8 0.099 0.716 <0.0001 0.013 0.013 0.019 0.0475 0.1052 0.2140 0.2213 0.777 0.1052 0.649 0.2706
Replication Stage
A total of 546 peripheral DNA blood samples (151 cases and 395 controls) from 17 European centers were included (Table 1). Seventy-one SNPs were included. There was one failure in the design of the primers, the one corresponding to the rs1982073. There were no failures for the genotyping process, with a global call rate of 97.59%. The genotypic data quality was evaluated, and all informative markers verified the Hardy-Weinberg equilibrium. 
Regarding clinical information some significant associations were observed. The control group was significantly older than cases (P < 0.0001) with a median of 6 years (95% confidence interval [CI]: 3.39–8.31). Patients with history of PVR in the fellow eye were more frequently cases (83%) than controls (17%). Pneumatic retinopexy was more frequently performed on patients that did not developed PVR (71%); scleral surgery (P = 0.0281), PPV (P < 0.0001), and tamponade-like air (P = 0.0153) or silicone oil (SO) (P < 0.0001) were more frequent in cases, while scleral surgery plus drainage (SS + d) (P = 0.0383), SF6 as tamponade (P = 0.0077), and cryotherapy (P = 0.0481) were significantly less frequent in cases. Regarding systemic and ocular diseases and treatment occurring simultaneously with the RD, very few patients reported inherited ocular disease or connective tissue disease (1 case versus 11 controls and 1 case versus 13 controls, respectively). Seven cases were receiving systemic nonsteroidal anti-inflammatory drugs (NSAIDs), 2 steroids, and 1 antiproliferant treatment when suffered from RD versus 21, 6, and 2 controls, respectively. One case was receiving topic steroids, while 7 controls were receiving ocular NSAIDs, and 15 steroids. In the same way as in the discovery stage, none of the patients received intravitreal Triamcinolone Acetonide (TA) in any surgery. Fifty one percent of patients who suffered from PVR following a RD surgery were operated on by an experienced surgeon (nontraining grade). There were no significant associations with sex, race, affected eye, family history of PVR, or phakic/aphakic status. There were no differences regarding the geographical localization or center where the patients came from. 
Single and Haplotypic Associations.
In the single analysis new significant associations were observed in PIK3CG (rs4460309, P = 0.0457), SMAD7 (rs6507877, P = 0.0345), and TNF locus (rs2256974, P = 0.02615; rs909253, P = 0.005917; rs1799964, P = 0.03152; and rs1800629, P = 0.04563) (Table 3). No associations were observed in TNFR2. One replication was detected, the rs7226855 in SMAD7 (P = 0.0064) (Table 3). Following permutation, all genes maintained their association: PIK3CG (P = 0.0470), SMAD7 (P = 0.070), and TNF locus (P = 0.0060) (Table 3). 
In multiple analyses, no haplotype was associated to PVR. One subhaplotype was significantly associated in the SMAD7, the one which implies the two first markers: rs4939826-rs7226855 (P = 0.0330; odds ratio [OR] = 1.4062; CI 95%: 1.0637–1.8590). However, this association was lost following permutation (Table 4). 
Finally, after corrections for multiple comparisons, replication was observed in two genes: SMAD7 (P = 0.047) and TNF locus (P = 0.044) (Table 5). 
Discussion
In the present study, we analyzed common genetic variants in genes implicated in the inflammatory cascade in order to investigate their role in the susceptibility to developing PVR following primary rhegmatogenous RD surgery. 
After the discovery stage, four genes maintained their significant association to the disease following a very stringent statistical analysis. This finding warranted investigation whether these associations were genuine or not, as is recommended in the literature. 23,24,41,42 In the replication stage, studying a new larger sample of DNA from patients across different countries in Europe, two of these four genes continued to be significant: the SMAD7 and the TNF locus
Limitations of this work must be discussed before analyzing our results. Despite the fact that the exact mechanisms responsible of PVR are not completely understood, it is widely accepted that inflammation plays an important role in its pathogenesis. 3 Although not every gene participating in the inflammatory cascade could be included, genes encoding for the key mediators and their signaling pathway molecules were investigated. Genetic variation in each gene was widely covered, taking into consideration that in addition to the parameters used for the SNPs selection (TagSNPs with a r 2 ≥ 0.85), previously described as functional SNPs and extragenic regions were also studied. 
Other important issue in an association study is the sample size. 24 When we analyze markers with a very subtle effect on the entire population, there is a significant risk that the genes may be below the threshold for detection. The power sample was greater than 80% for most of genes. In the replication stage TNFR2 did not reach 70%, as it would have been necessary to collect more than 800 samples to achieve this power. This would be extremely challenging for a low prevalence condition such as PVR. This could have been one of the reasons for not detecting replication in this gene. 
An inadequate phenotyping and stratification are other factors that could invalidate these kinds of studies. 41 In order to prevent this, very stringent exclusion and inclusion criteria for classification of patients were defined (e.g., PVR patients were included only once they have been operated on by PPV, confirming preoperatively the diagnosis by the membrane peeling; complexity of RDs was homogeneous as those patients with PVR grade C1 or higher were excluded). Although not all clinical variables related with a high risk of developing PVR were recorded, the classification process exhaustively covered others that could induce any kind of error during the phenotyping process and that could also introduce biases such as history of trauma, status of the lens, and so on. In addition to phenotyping, location is the other main source of subpopulation in these kind of studies. No differences regarding geographical origin or center where the patients came from were found as well as race, sex, and so on. Methodology of collection of samples was also carefully defined for the replication stage in order to avoid including same patients in those centers that had participated in the discovery stage. 
In the replication stage, there were some differences among clinical variables that worth to be discussed. Cases were younger than controls and this could be considered a confounding factor. However, this difference was only 6 years, making it unlikely that this is responsible for the differences found in the genetic profiles of cases and controls. Moreover, this difference should not be of concern as in another work carried out by our group we found that in people aged over 55 there was a risk for PVR (Pastor JC, Fernandez I, Rodriguez de la Rua E, et al., unpublished data, 2012). In younger patients (who had a higher rate of PVR), the posterior vitreous may be more frequently attached and is more difficult to peel. Although the difference in age between groups was not great, this could explain why non PVR controls are older than cases in our sample. A history of PVR in the fellow eye was more frequently observed in cases, as is expected since those patients with RD and history of PVR in the fellow eye were excluded. Regarding the systemic or ocular diseases and treatments receiving at the time of the RD, there were very few patients that reported these characteristics. Then it would not be appropriate to draw any conclusion about it. Regarding the differences observed in the intraoperative variables, it is important to point out that each procedure was recorded as positive or negative. In this way, one patient could have been operated on by more than one procedure (e.g., SS + d and PPV in a second time). This justifies that PPV or the usage of SO as tamponade are more frequent among cases. Cases could have required intravitreal procedures in order to peel membranes while scleral procedures could have been sufficient among controls. These differences could be considered a bias, as they could mean that we have included more complicated RDs in the case group, and then we could have an spurious association. However, there are some issues to emphasize. First, it is has been demonstrated that current tendency of retinal surgeons toward PPV for treating RDs is not based in a judgment of the degree of complexity of the RDs. In addition to that, if we take into account that PVR is per se a complication of the RD surgery, we could say that we did analyze more complicated RD in this group. The question that arises here is why these RDs are more complicated. Genetic profile could be one of the answers. In order to avoid this confounding factor, those procedures orientated to treat the RDs once the patient developed PVR should have probably been eliminated. In this way, only procedures in RDs clinically comparable would have been considered and this intríngulis would have been solved. Despite that, it worth to remark that phenotype was carefully defined, and well recognized experts on the vitreoretinal surgery were in charge of classifying the patients. Therefore, we consider that we can rely that patients were correctly classified as case or control. Since phenotyping was correctly performed, we consider that all mentioned above do not invalidate our work. 
It is important to remark that half of patients who suffered from PVR following RD surgery were operated on by an experienced surgeon. This allows us to downplay the influence of the surgeon's skill in the susceptibility to develop PVR, stressing also the importance of the genetic component. Finally, one of the major pitfalls of genetic association studies is the problem of multiple comparisons, which may throw up anomalous connections purely by chance. 24 In order to limit the presence of these spurious associations we have used a multiple comparison procedure based on the estimation of the false discovery rate (FDR). 35 The estimation of this rate of “false positives” was used to correct the original P values. We have set the threshold of significance level, traditionally 0.05, and we have considered that a association is statistically significant when the adjusted P value, and not the original P value, is less than this threshold. 
Threshold of significance is another important issue here. Ioannidis states that the threshold for declaring the presence or not of an effect can still be a subject of discussion of schools of statistics. 43 In our study, we observed P values well below the traditional 0.05 that Ioannidis admits for the replication data (0.0070 for SMAD7 and 0.0060 for TNF locus in the single analysis of replication data). Following the FDR estimation, we observed that 95% of these associations can be genuine, which can be consider a reliable result to continue working on these genes in future researches. 
Regarding the replication found in SMAD7 gene and the TNF locus, it reinforces the idea that prevention or even treatment of PVR should probably be targeted to the inflammation mediators and their signaling pathway molecules. 
High vitreous levels of TNF-α and its receptors (TNFR1 and 2) have been found in eyes with PVR 44 and local production of TNF-α has been suggested to occur in these eyes. 45 Keeping in mind the important role of TNF-α and its signaling pathway as early mediators in the inflammatory cascade, it makes sense that cytokines from the TNF-family have a protagonical role in the genesis of PVR. Our finding in the TNF locus reinforces this notion. 
Apoptosis may be another important mechanism in PVR, where TGF-β has been strongly implicated. 46 Amongst other numerous biological functions of TGF-β, of significance may be the stimulation of epithelial-mesenchymal cells, fibroblast- myofibroblast conversion, and the enhanced expression of extracellular matrix proteins 47 ; all key processes in the establishment of PVR. The TGF-β family members (TGFβ1–3) bind to their membrane receptors, and use the SMADs signaling pathway. SMAD2 and 3 (known as receptor-regulated SMADs) play an important role in the activation of TGF-β–dependent gene targets, with SMAD3 mediating the mainly profibrotic actions of TGF-β. SMAD7 is known to be inhibitory, blocking phosphorylation of SMAD2/3. 47,48 These events are evidence of potentially important roles that SMAD proteins could have in the pathogenesis of PVR. In fact, over expression of SMAD7 suppresses the fibrotic response of RPE cells to TGF-β2 in mice, inhibiting the RPE cells transition to myofibroblasts. 49 Our results highlight the possible role of SMAD7 in the development of PVR in humans. 
One question that arises from the above is why TGF-β was not significantly associated to the disease. One explanation could be that there were three markers (two in the TGF-β1 and one in the TGF-β2) without information either for failure in the design pipeline or during the genotyping process. That means that some regions of the gene were not exhaustively studied as there were some SNPs that could not be genotyped. In order to solve this problem, TGF-β should be studied by other method. 
In summary, we have confirmed that two genes previously implicated in the establishment of PVR are indeed significantly associated to the disease in Europeans undergoing retinal detachment surgery. These results may help us to understand the molecular basis of this complication and could potentially guide us to develop new strategies in the prevention or treatment of PVR. Further experimental studies on these genes are now warranted. 
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Footnotes
 Supported by grants from The Special Trustees of Moorfields Eye Hospital, the Health Foundation (London, England), and the National Institute for Health Research Biomedical Research Centre; the Portuguese grants from Fundação para a Ciência e a Tecnologia (FCT) (PTDC/SAU-ORG/110683/2009), through Unidade I and D Cardiovascular (51/94-FCT); the General R and D and I Office of the Government of Galicia (through Grant PGIDIT06PXIB208204PR) and the Fondo de Investigacion Sanitario (FIS) PI051437 project of the “Carlos III Health Institute,” Ministry of Health, Spanish Government.
Footnotes
 Disclosure: J. Rojas, None; I. Fernandez, None; J.C. Pastor, None; R.E. MacLaren, None; Y. Ramkissoon, None; S. Harsum, None; D.G. Charteris, None; J.C. Van Meurs, None; S. Amarakoon, None; J.M. Ruiz-Moreno, None; A. Rocha-Sousa, None; M. Brion, None; A. Carracedo, None
Footnotes
 See the Appendix for the members of the Genetics on PVR Study Group.
Appendix
The Genetics on PVR Study Group
Mário Alfaiate, Centro Hospitalar e Universitário de Coimbra, Portugal; 
Anna Boixadera, Hospital Vall d'Hebrón, Instituto de Cirugía ocular Avanzada (ICOAB), Barcelona, Spain; 
Rosa Maria Coco, Departament of Ophthalmology, IOBA, Valladolid, Spain; 
Miguel A. de la Fuente, Ophthalmology Department, Fundacion Jimenez Diaz University Hospital, Madrid, Spain; 
Carmen Desco, Fundación Oftalmológica del Mediterráneo, Valencia, Spain; 
Manuel Diaz-Llopis, Ophthalmology Department, University of Valencia, Valencia, Spain. Department of Ophthalmology, La Fe University Hospital, Valencia, Spain; 
Javier Elizalde, Institut Universitari Barraquer, Barcelona, Spain; 
Patricia Fernández-Robredo, Clinica Universidad de Navarra, Spain; 
João Figueira, Centro Hospitalar e Universitário de Coimbra, Portugal; 
Marta S. Figueroa, Hospital Universitario Ramón y Cajal, Madrid, Spain; 
Ester Frances, Department of Ophthalmology, La Fe University Hospital, Valencia, Spain; 
Jose Maria Gallardo, Hospital Reina Sofia, Cordoba, Spain; 
Alfredo Garcia-Layana, Clinica Universidad de Navarra, Spain; 
José García-Arumí, Hospital Vall d'Hebrón, Instituto de Microcirugía Ocular (IMO), Barcelona, Spain; 
Maria Teresa Garcia-Gutierrez. Molecular Biology Lab. IOBA, Valladolid, Spain; 
Paula Magro, Centro Hospitalar e Universitário de Coimbra, Portugal; 
Vicente Martínez-Castillo, Hospital Vall d'Hebrón, Instituto de Cirugía ocular Avanzada (ICOAB), Barcelona, Spain; 
Jorge Mataix, Fundación Oftalmológica del Mediterráneo, Valencia, Spain; 
Amparo Navea, Fundación Oftalmológica del Mediterráneo, Valencia, Spain; 
Elena Palacios, Fundación Oftalmológica del Mediterráneo, Valencia, Spain; 
Marta Pérez, Hospital Universitario Ramón y Cajal, Madrid, Spain; 
Sergio Recalde-Maestre, Clinica Universidad de Navarra, Spain; 
Enrique Rodriguez-de la Rua, Hospital Clinico Universitario de Valladolid, Valladolid, Spain. Hospital Universitario Puerta del Mar, Cadiz, Spain; 
Miguel Ruiz Miguel, Ophthalmology, Hospital Donostia, San Sebastián, Spain; 
David Salom, Department of Ophthalmology, La Fe University Hospital, Valencia, Spain; 
Maria Rosa Sanabria, Departament of Ophthalmology, IOBA, Valladolid, Spain; 
Xavier Valldeperas, Ophthalmology Department, Hospital Universitari Germans Trias, Barcelona, Spain; 
Sara Velilla, Ophthalmology, Hospital San Pedro, Logroño, Spain; and 
Lurdes Zamora, Jose Carreras Leukemia Institute-ICO Badalona, Hospital Universitari Germans Trias i Pujol, Barcelona, Spain. 
Table 1. 
 
Centers Involved in the Study
Table 1. 
 
Centers Involved in the Study
Country Center Discovery Stage Replication Stage
Cases PVR Controls RD Total Cases PVR Controls RD Total
England
 Moorfields Eye Hospital, London N - - - 32 118 150
 % Total - - - 5.9 21.6 27.5
Holland
 Rotterdam Eye Hospital, Rotterdam N - - - 39 89 128
 % Total - - - 7.1 16.3 23.4
Portugal
 Coimbra University Hospital, Coimbra  N - - - 6 1 7
 % Total - - - 1.1 0.2 1.3
 Sao Joao Hospital, Oporto N - - - 6 52 58
 % Total - - - 1.1 9.5 10.6
Spain
 Germans Trias i Pujol University Hospital, N - - - 11 11 22
  Badalona  % Total - - - 2.0 2.0 4.0
 Jiménez Díaz Foundation, Madrid N - - - 2 2 4
 % Total - - - 0.4 0.4 0.7
 Ophthalmological Foundation of Mediterranean, N - - 5 2 7
  Valencia  % Total - - 0.9 0.4 1.3
 San Millan San Pedro Hospital, Logroño N - - - 9 1 10
 % Total - - - 1.6 0.2 1.8
 Universitary Hospital La Fe, Valencia  N - - - 10 25 35
 % Total - - - 1.8 4.6 6.4
 University Hospital of Navarra  N - - - 11 19 30
 % Total - - - 2.0 3.5 5.5
 Universitay Hospital Ramón y Cajal, Madrid N - - - 2 3 5
 % Total - - - 0.4 0.5 0.9
 Barraquer Ophthalmology Centre  N 8 35 43 2 6 8
 % Total 1.8 7.8 9.5 0.4 1.1 1.5
 Donostia Hospital, San Sebastian N 7 35 42 2 0 2
 % Total 1.5 7.8 9.3 0.4 0 0.4
 IOBA (University Eye Institute), Valladolid N 28 22 50 10 15 25
 % Total 6.2 4.9 11.1 1.8 2.7 4.6
 Reina Sofia Universitary Hospital, Cordoba N 5 9 14 - - -
 % Total 1.1 2 3.1 - - -
 Universitary Hospital Pio del Rio Hortega N 7 10 17 - - -
 % Total 1.5 2.2 3.8 - - -
 University Hospital Vall d'Hebrón N 20 75 95 1 25 26
 % Total 4.4 16.7 21.1 0.2 4.6 4.8
 University Hospital of Valladolid N 32 28 60 2 8 10
 % Total 7.1 6.2 13.3 0.4 1.5 1.8
 Vissum, Alicante N 31 98 129 1 18 19
 % Total 6.9 21.8 28.7 0.2 3.3 3.5
TOTAL N 138 312 450 151 395 546
% Center 30.7 69.3 100.0 27.7 72.3 100.0
Table 2. 
 
Clinical Characteristics of the Replication Sample
Table 2. 
 
Clinical Characteristics of the Replication Sample
Replication Stage
Characteristic Case—PVR Control—RD Total P Value
% Type % Type
Sex
 Male 100 28.74 248 71.26 348 0.362
 Female 45 25.00 135 75.00 180
 Total 145 (6)° 27.46 383 (12)° 72.54 528
Race
 North-African 0 0 6 100 6 0.0644
 Asian 2 40.00 3 60.00 5
 European 133 26.44 370 73.56 503
 Hispanic 7 53.85 6 46.15 13
 Indian 5 41.67 7 58.33 12
 Subsaharian 2 50.00 2 50.00 4
 Total 149 (2)° 27.44 394 (1)° 72.56 543
RD family history
 No 142 27.52 374 72.48 516 0.932
 Yes 9 30 21 70 30
 Total 151 27.66 395 72.34 546
PVR family history
 No 149 27.49 393 72.51 542 0.4758*
 Yes 1 50 1 50 2
 Total 150 (1)° 27.57 394 (1)° 72.43 544
RD in fellow eye
 Unknown 5 38.46 8 61.54 13 0.5289*
 No 136 27.7 355 72.3 491
 Yes 10 23.81 32 76.19 42
 Total 151 27.66 395 72.34 546
PVR in fellow eye
 Unknown 5 38.46 8 61.54 13 0.0057*
 No 141 26.70 387 73.30 528
 Yes 5 100 0 0 5
 Total 151 27.66 395 72.34 546
Phakia/aphakia
 Unknown 7 33.33 14 66.67 21 0.2333
 Aphakia 19 38.78 30 61.22 49
 Pseudophakia 40 28.37 101 71.63 141
 Phakia 85 25.37 250 74.63 335
 Total 151 27.66 395 72.34 546
Retinopexy
 No 147 27.63 385 72.37 532 0.8268*
 Yes 4 28.57 10 71.43 14
 Total 151 27.66 395 72.34 546
Scleral surgery (SS)
 No 116 25.55 338 74.45 454 0.0281*
 Yes 34 38.2 55 61.8 89
 Total 150 (1)° 27.62 393 (2)° 72.38 543
SS + drainage
 No 131 29.18 318 70.82 449 0.0383*
 Yes 20 20.62 77 79.38 97
 Total 151 27.66 395 72.34 546
PPV
 No 15 10.87 123 89.13 138 <0.0001
 Yes 136 33.33 272 66.67 408
 Total 151 27.66 395 72.34 546
Air
 No 138 26.59 381 73.41 519 0.0153*
 Yes 13 48.15 14 51.85 27
 Total 151 27.66 395 72.34 546
SF6
 No 87 30.21 201 69.79 288 0.0077*
 Yes 64 24.81 194 75.19 258
 Total 151 27.66 395 72.34 546
C3F8
 No 116 26.54 321 73.46 437 0.3041*
 Yes 35 32.11 74 67.89 109
 Total 151 27.66 395 72.34 546
Silicone
 No 55 13.03 367 86.97 422 <0.0001*
 Yes 96 77.42 28 22.58 124
 Total 151 27.66 395 72.34 546
Laser
 No 35 13.41 226 86.59 261 <0.0001*
 Yes 116 40.7 169 59.3 285
 Total 151 27.66 395 72.34 546
Cryotherapy
 No 99 31.03 220 68.97 319 0.0481*
 Yes 52 22.91 175 77.09 227
 Total 151 27.66 395 72.34 546
Table 3. 
 
Single Associations in the Discovery and Replication Stages
Table 3. 
 
Single Associations in the Discovery and Replication Stages
Gene SNP Genotype Discovery Stage Replication Stage
Frequency, % Inheritance Model* OR CI 95% OR P Value Corrected P Value Frequency, % Inheritance Model* OR CI 95% OR P Value Corrected P Value
Control Case Lower Upper Control Case Lower Upper
EGF rs1024600 CC/CG 92.7 85.7 Recessive 0.02846 0.6870 - - - - - - - -
C→G GG 7.3 14.3 2.12 1.09 4.11 - - - - - - - -
IGF1 rs2195240 TT/CT 93.4 98.4 Recessive 0.23 0.05 0.98 0.017 0.1816 - - - - - - - -
T→C CC 6.6 1.6 - - - - - - - -
rs5742629 AA/AG 90.4 97.5 Recessive 0.25 0.07 0.84 0.0087 0.1816 - - - - - - - -
A→G GG 9.4 2.5 - - - - - - - -
IL1RN rs3087270 AA 47.6 56.8 Additive 0.72 0.53 0.99 0.0420 0.9132 - - - - - - - -
A→G AG 38.8 35.2 - - - - - - - -
GG 13.6 8 - - - - - - - -
MIF rs1007888 AA 30 19 Dominant 0.0201 0.2306 - - - - - - - -
A→G AG/GG 70 81 1.82 1.08 3.07 - - - - - - - -
rs243845 CC 45.4 29.6 Dominant 0.0023 0.0500 - - - - - - - -
C→T CT/TT 54.6 70.4 1.98 1.26 3.09 - - - - - - - -
rs243864 TT/GT 92.2 97.5 Recessive 0.30 0.09 1.01 0.026 0.3446 - - - - - - - -
T→G GG 7.8 2.5 - - - - - - - -
rs243866 GG/AG 92.4 97.5 Recessive 0.31 0.09 1.05 0.0317 0.3446 - - - - - - - -
G→A AA 7.6 2.5 - - - - - - - -
NFKB1 rs4698858 CC 45.1 32.3 Dominant 1.73 1.11 2.69 0.01435 0.2614 - - - - - - - -
C→G CG/GG 54.9 67.7 - - - - - - - -
NFKBIA rs17103274 TT 84.9 72.1 Dominant 0.0034 0.0515 - - - - - - - -
T→C CT/CC 15.1 27.9 2.17 1.3 3.61 - - - - - - - -
NFKBIB rs3136640 GG/CC 51.3 65.1 Overdominant 0.0087 0.0993 - - - - - - - -
G→C CG 48.7 34.9 0.57 0.37 0.87 - - - - - - - -
PDGFA rs7806249 TT/CC 52.4 62.9 Overdominant 0.04696 0.3917 - - - - - - - -
T→C CT 47.6 37.1 0.65 0.42 1.00 - - - - - - - -
PDGFRA rs7656613 TT/CC 65.1 51.4 Overdominant 0.1654 0.1884 - - - - - - - -
T→C CT 34.9 48.6 1.76 1.11 2.8 - - - - - - - -
SMAD3 rs6494634 CC 40.8 50.8 Additive 0.71 0.50 0.99 0.04016 0.8741 - - - - - - - -
C→T CT 49.3 43 - - - - - - - -
TT 9.9 6.2 - - - - - - - -
PIK3CG rs4460309 CC/CT - - - - - - - - 95.1 90.2 Recessive 0.04566 0.047
C→T TT - - - - - - - - 4.9 9.8 2.12 1.03 4.35
rs7226855 AA 56.3 36.2 Overdominant 2.27 1.48 3.48 0.00014 0.0015 34.8 22.8 Dominant 0.006382 0.0070
A→G 65.2 77.2
43.7 63.8 1.8 1.17 2.79
rs6507877 GG/AA 57.2 46.9 Overdominant 0.03446 0.0070
A→G AG 1.51 1.03 2.22
TGFB1 rs2241713 GG/CG 79.2 87.6 Recessive - - - - - - - -
G→C CC 20.8 12.4 0.54 0.29 0.99 0.03810 0.3175 - - - - -
TGFB2 rs1891467 AA 53.5 45 Additive 1.44 1.03 2.02 0.03447 0.25 - - - - - - - -
A→G AG 42.2 45.7 - - - - -
GG 4.3 9.3 - - - - -
rs2000220 AA 48.1 39.1 - - - - - - - - -
A→G AG 41.8 43.8 Addictive - - - - -
GG 10.1 17.2 1.41 1.04 1.90 0.02595 0.25 - - - - -
rs947712 GG 58.6 47.6 - - - - - - - -
G→A AG 35.4 38.9 Addictive - - - - -
AA 6.1 13.5 1.54 1.12 2.11 0.00787 0.1718 - - - - -
TNF locus rs2857706 AA/GG 75.2 62.2 Overdominant 0.0077 0.0837 - - - - - - - -
G→A AG 24.83 37.80 1.84 1.18 2.87 - - - - -
rs2229094 CT/CC 6.33 8.94 Dominant 2.02 1.31 3.11 0.0013 0.0283 - - - - - - - -
T→C TT 52.00 34.96 - - - - -
rs2256974 GG 64.55 74.59 Additive 0.64 0.42 0.97 0.0315 0.2783 64.7 74.7 Dominant 0.02615 0.0060
G→T GT 31.34 23.77 35.3 25.3
TT 4.10 1.64 0.62 0.40 0.95
rs909253 TT/CT - - - - - - - - 87.4 95.1 Recessive 0.0059 0.0060
T→C CC - - - - - - - 12.6 4.9 0.36 0.16 0.81
rs199964 TT/CT - - - - - - - - 93.6 97.9 Recessive 0.03152 0.0060
T→C CC - - - - - - - 6.4 2.1 0.31 0.09 1.06
rs1800629 GG/AA - - - - - - - - 76.3 67.8 Overdominant 0.04563 0.0060
G→A GA - - - - - - - 23.7 32.2 1.53 1.01 2.32
Table 4. 
 
Haplotypic Associations in the Discovery and Replication Stages
Table 4. 
 
Haplotypic Associations in the Discovery and Replication Stages
Gene N SNP Initial SNP Inheritance Model* Discovery Stage Replication Stage
Frequency, % OR CI 95% OR Multiple Association P Value Corrected by Gene Inheritance Model* Frequency, % OR CI 95% OR Multiple Association P Value Corrected by Gene
Control Case Inferior Superior P Value Corrected P Value Control Case Inferior Superior P Value Corrected P Value
IGF-IR 2 rs10794486 Additive 9.72 16.98 1.78 1.12 2.85 0.0125 0.0150 0.1760 - - - - - - - - -
3 rs7166287 Additive 6.69 14.42 2.49 1.37 4.52 0.0031 0.0050 - - - - - - - - -
4 rs7166287 Additive 2.74 7.78 3.19 1.36 7.49 0.0054 0.0110 - - - - - - - - -
rs10794486 Additive 2.10 7.94 4.15 1.61 10.70 0.0032 0.0043 - - - - - - - - -
5 rs7166287 Additive 2.54 6.90 4.19 1.37 12.83 0.0168 0.0180 - - - - - - - - -
rs10794486 Additive 2.01 7.82 4.18 1.59 10.98 0.0010 0.0020 - - - - - - - - -
6 rs7166287 Additive 2.51 6.72 3.88 1.27 11.86 0.0072 0.0080 - - - - - - - - -
rs10794486 Additive 2.0 7.27 3.36 1.30 8.66 0.0013 0.0030 - - - - - - - - -
IL-10 3 rs1800890 Additive 4.6 3.09 9.17 1.87 45.05 0.0015 0.0023 0.1700 - - - - - - - - -
IL-6 6 rs4719713 Additive 1.48 3.80 3.43 1.16 10.14 0.0533 0.0529 0.1100 - - - - - - - - -
8 rs22725336 Additive 1.06 3.29 3.75 1.02 13.75 0.0127 0.0112 - - - - - - - - -
0.46 3.18 9.98 1.99 49.95 0.0025 0.0010 - - - - - - - - -
1.42 3.81 3.49 1.17 10.42 0.0468 0.0360 - - - - - - - - -
MIF 2 rs1007888 Additive 7.07 11.60 1.90 1.12 3.24 0.0391 0.0430 0.1850 - - - - - - - - -
MMP2 2 rs9928731 Additive 33.88 42.54 1.43 1.02 1.99 0.0185 0.0180 0.2050 - - - - - - - - -
rs243845 Dominant 34.27 43.36 1.95 1.20 3.14 0.0127 0.0092 - - - - - - - - -
3 rs9928731 Additive 0.15 2.07 14.13 12.59 15.86 0.0267 0.0170 - - - - - - - - -
NFKB1 3 rs3774932 Dominant 2.36 5.81 2.69 1.19 6.07 0.0173 0.0230 0.0460 - - - - - - - - -
rs4698858 Dominant 18.68 10.01 0.49 0.28 0.87 0.0033 0.0030 - - - - - - - - -
rs11722146 Dominant 1.80 5.90 3.87 1.61 9.30 0.0026 0.0023 - - - - - - - - -
4 rs4698858 Dominant 18.46 10.82 0.55 0.32 0.95 0.0097 0.0063 - - - - - - - - -
0.84 3.27 4.89 1.32 18.11 0.0021 0.0047 - - - - - - - - -
rs11722146 Dominant 1.80 5.93 3.83 1.59 9.21 0.0025 <0.0001 - - - - - - - - -
5 rs2300540 Dominant 1.05 3.08 3.43 1.07 10.93 0.0326 0.0230 - - - - - - - - -
0.67 2.80 4.76 1.25 18.17 0.0081 0.0100 - - - - - - - - -
rs4698858 Dominant 0.82 3.45 5.25 1.48 18.67 0.0019 0.0022 - - - - - - - - -
6 rs3774932 Dominant 0.17 2.10 12.85 1.42 116.34 0.0020 0.0050 - - - - - - - - -
rs2300540 Dominant 1.05 3.17 3.37 1.06 10.67 0.0293 0.0320 - - - - - - - - -
0.67 2.71 4.46 1.16 17.16 0.0090 0.0070 - - - - - - - - -
NFKBIA 2 rs2007960 Dominant 8.04 14.48 2.47 1.44 4.24 0.0054 0.0037 0.0460 - - - - - - - - -
3 rs3138045 Dominant 8.02 13.64 2.59 1.49 4.52 0.0054 0.0020 - - - - - - - - -
6 rs7152826 Dominant 5.75 8.24 0.27 0.21 0.35 0.0493 0.0500 - - - - - - - - -
NFKBIB 2 rs11879872 Dominant 0.32 2.49 11.88 1.28 110.62 0.0333 0.0360 0.0830 - - - - - - - - -
PIK3CG 3 rs4460309 Dominant 0.01 1.14 15.8 × 104 15.8 × 104 15.8 × 104 0.0010 0.0410 0.0010 - - - - - - - - -
SMAD7 2 rs4939826 - - - - - - - - - Additive 41.85 49.69 1.40 1.06 1.86 0.0207 0.0330 0.3110
TGFB2 2 rs947712 Dominant 13.41 19.20 1.99 1.22 3.24 0.0205 0.0230 0.3180 - - - - - - - - -
rs2796821 Dominant 14.04 20.49 1.83 1.15 2.89 0.0189 0.0120 - - - - - - - - -
TNF locus 2 rs909253 Dominant 27.14 37.18 1.98 1.27 3.08 0.0024 0.0046 0.0050 - - - - - - - - -
rs2229094 Dominant 2.98 9.49 3.65 1.82 7.31 0.0001 <0.0001 - - - - - - - - -
3 rs2857706 Dominant 15.16 20.52 1.87 1.18 2.96 0.0484 0.0430 - - - - - - - - -
rs909253 Dominant 2.97 9.57 3.64 1.80 7.37 0.0001 0.0001 - - - - - - - - -
rs2229094 Dominant 2.97 9.53 4.08 2.01 8.28 0.0001 0.0001 - - - - - - - - -
4 rs2857706 Dominant 3.07 9.54 3.36 1.66 6.79 0.0002 0.0005 - - - - - - - - -
rs909253 Dominant 2.97 9.57 3.91 1.92 7.99 0.0001 0.0001 - - - - - - - - -
rs2229094 Dominant 2.98 9.53 4.15 2.04 8.45 0.0001 0.0002 - - - - - - - - -
5 rs2857602 Dominant 3.06 9.50 3.35 1.66 6.77 0.0002 0.0004 - - - - - - - - -
rs2857706 Additive 3.07 9.54 3.47 1.76 6.85 0.0002 0.0001 - - - - - - - - -
rs909253 Dominant - - - - - - - - -
- - - - - - - - -
6 - - - - - - - - -
- - - - - - - - -
- - - - - - - - -
- - - - - - - - -
7 - - - - - - - - -
rs2857706 Dominant - - - - - - - - -
- - - - - - - - -
- - - - - - - - -
8 - - - - - - - - -
rs2857706 Dominant - - - - - - - - -
- - - - - - - - -
9 - - - - - - - - -
TNFR2 6 rs12141399 Additive NA 1.04 NA NA NA 0.0093 <0.0001 0.0130 - - - - - - - - -
Table 5. 
 
Associations following the Correction for Single and Multiple Comparisons
Table 5. 
 
Associations following the Correction for Single and Multiple Comparisons
Gene Chr Extension Discovery Stage Replication Stage
Single Contrasts Haplotypic Contrasts Omnibus Test Single Contrasts Haplotypic Contrasts Omnibus Test
MinP Test, P Value, MinP Test, Value, MinP Test, P Value, MinP Test, Value, MinP Test, Value, MinP Test, P Value,
WYZ* P WYZ HFT P HFT § OMNI P OMNI # q-Value WYZ* P WYZ HFT P HFT § OMNI P OMNI # q-Value
CTGF 6q23.1 3.13 kpb (132311017–132314146) 6 0.0484 0.2096 0.1374 0.5432 0.2096 0.53 0.2409 - - - - - - -
EGF 4q25 99.37 kbp (111053499–111152860) 9 0.0214 0.1476 0.1686 0.749 0.328 0.47 0.2409 - - - - - - -
FGF2 4q26 71.53 kbp (123967313–124038840) 10 0.0674 0.68 0.1218 0.599 0.599 0.8 0.2667 - - - - - - -
HGF 7q21.1 68.01 kbp (81166258–81237388) 5 0.1326 0.605 0.1444 0.372 0.372 0.519 0.2409 - - - - - - -
IFNγ 12q14 4.972 kbp (66834816–66839787) 6 0.221 0.868 0.23 0.551 0.551 0.682 0.2526 - - - - - - -
IGF1 12q22-q23 84.65 kbp (101313809–101398471) 10 0.0087 0.154 0.1759 0.716 0.154 0.236 0.1854 - - - - - - -
IGF2 11p15.5 6.047 kbp (2106918–2125616) 5 0.0739 0.536 0.2921 0.735 0.536 0.734 0.2576 - - - - - - -
IGF-IR 15q26.3 308.7 kbp (97010302–97319034) 12 0.0574 0.706 0.0004 0.176 0.176 0.329 0.2056 - - - - - - -
IL10 1q31-q32 4.892 kbp (205007570–205012462) 9 0.0735 0.665 0.0233 0.17 0.17 0.238 0.1854 - - - - - - -
IL1α 2q12-q21 11.48 kbp (113247966–113259442) 7 0.2414 0.912 0.2356 0.48 0.48 0.619 0.2526 - - - - - - -
IL1β 2q13-q21 7.02 kbp (113303567–113310586) 5 0.1298 0.735 0.1649 0.494 0.494 0.641 0.2526 - - - - - - -
IL1RN 2q14.2 16.12 kbp (113591941–113608063) 9 0.042 0.47 0.1095 0.447 0.447 0.59 0.2526 - - - - - - -
IL-6 7p21 4.797 kbp (22732028–22738091) 8 0.1806 0.891 0.0151 0.11 0.11 0.162 0.1800 - - - - - - -
IL-8 4q13-q21 3.157 kbp (74825139–74828295) 5 0.054 0.25 0.5179 0.8 0.25 0.363 0.2135 - - - - - - -
MCP1 17q11.2-q21.1  1.923 kbp (29606409–29608329) 6 0.1538 0.805 0.2292 0.507 0.507 0.666 0.2526 - - - - - - -
MIF 22q11.2 845 bp (22369647–22567417) 7 0.0202 0.223 0.0434 0.185 0.185 0.284 0.2029 - - - - - - -
MMP2 16q13-q21 27.52 kbp (54070589–54098101) 8 0.0023 0.048 0.0185 0.205 0.048 0.113 0.1425 - - - - - - -
MMP9 20q12-q13 7.653 kbp (44070954–44078607) 7 0.0549 0.421 0.2193 0.579 0.421 0.528 0.2409 - - - - - - -
NFκB1 4q24 116 kbp (103641518–10357506) 9 0.0144 0.162 0.0088 0.046 0.046 0.079 0.1425 - - - - - - -
NFκBIA 14q13 3.228 kbp (34940467–34943694) 6 0.0035 0.059 0.0036 0.046 0.046 0.097 0.1425 - - - - - - -
NFκBIB 19q13.1 8.919 kbp (44082454–44091372) 7 0.0087 0.13 0.023 0.083 0.083 0.114 0.1425 - - - - - - -
PDGFα 7p22 21 bp (179285–179305) 5 0.047 0.367 0.0637 0.148 0.148 0.241 0.1854 - - - - - - -
PDGFRα 4q11-q13 68.97 kbp (54790204–54859171) 8 0.0165 0.197 0.5423 0.913 0.197 0.313 0.2056 - - - - - - -
PIK3 7q 41.67 kbp (106292977–106334801) 10 0.0551 0.611 <0.0001 0.001 0.001 0.009 0.0300 0.0457 0.0470 0.5491 0.982 0.0456 0.331 0.1866
SMAD3 15q21-q22 129.3 kbp (65145249–65274586) 10 0.0402 0.551 0.0188 0.371 0.371 0.747 0.2576 - - - - - - -
SMAD7 18q21.1 30.86 kbp (44700222–44731079) 7 0.0001 0.002 0.0603 0.313 0.002 0.004 0.0250 0.0064 0.0070 0.0554 0.311 0.0064 0.047 0.0397
TGFβ1 19q13.1 23.17 kbp (46528490–46551655) 6 0.0381 0.3 0.1671 0.397 0.3 0.434 0.2409 - - - - - - -
TGFβ2 1q41 95.1 kbp (216586200–216684584) 9 0.0079 0.151 0.035 0.318 0.151 0.237 0.1854 - - - - - - -
TNF 6p21.3 2.763 kbp (31651328–31654090) 11 0.0013 0.034 0.0004 0.005 0.005 0.005 0.0250 0.0059 0.0060 0.0870 0.416 0.0059 0.044 0.0397
TNFR2 1p36.22 42.22 kbp (12149647–12191872) 8 0.099 0.716 <0.0001 0.013 0.013 0.019 0.0475 0.1052 0.2140 0.2213 0.777 0.1052 0.649 0.2706
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