Determination of the association of ETS1 and WDFY4 gene polymorphisms with systemic lupus erythematosus in an Iranian population

Document Type: Original Article

Authors

1 Immunology Department, Shahid Sadoughi University of Medical Sciences (International Campus), Yazd, Iran

2 Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran

3 Genetic Department, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

4 Department of Epidemiology and Biostatistics, school of public health, Tehran University of Medical Sciences, Tehran, Iran

Abstract

Systemic Lupus Erythematosus (SLE) is a heterogeneous complex relapsing-remitting autoimmune disease. The role of genetics is obvious in predisposition of the disease. Several Single Nucleotide Polymorphisms (SNPs) in ETS1 and WDFY4 showed association with SLE in genome-wide association studies. The aim of this study was to examine the association of the SNPs in ETS1 and WDFY4 genes with SLE in an Iranian population. This study was performed on 280 patients that were not related to one another, and 281 healthy control subjects matched based on age, sex, and ethnicity, all of which were of Iranian origin. Rs10893872 and rs1128334 in the ETS1 gene, and rs877819 and rs707397 in the WDFY4 gene were genotyped using MGB TaqMan Allelic Discrimination Real-Time PCR. Our results showed no association in all mentioned SNPs with the susceptibility and clinical features of SLE in the Iranian population. The results were not consistent with genome-wide association studies performed on Asian and Caucasian populations.

Keywords


Introduction_________________________

Systemic Lupus Erythematous (SLE) is a relapsing- remitting autoimmune disorder which involves many different organs and can be characterized by several immunological abnormalities. These include the presence of hyper-reactive T and B cells and the production of an array of auto-antibodies against serological, intra- cellular, nucleic acid and cell surface antigens [1, 2]. The prevalence of SLE is approximately 1 in 2,500 Europeans with asex ratio of 9:1. The disease is more frequent in people with non-European ancestry [2, 3]. Davatchi et al. reported a prevalence of 0.04% for SLE in a WHO-ILAR COPCORD study conducted inIran.

Considering the wide range of clinical manifestations and the heterogeneous nature of the SLE phenotype, the American College of Rheumatology (ACR) has defined Eleven clinical criteria to identify patients, namely malar rash, discoid rash, photosensitivity, oral ulcers, non-erosive arthritis, pleuritis, renal disorders, neurologic disorders, hematologic disorders, immunologic disorders and, positive antinuclearantibodies [4].

Despite the fact that the exact etiology of SLE remains unclear, strong genetic linkage has been well accepted for this disease. Its heritability is estimatedto be approximately 66%, with concordance rates of 24% to 57% in monozygotic twins and 2% to 5% in dizygotic twins5. The role of genetic predisposition is highly attributable to the disease. According to recent studies, SLE is characterized as a polygenic genetic model. As many as 100 genes could be involved, and every gene may have only a moderate effect size [6-8]. According to recent genome-wide association studies (GWAS), ETS1 and WDFY4 were introduced as novel predisposing genes forSLE [9, 10].

V-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1) is a negative regulator of B-cell differentiation and T helper 17 (Th17) cell proliferation. It has been shown that Patients with SLE present a reduced expression of ETS1, which might contribute to abnormal B-cell differentiation into auto- antibody secreting plasma cells and increased number of Th17 cells 3. Moreover, two recent genome-wide association studies on an Asian population (SLE patients from Hong Kong, Mainland China, Thailand, and China Han population) indicated that, variants (rs6590330, rs7932088, rs10893872, rs4937333, rs1128334) of the ETS1 gene have significant association with SLE [9, 10], confirming the SLE-like disease observed in ETS1-deficientmice [11].

WDFY family member 4 (WDFY4) is a huge protein with unknown functions. WDFY4 is expressed predominantly in immune tissues such as lymph nodes, the spleen, the thymus and the tonsils (http:// www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.287379). Two recent genome-wide studies in Asia have identified several variants (rs7097397, rs10857650, rs877819) of this gene associated withSLE [9, 10].

Considering the differences in genetic and environmental factors affecting different populations, as well as the need for replication studies in various populations, in this study, we have investigated the association of ETS1 (rs10893872, rs1128334) and WDFY4 (rs877819, rs707397) polymorphisms with SLE susceptibility in the Iranian population. We have also investigated the association of these polymorphisms with different clinical features of thepatients.

Materials andMethods__________________

Subjects

The study population was made up of 280 patients (who were not related to one another) with SLE who were recruited from the outpatient clinic of the Rheumatology Research Center, Shariati Hospital, Tehran University of Medical Sciences. All SLE patients met the revised 1982 American College of Rheumatology (ACR) classification criteria for SLE.

281 healthy control subjects had no clinical evidence or family history of any types of rheumatologic or autoimmune disorders 4. The SLE patient group had a mean age of 36.77±11.72 years (244 females and 36 males) and the healthy control group had a mean ageof 38.04±12.71 years (246 females and 35 males). All patients and healthy controls had Iranian ancestry. Healthy controls were matched with patients with regard to their gender, ethnicity, and age. To determine the association between two SNP variations and clinical features of SLE, all of the clinical manifestations of the patients were recorded. Informed consent was obtained from all of the subjects. The Ethical Committee of Tehran University of Medical Sciences approved thisstudy.

Genotyping (DNA preparation andanalysis)

Genomic DNA was extracted from peripheral blood leukocytes using a phenol-chloroform method [12]. The extracted DNA was stored at -20°C until it was analyzed. Approximately 30ng of the genomic DNA of each sample was used for genotyping. Amplification was performed in 10ml reaction volume, containing 5ml of the TaqMan Genotyping master mix (PN, 4371355), 0.25ml of the TaqMan Genotyping assay mix (PN, 4351376), 0.25ml of distilled water, and 4.5ml of genomic DNA. The genotyping of ETS1 (rs10893872, rs1128334) and WDFY4 (rs877819, rs707397) were performed using Real Time PCR using the TaqMan allelic discrimination method (Applied Biosystems, Foster City, CA, USA). The characteristics of the selected SNPs are summarized in Table1.

Statisticalanalysis

The distribution of genotypes in the control subjects was examined for deviation from Hardy Weinberg equilibrium using the c2 test. Analysis of the data was carried out using the IBM™ SPSS version 20 (SPSS Inc., USA). The genotypic and allelic distribution between patients and controls was assessed by the c2 test and by binary logistic regression. The Odds Ratio (OR), with 95% Confidence Intervals (95% CI), was calculated from logistic regression analysis. P-values of less than 0.05 were considered statistically significant. The association of SNPs to clinical manifestations was also determined by the c2 test. We used the Benjamini- Hochberg method to control false discovery rate (FDR) for multiplecomparisons.

Results_______________________________

Distribution of ETS1 and WDFY4 SNP genotypes among patients andcontrols

Distribution of rs10893872 and rs1128334 genotypes in the ETS1 gene and rs877819 and rs7097397 genotypes in the WDFY4 gene did not show any significant deviation from the Hardy-Weinberg equilibrium in the healthy control subjects. None of the SNPs in either gene had significant distribution in patients by comparison to the control group (Table 2), however, we observed a mild shift to G allele and GG genotype in WDFY4 (rs7097397) in comparison to the control group.

Table 1. Characteristics of selectedSNPs

Genes

SNPs

Minorallele

PolymorphismType

Location/Exon

PositionGRCh37

ETS1

rs10893872

C

C/T, TransitionSubstitution

Intragenicregion

Chr.11128325553

ETS1

rs1128334

T

C/T, TransitionSubstitution

UTR-3

Chr.11128328959

WDFY4

rs877819

A

A/G, TransitionSubstitution

Intron

Chr.1050042951

WDFY4

rs7097397

A

A/G, TransitionSubstitution

MissenseMutation

Chr.1050025396

We next investigated the association of these four SNPs with the clinical manifestations of SLE. Such manifestations include photo-sensitivity, malar rash, discoid rash, oral ulcers, arthritis, pleuritis, pericarditis, proteinuria, seizures, leucopenia, anti-ds DNA, and ANA. After a false discovery rate (FDR) test, we did not detect any correlation between these SNPs and the mentioned clinical manifestations (Table 3 and4).

Discussion___________________________

Systemic lupus erythematosus (SLE) is a complex autoimmune disease, which is clinically heterogeneous with a wide range of clinical manifestations, which differ from patient to patient [13]. It is now widely accepted that genetic components play important roles in the abnormal immune responses and pathogenesis of SLE, thus, people carrying the special genes are susceptible to the disease. Studies in animal models have also confirmed that a large part of susceptibility to SLE is due to genetic predisposition [14]. However, SLE does not follow the simple Mendelian inheritance and no major single gene governs the pathogenesis, instead a polygenic model is expected, as genome-wide association studies have identified more than 30 associatedloci [3, 15].

ETS1 is a member of the ETS family of transcription factors defined by the conserved DNA-binding domain known as ETS, which is a winged helix-turn-helix motif, located on chromosome number [11]. The protein contains 485 amino acids, which function as transcriptional activators and suppressors of numerous genes (http://www.ncbi.nlm.nih.gov/gene/ 2113) [16]. ETS1 regulates lymphocyte differentiation and development through regulating the B-cell differentiation and T helper-17 proliferation [16, 17]. ETS1 deficient Th-1 cells show increased and reduced secretion of IL-10 and IL-2 respectively. ETS1 deficient B-cells show enhanced differentiation to IgM secreting plasma cells and are hyper-responsive to TLR9, which indicates the possible involvement of ETS1 in the pathogenesis ofSLE [18].

Kathleen et al. showed that a microsatellite repeat polymorphism in ETS1 3` flanking region is associated with SLE phenotypes [19]. Two genome-wide association studies on an Asian population also demonstrated a high association of rs6590330 and rs10893872 with SLE [9, 10].

Table 2. Distribution of ETS1 and WDFY4 SNP alleles and genotypes among patients andcontrols

Odds Ratio

(CI 95%)

Adj.Pa

P

Controls N

(%)

Patients N

(%)

Alleles/ Genotypes

SNPs

Genes

1

 

Reference

278(49.46)

277(49.46)

T

 

0.99(0.79-1.26)

0.96

0.96

284(50.53)

283(50.53)

C

1

 

Reference

74(26.3)

73(26.1)

TT

rs10893872

ETS1

1.02(0.682-1.530)

0.99

0.918

130(46.3)

131(46.8)

CT

 

 

1.00(0.636-1.573)

0.99

0.998

77(27.4)

76(27.1)

CC

 

 

 

 

 

0.21

0.28

HWE

 

 

1

 

Reference

512(91.10)

497(88.75)

C

 

 

1.30(0.88-1.19)

0.38

0.19

50(8.89)

63(11.25)

T

 

 

1

 

Reference

233(82.9)

220(78.6)

CC

rs1128334

ETS1

1.31(0.854-2.017)

0.43

0.215

46(16.4)

57(20.4)

CT

 

 

1.59(0.263-9.598)

0.82

0.614

2(0.7)

3(1.1)

TT

 

 

 

 

 

0.87

0.74

HWE

 

 

1

 

Reference

240(42.70)

228(40.71)

G

 

 

1.08(0.86-1.38)

0.66

0.50

322(57.29)

332(59.28)

A

 

 

1

 

Reference

56(19.9)

45(16.1)

GG

rs877819

WDFY4

1.34(0.847-2.126)

0.43

0.211

128(45.6)

138(49.3)

AG

 

 

1.24(0.768-2.017)

0.60

0.375

97(34.5)

97(34.6)

AA

 

 

 

 

 

0.25

0.73

HWE

 

 

1

 

Reference

289(51.42)

318(56.78)

G

 

 

0.81(0.64-1.02)

0.28

0.07

273(48.57)

242(43.21)

A

 

 

1

 

Reference

71(25.3)

94(33.6)

GG

rs7097397

WDFY4

0.67(0.453-0.985)

0.34

0.042

147(52.3)

130(46.4)

AG

 

 

0.671(0.418-1.078)

0.39

0.099

63(22.4)

56(20.0)

AA

 

 

 

 

 

0.43

0.37

HWE

 

 

                 

 

Table3.DistributionoftheETS1(rs1128334)and(rs10893872)genotypesamongSLEphenotypes

Adj.P*

rs10893872 Genotype

Adj.P*

P

rs1128334 Genotype

Frequency

Clinical features

P

TT

(%)

CT (%)

CC (%)

CC (%)

CT

(%)

TT

(%)

N

(%)

0.33

0.25

54

(26.0)

96

(47.1)

56

(26.9)

0.33

0.25

159

(76.4)

46

(22.1)

3

(1.4)

208

(74.3)

Photo-sensitivity

0.09

0.03

33

(28.9)

51

(44.7)

30

(26.3)

0.09

0.03

81

(71.1)

31

(27.2)

2

(1.8)

114

(40.9)

Malar rash

0.49

0.41

5

(33.3)

5

(33.3)

5

(33.3)

0.49

0.41

10

(66.7)

5

(33.3)

0

(0.0)

15

(5.4)

Discoid rash

0.89

0.89

20

(25.0)

38

(47.5)

22

(27.5)

 

0.89

64

(80.0)

15

(18.8)

1

(1.3)

80

(28.7)

Oral ulcer

0.09

0.01

52

(25.5)

95

(46.6)

57

(27.9)

0.89

0.01

162

(79.4)

42

(20.6)

0

(0.0)

204

(72.9)

Arthritis

0.74

0.68

16

(30.8)

22

(42.3)

14

(26.9)

0.09

0.68

42

(80.8)

9

(17.3)

1

(1.9)

52

(18.6)

Pleuritis

0.14

0.08

5

(33.3)

5

(33.3)

5

(33.3)

0.74

0.08

12

(80.0)

2

(13.3)

1

(6.7)

15

(5.4)

Pericarditis

0.14

0.08

37

(30.3)

53

(43.4)

32

(26.2)

0.14

0.08

98

(80.3)

21

(17.2)

3

(2.5)

122

(43.0)

Proteinuria

0.14

0.07

5

(25.0)

8

(40.0)

7

(35.0)

0.14

0.07

12

(60.0)

8

(40.0)

0

(0.0)

20

(7.2)

Seizures

0.09

0.03

27

(23.5)

56

(48.7)

32

(27.8)

0.09

0.03

99

(86.1)

15

(13.0)

1

(9.0)

115

(41.1)

Leukopenia

0.33

0.25

66

(25.0)

125

(47.3)

73

(27.7)

0.33

0.25

207

(78.4)

54

(20.5)

3

(1.1)

264

(95.3)

Anti- dsDNA

0.09

0.03

73

(26.4)

128

(46.2)

76

(27.4)

0.09

0.03

217

(78.3)

57

(20.6)

3

(1.1)

277

(98.9)

ANA

 

Table4.DistributionoftheWDFY4(rs877819)and(rs7097397)genotypesamongSLEphenotypes

Clinical features frequency

rs877819 Genotype

P

Adj.P*

rs7097397 Genotype

P

Adj.P*

N (%)

GG (%)

GA (%)

AA (%)

GG (%)

GA

(%)

AA

(%)

Photo-sensitivity

208

(74.3)

34

(16.3)

102

(49.0)

72

(34.6)

0.97

0.97

67

(32.2)

99

(47.6)

42

(20.2)

0.70

0.85

Malar rash

114

(40.9)

19

(16.7)

55

(48.2)

40

(35.1)

0.94

0.97

29

(25.4)

66

(57.9)

19

(16.7)

0.005

0.06

Disciod rash

15

(5.4)

4

(26.7)

7

(46.7)

4

(26.7)

0.49

0.65

6

(40.0)

4

(26.7)

5

(33.3)

0.23

0.69

Oral ulcer

80

(28.7)

15

(18.8)

31

(38.8)

34

(42.5)

0.07

0.24

29

(36.3)

36

(45.0)

15

(18.8)

0.83

0.90

Arthritis

204

(72.9)

34

(16.7)

102

(50.0)

68

(33.3)

0.73

0.87

67

(32.8)

88

(43.1)

49

(24.0)

0.019

0.11

Pleuritis

52

(18.6)

7

(13.5)

23

(44.2)

22

(42.3)

0.43

0.65

19

(36.5)

21

(40.4)

12

(23.1)

0.61

0.85

Pericarditis

15

(5.4)

0

(0.0)

7

(46.7)

8

(53.3)

0.12

0.29

7

(46.7)

5

(33.3)

3

(20.0)

0.49

0.85

Proteinuria

122

(43.0)

19

(15.6)

56

(45.9)

47

(38.5)

0.08

0.24

39

(32.0)

56

(45.9)

27

(22.1)

0.71

0.85

Seizures

20

(7.2)

1

(5.0)

10

(50.0)

9

(45.0)

0.07

0.24

9

(45.0)

10

(50.0)

1

(5.0)

0.19

0.69

Leukopenia

115

(41.1)

15

(13.0)

50

(43.5)

50

(43.5)

0.033

0.24

38

(33.0)

55

(47.8)

22

(19.1)

0.91

0.91

Anti-dsDNA

264

(95.3)

45

(17.0)

126

(47.7)

93

(35.2)

0.17

0.34

87

(33.0)

124

(47.0)

53

(20.0)

0.61

0.85

ANA

277

(98.9)

45

(16.2)

137

(49.5)

95

(34.3)

0.46

0.65

92

(33.2)

129

(46.6)

56

(20.2)

0.42

0.85

Jing Zhang et al. also examined serum IL-17 levels from 283 SLE cases, and reported a significant correlation between previously determined risk variants in ETS1 and the concentration of IL-17 in their serum [20]. It is also reported that genetic variants of ETS1 affect STAT1 binding. The results of the genome-wide association study on an Asian population was replicated for a Caucasian population for ETS1 21. In our study, we adopted rs1128334 instead of rs6590330 because the study of Yang et.al. show that it is in 3`UTR and correlates with the expression of ETS1 [10]. Rs10893872 has a high linkage disequilibrium (LD) with rs4937333 [10]. Our results did not confirm previously presented data in Asian and Caucasian populations. We observed no significant association between any of the selected SNPs and SLE in Iranian population (Table 2). There were also no significant correlation between the clinical features of SLE and the studied SNPs, which contrasts with the study performed on the Hanpopulation [22].

WDFY4 is a 3184 amino acid protein with unknown functions, which is expressed in secondary immune tissues. The protein includes WD40 and BEACH (Beige and Chediak-Higashi) domains (http://www.ncbi.nlm. nih.gov/gene/57705). WD40is found in a number of eukaryotic proteins and has many functions including adaptor/regulatory modules in signal transduction, pre-mRNA processing, and cytoskeleton assembly. The domain typically contains  a GH dipeptide near the N-terminus and a WD dipeptide at the C-terminus, between which a conserved core exists that creates a propeller-like platform to bind other proteins either stably or reversibly (http://www.ncbi.nlm.nih.gov/Structure/ cdd/ cddsrv.cgi?uid=100117). The BEACH domain is important in membrane trafficking (http://www.ncbi. nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=207648).

We decided to use rs7097397 and rs877819 in the study, due to the results of genome-wideassociation studies on Asian populations, in which both of the SNPs showed association with SLE [9, 10]. Rs7097397 is in the coding region Arg1816Gln of the gene and can be considered as a functional SNP, while rs877819 has been shown to change the binding affinity of the Yin Yang1 transcription factor and down regulate WDFY4 10. Our results were not consistent with those of the GWA studies on an Asian population as there were no associations between both SNPs with the disease (Table2).

In conclusion, our results did not confirm the results of other association studies performed on Asian and Caucasian populations. We did not observe correlation between the SNPs and Systemic Lupus Erythematous, or between the SNPs and SLE clinical features. This data indicates the genetic and environmental differences within differentpopulations.

Conflict ofinterests

Authors have no conflict ofinterests.

Acknowledgment

This work was partially supported by a research grant [grant number: 90-04-41-14909] from the Deputy of Research, Tehran University of Medical Sciences.

Bengtsson A, Rylander L, Hagmar L, Nived O, Sturfelt G. Risk factors for developing systemic lupus erythematosus: a case-control study in southern Sweden. Rheumatology. 2002;41(5):563-71.

Graham DC, Morris DL, Bhangale TR, Criswell LA, Syvanen A-C, Ronnblom L, et al. Association of NCF2,IKZF1, IRF8, IFIH1, and TYK2 with systemic lupus erythematosus. PLoS Genet. 2011;7(10):e1002341-e.

Guerra SG, Vyse TJ, Graham DSC. The genetics of lupus: a functional perspective. enzyme. 2012;2:3.

Tan EM, Cohen AS, Fries JF, Masi AT, Mcshane DJ,Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis & Rheumatism. 1982;25(11):1271-7.

Ahmad YA, Bruce IN. Genetic epidemiology: systemic lupus erythematosus. Arthritis Res. 2001;3(6):331-6.

Yuan Y, Luo X, Shen N. Current advances in lupus genetic and genomic studies in Asia. Lupus.2010;19(12):1374-83.

Thibault Flesher DL, Sun X, Behrens TW, Graham RR, Criswell LA. Recent advances in the genetics of systemic lupus erythematosus. 2010.

Nakabayashi K, Tajima A, Shirasawa S. [Genome-wide association studies: recent advances and future directions]. Nihon rinsho Japanese journal of clinical medicine. 2009;67(3):469-76.

Han J-W, Zheng H-F, Cui Y, Sun L-D, Ye D-Q, Hu Z, et al. Genome-wide association study in a Chinese Han population identifies nine new susceptibility loci for systemic lupus erythematosus. Nature genetics.2009;41(11):1234-7.

Yang W, Shen N, Ye D-Q, Liu Q, Zhang Y, Qian X-X, et al. Genome-wide association study in Asian populations identifies variants in ETS1 and WDFY4 associated with systemic lupus erythematosus. PLoS Genet.2010;6(2):e1000841.

Pan H, Leng R, Tao J, Li X, Ye D. Ets-1: a new player in the pathogenesis of systemic lupus erythematosus?Lupus. 2011;20(3):227-30.

Roe BA, Crabtree J, Khan A. Methods for DNA isolation. Part III. Protocols for recombinant DNA isolation, cloning, and sequencing [Internet edition] Norman, OK: University of Oklahoma. 1995:2488-98.

Singh A, Kamen DL. Potential benefits of vitamin D for patients with systemic lupus erythematosus. Dermato- endocrinology. 2012;4(2):146-51.

Kono DH, Theofilopoulos AN, editors. Genetics of SLE in mice. Springer seminars in immunopathology; 2006: Springer.

Rhodes B, Vyse T. The genetics of SLE: an update in the light of genome-wide association studies. Rheumatology.2008;47(11):1603-11.

Wang D, John SA, Clements JL, Percy DH, Barton KP, Garrett-Sinha LA. Ets-1 deficiency leads to altered B cell differentiation, hyperresponsiveness to TLR9 and autoimmune disease. International immunology.2005;17(9):1179-91.

Moisan J, Grenningloh R, Bettelli E, Oukka M, Ho I-C. Ets-1 is a negative regulator of Th17 differentiation. The Journal of experimental medicine. 2007;204(12):2825-35.

Leng R-X, Pan H-F, Chen G-M, Feng C-C, Fan Y-G, Ye D-Q, et al. The dual nature of Ets-1: focus to the pathogenesis of systemic lupus erythematosus.Autoimmunity reviews. 2011;10(8):439-43.

Sullivan KE, Piliero LM, Dharia T, Goldman D, Petri MA. 3'polymorphisms of ETS1 are associated with different clinical phenotypes in SLE. Human mutation.2000;16(1):49.

Zhang J, Zhang Y, Zhang L, Yang J, Ying D, Zeng S, et al. Epistatic Interaction between Genetic Variants in Susceptibility Gene ETS1 Correlates with IL-17 Levels in SLE Patients. Annals of human genetics.2013;77(4):344-50.

Wang C, Ahlford A, Järvinen TM, Nordmark G, Eloranta M-L, Gunnarsson I, et al. Genes identified in Asian SLE GWASs are also associated with SLE in Caucasian populations. European Journal of Human Genetics. 2013;21(9):994-9.

He C, Liu Y, Cheng Y, Gao J, Pan T, Han J, et al.TNIP1, SLC15A4, ETS1, RasGRP3 and IKZF1 are associated with clinical features of systemic lupus erythematosus in Chinese Han population. Lupus. 2010.