Rheumatol Int DOI 10.1007/s00296-017-3745-y

Rheumatology INTERNATIONAL

GENES AND DISEASE

Association study of CD154 polymorphisms and serum CD154 level with systemic lupus erythematous in Chinese population Yang Xiang1 · Jing Guo2 · You‑Fan Peng1 · Hua‑Tuo Huang1 · Yan Lan2 · Ye‑Sheng Wei1 

Received: 5 February 2017 / Accepted: 17 May 2017 © Springer-Verlag Berlin Heidelberg 2017

Abstract  The aim of this study was to investigate the association of three polymorphisms of CD154 with risk of SLE in Chinese population. The study population comprised 770 Chinese individuals, including 350 SLE patients and 420 healthy controls. The gene polymorphism was measured using Snapshot SNP genotyping assays and confirmed by sequencing. Serum CD154 (sCD154) level was measured by ELISA. Compared with control group, sCD154 levels were significantly increased in case group (P < 0.001). The minor C allele of rs1126535 was associated with a significantly increased risk of SLE as compared to the major T allele (P < 0.001). Furthermore, an increased frequency of C-G-A haplotype was also detected in case group which associated with an increased risk of SLE (P  = 0.009). Notably, patients carrying rs1126535CT/CC genotypes had a higher sCD154 level compared with that carrying rs1126535TT genotype (P < 0.05). Unfortunately, analyses on the association between rs1126535 and several clinical manifestations of SLE failed to find any significant results. In conclusion, these results indicated that CD154 gene polymorphisms may associate with the risk of SLE and may play regulation role in the expression of sCD154 in SLE patients.

* Yan Lan [email protected] Ye‑Sheng Wei [email protected] 1

Department of Clinical Laboratory, The Affiliated Hospital of Youjiang Medical University for Nationalities, 533000 Baise, Guangxi, China

2

Department of Dermatology, the Affiliated Hospital of Youjiang Medical University for Nationalities, 533000 Baise, Guangxi, China





Keywords  CD154 · Systemic lupus erythematous · Single nucleotide polymorphism

Introduction Systemic lupus erythematous (SLE) is a severe multisystem autoimmune inflammatory disorder. And it is characterized by autoantibody production and immune complex deposition that contribute to multiple tissue damage [1]. It showed several kinds of immunological abnormalities, including T-cell activation and anergy, dysregulation of cytokine production, and polyclonal B-cell activation [2, 3]. The exact pathogenic mechanism of SLE remains unclear. Nongenetic risk factors, such as environmental factors, infection, hormones and cytokines deregulations [4, 5] were reported to contribute to the pathogenesis of SLE. While, the existing reports indicated that genetic factors may play more important roles in the development of SLE [6–8]. CD154 (CD40 ligand, CD40L) is a protein that is primarily expressed on the surface of activated ­CD4+ T cells, and it is a member of the TNF superfamily of molecules. It binds to its preferred receptor CD40 on antigen-presenting cells (APC) thereby leading to many effects [9]. Current evidence shows that the co-stimulatory (CD40-CD154) system plays crucial roles in the development, progression in various immunological disorders [10–13]. Published reports indicated that CD154 was involved in the progression of SLE and maybe therapeutic target for SLE [14–16]. Besides, sCD154 levels were reported to be significantly increased in SLE patients and related to the activity of SLE [17–19]. However, the exact mechanism of the abnormal expression of CD154 in SLE patients needs to be further explored, and genetic factors may be contribute to this progress [20].

13



Rheumatol Int

The gene encoding CD154 is located on chromosome Xq26 in humans, including five exons and four introns. Recently, a number of polymorphisms in the CD154 gene have been identified [21, 22]. Investigations on CD154 gene polymorphism and SLE have been conducted and a dinucleotide repeat polymorphism which can regulate the expression of sCD154 in SLE patients have been confirmed [23, 24]. But, the association between CD154 gene single nucleotide polymorphisms and Chinese SLE susceptibility remains unclear and little is known about the association between CD154 SNPs and expression of sCD154 in Chinese SLE patients. Therefore, the aim of the present study was to analyze, for the first time, the role of three CD154 SNPs (rs1126535 T/C, rs7050168 A/G and rs3092921G/A) in Chinese SLE patients and to assess the association between CD154 gene polymorphisms and the expression of sCD154.

(according to dbSNP from 1000 Genomes Project, https:// ncbi.nlm.nih.gov/variation/view/). Second, basing on genotyping putative functional SNPs,we selected three SNPs with different putative functions (rs1126535T/C, synonymous codon, chromosome X:136648396; rs7050168A/G, intron variant, chromosome X: 136656589; rs3092921G/A, downstream variant 500B, chromosome X: 136660841) to study.

Materials and methods

Determination of CD154 genotype

Study subjects

The methods of Snapshot SNP genotyping assays technique and DNA sequencing were taken to detect the allele and genotype frequencies of the CD154 gene. The PCR primers were designed based on the GenBank reference sequence (accession no. NC_000023.11) (Table 1). To confirm the genotyping results, PCR-amplified DNA samples were examined by DNA sequencing and the results were 100% concordant.

Our study adopted a retrospective design. This study consisted of 350 SLE patients (70 males aged 25–76 years and 280 females aged 11–81 years). All SLE patients were consecutively selected. An established diagnosis of SLE was determined according to clinical manifestations and autoimmune antibody tests. All SLE patients recruited into the study met the 1997 revised American College of Rheumatology (ACR) SLE criteria. They were recruited from the Department of Dermatology Affiliated Hospital of Youjiang Medical University for Nationalities, Guangxi, China between January 2013 and September 2015. The 420 control subjects were matched to the patients on the basis of age and gender (90 males aged 15–65 years and 330 females aged 11–82 years), and underwent a routine medical check-up in the outpatient clinic of the Department of Internal Medicine, Affiliated Hospital of Youjiang Medical University for Nationalities, Guangxi, China during the same period. According to the thorough clinical and laboratory evaluation, none of them were found to have any medical condition. All study subjects were Chinese and resided in the same geographic area in Guangxi China. The study was performed with the approval of the ethics committee of the Affiliated Hospital of Youjiang Medical University for Nationalities, and written informed consent was obtained from all the subjects. SNP selection According to flowing standards, we selected these three SNPs. First, the minor allele frequency is greater than 10%

13

DNA extraction Whole-blood samples were collected immediately after the subjects were determined. And the samples were stored at −70 °C until use. Genomic DNA was extracted from blood leucocytes by using a whole-blood genome DNA extraction reagent kit [Axygene Biotechnology (Hangzhou) Limited (Hangzhou City, China)], following the manufacturers’ instructions.

Serum CD154 determinations The plasma samples of SLE patients were collected within 12 h after they were admitted to hospital. After an overnight fasting, venous blood samples of control population were collected. Plasma samples from the patients and healthy controls were separated from venous blood at room temperature, and stored at −70 °C until use. The quantity determination of serum CD154 levels was performed by enzyme-linked immunesorbent assay (ELISA) kits (RayBiotech, America) following the manufacturer’s protocol. Developed color reaction was measured as OD420 units on an ELISA reader (RT-6000, China). The concentration of plasma CD154 was determined by using standard curve constructed with the kit’s standards over the range of 0–1000 pg/ml. Statistical analysis The SPSS statistical software package version 17.0 was used for the statistical analysis. Continuous variables were displayed as mean ± SD. If the data were normally distributed, the Student’s t test was used; otherwise,

Rheumatol Int Table 1  The primer sequences used for detecting the different SLE SNPs

Reference SNP ID

PCR primers

rs1126535 T/C synonymous codon X:136648396

F:5′-GGACTGCCCATCAGCATGAAA-3′ R:5′-TCCATCATTTGGGTAGAACCAACCT-3′ EF:5′-TTTTTTTTTTTTACTTTTTGCTGTGTATCTTCATAGAAGG-3′

rs7050168 A/G intron variant X:136656589

F:5′-CTTTTCCCCTGGGACCCAATTT-3′ R:5′- AATACCCCATGAGGCCCATTTC-3′ EF:5′-TT CCTCCTTTTGTCCCTCTAAGTCCCA-3′

rs3092921G/A downstream vari- F:5′-GGAAGTATTGGCCCCCCAGTCT-3′ ant 500B X:136660841 R:5′-CCATGAGCCCAGGGAGATAACC-3′ EF:5′- TTTTTTTTTTTGGGTTTCTTACATGAAAAATTCCTCT-3′

Mann–Whitney U test was used. Categorical variables were expressed as proportions and compared by using chisquared test. Hardy–Weinberg equilibrium (HWE) was tested by chi-squared test. The association of the polymorphisms and risk of SLE was evaluated by odds ratios (ORs) with 95% confidence interval (CIs). For multivariate analyses, logistic regression analyses were used to adjust for possible confounders, including age, gender. Statistical significance was accepted at the P < 0.05 level.

Results Clinical characteristics of the study participants The clinical characteristics of the study participants were summarized in Table 2. SLE patients were significantly more likely to have high family medical history, positive autoantibodies rate, hematologic abnormalities, renal and neurologic disorder, but low levels of complements (both P < 0.05). Besides, compared to control group sCD154 Table 2  Clinical characteristics of the subjects

Variable

levels were significantly increased in case group (SLE vs. control = 9.82 ± 3.92 vs. 4.31 ± 1.61, P < 0.001) (Table 2; Fig. 1). The genotype and allele frequencies of CD154 gene All of the polymorphisms have three genotypes, respectively (Fig. 2). The genotype and allele frequencies of CD154 gene polymorphisms in case and control group were shown in Table 3. All of the polymorphisms among the controls and the cases were in Hardy–Weinberg equilibrium (both P > 0.05). The distribution of rs1126535T/C polymorphism was significantly different between the two groups (P < 0.05). Minor C allele of rs1126535 was associated with a significantly increased risk of SLE compared with major T allele (CT vs. TT: AOR = 1.7, 95% CI 1.2– 2.5, P = 0.021; CC vs. TT: AOR = 1.9, 95% CI 1.5–3.1, P = 0.015). Dominant and recessive model also found to be significant associated with increased risk (CC + CT vs. TT: AOR = 2.2, 95% CI 1.4–2.5, P = 0.008; CC vs. CT + TT: AOR = 1.8, 95% CI 1.3–3.1, P = 0.023). However, the left SLE patients n = 350 (%)

Control subjects n = 420 (%)

P value

Age, year (mean ± SD)

36.7 ± 13.4

35.9 ± 10.9

0.377

Gender (M/F) Active disease Renal disorder Neurologic disorder Thrombocytopenia Leukopenia Fever Autoantibody positive

70/280 260 (74.3) 119 (34.0) 91 (26.1) 147 (42.0) 179 (51.1) 205 (87.2) 330 (94.3)

90/330 – – – – – 41 (9.8) 23 (5.4)

0.627 – – – – – 0.000 0.000

Family medical history (SLE) Low levels of complements Hyperprolactinemia Estradiol metabolic disorder History of streptococcus infection

96 (27.4) 187 (53.6) 79 (22.6%) 65 (18.6%) 85 (24.3)

3 (0.7) – – – 120 (28.6)

0.000 – – – 0.180

sCD154 (ng/ml)

9.82 ± 3.92

4.31 ± 1.61

0.000

13



Rheumatol Int

two polymorphisms showed no significant differences in the genotype and allele frequencies between the groups. Haplotype analysis of the CD154 gene Haplotype analyses were performed using Haploview software and the major haplotype frequencies were shown in Table  4. T-A-G haplotype accounted for 86.1 and 88.3% in case and control group, respectively. By haplotype analyses, we found the C-G-A haplotype was significantly increased in case group (P = 0.010). And C-G-A haplotype was associated with a significantly increased risk of SLE (OR = 2.2; 95% CI 1.2–4.1; P = 0.009).

CD154 rs1126535 polymorphism and clinical manifestations

Fig. 1  The sCD154 levels in SLE patients and normal control subjects. Compared with the control subjects, the sCD154 was significantly increased in patients with SLE [(9.82 ± 3.92 ng/mL, n = 350) vs. (4.31 ± 1.61 ng/mL, n = 420); P < 0.001]

We conducted stratification analysis by comparing genotype frequencies between positive and negative patients in specific clinical and laboratory features. However, we failed to detect any marked associations between rs1126535

Fig. 2  Sequencing map of genotypes for CD154 gene polymorphisms. Sequencing map showed CC, CT and TT genotypes of rs1126535 T/C; AA, AG and GG genotypes of rs7050168 A/G; GG, GA and AA genotypes of rs3092921G/A, respectively (From top to bottom)

13

Rheumatol Int Table 3  The genotype and allele frequencies of CD154 Polymorphism rs1126535 T/C  TT  CT  CC Dominate model Recessive model Allele C frequency rs7050168 A/G  AA  AG  GG Dominate model Recessive model Allele G frequency rs3092921G/A  GG  GA  AA Dominate model Recessive model Allele A frequency

Control subjects n = 420 (%)

SLE patients n = 350 (%)

OR (95% CI)

P

AOR (95% CI)

P*

363 (86.4) 36 (8.6) 21 (5.0) 57 (13.6) 399 (95.0) 78 (9.3)

270 (77.1) 47 (13.5) 33 (9.4) 80 (22.9) 317 (90.6) 113 (16.1)

1.0 (Ref.) 1.8 (1.1–2.8) 2.1 (1.2–3.7) 1.9 (1.3–2.7) 2.0 (1.1–3.5)

– 0.016 0.009 0.001 0.017

1.0 (Ref.) 1.7 (1.2–2.5) 1.9 (1.5–3.1) 2.2 (1.4–2.5) 1.8 (1.3–3.1)

– 0.021 0.015 0.008 0.023

384 (91.4) 25 (6.0) 11 (2.6) 36 (8.6) 395 (97.4) 47 (5.6)

312 (89.1) 26 (7.5) 12 (3.4) 38 (10.9) 338 (96.6) 50 (7.1)

1.0 (Ref.) 1.3 (0.7–2.3) 1.3 (0.6–3.1) 1.3 (0.8–2.1) 1.3 (0.6-2.9)

– 0.394 0.486 0.284 0.566

1.0 (Ref.) 1.2 (0.5–2.1) 1.4 (0.4–3.6) 1.3 (0.7–2.3) 1.2 (0.7–3.1)

– 0.421 0.325 0.342 0.389

359 (85.5) 43 (10.2) 18 (4.3) 61 (14.5) 402 (95.7)

290 (82.9) 41 (11.7) 19 (5.4) 60 (17.1) 331 (94.6)

1.0 (Ref.) 1.2 (0.7–1.9) 1.3 (0.7–2.5) 1.2 (0.8–1.8) 1.3 (0.7–2.5)

– 0.475 0.428 0.826 0.460

1.0 (Ref.) 1.3 (0.6–2.1) 1.2 (0.6–2.1) 1.2 (0.7–2.1) 1.4 (0.6–2.7)

– 0.384 0.415 0.628 0.389

79 (9.4)

79 (11.3)

OR odds ratio, AOR adjusted odds ratio, 95% CI 95% confidence interval * The AOR on the basis of risk factors such as age, gender

Table 4  Haplotype distribution in the patients with SLE and the controls Haplotypes

SLE patients 2n = 700 (%)

Controls 2n = 840 (%)

OR (95% CI)

P value

OR (95% CI)

P value

TAG CAA

603 (86.1) 34 (4.9)

742 (88.3) 49 (5.8)

0.821 (0.6–1.1) 0.824 (0.5–1.3)

0.198 0.398

1.0 (Ref.) 0.9 (0.5–1.3)

– 0.491

TGA TAA

13 (1.9) 21 (3.0)

14 (1.7) 19 (2.3)

1.116 (0.5–2.4) 1.336 (0.7–2.5)

0.777 0.365

0.9 (0.5–2.4) 1.4 (0.7–2.6)

0.732 0.337

CGA

29 (4.1)

16 (1.9)

2.226 (1.2–4.1)

0.010

2.2 (1.2–4.1)

0.009

OR odds ratio, 95% CI confidence interval

polymorphism and the listed clinical manifestations of SLE (all P > 0.05, Table 5). In addition, according to another stratification analysis, the rs1126535T/C polymorphism was associated with high disease activity (Table 6). Association between CD154 gene polymorphisms and sCD154 levels Associations between CD154 gene polymorphisms and sCD154 levels were further investigated. Genotype at rs1126535 polymorphism was significantly associated with sCD154 levels in SLE patients. SCD154 levels were significantly higher in individuals with homozygous CC

genotypes (12.78 ± 4.35 ng/ml, n  = 33) or heterozygous CT genotypes (13.25 ± 3.98 ng/ml, n = 47) than individuals with homozygous TT genotypes (8.87 ± 3.30 ng/ml, n  = 270) (both P < 0.05, respectively) (Fig. 3a). Individuals with CC and TC genotypes showed no difference in sCD154 levels (P > 0.05). However, the differences were not observed in the control group (Fig. 3b).

Discussion In the present study, we analyzed three SNPs (rs1126535 T/C, rs7050168 A/G and rs3092921G/A) of CD154

13



Rheumatol Int

Table 5  Analysis of CD154 rs1126535 polymorphism in SLE by clinical features

Variables

N (Yes/No)

Control 363 (86.4) SLEDAI (score)  0–4 75 (83.3)  5–9 55 (76.4)  10–14 78 (75.7)  ≥15 62 (72.9)  Total

270 (77.1)

TC, n = 47

CC, n = 33

37.1 ± 13.3 52.06 ± 3.58 35.56 ± 2.71 53/217 195/75 66/204 69/201 99/171 199/71 36/324 326/34 86/184 68/202 43/227 46/230 120/150 145/125 46/224 54/216 102/168 194/76 79/191 69/201 252/18

34.6 ± 12.8 52.15 ± 1.98 35.46 ± 1.79 9/38 38/9 17/30 13/34 18/29 35/12 7/40 41/6 17/30 13/34 8/39 9/38 16/31 21/26 10/37 15/32 21/26 34/13 19/28 13/34 43/4

36.6 ± 14.5 54.62 ± 3.41 36.18 ± 3.19 8/21 27/6 13/20 11/22 14/19 29/4 6/27 28/5 16/27 10/23 4/29 6/27 11/22 13/20 9/24 10/23 10/23 22/11 9/24 14/19 28/5

ACA-positive

74/276

51/219

14/33

9/24

P value

TC, N (%)

CC, N (%)

36 (8.6)

21 (5.0)

Ref.

9 (10.0) 10 (13.9) 16 (15.5) 12 (14.1)

6 (6.7) 7 (9.7) 9 (8.8) 11 (13.0)

0.725 0.082 0.028 0.004

47 (13.4)

33 (9.5)

0.003

gene between 350 SLE patients and 420 healthy controls in Chinese population. Our results suggested that rs1126535 C allele was associated with increased risk of SLE (P < 0.001). SCD154 was significantly increased in case group (P < 0.001). The minor C allele of rs1126535 was associated with the abnormal expression of sCD154 in case group. Briefly, patients carrying CC or CT genotypes had significant higher sCD154 levels than

13

TT, n = 270 – – – 70/280 260/90 96/254 93/257 131/219 263/87 49/301 205/145 119/231 91/259 55/295 61/289 147/203 179/171 65/285 79/271 133/217 250/100 107/243 96/254 323/27

Genotypes of rs1126535 T/C TT, N (%)

P value

Age (mean ± SD) CRP(mean ± SD, mg/L) ESR(mean ± SD, mm/h) Sex (M/F) Active disease Family medical history Oral ulcer Sun allergy Arthritis Serositis Fever Renal disorder Neurologic disorder Eye or oral dry Raynaud’s phenomenon Thrombocytopenia Leukopenia Estradiol metabolic disorder Hyperprolactinemia Anti-Smith antibody-positive Anti-dsDNA antibody-positive Low levels of C4 Low levels of C3 ANA-positive

Table 6  Association between rs1126535 T/C polymorphism and disease activity SLEDAI

Genotypes of rs1126535 T/C

>0.05 >0.05 >0.05 0.587 0.092 0.068 0.624 0.805 0.204 0.247 0.486 0.698 0.787 0.822 0.933 0.234 0.190 0.345 0.117 0.422 0.815 0.281 0.122 0.221 0.159

individuals carrying TT genotypes (P > 0.05). Haplotypes of these three SNPs were constructed. The C-G-A haplotype was associated with significantly increased risk of SLE (P  = 0.009). Additionally, rs1126535 polymorphism was associated with high SLE activity. These findings indicated that CD154 rs1126535C/T polymorphism may play a role in the progression of SLE. Published reports showed that sCD154 was significantly increased in SLE patients. Several factors, such as estrogens, phosphorylated PYK2 were reported to exert influence on the expression of sCD154 in SLE patients [25–29]. Genetic factors were reported to play more important roles in the regulation of CD154 expression [30]. In a recent study, Citores et al. conducted a research to explore the relationship between the dinucleotide repeat polymorphism in the 3′UTR of the CD154 gene and protein expression in Spain SLE patients; and they found that the CD154 gene 3′UTR microsatellite is associated with SLE and the most represented alleles in patients were accompanied by a more prolonged protein expression in activated lymphocytes from controls [24]. In addition, another association analysis between haplotype structure of CD40-CD154 and

Rheumatol Int Fig. 3  Association between sCD154 levels and rs1126535 T/C polymorphism. SLE patients with TT homozygous (8.87 ± 3.30 ng/ml, n = 270) showed significantly lower sCD154 levels than that of with CC homozygous (12.78 ± 4.35 ng/ml, n = 33) or CT heterozygotes (13.25 ± 3.98 ng/ml, n = 47), respectively [both P < 0.05, (a)]. This significant difference did not observe in the control group [both P > 0.05, (b)]

SLE suggested that the synonymous coding rs1126535 polymorphism of CD154 gene was not associated with the European-Caucasians SLE patients, but the haplotype tagging SNPs (including rs1126535) can facilitate analysis for susceptibility in other autoimmune diseases [23]. These studies indicated that polymorphisms of CD154 gene might be involved in the progression of SLE. However, the association between CD154 gene polymorphisms and Chinese SLE susceptibility remains unclear and little is known about the relationship between CD154 gene polymorphisms and the expression of CD154 in Chinese population. Therefore, we conducted this study to figure out the roles of three CD154 gene polymorphisms in a Chinese population. Inconstant with previous study, our study demonstrated that the rs1126535C/T polymorphism was associated with the increased risk of SLE. A plausible explanation for this difference was that the distributions of CD154 gene polymorphisms may be distinct from specific population, various ethnicities and geographic region. And this distinction may result in different population with different susceptibility to disease. In addition, our study suggested that rs1126535C/T polymorphism was associated with the expression of sCD154. Patients carrying CT or CC genotypes seem to exhibit relative high levels of sCD154 compared with those carrying TT genotypes in case group. It may be that the synonymous SNP of rs1126535 which is located in the exon of CD154 gene may exert influences on the splicing of exon, thereby affecting the expression of sCD154. However, the differences did not exist in the control group. A possible explanation was that the CD154 expression is determined both by genetic and inducing factors. However, it should be noted that our study has several limitations. First, our data were collected retrospectively, the potential diagnostic mistakes was unavoidable. Besides, all patients may not give the same battery of diagnostic clinical and laboratory tests. Therefore, ascertainment

bias in diagnosis may make the interpretation of the data unclear. Second, the sample size in the present study was relatively small; further studies with larger sample size are required to confirm the role of these CD154 polymorphisms in SLE patients and associations between these polymorphisms and disease activity. Generally, our study indicates that the rs1126535C/T polymorphism of CD154 gene was involved in the progression of Chinese SLE patients, probably by affecting the expression of CD154. Acknowledgements This study was supported by National Natural Science Foundation of China (No. 81260234, No. 81560552). Compliance with ethical standards  Conflict of interest Author Yan Lan has received research grants from National Natural Science Foundation of China (grant number 81260234). Author Ye-Sheng Wei has received research grants from National Natural Science Foundation of China (grant number 81560552). All the authors declare that they have no conflict of interest. Funding  This study was funded by National Natural Science Foundation of China (grant number 81260234, 81560552). Informed consent  Informed consent was obtained from all individual participants included in the study.

References 1. Aringer M et al (2016) Toward new criteria for systemic lupus erythematosus-a standpoint. Lupus 25(8):805–811 2. Wu M et al (2016) The Role of gammadelta T Cells in systemic lupus erythematosus. J Immunol Res 2016:2932531 3. Araujo JA et al (2016) Th17 cells and CD4(+) multifunctional T cells in patients with systemic lupus erythematosus. Rev Bras Reumatol (Rio J) 56(1):28–36 4. Andrade D et al (2015) Interferon-alpha and angiogenic dysregulation in pregnant lupus patients who develop preeclampsia. Arthritis Rheumatol 67(4):977–987

13

5. Zhu X et al (2015) Role of abnormal anterior pituitary hormones-growth hormone and prolactin in active systemic lupus erythematosus. Int J Clin Exp Med 8(10):19223–19231 6. Abdallah E, Waked E, Abdelwahab MA (2016) Evaluating the association of interleukin-10 gene promoter -592 A/C polymorphism with lupus nephritis susceptibility. Kidney Res Clin Pract 35(1):29–34 7. Al-Rayes H et al (2016) Apolipoprotein E Gene polymorphisms in saudi patients with systemic lupus erythematosus. Clin Med Insights Arthritis Musculoskelet Disord 9:81–87 8. Shahin RM et al (2016) Contribution of toll-like receptor 9 gene single-nucleotide polymorphism to systemic lupus erythematosus in Egyptian Patients. Immunol Invest 45(3):235–242 9. Senhaji N et al (2015) The contribution of CD40/CD40L axis in inflammatory bowel disease: an update. Front Immunol 6:529 10. Oxer DS et al (2011) PPARgamma expression is increased in systemic lupus erythematosus patients and represses CD40/ CD40L signaling pathway. Lupus 20(6):575–587 11. Liu MF et al (2001) Expression of CD40 and CD40 ligand among cell populations within rheumatoid synovial compartment. Autoimmunity 34(2):107–113 12. Nakamura H, Kawakami A, Eguchi K (2006) Mechanisms of autoantibody production and the relationship between autoantibodies and the clinical manifestations in Sjogren’s syndrome. Transl Res 148(6):281–288 13. Toubi E, Shoenfeld Y (2004) The role of CD40-CD154 interactions in autoimmunity and the benefit of disrupting this pathway. Autoimmunity 37(6–7):457–464 14. Xu H et al (2015) Increased frequency of circulating follicular helper T cells in lupus patients is associated with autoantibody production in a CD40L-dependent manner. Cell Immunol 295(1):46–51 15. Willis R et al (2014) Effects of statins on proinflammatory/prothrombotic biomarkers and on disease activity scores in SLE patients: data from LUMINA (LXXVI), a multi-ethnic US cohort. Clin Exp Rheumatol 32(2):162–167 16. Willis R et al (2012) Effect of hydroxychloroquine treatment on pro-inflammatory cytokines and disease activity in SLE patients: data from LUMINA (LXXV), a multiethnic US cohort. Lupus 21(8):830–835 17. Goules A et al (2006) Elevated levels of soluble CD40 ligand (sCD40L) in serum of patients with systemic autoimmune diseases. J Autoimmun 26(3):165–171

13

Rheumatol Int 18. Ciferska H et al (2007) The levels of sCD30 and of sCD40L in a group of patients with systemic lupus erythematodes and their diagnostic value. Clin Rheumatol 26(5):723–728 19. Urquizu-Padilla M et al (2009) Serum levels of soluble CD40 ligand at flare and at remission in patients with systemic lupus erythematosus. J Rheumatol 36(5):953–960 20. Chen JM et al (2015) The association of CD40 polymorphisms with CD40 serum levels and risk of systemic lupus erythematosus. BMC Genet 16:121 21. Lee SH et al (2014) The Interaction Between Allelic Variants of CD86 and CD40LG: a common risk factor of allergic asthma and rheumatoid arthritis. Allergy Asthma Immunol Res 6(2):137–141 22. Teruel M et al (2012) Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis. Arthritis Res Ther 14(3):R154 23. Chadha S et al (2005) Haplotype structure of TNFRSF5 TNFSF5 (CD40-CD40L) and association analysis in systemic lupus erythematosus. Eur J Hum Genet 13(5):669–676 24. Citores MJ et al (2004) The dinucleotide repeat polymorphism in the 3′UTR of the CD154 gene has a functional role on protein expression and is associated with systemic lupus erythematosus. Ann Rheum Dis 63(3):310–317 25. Wang N, Peng XB, Zeng K (2007) Effect of prolactin on CD40/ CD154 expression on peripheral blood mononuclear cells in patients with systemic lupus erythematosus. Nan Fang Yi Ke Da Xue Xue Bao 27(9):1382–1384 26. Rider V et al (2001) Estrogen increases CD40 ligand expression in T cells from women with systemic lupus erythematosus. J Rheumatol 28(12):2644–2649 27. Li X et al (2006) Estrogen does not regulate CD154 mRNA stability in systemic lupus erythematosus T cells. Lupus 15(12):852–857 28. Wang M et al (2009) Functional activation of proline-rich tyrosine kinase2 (PYK2) in peripheral blood mononuclear cells from patients with systemic lupus erythematosus. BMC Musculoskelet Disord 10:141 29. Wang M, Zhang W, Zhang Y (2009) Activation signal transduction by proline-rich tyrosine kinase 2 (PYK2) in peripheral blood mononuclear cells from patients with systemic lupus erythematosus. Hybridoma (Larchmt) 28(5):333–339 30. Peeva E et al (2006) Cutting edge: lupus susceptibility interval Sle3/5 confers responsiveness to prolactin in C57BL/6 mice. J Immunol 177(3):1401–1405