ORIGINAL ARTICLE

Association of CD4 enhancer gene polymorphismswith rheumatoid arthritis in Egyptian female patients

Yousri M. Hussein • Shereen A. El Tarhouny •

Randa H. Mohamed • Amal S. El-Shal •

Amany M. Abul-Saoud • Manal Abdo

Received: 19 December 2010 / Accepted: 8 May 2011 / Published online: 28 May 2011

� Springer-Verlag 2011

Abstract CD4 is a candidate gene in autoimmune dis-

eases, including rheumatoid arthritis (RA). Because the

CD4 receptor is crucial for appropriate antigen responses

of CD4? T cells, changes in CD4 expression and CD4?

T-cell activity may influence tolerance or tissue destruction

in autoimmune diseases and contribute to their risk. We

analyzed two polymorphisms of the CD4 in 172 female

Egyptian patients with RA and in 112 matched healthy

control. Genotyping of CD4-11743 and CD4-10845 was

determined by restriction fragment length polymorphism-

polymerase chain reaction (PCR–RFLP). Subjects with the

CC genotype of CD4-11743 were significantly more likely

to develop RA (OR = 2.7, P = 0.03) and more likely to

have sever RA (OR = 2.7, P = 0.024). Carrier of A allele

of CD4-10845 was significantly more likely to develop

sever RA (OR = 3.7, P = 0.000). CD4-11743 genetic

polymorphisms are associated with the susceptibility and

severity of RA, and CD4-10845 genetic polymorphisms are

associated with the severity of RA.

Keywords CD4 � Gene � Polymorphism �Rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA), one of the most common sys-

temic autoimmune diseases, is characterized by chronic

joint inflammation and subsequent joint destruction [1]. It

has a worldwide prevalence of approximately 1% and is

always observed to affect women 2–3 times more fre-

quently than men [2]. Both environmental and genetic

factors contribute to this pathogenesis, but the complete

etiological picture remains ambiguous [3].

Previous studies have shown that T cells play an

important role in the development of such autoimmune

disease [4]. The balance between regulatory T cells and

proinflammatory effector T cells has been shown to be of

critical importance in the development and persistence of

autoimmune diseases [5].

CD4? T cells play the main role in the development of

most human autoimmune diseases as RA [6]. There is

substantial evidence to support the involvement of T

lymphocytes in the pathogenesis of rheumatoid arthritis

(RA), including the presence of CD4? T cells in RA

synovium. Although the mechanisms that prevent autoim-

munity remain poorly understood, substantial evidence

supports the existence of a group of regulatory T cells that

contribute to peripheral tolerance via an active, dominant

mechanism [7].

The CD4 receptor is glycoprotein expressed on the

surface of helper T cells colocalizes with the T-cell

receptor (TCR) and major histocompatibility complex

(MHC) class II molecules and interacts with the non-anti-

gen-binding regions on the MHC class II molecule in

antigen recognition [6]. Theoretically, any malfunction of

the CD4 receptor could potentially allow for improper

activation and proliferation of CD4? T cells [8]. Inhibition

of CD4–MHC class II interaction severely impairs the

Y. M. Hussein � S. A. El Tarhouny (&) �R. H. Mohamed � A. S. El-Shal

Medical biochemistry department, Faculty of medicine,

Zagazig University, Zagazig, Egypt

e-mail: shereeneltarhony@hotmail.com

A. M. Abul-Saoud � M. Abdo

Rheumatology and rehabilitation department,

Faculty of medicine, Zagazig University, Zagazig, Egypt

123

Rheumatol Int (2012) 32:2325–2330

DOI 10.1007/s00296-011-1959-y

response of T cells to antigen exposure, and low CD4 cell-

surface expression on Th lymphocytes results in impaired

TCR response to low-level antigenic stimulation [9].

The human CD4 gene maps to chromosome 12p13 in a

gene-dense region [10] and its expression is regulated

primarily at the transcriptional level [11]. The CD4

enhancer and promoter seem to be the major regulatory

regions for CD4 transcription in T cells [12]. Therefore, an

analysis of the CD4 enhancer polymorphism can provide

insights into regulation of its expression. Clearly, the

molecular mechanisms controlling CD4 gene expression

are very complex and are controlled by many different

signals as the thymocyte develop [8].

In view of the importance of CD4 in the functional

performance of T cells, as well as its consequent crucial

effect in the immune response of an individual, we

hypothesized that genetic variations in the CD4 enhancer

may be associated with the incidence and severity of RA.

Materials and methods

Study population

The study included 172 female patients (mean age

47.39 ± 9.3 years) with RA diagnosed according to the

criteria of American College of Rheumatology [13] under

the care of the Division of Rheumatology of the Zagazig

University Hospital, Egypt.

Subjects enrolled in the study underwent routine bio-

chemical blood analysis. X-rays of the hands and feet were

obtained in all patients. The evaluation of subjects included

physical examination, with particular focus on the pattern

of joint involvement, the presence of nodules and other

extra-articular features (such as vasculitis, anemia, subcu-

taneous nodules, vasculitis manifestation, and organ

involvement), and laboratory features such as rheumatoid

factor (RF). Disease activity was determined on the basis of

defined parameters (the number of swollen and tender

joints, ESR, Health Assessment Questionnaire (HAQ), and

CRP) and a global physician’s assessment. Disease severity

was determined on the basis of defined parameters (RF and

X-ray erosion). Patients were deemed to have an erosive

arthropathy if one or more definitive erosions were

apparent in any of the peripheral joints that have been

identified to be predictive of disease progression [13].

One hundred and twelve healthy women (mean age

48.21 ± 8.94 years) of same ethnic origin and no family

history were used as a healthy control group. They never had

any signs or symptoms of RA, other arthritis, or joint diseases

(pain, swelling, tenderness, or restriction of movement) at

any site based on their medical history and a thorough

examination conducted by an experienced physiatrist.

In this study, all subjects were Egyptians from the

Sharkia governate of Egypt. Also, the study was approved

by the ethical committee of the Zagazig University, and

informed consent was obtained from them.

Biochemical analysis

Blood samples were drawn from all subjects after an overnight

fast. Sera were separated immediately and stored at -20�C.

CRP is assayed by high-sensitivity enzyme-linked immuno-

sorbent assay (ELISA). IgM RF was measured by latex

agglutination. RF was considered positive above 20 IU/ml.

Isolation of DNA

Genomic DNA was extracted from EDTA whole-blood

sample using a spin column method according to the protocol

(QIAamp Blood Kit; Qiagen GmbH, Hilden, Germany).

Detection of two CD4 polymorphic sites

CD4-11743 A/C and CD4-10845A/G polymorphisms were

analyzed by polymerase chain -restriction fragment length

polymorphism reaction (PCR–RFLP) method as previously

describe by Sui-Foon et al. [14].

The region surrounding CD4-11743A/C polymorphism

was amplified with following forward primers 50-TCA

GAT ATT CTC TGC TCA GCC CA-30 and reverse

primers 50-TTC CAG TCT GAA AAA AGT GG-30.Twenty-five microliters of reaction mixture contains

genomic DNA samples (100 ng), 200 lmol/L dNTPs,

1.5 mM MgCl2, 1X Taq polymerase buffer, 50 pmol of

each primer, and 0.5 unit of Taq DNA polymerase

(Amersham, Paris, France). The amplification protocol

comprised initial denaturation at 95�C for 5 min, 18 cycles

at 95�C for 30 s, touchdown 60-51�C for 30 s, and 72�C

for 30 s, 17 cycles at 95�C for 30 s, 51�C for 30 s, and

72�C for 30 s, and final extension at 72�C for 7 min. The

302-base pair (bp) PCR products were digested with MwoI

for 3 h at 37�C yielded 302 bp for A allele and 205- and

97-bp fragments when C allele was present.

The region surrounding the CD4-10845A/G polymor-

phism was amplified with following forward primers 50-GAA ATG AGA AGT AGC ACA CAG T-30 and reverse

primers 50-AAA AGT TAA GCA GAA TCA GGC-30.Two fragments measuring 202 and 98 bp would be

present if the product is able to be excised. The uncut band

showed up as a 300-bp length on the gel. The 300-base pair

(bp) PCR products were digested with HaeII for 3 h at

37�C yielded 300 bp for A allele and 202-bp and 98-bp

fragments when G allele was present. Quality control

measures include blinded analyses, replicates of 10% of

samples, and positive controls (blood-derived DNA from

2326 Rheumatol Int (2012) 32:2325–2330

123

all known genotypes), and negative controls for contami-

nation (no DNA) were run routinely with patient samples.

Statistical analysis

The results for continuous variables were expressed as

means ± SD. The means of the three-genotype groups were

compared in a one-way analysis of variance (ANOVA). The

statistical significance of differences in frequencies of

variants between the groups were tested using the chi-

square (v2) test. In addition, the odds ratios (ORs) and 95%

confidence intervals (CIs) were calculated as a measure of

the association of the CD4-11743 and CD4-10845 geno-

types of CD4 enhancer gene polymorphism. A difference

was considered significant at P \ 0.05. All data were

evaluated using SPSS version 10.0 of windows (SPSS Inc.,

Chicago, IL, version 10, USA).

Results

The genotype and allele frequencies of the CD4-11743A/C

and CD4-10845A/G polymorphisms of patients with RA

and controls are shown in (Tables 1 and 2).

The frequencies of CC genotype of CD4-11743A/C

were significantly increased in patients with RA compared

with control group (60% vs. 44.7%, P = 0.03). Subjects

with the CC genotype were significantly more likely to

develop RA (OR = 2.7, 95% CI = 1–6.8, P = 0.03).

However, there were no significant differences between

CD4-10845 polymorphisms in RA and control group.

Association of CD4 genotypes and allelic frequencies with

RF in rheumatoid arthritis patients is shown in (Table 3).

Table 1 Patient’s characteristics

RA patients

No. 172

Age 47.39 ± 9.3

Disease duration 10. 65 ± 7.9

No. swollen joint 5.1 ± 4.3

No. tender joints 13.1 ± 7.13

ESR (mm/h) 47.5 ± 32.9

RF 77.5 ± 79.3

CRP (lg/ml) 16.17 ± 14.9

Table 2 Distribution of CD4

genotypes in RA patients and

control group

Controls (No. = 112) RA (No. = 172) OR Confidence

interval (CV)

P

No. % No. %

CD4-11743

AA 18 16 14 8

AC 44 39.3 55 32 0.6 0.2–1.6 0.3

CC 50 44.7 103 60 2.7 1–6.8 0.03

C allele 144 64.3 261 75.9 0.6 0.3–1 0.06

CD4-10845

GG 15 13.4 20 11.6

AG 42 37.5 64 37.2 0.9 0.4–2.3 0.9

AA 55 49.1 88 51.2 0.8 0.4–2.1 0.7

A allele 152 67.9 240 69.8 0.9 0.5–1.6 0.7

Table 3 Association of CD4

genotypes with RF in

rheumatoid arthritis

RF negative (No. = 54) RF positive (No. = 118) OR Confidence

interval (CV)

P

No. % No. %

CD4-11743

AA 5 9.25 20 16.9

AC 16 29.6 48 40.7 0.7 0.28–1.8 0.4

CC 33 61.1 50 42.4 2.7 1.1–6.7 0.024

C allele 82 75.9 148 62.7 1.8 1–3.4 0.04

CD4-10845

GG 34 62.9 8 6.8

AG 15 27.8 32 27.1 8.7 3.4–22.3 \0.001

AA 5 9.25 78 66.1 7.5 4–13.9 \0.001

A allele 25 23 188 79.7 3.7 2.5–5.6 \0.001

Rheumatol Int (2012) 32:2325–2330 2327

123

The frequencies of CC genotype of CD4-11743A/C

were significantly increased in patients with RF-negative

compared with RF-positive group (61.1% vs. 42.4%

P = 0.024). Subjects with the CC genotype were signifi-

cantly more likely to have sever RA (OR = 2.7, 95%

CI = 1.1–6.7, P = 0.024). The frequencies of GG geno-

types of CD4-10845 polymorphisms were significantly

increased in patients with RF negative compared with RF-

positive patients (62.9 vs. 6.8% P \ 0.001). Carrier of A

allele of CD4-10845 was significantly more likely to

develop sever RA (OR = 3.7, P \ 0.001).

Parameters of disease activity in relation to CD4 geno-

types are shown in (Table 4).

There was a significant increase in number of tender joint

in patients carry CC genotype as compared with patients

carry AA genotype of CD4-11743 and also significant

increase in number of tender joint in patients carry GG

genotype as compared with patients carry AA genotype of

CD4-10845. There was a significant decrease in RF in

patients carry AG and GG variants when compared with

those carry AA genotype of CD4-10845A/G polymorphism.

Discussion

Tolerance to ‘‘self’’ is a major immune regulatory mecha-

nism that protects the body’s own tissues from immune-

mediated damages [15]. The development of autoimmune

diseases requires the breakdown of immunologic self-tol-

erance that usually controls self- and non-self-discrimina-

tion [16]. The primary mechanism that leads to tolerance to

self-antigens is the thymic deletion of self-reactive T cells

(negative selection). However, because some self-reactive

T cells escape this process physiologically and autoreactive

CD4? T cells are present in the peripheral circulation of

healthy individuals, where they retain their capacity to

initiate autoimmune inflammation negative selection in the

thymus is not sufficient to prevent the activation of self-

reactive T cells in the periphery [17]. Interestingly, CD4 is

a candidate gene in autoimmune diseases, because the CD4

receptor is crucial for appropriate antigen responses of

CD4 T cells [18].

Most human autoimmune diseases require CD4? T cells

for their development including Type 1 diabetes mellitus

(T1DM) [19, 20], vitiligo [8], and rheumatoid arthritis

(RA) which are considered one of the most common

autoimmune diseases[21]. RA is assumed to be an auto-

immune disease, based on the presence of autoantibodies in

the serum and remarkable lymphocyte infiltration into the

synovium [21].

Among the infiltrating cells, CD4? T cells are the main

cell population and are considered to play a crucial role in

the pathogenesis of RA [22]. CD4 is a coreceptor that

assists the T-cell receptor (TCR) to activate its T cell fol-

lowing an interaction with an antigen-presenting cell.

Using its portion that resides inside the T cell, CD4

amplifies the signal generated by the TCR by recruiting a

tyrosine kinase lck enzyme, which is essential for activat-

ing many molecules involved in the signaling cascade of an

activated T cell [23]. Also, they recognize antigen on target

cells in association with class II major histocompatibility

complex (MHC) molecules and interact directly with it on

the surface of the antigen-presenting cell using its extra-

cellular domain [24]. Inhibition of CD4–MHC class II

interaction severely impairs the T-cell response to antigen

exposure [25], and low CD4 cell-surface expression on Th

lymphocytes results in impaired TCR response to low-level

antigenic stimulation [9]. So, the genetic regulation of the

CD4? T-cell activity may influence tolerance or tissue

destruction in RA.

Immune regulation in RA might be achieved via various

mechanisms. Skewing from Th1- to Th2-type CD4? T

cells [26] or toward IL-10-producing T regulatory cells

type 1 [27] could induce a shift from proinflammatory to

anti-inflammatory responses. In recent years, attention has

focused on another subset of regulatory CD4? T cells that

Table 4 Parameters of disease activity and severity in relation to CD4 genotypes

CD4-11743 (n = 172) CD4-10845 (n = 172)

AA (n = 14) AC (n = 55) CC (n = 103) AA (n = 88) AG (n = 64) GG (n = 20)

ESR (mm/h) 45.3 ± 21.7 45.8 ± 21.3 49.3 ± 41.1 44.9 ± 26.2 44.5 ± 19.2 55.2 ± 50.1

RF 87.1 ± 88.3 83.6 ± 62.8 70 ± 81.9 117.2 ± 87.3 51.6 ± 39.3§ 21.7 ± 34.5§*

CRP (mg/l) 14.7 ± 10.1 16.9 ± 19.2 16.5 ± 14.5 17.9 ± 14.4 15 ± 8.4 13.6 ± 9.4

No. of swollen joints 4.2 ± 3.2 6 ± 4.4 5.1 ± 4.7 4.9 ± 4.1 4.6 ± 3.9 5.8 ± 4.9

No. of tender joint 11.1 ± 5.5 12.9 ± 6.6 14.1 ± 7.9# 10.4 ± 5.9 13.7 ± 5.9§ 17.8 ± 7.9§*

HAQ 1.6 ± 0.9 1.7 ± 0.7 1.3 ± 0.75 1.3 ± 0.8 1.8 ± 0.6§ 1.7 ± 0.8§

# Significant difference from AA of CD4-11743§ Significant difference from AA of CD4-10845

* Significant difference from AG of CD4-10845

2328 Rheumatol Int (2012) 32:2325–2330

123

are naturally occurring and can be identified by their con-

stitutive low-level expression of the IL-2 receptor a chains

(CD25) [28–30].

Activated memory CD4? T cells recruited in the RA

synovium have been thought to drive the synovial

inflammation by stimulating the production of proin-

flammatory cytokines by macrophages and fibroblasts,

activating B cells, and promoting the formation of

osteoclasts [1].

Many inflammatory responses in the synovium have

been suggested to be T cell driven [31]. However, the

number of CD4? T cells in ST and the score for knee pain

have been observed to be negatively correlated [32]. Fur-

thermore, the treatment of RA patients with depleting anti-

CD4 antibodies has shown reduction in the number of

accumulating T cells without a marked clinical improve-

ment [33].

Accumulated evidence has shown that the repertoire of

CD4? T cells in patients with RA is distinct [34, 35]. Cell–

cell contact is necessary both at the inductive phase of

T-cell activation in RA and at the effector phase, in which

T cells indirectly mediate autoantibody production, joint

inflammation, and bone resorption [25].

In our study, we found that the frequencies of CC

genotype were significantly increased in patients with RA

compared with control group. However, there were no

significant differences between CD4-10845 polymor-

phisms in RA and control group.

In accordance with our data, Sui-Foon et al. [14] found a

higher frequency of the CD4-11743C allele and a lower

frequency of the CD4-11743A allele, compared with the

controls. In addition, they suggested that CD4-11743C

allele increased the risk of development of RA.

On the contrary, Sui-Foon et al. [14] found that patients

with RA had a significantly higher frequency of the CD4-

10845 AA genotype compared with the controls. Similarly,

further comparison of the allele frequencies between

patients with RA and controls are made and they revealed

that the former had a higher frequency of the A allele and a

lower frequency of the G allele. Interestingly, they added

that CD4-10845 AA genotype and CD4-10845A allele

increased the risk of RA development.

In addition, Kristiansen et al. [35] and Zamani et al. [36]

found that genetic polymorphisms at the CD4 enhancer

locus have been shown to be associated with the risk of

development of rheumatoid arthritis and systemic lupus

erythematosus [14].

However, Donda et al. [37] found that CD4 promoter

and enhancer are turned out to be not responsible for

lineage-specific CD4 gene expression. These findings are

in agreement with Ellmeier et al. [11] who suggested that a

regulatory element or a mutation linked to the CD4 pen-

tanucleotide repeat.

A silencer and some other disputable cis-acting elements

have been identified in proximity that might be involved in

the gene expression in the early thymic precursors. This

pentanucleotide itself confers susceptibility. It is a poly-

pyrimidine sequence, which may affect CD4 gene expres-

sion, through an as yet unknown mechanism [8]. Thus, the

molecular mechanisms controlling CD4 gene expression

are very complex and are controlled by many different

signals beside CD4 enhancer as the thymocyte develops

which need more in-depth studies concerning both CD4

enhancer and CD4 silencer.

Conclusion

Genetic polymorphisms at the CD4 enhancer gene are one

of important factors which associated with susceptibility

and severity of RA and can serve as a genetic marker for

the risk of development of RA. Similar studies in other

populations and with larger sample size are needed. Also,

functional studies on the role and function of the poly-

morphism of CD4 enhancer and CD4 pentanucleotide

repeat, silencer, and other disputable cis-acting elements

that affect CD4 gene expression will be also necessary.

Acknowledgments This work was funded by support of academic

research in Zagazig University Projects, Zagazig University Post-

graduate & Research Affairs.

Conflict of interest No conflict of interest.

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