ORIGINAL ARTICLE

High expression of TNF alpha is associated with 2308 and 2238TNF alpha polymorphisms in knee osteoarthritis

Jose Francisco Munoz-Valle • Edith Oregon-Romero • Hector Rangel-Villalobos •

Gloria Esther Martınez-Bonilla • Eduardo Castaneda-Saucedo • Lorenzo Salgado-Goytia •

Marco Antonio Leyva-Vazquez • Berenice Illades-Aguiar • Luz del Carmen Alarcon-Romero •

Monica Espinoza-Rojo • Isela Parra-Rojas

Received: 4 April 2012 / Accepted: 13 October 2012 / Published online: 30 October 2012

� Springer-Verlag Italia 2012

Abstract Knee osteoarthritis (OA) is a common chronic

degenerative disease characterized by the loss of articular

cartilage components due to an imbalance between extra-

cellular matrix destruction and repair. The proinflammatory

cytokines involved in OA, TNFa and IL1b, are considered

the major implicated. The aim of this study was to inves-

tigate the relationship between TNFa -308 and -238

polymorphisms with messenger RNA (mRNA) and soluble

TNFa expression in knee OA patients and healthy subjects

(HS). Case–control study involved 50 knee OA patients

classified according to 1986 ACR Classification Criteria, as

well as 100 HS. The Western Ontario and McMaster

Universities Osteoarthritis Index and Lequesne disability

index were applied to OA patients. The -308 and -238

polymorphisms were determined by polymerase chain

reaction–restriction fragment length polymorphism tech-

nique. The TNFa mRNA expression was quantified by

real-time PCR using TaqMan method. The sTNFa levels

were measured by enzyme-linked immunosorbent assay.

The TNFa mRNA expression in knee OA patients was

higher than in HS (1.56-fold). In addition, the TNFamRNA expression was higher in carriers of G allele in the

knee OA group for both polymorphisms. The sTNFa levels

were increased in G/G versus G/A genotypes in both

studied polymorphisms (p \ 0.05). However, the TNFa-308 and -238 genotypes did not show statistical differ-

ences between groups. The G allele of TNFa -308 and

-238 polymorphisms is associated with high mRNA and

soluble expression in knee OA patients. However, it is not

a marker of susceptibility in Western Mexico. Further

studies are necessary to confirm these findings.

Keywords Knee osteoarthritis � TNF alpha expression �TNF alpha levels � TNF alpha polymorphisms

Introduction

Knee osteoarthritis (OA) is a common chronic degenera-

tive disease characterized by the loss of articular cartilage

components due to an imbalance between extracellular

matrix destruction and repair [1]. Although articular car-

tilage breakdown is a major characteristic of OA, other

joint tissues such as the synovial membrane and subchon-

dral bone actively participate in the progression of the

disease [2].

Jose Francisco Munoz-Valle and Edith Oregon-Romero contributed

equally to this work.

J. F. Munoz-Valle (&) � E. Oregon-Romero

Grupo de Inmunogenetica Funcional, Departamento de Biologıa

Molecular y Genomica, Centro Universitario de Ciencias de la

Salud, Universidad de Guadalajara, Insurgentes 244-1, Colonia

Lomas de Atemajac, C.P. 45178 Zapopan, Guadalajara, Jalisco,

Mexico

e-mail: biologiamolecular@hotmail.com

H. Rangel-Villalobos

Instituto de Investigacion en Genetica Molecular, Centro

Universitario de la Cienega, Universidad de Guadalajara,

Ocotlan, Jalisco, Mexico

G. E. Martınez-Bonilla

Servicio de Reumatologıa, OPD Hospital Civil de Guadalajara

‘‘Fray Antonio Alcalde’’, Guadalajara, Jalisco, Mexico

E. Castaneda-Saucedo � L. Salgado-Goytia �M. A. Leyva-Vazquez � B. Illades-Aguiar �L. d. C. Alarcon-Romero � M. Espinoza-Rojo � I. Parra-Rojas

Unidad Academica de Ciencias Quımico Biologicas,

Universidad Autonoma de Guerrero, Chilpancingo,

Guerrero, Mexico

123

Clin Exp Med (2014) 14:61–67

DOI 10.1007/s10238-012-0216-3

Among the proinflammatory cytokines involved in OA,

TNFa and IL1b are considered the major implicated. Both

cytokines are produced by chondrocytes, mononuclear

cells, osteoblasts and synovial tissues. In OA patients,

TNFa levels are elevated in the synovial fluid, synovial

membrane, subchondral bone and cartilage [2].

The TNFa gene is located within the human leukocyte

antigen class III region. Previous reports have suggested

that the endogenous production of TNFa may be associated

with TNFa polymorphisms influencing the messenger

RNA (mRNA) and protein expression [3–6]. The TNFa-308 and -238 polymorphisms (G/G genotype) have been

associated with high mRNA expression and soluble TNFalevels in rheumatoid arthritis (RA). Besides, in human

blood leukocytes the G allele of TNFa -308 polymor-

phism was associated with higher mRNA expression

compared to the A allele [7].

The aim of this study was to investigate the association

of the TNFa -308 and -238 polymorphisms with the

mRNA expression and soluble TNFa levels in knee OA

patients.

Materials and methods

Patients and healthy subjects

We selected 50 OA patients attending the Hospital Civil

‘‘Fray Antonio Alcalde,’’ Rheumatology Department. All

patients fulfilled the 1986 classification criteria for knee

OA of the American College of Rheumatology. The

diagnosis of OA was based on clinical evaluations,

excluding metabolic causes. Idiopathic OA patients were

selected. WOMAC and Lequesne disability indexes were

applied to OA patients at the beginning of the study [8, 9].

As a control group, 100 healthy subjects (HS, between the

ages of 22 and 67), residents from Guadalajara, Jalisco,

Mexico, who had the same ethnic and geographical back-

ground than the OA patients, were included. The inclusion

criteria for the control group were as follows: [18 years

old, clinically healthy individuals. All participants were of

the Mexican Mestizo population. According to the National

Institute of Anthropology, the definition of a Mexican

Mestizo states that the individual must be born in the

country, having a Spanish last name, with a family history

of Mexican ancestors and at least back to the third gener-

ation [10].

Ethical considerations

An informed written consent was obtained from all

subjects before enrollment in the study, according to the

ethical guidelines of the 2008 Declaration of Helsinki. The

study was approved by the Ethics Committee of the Hos-

pital Civil ‘‘Fray Antonio Alcalde.’’ Registration number:

CI-25011.

PCR–RFLP of TNFa promoter polymorphism

Genomic DNA was extracted from 3 mL of peripheral

blood leukocyte according to Miller method [11]. Ampli-

fication of TNFa -308 and -238 promoter region was

performed by PCR in a Thermal Cycler (iCyclerTM BIO-

RAD Life Science Research Products) using the following

primers: for the TNFa -308 polymorphism, 50-AGG CAA

TAG GTT TTG AGG GCC AT-30 (Forward) and 50-TCC

TCC CTG CTC CGA TTC CG-30 (Reverse) [12]; and for

the TNFa -238 polymorphism, 50-AGA AGA CCC CCC

TCG GAA CC-30 (Forward) and 50-ATC TGG AGG AAG

CGG TAG TG-30 (Reverse) [13]. The amplification con-

ditions for both TNFa polymorphism were reported pre-

viously [14]. The electrophoretic TNFa genotype

restriction patters were as follows: For TNFa -308 poly-

morphism, digestion fragments of 87 and 20 bp represent

the wild-type genotype (G/G). Fragments of 107, 87 and

20 bp represent the heterozygote genotype (G/A). The

107-bp fragment represents the homozygote genotype (A/

A). For the TNFa -238 polymorphism, fragments of 152

and 133 bp represent the wild genotype (G/G). Fragments

of 152, 133 and 19 bp represent the heterozygote genotype

(G/A). A unique 152-bp fragment represents the homozy-

gote genotype (A/A). The experiments were made in

duplicate, and for each genotype (n = 3), random samples

were sequenced using an ABIPRISM 310 Sequencer

(Applied Biosystems) in order to confirm the above results.

RNA extraction and reverse transcription

Peripheral blood was collected in EDTA blood collection

tubes (BD Vaccutainer, NJ, USA). Immediately, the PBMC

were isolated using dextran reagent (Sigma Chemical Co,

St. Louis MO, USA), and the total RNA was immediately

obtained using Trizol reagent (InvitrogenTM, Carlsbad, CA,

USA) according to Chomiczyki and Sacchi method [15].

The maximum amount of time between drawing blood and

beginning procedures for mRNA extraction from the OA

and control samples was 1 h. RNA concentration was

determined by spectrophotometry, and RNA integrity was

corroborated on 1 % agarose gel. The cDNA synthesis was

performed using oligo(dT)12–18 primer. Briefly, 2 lg of

total RNA was used in a reaction containing RNase-free

water to 9 and 1 lL oligo(dT)12–18 primer. The reaction

was incubated at 75 �C for 15 min followed by 5 min on

ice. After that, 4 lL of 5X first-strand buffer, 4 lL 2.5 mM

of each dNTPs (InvitrogenTM, Carlsbad, CA, USA),

0.25 lL of 10 U/lL RNase inhibitor, 1 lL of 200 U/lL

62 Clin Exp Med (2014) 14:61–67

123

Moloney murine leukemia virus reverse transcriptase and

0.75 lL RNase-free water were added. The reactions were

carried out at 75 �C for 1 h followed by 5 min of incuba-

tion at 95 �C. The cDNA samples were stored at -80 �C

until the real-time PCR assays.

Real-time PCR for TNFa

The TNFa and glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) expression was quantified using ABI Prism 7500

Sequence Detection System (Applied Biosystems), according

to the manufacturer’s protocol. Reaction for the real-time

PCR using TaqMan detection consisted of 10 lL of a 20X

TaqMan buffer (Applied Biosystem, Foster City, CA, USA);

1 lL of a mixture containing 5X TaqMan MGB probes and

18 lM of each primer (Applied Biosystem, Foster City, CA,

USA); 2 lL of cDNA; and 20 lL of water. The reactions

were performed in MicroAmp 96-well plate capped with

MicroAmp optical caps. All samples were incubated at 50 �C

for 2 min and 95 �C for 10 min and then cycled at 95 �C for

15 s and 60 �C for 1 min for 50 cycles. Controls with no

template cDNA were performed in each assay. The mRNA

expression levels were quantified using the critical threshold

value (Ct). Relative gene expression levels were obtained

using the 2�DDCt method (expressed as relative expression

units). Each sample was tested in triplicate.

sTNFa quantification

The sTNFa levels (R&D Systems, Minneapolis, MN, USA)

were measured using serum samples from OA patients and

HS by ELISA. The range of detection was 15.6–1,000 pg/mL,

and the sensitivity of the assay was\4.4 pg/mL. The sTNFaproduction was calculated from a standard curve of the cor-

responding recombinant human TNFa.

Study design and statistics

The study was a case–control type. Differences in genetic

and allelic frequencies between groups were studied using

chi-square test (v2), odds ratio (OR) and 95 % confidence

intervals (95 % CI). If the number in any cell was \5,

Fisher’s exact test was performed. The Student’s t test was

used to compare the two groups. Mann–Whitney U test was

used to evaluate the association between carriership of

TNFa -308, TNFa -238 and sTNFa levels. We used v2

test for comparison of TNFa mRNA expression propor-

tions. Differences in sTNFa levels were evaluated by

Pearson’s correlation (rho). Probability values less than

0.05 were considered statistically significant. The analysis

was performed using SPSS version 10.0, Epi Info version

2002 and GraphPad Prism 5 software.

Results

The mean age of OA patients was 55 years (range 31–86), and

the HS mean age was 40 years (range 22–67). Forty-four OA

patients were treated with nonsteroidal anti-inflammatory

drugs (NSAIDs). The clinical and demographic characteris-

tics of the OA patients in relationship to TNFa -308 and

-238 genotypes are shown in Table 1.

Table 1 Demographic and clinical characteristics according to TNFa genotypes in OA patients

TNFa -308 TNF a -238

All patients

(n = 50)

G/G Genotype

(n = 44)

G/A Genotype

(n = 6)

p G/G Genotype

(n = 47)

G/A Genotype

(n = 3)

p

Demographics

Age, years 55 (31–86) 55 (31–85) 51 (44–72) – 55 (31–85) 55 (45–68) –

Men/women 2/48 2/42 0/6 – 2/45 0/3 –

Disease status

Disease duration, years 5 (0.5–20) 5 (0.5–20) 1 (1–1) NS 3 (0.5–15) 15 (10–20) 0.016

Drug treatment

NSAIDs 42/50 41/44 5/6 – 42/47 2/3 –

Clinical assessment

WOMAC Total score 34.97 (2–72) 33.39 (2–72) 45 (29–64) NS 35.41 (2–72) 29 (23–34) NS

WOMAC-Pain score 7.88 (1–15) 7.52 (1–15) 10.16 (6–13) NS 7.85 (1–15) 8.33 (8–9) NS

WOMAC-Stiffness score 2.90 (0–7) 2.76 (0–7) 3.83 (2–6) NS 3.02 (0–7) 1.33 (1–2) NS

WOMAC-Function score 24.18 (1–52) 23.10 (1–52) 31 (17–48) NS 24.53 (1–52) 19.33 (13–24) NS

Lequesne score 11.88 (3–21) 11.36 (3–18) 15.4 (8–21) 0.028 11.97 (3–21) 10.66 (8–13) NS

Values represent the mean, minimum and maximum scores

NS not significant, NSAIDs nonsteroidal anti-inflammatory drugs, WOMAC Western Ontario and McMaster Universities Osteoarthritis Index

Clin Exp Med (2014) 14:61–67 63

123

Our population was in Hardy–Weinberg equilibrium

(p [ 0.05). Allelic and genotypic frequencies of TNFa-308 and -238 polymorphisms in OA and HS are shown in

Table 2. The allelic frequencies in OA and HS groups were

as follows: 94 versus 97 % for -308G and 6 versus 3 % for

-308A; and 97 versus 94 % for -238G and 3 versus 6 % for

-238A. The TNFa -308 and -238 genotypes did not show

statistical differences between groups.

To investigate the functional impact of the TNFa -308

and -238 variants, the quantitative TNFa mRNA expres-

sion among the different genotypes for both polymor-

phisms (G/G, G/A) was analyzed. The A/A genotype was

not found in the studied population. The TNFa mRNA

expression in OA patients was higher than in HS (1.56-

fold) (Fig. 1). When we analyzed the TNFa mRNA

expression according to each genotype in both polymor-

phisms, we found that the G/G OA carriers expressed

higher mRNA in comparison with HS G/G carriers

(Fig. 2a, b). However, OA patients with -308 G/G geno-

type expressed slightly less TNFa mRNA than patients

with G/A genotype. In contrast, OA patients carrying the

-238 G/G genotype expressed higher TNFa mRNA levels

than patients with -238 G/A genotype (Fig. 2a, b).

Finally, we analyzed the sTNFa levels in OA patients

and HS. sTNFa levels in OA patients (55.26 pg/mL) were

significantly higher than the levels in HS (6.20 pg/mL)

(Fig. 1; p = 0.001). When the OA patients were classified

according to -308 and -238 G/G and G/A genotypes, we

observed that OA patients have increased sTNFa levels

when compared to HS, irrespective of the -308 or -238

genotypes (Fig. 2c, d; p \ 0.05). Rheumatoid factor

(20.22 IU/mL), erythrocyte sedimentation rate (25.57 mm/h)

and C-reactive protein (0.62 mg/dL) were not associated with

TNFa levels in OA patients.

Discussion

The genotypic frequencies for both TNFa -308 and -238

polymorphisms are similar to other cohorts, except in

Danish population, including the low frequency of A/A

genotype [16–21]. This study shows that the G alleles of

the TNFa -308 and -238 polymorphisms are associated

with high expression of mRNA and soluble TNFa in knee

OA patients. In addition, the G allele for both polymor-

phisms is predominant but is not a susceptibility marker in

the studied population. Similar results were obtained by

Sezgin et al., in a Turkish population. They did not find a

correlation between -308 polymorphism and OA [22].

In the cohort investigated, we found a significant higher

TNFa mRNA expression in OA patients carrying the TNFa

Table 2 Genotypic and allelic

frequencies of TNFa -308 and

-238 polymorphisms

OA osteoarthritis, HS healthy

subjects, p [ 0.05

TNFa genotypes

and alleles

Frequency

TNFa -308 TNFa -238

OA %(n = 50) HS %(n = 100) OA %(n = 50) HS %(n = 100)

G/G 88 (44) 93 (93) 94 (47) 89 (89)

G/A 12 (6) 7 (7) 6 (3) 11 (11)

A/A 0 (0) 0 (0) 0 (0) 0 (0)

G 94 (94) 97 (193) 97 (97) 94 (189)

A 6 (6) 3 (7) 3 (3) 6 (11)

(A)

(B)

Fig. 1 Relative mRNA expression and sTNFa levels in OA patients

and HS. a Relative TNFa mRNA expression in OA patients and HS.

b sTNFa levels in OA patients and HS. OA osteoarthritis, HS healthy

subjects. Dark shaded square represents TNFa mRNA expression in

OA. Light shaded square represents TNFa mRNA expression in HS.

sTNFa levels in OA are represented by a (circle), sTNFa levels in HS

are represented by a (filled square), and mean value is represented by

a (long line)

64 Clin Exp Med (2014) 14:61–67

123

-308 or -238 G alleles when compared with HS. OA

carriers of the -238 G/G genotype had higher mRNA

expression than carriers of the -238 G/A genotype.

However, a slightly higher TNFa mRNA expression was

found in the carriers of -308 G/A genotype when com-

pared to the -308 G/G carriers. This finding is possibly a

reflection of the G allele impact that influences TNFamRNA expression. We previously demonstrated in RA

patients that TNFa -308 G/G and -238 G/G polymor-

phisms are related with high TNFa mRNA expression and

high sTNFa concentrations [14]. Our results for the -238

polymorphism in OA patients are consistent with our pre-

vious findings in RA patients, in which the mRNA levels of

-238 G/G carriers were higher than in G/A carriers. In

contrast, mRNA levels in OA patients carrying the -308

G/A genotype are slightly higher than levels in G/G car-

riers, whereas in RA patients mRNA levels were higher in

G/G carriers than in G/A carriers [14]. These findings are

strongly related to the results from Haddy et al., who

studied healthy families from France and found that indi-

viduals carrying the TNFa -308 G/G genotype had higher

sTNFa levels than -308 G/A carriers [23]. In line with

these observations, in a recent report Helmig et al. studied

178 Germans and found that the -308 G allele is associ-

ated with a significant higher TNFa mRNA expression

compared to the A allele [7]. To our best knowledge, there

have been no studies which analyze the relationship of

TNFa-238 polymorphism with mRNA expression and

TNFa soluble levels.

Information on the variation of TNFa is reported. Haddy

et al. investigated the effect of age and gender on plasma

TNFa levels in healthy families (offspring and parents).

The study showed that TNFa concentrations decreased

with age only in offspring (both genders) but not in parents

(women and men). In the adults, TNFa levels were con-

stants in women, and it diminished a few in men after

45 years old (without statistical significance) [23]. It must

be considered that TNFa results could be affected by the

NSAID treatment because an animal model study of OA

showed that NSAID drugs (Loxoprofen and Ketoprofen)

suppressed proinflammatory cytokines, including TNFa[24].

Previous studies have demonstrated the association

between TNFa -308 and -238 polymorphisms in some

rheumatic diseases [25–28]. But until now, this is the first

study to address the correlation between the TNFa -308

and -238 polymorphism with TNFa mRNA expression

and soluble TNFa levels and its possible role as suscepti-

bility markers in knee OA patients. We found no associa-

tion of the TNFa polymorphisms with knee OA; in this

sense we considered that the genetic background is an

important determinant of the inter-population genetic

(A) (B)

(D)(C)

Fig. 2 sTNFa levels and

relative mRNA expression in

OA patients and HS. a Relative

TNFa mRNA expression in OA

patients and HS in TNFa -308

polymorphism; G/A and G/G

carriers. b Relative TNFamRNA expression in OA

patients and HS in TNFa -238

polymorphism; G/A and G/G

carriers. OA osteoarthritis, HS

healthy subjects. Dark shaded

square represents TNFa mRNA

expression in OA. Light shaded

square represents TNFa mRNA

expression in HS. c sTNFalevels in OA patients and HS in

TNFa -308 polymorphism;

G/A and G/G carriers. d sTNFalevels in OA patients and HS in

TNFa -238 polymorphism;

G/A and G/G carriers. sTNFalevels in OA are represented by

a (circle), sTNFa levels in HS

are represented by a (filled

square), and mean value is

represented by a (long line)

Clin Exp Med (2014) 14:61–67 65

123

variability of the TNFa -308 and TNFa -238 polymor-

phisms and the peculiar characteristics of the Mexican

population. The population of the Western Mexico is

considered Mexican Mestizo and has been estimated that

the paternal ancestry in Western Mexican Mestizos is

mainly European (60–64 %), followed by Amerindian

(25–21 %) and African (15 %) [29, 30].

TNFa, together with IL 1b, is considered one of the most

important players in OA pathophysiology. TNFa is elevated

in synovial fluid, synovial membrane, subchondral bone and

cartilage [31, 32]. TNFa is associated with driving the

inflammatory cascade and stimulating the release of MMP-

1, MMP-3 and MMP-13 which are key regulators of carti-

lage destruction [33–35]. Previous results support the role of

TNFa in knee OA. Satannus et al. suggested that serum level

of TNFa is associated with knee cartilage loss and joint

space narrowing in older people [36]. Likewise, TNFa is

correlated with pain and also is associated with the total

WOMAC score including their subscales (pain, stiffness and

physical function). However, TNFa did not correlate with

the radiographic grading [37]. These findings suggest that

TNFa could be blocked to improve the inflammation pro-

cess, pain and physical function in OA patients.

The importance of our study is based on the relationship

found between the G allele of the polymorphism of TNFa-308 and -238 with high expression of mRNA and sol-

uble TNFa levels in knee OA. However, the limitations of

our study are the low heterozygosity found in the studied

population and that it was not possible to identify any

individual with the homozygous A/A genotype for both

studied polymorphisms.

Conclusions

In conclusion, this study showed that the G allele of TNFa-308 and -238 polymorphisms is associated with high

mRNA and soluble expression in knee OA. However, it is

not a marker of susceptibility in Western Mexico. Further

studies are necessary to confirm these findings.

Acknowledgments This work was supported by Grant No. 69235 to

JFMV of the CONACYT (Fondo Sectorial Secretarıa de Salud-IMSS-

ISSSTE CONACYT, Mexico-Universidad de Guadalajara) and Grant

No. 147778 of the Fondo Mixto CONACYT-Gobierno del Estado de

Guerrero 2010-01.

Conflict of interest None.

References

1. Orita S, Koshi T, Mitsuka T, Miyagi M, Inoue G, Arai G,

Ishikawa T, Hanaoka E, Yamashita K, Yamashita M, Eguchi Y,

Toyone T, Takahashi K, Ohtori S (2011) Associations between

proinflammatory cytokines in the synovial fluid and radiographic

grading and pain-related scores in 47 consecutive patients with

osteoarthritis of the knee. BMC Musculoskelet Disord 12(1):144

2. Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi

H (2011) Role of proinflammatory cytokines in the pathophysi-

ology of osteoarthritis. Nat Rev Rheumatol 7(1):33–42

3. Pociot F, Briant L, Jongeneel CV, Molvig J, Worsaae H, Abbal M,

Thomsen M, Nerup J, Cambon-Thomsen A (1993) Association of

tumor necrosis factor (TNF) and class II major histocompatibility

complex alleles with the secretion of TNF-alpha and TNF-beta by

human mononuclear cells: a possible link to insulin-dependent

diabetes mellitus. Eur J Immunol 23:224–231

4. Turner DM, Grant SC, Lamb WR, Brenchley PE, Dyer PA,

Sinnott PJ, Hutchinson IV (1995) A genetic marker of high TNF-

alpha production in heart transplant recipients. Transplantation

60:1113–1117

5. Wilson AG, Symons JA, Mcdowell TL, McDevitt HO, Duff GW

(1997) Effects of a polymorphism in the human tumor necrosis

factor alpha promoter on transcriptional activation. Proc Natl

Acad Sci USA 94(7):3195–3199

6. Uglialoro AM, Turbay D, Pesavento PA, Delgado JC, McKenzie

FE, Gribben JG, Hartl D, Yunis EJ, Goldfeld AE (1998) Identi-

fication of three new single nucleotide polymorphisms in the

human tumor necrosis factor-alpha gene promoter. Tissue Anti-

gens 52:359–367

7. Helmig S, Aliahmadi N, Stephan P, Dohrel J, Schneider J (2011)

TNF-a -308 genotypes are associated with TNF-a and TGF-b1

mRNA expression in blood leucocytes of humans. Cytokine

53(3):306–310

8. Bellamy N, Buchanan WW, Goldsmith CH (1988) Validation

study of WOMAC: a health status instrument for measuring

clinically important patient relevant outcomes to antirheumatic

drug therapy in patients with osteoarthritis of the hip or knee.

J Rheumatol 15:1833–1840

9. Lequesne MG, Mery C, Samson M, Gerard P (1987) Indexes of

severity for osteoarthritis of the hip and knee. Validation—value

in comparison with other assessment test. Scand J Rheumatol

65:85–89

10. Gorodezky C, Alaez C, Vazquez-Garcıa MN, de la Rosa G,

Infante E, Balladares S, Toribio R, Perez-Luque E, Munoz L

(2001) The genetic structure of Mexican Mestizos of different

locations: tracking back their origins through MHC genes, blood

groups systems, and microsatellites. Hum Immunol 62:979–991

11. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out

procedure for extracting DNA from human nucleated cells.

Nucleic Acids Res 16:1215

12. Verity DH, Wallace GR, Vaughan RW, Kondeatis E, Madanat W,

Zureikat H, Fayyad F, Marr JE, Kanawati CA, Stanford MR (1999)

HLA and tumour necrosis factor (TNF) polymorphisms in ocular

Behcet’s disease. Tissue Antigens 54:264–272

13. Fargion S, Valenti L, Dongiovanni P, Scaccabarozzi A, Fracan-

zani AL, Taioli E, Mattioli M, Sampietro M, Fiorelli G (2001)

Tumor necrosis factor alpha promoter polymorphisms influence

the phenotypic expression of hereditary hemochromatosis. Blood

97:3707–3712

14. Oregon-Romero E, Vazquez-Del Mercado M, Ruiz-Quezada SL,

Navarro-Hernandez RE, Rangel-Villalobos H, Martınez-Bonilla

G, Bernard-Medina AG, Armendariz-Borunda J, Garcıa-Banue-

los J, Munoz-Valle JF (2008) Tumor necrosis factor a -308 and

-238 polymorphism in rheumatoid arthritis: association with

messenger RNA expression and sTNF-a. J Investig Med 56(7):

937–943

15. Chomiczyki P, Sacchi N (1987) Single step method of RNA

isolation by acid guanidinium thiocyanate-phenol-chloroform

extraction. Annal Biochem 162:156–159

66 Clin Exp Med (2014) 14:61–67

123

16. Moos V, Rudwaleit M, Herzog V, Hohlig K, Sieper J, Muller B

(2000) Association of genotypes affecting the expression of

interleukin-1beta or interleukin-1 receptor antagonist with

osteoarthritis. Arthritis Rheum 43(11):2417–2422

17. de Maat MP, Bladbjerg EM, Hjelmborg JB, Bathum L, Jespersen

J, Christensen K (2004) Genetic influence on inflammation

variables in the elderly. Arterioscler Thromb Vasc Biol 24(11):

2168–2173

18. Rodrıguez-Carreon AA, Zuniga J, Hernandez-Pacheco G, Rod-

rıguez-Perez JM, Perez-Hernandez N, Montes de Oca JV, Cardiel

MH, Granados J, Vargas-Alarcon G (2005) Tumor necrosis fac-

tor-alpha -308 promoter polymorphism contributes indepen-

dently to HLA alleles in the severity of rheumatoid arthritis in

Mexicans. J Autoimmun 24(1):63–68

19. Berdeli A, Tabel Y, Celik HA, Ozyurek R, Dogrusoz B, Aydin

HH (2006) Lack of association between TNFalpha gene poly-

morphism at position -308 and risk of acute rheumatic fever in

Turkish patients. Scand J Rheumatol 35(1):44–47

20. Merza M, Farnia P, Anoosheh S, Varahram M, Kazampour M,

Pajand O, Saeif S, Mirsaeidi M, Masjedi MR, Velayati AA,

Hoffner S (2009) The NRAMPI, VDR and TNF-alpha gene

polymorphisms in Iranian tuberculosis patients: the study on host

susceptibility. Braz J Infect Dis 13(4):252–256

21. Liu C, Wang J, Zhou S, Wang B, Ma X (2010) Association

between -238 but not -308 polymorphism of Tumor necrosis

factor alpha (TNF-alpha)v and unexplained recurrent spontane-

ous abortion (URSA) in Chinese population. Reprod Biol

Endocrinol 8:114

22. Sezgin M, Barlas IO, Ankarali HC, Altintas ZM, Turkmen E,

Gokdogan T, Sahin G, Erdal ME (2008) Tumour necrosis factor

alpha -308G/A gene polymorphism: lack of association with

knee osteoarthritis in a Turkish population. Clin Exp Rheumatol

26(5):763–768

23. Haddy N, Sass C, Maumus S, Marie B, Droesch S, Siest G,

Lambert D, Visvikis S (2005) Biological variations, genetic

polymorphism and familial resemblance of TNF-a and IL-6

concentrations: STANISLAS cohort. Eur J Hum Genet 13:

109–117

24. Orita S, Ishikawa T, Miyagi M, Ochiai N, Inoue G, Eguchi Y,

Kamoda H, Arai G, Suzuki M, Sakuma Y, Oikawa Y, Toyone T,

Aoki Y, Takahashi K, Ohtori S (2012) Percutaneously absorbed

NSAIDs attenuate local production of proinflammatory cytokines

and suppress the expression of c-Fos in the spinal cord of a rodent

model of knee osteoarthritis. J Orthop Sci 17(1):77–86

25. Lee YH, Ji JD, Bae SC, Song GG (2010) Associations between

tumor necrosis factor-alpha (TNF-alpha) -308 and -238 G/A

polymorphisms and shared epitope status and responsiveness to

TNF-alpha blockers in rheumatoid arthritis: a metaanalysis

update. J Rheumatol 37(4):740–746

26. Ozen S, Alikasifoglu M, Bakkaloglu A, Duzova A, Jarosova K,

Nemcova D, Besbas N, Vencovsky J, Tuncbilek E (2002)

Tumour necrosis factor alpha G ? A -238 and G ? A -308

polymorphisms in juvenile idiopathic arthritis. Rheumatology

(Oxford) 41(2):223–227

27. Sousa E, Caetano-Lopes J, Pint P, Pimentel F, Teles J, Canhao H,

Rodrigues A, Resende C, Mourao AF, Ribeiro C, Pinto TL, Rosa

CM, da Silva JA, Branco J, Ventura F, Queiroz MV, Fonseca JE

(2009) Ankylosing spondylitis susceptibility and severity–con-

tribution of TNF gene promoter polymorphisms at positions

-238 and -308. Ann N Y Acad Sci 1173:581–588

28. Pan HF, Leng RX, Wang C, Qin WZ, Chen LL, Zha ZQ, Tao JH,

Ye DQ (2011) Association of TNF-a promoter -308 A/G poly-

morphism with susceptibility to systemic lupus erythematosus: a

meta-analysis. Rheumatol Int. doi:10.1007/s00296-011-1924-9

29. Lisker R, Ramırez E, Gonzalez-Villalpando C, Stern MP (1995)

Racial admixture in a mestizo population from Mexico City. Am

J Hum Biol 7(2):213–216

30. Rangel-Villalobos H, Munoz-Valle JF, Gonzalez-Martın A,

Gorostiza A, Magana MT, Paez-Riberos LA (2008) Genetic

admixture, relatedness, and structure patterns among Mexican

populations revealed by the Y-chromosome. Am J Phys

Anthropol 135(4):448–461

31. Vignon E, Balblanc JC, Mathieu P, Louisot P, Richard M (1993)

Metalloprotease activity, phospholipase A2 activity and cytokine

concentration in osteoarthritis synovial fluids. Osteoarthr Cartil

1(2):115–120

32. Smith MD, Triantafillou S, Parker A, Youssef PP, Coleman M

(1997) Synovial membrane inflammation and cytokine produc-

tion in patients with early osteoarthritis. J Rheumatol 24(2):

365–371

33. Lefebvre V, Peeters-Joris C, Vaes G (1990) Modulation by

interleukin 1 and tumor necrosis factor alpha of production of

collagenase, tissue inhibitor of metalloproteinases and collagen

types in differentiated and dedifferentiated articular chondro-

cytes. Biochim Biophys Acta 1052(3):366–378

34. Reboul P, Pelletier JP, Tardif G, Cloutier JM, Martel-Pelletier J

(1996) The new collagenase, collagenase-3, is expressed and

synthesized by human chondrocytes but not by synoviocytes. A

role in osteoarthritis. J Clin Invest 97(9):2011–2019

35. Westacott CI, Barakat AF, Wood L, Perry MJ, Neison P, Bisbinas

I, Armstrong L, Millar AB, Elson CJ (2000) Tumor necrosis

factor alpha can contribute to focal loss of cartilage in osteoar-

thritis. Osteoarthr Cartil 8(3):213–221

36. Stannus O, Jones G, Cicuttini F, Parameswaran V, Quinn S,

Burgess J, Ding C (2010) Circulating levels of IL-6 and TNF-aare associated with knee radiographic osteoarthritis and knee

cartilage loss in older adults. Osteoarthr Cartil 18(11):1441–1447

37. Orita S, Koshi T, Mitsuka T, Miyagi M, Inoue G, Arai G,

Ishikawa T, Hanaoka E, Yamashita K, Yamashita M, Eguchi Y,

Toyone T, Takahashi K, Ohtori S (2011) Associations between

proinflammatory cytokines in the synovial fluid and radiographic

grading and pain-related scores in 47 consecutive patients with

osteoarthritis of the knee. BMC Musculskelet Disord 12:144

Clin Exp Med (2014) 14:61–67 67

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