Thymidylate synthase genetic polymorphism and plasma total homocysteine level in a group of Turkish patients with rheumatoid arthritis: relationship with disease activity and methotrexate toxicity

Borman, Pınar; Taşbaş, Özgur; Karabulut, Halil; Tukun, Ajlan; Yorgancıoğlu, Rezan

doi:10.1016/j.rbr.2014.12.001

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Original articles • Rev. Bras. Reumatol. 55 (6) • Nov-Dec 2015 • https://doi.org/10.1016/j.rbr.2014.12.001 copy

Thymidylate synthase genetic polymorphism and plasma total homocysteine level in a group of Turkish patients with rheumatoid arthritis: relationship with disease activity and methotrexate toxicity

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Background:

The polymorphism of thymidylate synthase (TS) gene and homocysteine are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine homocysteine levels and the frequency of TS gene triple repeat (TS3R) and double repeat (TS2R) polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity and disease activity.

Methods:

Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ± 12.5 and 46.2 ± 13.4 years were enrolled for the study. Demographic characteristics were obtained and a number of patients with MTX-related adverse affects were recorded in the patient group. The homocysteine levels and TS2R/TS3R polymorphisms of the TS gene were analyzed and the distribution of genotypes according to MTX toxicity and disease activity was determined.

Results:

The demographic properties were similar between the patient and control subjects. Folic acid supplementation with a mean dose of 5 mg folic acid/week was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment. The respective frequency of TS2R and TS3R polymorphisms was found to be similar in the patient and control groups. TS2R and TS3R gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. The mean homocysteine level was also similar in patients with and without TS gene polymorphism, but was found to be higher (12.45 μmol/L vs 10.7 μmol/L) in patients with MTX-related side effects than in patients without side effects. The mean level of homocysteine was correlated with levels of ESR in the patient group.

Conclusions:

In conclusion, homocysteine levels might affect the disease activity and toxicity of MTX but 2R and 3R polymorphisms in the TS gene were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible for the MTX toxicity in patients suffering from RA.

Keywords
Rheumatoid arthritis; Thymidylate synthase; Homocystein; Polymorphism; Methotrexate

	Patients (n = 64)	Controls (n = 31)
Age (mean ± SD) (years)	48.7 ± 12.5	46.2 ± 13.4
Sex (female/male)	53/11	26/05
BMI (kg/m²)	27 ± 5	27.5 ± 4.7
Duration of the disease (years)	6.5 (0.50–34)
Duration of MTX treatment (years)	2 (0.2–10)
Concurrent DMARD treatment (n) (%)	34 (53.1%)
Rheumatoid factor positivity (n) (%)	41 (64.1%)
Homocysteine (μmol/L)(median)	11.2 (5–146)	10.1 (6–18)

	Control (n = 31) n (%)	RA (n = 64) n (%)	p	Odds ratio (% CI)
TS 2R2R	6 (19.4%)	14 (21.9%)	–	1.000^a a Reference category.
TS 2R3R	20 (64.5%)	33 (51.6%)	0.539	0.707 (0.234–2.137)
TS 3R3R	5 (16.1%)	17 (26.6%)	0.593	1.457 (0.366–5.801)
2R3R + 3R3R	25 (80.6%)	50 (78.1%)	0.778	0.857 (0.294–2.499)

	Adverse events (−) (n = 28)	Adverse events (+) (n = 36)	p	Odds ratio (95% CI)
TS 2R2R	4 (14.3%)	10 (27.8%)	–	1.000^a a Reference category.
TS 2R3R	14 (50.0%)	19 (52.8%)	0.375	0.543 (0.141–2.093)
TS 3R3R	10 (35.7%)	7 (19.4%)	0.098	0.280 (0.062–1.266)
2R3R + 3R3R	24 (85.7%)	26 (72.2%)	0.195	0.433 (0.120–1.567)

Variables	Adverse effect (−) (n = 13)	Adverse effect (+) (n = 17)	p
TS 2R2R	3 (23.1%)	7 (41.2%)
TS 2R3R	5 (38.5%)	7 (41.2%)
TS 3R3R	5 (38.5%)	3 (17.6%)
2R3R + 3R3R	10 (76.9%)	10 (58.8%)	0.440

Variables	2R2R (n = 14)	2R3R (n = 33)	3R3R (n = 17)	p
HAQ (median)	0.8 (0–3)	0.6 (0–3)	0.75 (0–2.5)	0.695
DAS28 (median)	4.0 (2.1–7.5)	4.0 (1.7–7.8)	4.5 (1.9–6.9)	0.909
ESR (median)	29.0 (10.0–120.0)	30.0 (2.0–82.0)	25.0 (7.0–65.0)	0.695
CRP (median)	0.7 (0.3–10.0)	0.7 (0.1–10.5)	0.8 (0.2–4.8)	0.409

	Homocystein	p
Adverse effects		0.024
Absent	10.7 (5.0–33.0)
Present	12.45 (6.8–146.0)

	Homocystein	p
		0.893^a a Variance analysis among genotypes.
TS 2R2R	11.3 (5.2–146.0)
TS 2R3R	11.0 (5.2–50.0)
TS 3R3R	11.0 (5.0–42.0)
2R3R + 3R3R	11.0 (5.0–50.0)	0.655^b b Comparison between genotypes with risky allele and reference genotype.

Author	RA patients (n)	Studied polymorphisms	MTX efficacy	MTX toxicity	Receiving folic acid
Dervieux et al.²⁶26 Kawakami K, Omura K, Kanehira E, Watanabe Y. Polymorphic tandem repeats in the TS gene is associated with its protein expression in human gastrointestinal cancers. Anticancer Res. 1999;19:3249-52.	255	TS 2R/3R (and other genes)	ns	Increased toxicity	Not known
Dervieux et al.²2 Dervieux T, Furst D, Lein DO, Capps R, Smith K, Walsh M, et al. Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier aminoimidazole carboxamid ribonucleotide transformylase and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis. Arthritis Rheum. 2004;50:2766-74.	108	TS 2R/3R (and other genes)	Increased MTX response	Not studied	1 mg/day
Takatori et al.¹⁰10 Takatori R, Takahushi K, Tokunaga D, Hojo T, Fujioka M, Asano T, et al. ABCB1, C3435T polymorphism influences methotrexate sensitivity in rheumatoid arthritis patients. Clin Exp Rheumatol. 2006;24:546-54.	124	TS 6 bp deletion polymorphism (and other genes)	ns	ns	Not known
Derwieux et al.⁶6 Dervieux T, Greenstein N, Kremer J. Pharmacogenomic and metabolic biomarkers in the folate pathway and their association with methotrexate effects during dosage escalation in rheumatoid arthritis. Arthritis Rheum. 2006;54:3095–103.	48	TS 2R/3R (and other genes)	ns	ns	+ (95% of patients)
Weisman et al.¹⁸18 Weisman MH, Furst DE, Park GS, Kremer JM, Smith KM, Wallace DJ. Risk genotypes in folate-dependent enzymes and their association with methotrexate-related side effects in rheumatoid arthritis. Arthritis Rheum. 2006;54(2):607-12.	214	TS2R/3R 3R (and other genes)	ns	Increased alopecia	+ (81% of patients)
Grabar et al.⁸8 Grabar BP, Logar D, Lestan B, Dolzan V. Genetic determinants of methotrexate toxicity in rheumatoid arthritis patients: a study of polymorphisms affecting methotrexate transport and folate metabolism. Eur j Clin Pharmacol. 2008;64:1057-68.	213	TS 2R/3R (and other genes)	ns	Increased bone marrow toxicity	+ (62–91% patients)
Ghodke et al.⁵5 Ghodke Y, Chopra A, Joshi K, Patwardhan B. Are thymidilate synthase and methylene tetrahydrofolate reductase genes linked with methotrextae response (efficacy, toxicity) in Indian (Asian) rheumatoid arthritis patients?. Clin Rheumatol. 2008;27:787-9.	34	TS 2R/3R and 6bp deletion	ns	ns	Not known
Inoue et al.¹¹11 Inoue S, Hashiguchi M, Takagi K, Kawai S, Mochizuki M. Preliminary study to identify the predictive factors for the response to methotrexate therapy in patients with rheumatoid arthritis. J Pharmaceutical Soc Japan. 2009;129(7):843-9.	36	TS 6bp deletion and TS 2R/3R	6bp deletion related with CRP and better response to MTX	Not studied	5 mg/week
Kumagai et al.⁹9 Kumagai K, Hiyama K, Oyama T, Maeda H, Kohno N. Polymorphisms in the thymidylate synthase and methylenetetrahydrofolate reductase genes and sensitivity to the low-dose methotrexate therapy in patients with rheumatoid arthritis. Int J Mol Med. 2003;11:593-600.	105	TS 2R/3R and 6bp deletion	Poor response to MTX	ns	Not known
Ranganathan et al.¹³13 Ranganathan P, Culverhouse, Marsh S. Methotrexate gene polymorphisms and their effects on MTX toxicity in Caucasian and African American patients with rheumatoid arthritis. J Rheumatol. 2008;5:559-69.	222	TS 1494 of TTAAAG (and other genes)	Not studied	ns	1–3 mg/day
Zeng et al.⁷7 Zeng QY, Wang YK, Xiao ZY, Chen SB. Pharmacogenetic study of 5,10 MTHFR C677T and thymidylate syntase 3R/2R gene polymorphisms and methotrexate-related toxicity in Chinese Han patients with inflammatory arthritis. Ann Rheum Dis. 2008;67(8):1193-4.	85	TS 3R/2R	Not studied	ns	–
Present study	64	TS 2R/3R	ns	ns	5 mg/week

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[1] *Corresponding author. E-mail: pinarborman@gmail.com (P. Borman).