Molecular Vision 2004; 10:458-461 <http://www.molvis.org/molvis/v10/a58/> Received 23 April 2004 | Accepted 14 July 2004 | Published 15 July 2004

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Null type of glutathione S-transferase M1 polymorphism is associated with early onset pterygium

Yi-Yu Tsai,^1,2 Huei Lee,³ Sung-Huei Tseng,⁴ Ya-Wen Cheng,² Chang-Hai Tsai,⁵ Yu-Hsiao Wu,⁶ Fuu-Jen Tsai^6,7
 
 

¹Department of Ophthalmology and ⁶Department of Medical Genetics, and ⁷College of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan; ²Institute of Medicine and ³Institute of Toxicology, Chung Shan Medical University, Taichung, Taiwan; ⁴Department of Ophthalmology, National Cheng Kung University Hospital, Tainan, Taiwan; ⁵Taichung Healthcare and Management University, Taichung, Taiwan

Correspondence to: Fuu-Jen Tsai, MD, PhD, Department of Medical Genetics, China Medical University Hospital, Number 2, Yuh Der Road, Taichung, Taiwan; Phone: 886-4-22052121-7080; FAX: 886-4-22033295; email: d0704@www.cmuh.org.tw

Abstract

Purpose: To investigate the association of glutathione S-transferase M1 (GSTM1) null genotype with pterygium.

Methods: One hundred and twenty seven pterygium patients and 102 volunteers without pterygium were enrolled in this study. Polymerase chain reaction based analysis was used to resolve the GSTM1 null and positive genotypes.

Results: There was no significant difference between total pterygium and the control group. When stratified by the age, there was a significantly higher frequency of the GSTM1 null genotype in younger patients. The difference was not significant between patients and controls older than 60 years old.

Conclusions: These results indicate GSTM1 null genotype is associated with early onset pterygium, but not associated with late onset pterygium.

Introduction

Pterygium is a chronic condition characterized by the encroachment of a fleshy triangle of conjunctival tissue into the cornea. It is composed of epithelium and highly vascular, subepithelial, loose connective tissue. Pterygium has long been considered a chronic degenerative condition. However, after abnormal expression of the p53 protein being found in epithelium, pterygium is now considered to be a UV related uncontrolled cell proliferation, similar to a tumor [1-5].

UV light induces the formation of radical oxygen species (ROS) such as hydroxyl and superoxide radicals, hydrogen peroxide, and singlet oxygen [6,7]. ROS is very harmful to cells because they injury cellular proteins, lipids, and DNA, termed oxidative stress [6,7].

Glutathione S-transferase (GST) is one of the antioxidant defense enzymes, which contribute to the protection against ROS [6,8]. In humans, the GST family can be divided into classes of α, μ, π, σ, κ, and θ. The μ family GST (GSTM), on chromosome 1p13.3, comprises GSTM from 1 to 6. There are three alleles at the GSTM1 locus: GSTM1 0, GSTM1 A, and GSTM1 B. GSTM1 0 is a deletion and homozygotes (GSTM1 0/0, GSTM1 null genotype) express neither mRNA nor GSTM1 protein. Alleles GSTM1 A and GSTM1 B encode monomers that form homo- and heterodimeric enzymes [9]. In GSTM1 polymorphisms, GSTM1 0/0 is called GSTM1 null, and GSTM1 0/A, 0/B, A/A, A/B, and B/B are included in GSTM1 positive [6,9-15]. GSTM1 null genotype was reported to be associated with cutaneous UV sensitivity and cancer formation [6,10-15].

Because there is evidence that hereditary factors play a role in the development of pterygium [16,17], and GSTM1 null type is associated with cutaneous UV sensitivity, it is logical to suspect the correlation between pterygium formation and GSTM1 null type. Therefore, we aimed to test the hypothesis that lack of GSTM1 (GSTM1 null type) contributes to susceptibility of pterygium formation.

Methods

Patients

A total of 127 pterygium patients (71 males and 56 females) were enrolled in the study with ages ranging from 35 to 90 years (mean, 64.6 years). Patients included in this study were apex of pterygium invading the cornea for more than 1 mm. Volunteers (102) age 50 years or more without pterygium were enrolled as the control group. There were 63 males and 39 females in the control group (age ranges from 50 to 83 years with an average of 64.2 years). This study was carried out with approval from the Human Study Committee of the China Medical University Hospital and National Cheng Kung University Hospital. Informed consent was obtained from all individuals who participated in this study.

Methods

Genomic DNA was prepared from peripheral blood by use of a DNA Extractor WB kit (Wako, Japan). Polymerase chain reactions (PCRs) were carried out in a total volume of 25 μl containing genomic DNA, 2-6 pmol of each primer, 1X Taq polymerase buffer (1.5 mM MgCl₂), and 0.25 units of AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA). The method of determining the GSTM1 null or positive types was the same as a previous study [18]. Primers for GSTM1 were sense: 5'-AGC TGC CCT ACT TGA TTG ATG G-3' and antisense: 5'-CTG GGG ACA CTC ACA AAT TCT G-3'. PCR amplification was performed in a programmable thermal cycle GeneAmp PCR System 2400 (Perkin Elmer). Cycling conditions for PCR was set as follows; one cycle at 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s, and one final cycle for extension at 72 °C for 10 min. The PCR product (4 μl) was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The CYP1A1 gene was used as internal positive control and the primer used were sense: 5'-GCA TTG AGC TTG CAT GCT TG-3' and antisense: 5'-TAG GAG TCT TGT CTC ATG CCT-3'. PCR products were analyzed for the presence of the 293 bp PCR product (indicative of the presence of the GSTM1 gene) and of the 583 bp of the CYP1A1 gene (Figure 1).

Statistical analysis for the distributions of GSTM1 positive and null genotypes in the pterygium and control groups was done using the Chi-squared test.

Results

There were no significant differences between both groups in age and sex.

The percentage of GSTM1 null type in case and control group was 64.6% and 59.8%, respectively. There was no significant difference between both groups (p=0.50).

After further stratification by age of 60 years, there was a significantly higher frequency of the GSTM1 null genotype in young patients when comparing to the young individuals in control group (p=0.007, Table 1). Because in young group, range of age in patients was 35 to 60 years, and controls were 50 to 60 years, further stratified by the age between 50 and 60 years was evaluated. After excluding 6 patients less than 50 years old, who were all GSTM1 null, there was still a significantly higher frequency of the GSTM1 null genotype in patients group (p=0.036, Table 1). The difference was not significant in old patients and controls.

Discussion

Although the pathogenesis of pterygia is still poorly understood, epidemiologic evidence suggests that UV irradiation plays the most important role [3-5]. The noxious effects of UV irradiation is partly due to the formation of ROS, which cause oxidative stress [6,7].

GSTM1, an antioxidant defense enzyme, protects cells against oxidative stress, either by direct inactivation of peroxidized lipids and DNA or by detoxification of xenobitics, which are known co-factors for radical formation [6].

GSTM1 shows a polymorphic expression, and null genotype results from homologous unequal crossing over between two highly identical regions that flank the GSTM1 gene, resulting in a 15 kb deletion that contains the entire GSTM1 gene [19]. Because of no GSTM1 expression, carriers of GSTM1 null type were reported to suffer from more intense inflammatory reactions after UV irradiation and have a high prevalence of skin cancer [6,14,15]. Because the GSTM1 null phenotype was associated with UV related cutaneous cancer, the association between GSTM1 null type and pterygium is possible.

In our series, the frequency of GSTM1 null type was statistically higher in young pterygium patients, but the difference was not present in old patients. Moreover, all pterygium patients younger than 50 years were found to be GSTM1 null genotype in our series. Hence, we suggest that the GSTM1 null genotype is associated with early onset pterygium, but not associated with late onset pterygium.

The GSTM1 null type was reported to be associated with cutaneous photosensitivity [14,15], so GSTM1 null may be associated with the photosensitivity of corneal limbal cells. Hence, corneal limbal cells in GSTM1 null type is more susceptible to UV mediated oxidative stress than GSTM1 positive genotype, and if people with GSTM1 null type, they have the risk of pterygium formation earlier than other genotypes.

Threlfall and English reported that pterygium formation was mainly related to the dose of UV irradiation [3] and in our series, the risk effect of GSTM1 null type was not found in late onset pterygium. We proposed no matter what the GSTM1 polymorphism is, when the ocular sun exposure dose is enough for pterygium to form, pterygium will develop. However, the dose in patients with GSTM1 null type is less than patients with positive type.

There were several limitations in our series. First, we only evaluated the relationship between GSTM1 null and pterygium, and did not analyze the relationship between other GSTM1 genotypes in the GSTM1 positive and pterygium. Though there are several genotypes in GSTM1 polymorphisms, only GSTM1 null is considered to be defective in antioxidation and other GSTM1 polymorphisms are not [20]. Hence, in most reports about GSTM1 polymorphisms and susceptibility of disease, GSTM1 polymorphisms were divided into null and positive types as in our study [6,10-15]. Second, the age for stratification was not the onset time of pterygium, but was the time of diagnosis. Though onset time is better, it was impossible to get the data accurately. However, even if part of the patients were misclassified, our result still showed there was an association between GSTM1 null and early onset pterygium. We suggest if all late onset pterygium patients can be excluded from the young pterygium group, we could find a stronger association between GSTM1 null and early onset pterygium. The incidence of late onset pterygium is higher than early onset clinically, so most of the patients in our old patients group had late onset pterygium. Hence, even though there was early onset pterygium patients included in old patients group, the bias was small. Third, ultraviolet radiation, immunoinflammatory process, virus infection, and genetic factors were reported to be related to pterygium [4]. However, we did not analyze sun exposure and other factors in our series. Not only are sun exposure and other factors difficult to evaluate by questionnaires [3], but also we do not intend to assert that GSTM1 null is a direct cause of pterygium. We only investigated whether there was an association between GSTM1 null and pterygium. Hence, due to the fact that the above factors may be confounding factors, our results showed only that there was an association between GSTM1 null and early onset pterygium. We do not imply a causal relationship between GSTM and early onset pterygium. Sun exposure and other factors are unlikely to explain all 6 patients less than 50 years old who were GSTM1 null. We suggest GSTM1 null may play a role in causing early onset pterygium.

This study could be the basis of future surveys. Further studies on other genes related to UV-mediated oxidative stress are suggested for the detection of a genetic predisposition to pterygium formation and its recurrence. In conclusion, GSTM1 null genotype is associated with early onset pterygium and antioxidant defense enzymes may play a role in pterygium formation.

Acknowledgements

The authors would like to thank Miss Ming-May Wong, Department of Ophthalmology, National Cheng Kung University Hospital.

References

1. Tan DT, Lim AS, Goh HS, Smith DR. Abnormal expression of the p53 tumor suppressor gene in the conjunctiva of patients with pterygium. Am J Ophthalmol 1997; 123:404-5.

2. Chowers I, Pe'er J, Zamir E, Livni N, Ilsar M, Frucht-Pery J. Proliferative activity and p53 expression in primary and recurrent pterygia. Ophthalmology 2001; 108:985-8.

3. Threlfall TJ, English DR. Sun exposure and pterygium of the eye: a dose-response curve. Am J Ophthalmol 1999; 128:280-7.

4. Detorakis ET, Drakonaki EE, Spandidos DA. Molecular genetic alterations and viral presence in ophthalmic pterygium. Int J Mol Med 2000; 6:35-41.

5. Coroneo MT, Di Girolamo N, Wakefield D. The pathogenesis of pterygia. Curr Opin Ophthalmol 1999; 10:282-8.

6. Kerb R, Brockmoller J, Reum T, Roots I. Deficiency of glutathione S-transferases T1 and M1 as heritable factors of increased cutaneous UV sensitivity. J Invest Dermatol 1997; 108:229-32.

7. Halliwell B, Gutteridge JMC. Oxidative stress and antioxidant protection: some special cases. In: Halliwell B, Gutteridge JMC, editors. Free Radicals in Biology and Medicine. 3rd ed. Oxford: Clarendon Press; 1999. p. 530-533.

8. Halliwell B, Gutteridge JMC. Antioxidant defences. In: Halliwell B, Gutteridge JMC, editors. Free Radicals in Biology and Medicine. 3rd ed. Oxford: Clarendon Press; 1999. p. 105-245.

9. Mannervik B, Awasthi YC, Board PG, Hayes JD, Di Ilio C, Ketterer B, Listowsky I, Morgenstern R, Muramatsu M, Pearson WR, Pickett CB, Sato K, Widersten M, Wolf RC. Nomenclature for human glutathione transferases. Biochem J 1992; 282:305-6.

10. Bardakci F, Canbay E, Degerli N, Coban L, Canbay EI. Relationship of tobacco smoking with GSTM1 gene polymorphism in laringeal cancer. J Cell Mol Med 2003; 7:307-12.

11. Hirvonen A, Husgafvel-Pursiainen K, Anttila S, Vainio H. The GSTM1 null genotype as a potential risk modifier for squamous cell carcinoma of the lung. Carcinogenesis 1993; 14:1479-81.

12. Lafuente A, Pujol F, Carretero P, Villa JP, Cuchi A. Human glutathione S-transferase mu (GST mu) deficiency as a marker for the susceptibility to bladder and larynx cancer among smokers. Cancer Lett 1993; 68:49-54.

13. Katoh T, Nagata N, Kuroda Y, Itoh H, Kawahara A, Kuroki N, Ookuma R, Bell DA. Glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genetic polymorphism and susceptibility to gastric and colorectal adenocarcinoma. Carcinogenesis 1996; 17:1855-9.

14. Strange RC, Lear JT, Fryer AA. Polymorphism in glutathione S-transferase loci as a risk factor for common cancers. Arch Toxicol Suppl 1998; 20:419-28.

15. Ollier W, Davies E, Snowden N, Alldersea J, Fryer A, Jones P, Strange R. Association of homozygosity for glutathione-S-transferase GSTM1 null alleles with the Ro+/La- autoantibody profile in patients with systemic lupus erythematosus. Arthritis Rheum 1996; 39:1763-4.

16. Zhang JD. An investigation of aetiology and heredity of pterygium. Report of 11 cases in a family. Acta Ophthalmol (Copenh) 1987; 65:413-6.

17. Hilgers JHCh. Pterygium: Its incidence, heredity and etiology. Am J Ophthalmol 1960; 635-644.

18. Nicholl DJ, Bennett P, Hiller L, Bonifati V, Vanacore N, Fabbrini G, Marconi R, Colosimo C, Lamberti P, Stocchi F, Bonuccelli U, Vieregge P, Ramsden DB, Meco G, Williams AC. A study of five candidate genes in Parkinson's disease and related neurodegenerative disorders. European Study Group on Atypical Parkinsonism. Neurology 1999; 53:1415-21.

19. Xu S, Wang Y, Roe B, Pearson WR. Characterization of the human class Mu glutathione S-transferase gene cluster and the GSTM1 deletion. J Biol Chem 1998; 273:3517-27.

20. Heagerty AH, Fitzgerald D, Smith A, Bowers B, Jones P, Fryer AA, Zhao L, Alldersea J, Strange RC. Glutathione S-transferase GSTM1 phenotypes and protection against cutaneous tumours. Lancet 1994; 343:266-8.