Objectives: Graft-versus-host disease is a major problem after bone marrow transplant. GSTM1, GSTT1, and GSTO2 are important genes that interfere with xenobiotic and drug metabolism. Polymorphisms of these genes may influence the metabolism of immunosuppressive drugs given for inhibition of graft-versus-host disease and may influence their susceptibility to diseases, which bone marrow transplant could alleviate.

Materials and Methods: We examined the polymorphisms of 2 groups: The first group was composed of 88 patients who had undergone a bone marrow transplant and 100 otherwise healthy persons; the second group was composed of 54 patients without graft-versus-host disease and 34 patients with graft-versus-host disease. We used polymerase chain reaction–restriction fragment length polymorphism method for genotyping GSTO2 and also for multiplexing polymerase chain reactions for GSTT1 and GSTM1 genotypes.

Results: No significant association existed between the genotypes GSTO2 (DD: P = .458, OR 0.422), GSTM1 (P = .349, OR 1.52), or GSTT1 (P = .887, OR 1.086), and the incidence of GVHD. Moreover, we saw no association between these polymorphisms and the problems that lead to bone marrow transplant (GSTO2: DD, P = .181, OR 0.465; GSTM1: P = .699, OR 0.892; GSTT1: P = .656, OR 0.845). We showed that men have more bone marrow transplants than do women (P = .019, OR 2.034).

Conclusions: Our results show that these poly-morphisms may have no effect on the metabolism of drugs used to treat graft-versus-host disease and also, may play no significant role in creating the problems that lead to bone marrow transplant.

Key words : Graft-versus-host disease, Bone marrow transplant, Glutathione S-transferase

Introduction

Bone marrow transplant (BMT) is a therapy for patients with certain cancers and other diseases (eg, leukemia, lymphoma, aplastic anemia, immune deficiency disorders, and some solid- tumor cancers) that has been used successfully since 1968.1 Graft-versus-host disease (GVHD) poses significant mor-bidity and mortality after bone marrow transplant and is a long-term complication of BMT. Graft-versus-host disease is an immune process with an outstanding role of the T cell. It may be treated with immunosuppressive agents (eg, cyclosporine, corti-costeroids, tacrolimus, and mycophenolate mofetil). Despite advances in immunosuppressive regimens, GVHD remains a frequent complication of allogeneic BMT.^1,2 Acute GVHD usually occurs within 2 to 10 weeks after the transplant.³ Rates of GVHD vary from 20% to 50% among related donors, and between 60% to 80% of unrelated donors and recipients.³ The greater the mismatch between the donor and the recipient, the greater the risk of GVHD.

The glutathione S- transferase (GST; EC:2.5.1.18) family is an enzyme in phase II detoxification that is important to xenobiotic and drug metabolism, which catalyzes binding of glutathione to these substrates and cause more solubility in the water, to be excreted from the body through bile or urine.⁴ Mutation in these genes affects detoxification and may cause cancer and some diseases.⁵ One member of this family is GSTO2 (OMIM: 612314), which has a Cys in the active site that plays an important role in arsenic reduction. GSTO2 is expressed in whole body in the liver, the kidneys, the skeletal muscles, and the prostate gland.⁶ GSTM1 (OMIM: 138350) and GSTT1 (OMIM: 600436) are other family members that have 2 forms: null and active. Null GSTM1 and GSTT1 do not have any detoxification activities. GSTM1 has 8 exons and its length equals 5.9 kb. GSTT1 has 6 exons and its length is 8.1 kb.^7-9 GSTO2 (N142D) genetic poly-morphism is associated with colorectal cancer,¹⁰ gastric cancer,¹¹ but it is not associated with ovarian cancer¹²; additionally, it has been shown that this polymorphism plays a role in asthma^13,14 and liver carcinoma.¹⁵

Several studies have investigated GSTM1 and GSTT1 polymorphisms with cancer risk and have found significant associations between genetic polymorphism of these genes and colorectal can-cer,^16,17 hepatic veno-occlusive disease,¹⁸ and the incidence of GVHD.¹⁹ The association of GVHD and the susceptibility of disease that leads to BMT with GSTO2 has yet to be examined. We aimed to explore the effect of GSTO2 (N142D), GSTM1, and GSTT1 on immunosuppressive drugs metabolism, and its effect on the incidence of GVHD and how it influences disease susceptibility that would lead to BMT.

Materials and Methods

Buffy coat samples from 88 bone marrow trans-planted patients were collected from Namazi Hospital in Shiraz, Iran, between 2007 and 2011. We investigated stem cell transplant recipients, graft outcomes, and GVHD episode(s) for 12 weeks after transplant.

Patients were divided into 2 groups according to the presence or absence of a GVHD episode. We had 100 otherwise healthy persons as a normal popu-lation from Shiraz Transfusion Organization as controls, and 88 transplanted patients as the case group, who were used to evaluate the effect of these polymorphisms on disease susceptibility that could lead to BMT.

We obtained information from the patient’s medical records. The study was approved by the Ethics Committee of Shiraz University. Written informed consent was obtained from all participants. All of the protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration.

Immunosuppression consisted of busulfan 16 mg/kg or Busulfex intravenous (80% of oral dose) and cyclophosphamide 120 to 200 mg/kg in leukemia patients (acute myelogenous leukemia, acute lymphogenous leukemia, chronic myelogenous leukemia, and cyclophosphamide 60 to 120 mg/kg +ATG 90 kg/mg for severe aplastic anemia, Cooley anemia, and Fanconi anemia. Cyclosporine and methotrexate were given for GVHD prophylaxis. Prophylactic antibiotic, antifungal, and antiviral drugs were prescribed for all patients. We irradiated the blood products with gamma rays to prevent post-transfusion GVHD. Signs and symptoms for blood and marrow transplant criteria were identified by hematologists of the European group. Acute GVHD was based on what happened 2 to 10 weeks after BMT according to consensus on acute GVHD grading in 1994.20 All patients and controls were Iranian.

DNA extraction
Buffy coats of whole blood from patients who had undergone a bone marrow transplant were available in the sample bank of Shiraz Transplant Research Center. Genomic DNA was extracted from the Buffy coats using a DNP kit (CinnaGen Co. Shahrak Gharb, Tehran, Iran) according to manufacturer’s instruction.

Genotyping
We determined the genotypic analysis for the GSTO2 N142D polymorphism using polymerase chain reaction–restriction fragment length polymorphism method and evaluated the polymorphisms and laboratory quality control as previously described.^10,11

We used a multiplex polymerase chain reaction to detect genetic polymorphisms for GSTT1 and GSTM1 and β-globin as an internal control.^21,22 Using specific primers for GSTT1, GSTM1, and β-globin, amplified fragments showed 459, 219, and 268 bp bands on gel electrophoresis.

A negative control containing all reagents but water instead of DNA template was included in each amplification set. To test for contamination, negative controls (tubes containing the polymerase chain reaction mixture without the DNA template) were incubated in every run. We lost 2 samples of DNA for the GSTM1 and the GSTT1 polymorphisms.

Statistical analyses
Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 17.0, IBM Corporation, Armonk, NY, USA). We used the t test for continuous variables. We used the chi-square test for each polymorphism of GSTO2, GSTM1, and GSTT1 to see if the sample groups demonstrated the Hardy-Weinberg principle. We used odds ratios and 95% confidence intervals to determine the association between genetic polymorphisms of GSTO2, GSTM1, and GSTT1 GVHD after BMT. A P value < .05 was considered statistically significant, and all P values were 2-tailed.

Results

Among 88 consecutive recipients, 59 (67%) were male (aged, 7-52 y) and 29 (33%) were female (aged, 6-46 y). We determined the alleles and genotypic frequencies for 88 persons (ie, 34 GVHD and 54 non-GVHD bone marrow transplant recipients). The mean ages of the patients with GVHD and non-GVHD were 19 ± 12.3 and 23.7 ± 11.7. Our population met the Hardy–Weinberg principle (χ2 = 0.724, df = 1; P > .05).

We investigated these polymorphisms and incidence of GVHD. We found no statistically significant association between GSTO2 (N142D), GSTM1, and GSTT1 polymorphisms, and the incidence of acute GVHD (Table 1). We also found no association between age (OR 0.964; P = .132), sex (OR 1.303; P = .575), and cytomegalovirus (OR 0.438; P = .467) with GVHD (data not shown).

Our second study included 88 bone marrow transplanted patients (mean age, 22.7 ± 11.9 y) and 100 healthy persons (mean age, 24.2 ± 4.1 y). Our population met the Hardy-Weinberg principle (χ2 = 1.20, df = 1; P > .05). There was no statistically significant association between GSTO2 (N142D), GSTM1, and GSTT1 polymorphisms and conditions that lead to BMT (Table 2). After classification according to their gender, we observed that men more often had BMT than women (P = .019; OR 2.034).

Discussion

Graft-versus-host disease occurs after a bone marrow or stem cell transplant in which a person receives bone marrow tissue or cells from a donor. The newly transplanted cells regard the recipient’s body as foreign. When this happens, the newly transplanted cells attack the recipient’s body.¹

Several studies show that increasing the D allele of GSTO2 may reduce the risk of colorectal and gastric cancers.^10,11 In 2010, no association was found between GSTM1 and GSTT1 polymorphism with liver acute rejection.²³

Many genes also affect the incidence of GVHD. The cells and cytokines of the immune system play a major role in GVHD pathogenesis. The human leukocyte antigen matching of the donor and the recipient is the main cause of GVHD, but studies about CD31 mismatching show that the CD31 polymorphisms between the recipient and donor have no role in the development of GVHD.²⁴

A study in 2003 showed that IL10-592A/A compared with C/C genotype was associated with acute GVHD.25 In 2002, Middleton and associates²⁶ examined the association between vitamin D receptor polymorphisms and GVHD. They found a significant association between GVHD and vitamin D receptor polymorphism. In addition, they showed that the vitamin D receptor polymorphisms affects the survival in human leukocyte antigen-matched sibling allogeneic BMT.

In 2013, we examined the association of the GSTO2 polymorphism and hepatic failure that would lead to a liver transplant. We showed that augmenting D allele may increase the risk of hepatic failure that leads to a liver transplant. So persons with the mutant genotypes—ND and DD genotype for GSTO2—are more prone to develop hepatic failure. We also saw that the male sex was more sensitive than the female sex to develop hepatic failure that could lead to a liver transplant.²⁷

Another study conducted in the Iranian population showed that the GSTO2 polymorphism N142D was not associated with acute renal rejection. It should be noted that the combined effect of a deceased-donor kidney and DD genotypes of GSTO2 polymorphism results in a significant increase in risk of acute rental rejection (OR 3.82; P = .02). This points to the reduced function of the DD genotypes as antioxidant enzymes involved in phase II detox-ification in increased acute inflammatory conditions.²⁸ In 2007, Kim and associates showed that GSTA1 A*/A* decreased the risk of GVHD, especially in skin GVHD when a patient using busulfan/cyclophosphamide conditioning.²⁹ In 2010, another study showed that the GSTA1, GSTP1, and GSTM1 genotype in children with congenital hemoglobinopathies may allow better adjustment to the busulfan dosage than patients with GVHD.³⁰

The association of GVHD with the GSTO2 (N142D) polymorphism has not been examined. In this study, we observed that the polymorphisms of GSTO2, GSTM1, and GSTT1 were not associated with the incidence of GVHD, nor did they have any association that would lead to BMT.

CYP3A is the major cytochrome in the liver,³¹ which is sensitive to the progesterone hormone.^32,33 This hormone causes CYP3A to work well (30%-40%),^31-33 and because this hormone is secreted in women more than it is in men, it is possible that detoxification is done in women in a better level of operation than men (30%-40%)31-33; therefore, they are less likely affected by conditions that leads to BMT.

Our results point to the reduced function of DD genotype in GSTO2 and null genotype in GSTM1 and GSTT1 as antioxidant enzymes do not involve the incidence of GVHD, leading to BMT. Because the association of GVHD with the GSTO2 (N142D) polymorphism has not been examined before in any study, and this is the first study to examine this, further studies are needed to determine role of these enzymes in incidence of GVHD.

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