The Association Between Azathioprine Genetic Polymorphisms, Clinical Efficacy and Adverse Drug Reactions Among Egyptian Patients with Autoimmune Diseases

Introduction

Autoimmune disorders are a group of heterogeneous diseases that occur when the body tissues are attacked by its own immune system.¹ During the pathogenesis of these disorders, both T cells and B cells are activated even without any precipitating cause or infection.² The guidelines for treating these disorders aimed to change the thresholds of immune activation by using immunosuppressants.³ Azathioprine (AZA) is used in the treatment of various autoimmune diseases including systemic lupus erythematosus (SLE). Despite AZA efficacy in treating manifestations of autoimmune diseases, its use is associated with myelosuppression, gastric toxicity, pancreatitis, and liver toxicity. Inter-individual variability to AZA effect and outcomes is related to its genetic polymorphism. AZA is activated by the liver to 6-mercaptopurine (6-MP) through a glutathione-dependent process.⁴

Thiopurine S-methyltransferase (TPMT) is the enzyme responsible for catalyzing the methylation of 6-MP. It competes with xanthine oxidase and hypoxanthine-guanine phosphor-ribosyl-transferase to detect the percent of 6-MP metabolized to the active metabolite 6-thioguanine nucleotide (6-TGN) metabolite: the main component responsible for immunosuppressive effect.⁵ Many studies have proved that there are many genetic polymorphisms in the gene encoding for TPMT. Recently, the number of mutations in this specific gene reached 37 mutations.⁶ Patients were categorized into: (i) intermediate TPMT activity which is heterozygous for a mutant TPMT allele represents 4–11% of patients. (ii) very low or absent TPMT activity which is homozygous or compounds heterozygous about 1 in 300 patients.^7–9 Intermediate TPMT activity patients can accumulate 50% more from 6-TGNs in comparison with patients that have normal or high TPMT activity. These individuals will have a high risk of adverse drug reactions (ADRs) caused by AZA. High doses of 6-TGNs will be accumulated in patients with deficient TPMT activity which may result in fatal myelosuppression.¹⁰ Another study concluded that very few patients who have two inactive TPMT alleles are homozygous deficient, experienced severe bone marrow suppression; a large percentage of those who are heterozygous encounter moderate to severe bone marrow suppression and those who are homozygous for wild type TPMT alleles have reduced the risk of bone marrow suppression.¹¹ Detecting TPMT genotypes or phenotypes before starting AZA therapy is documented in many updated treatment protocols.^11–13 In autoimmune diseases, patients with homozygous wild type should receive full initial doses, while heterozygous patients required lower doses (30–70% of the recommended dose) and substantially very small doses or other drugs are required for the rare homozygous-deficient patients.¹⁴ TPMT protective testing has a great advantage as the doses can be tolerated on the basis of TPMT status. This decreases the probability of acute myelosuppression without affecting disease progression.¹⁵ To our knowledge, only one study investigated the frequency of TPMT gene polymorphisms (TPMT*3A and TPMT*3C alleles) in Egypt.¹⁶ However, no studies investigated the frequency of TPMT*3G or the association between TPMT polymorphisms and AZA clinical efficacy and ADRs among Egyptian patients with autoimmune diseases. This study aimed to detect the frequencies of allelic variants (TPMT*3A, TPMT*3C and TPMT*3G) in the TPMT genes among Egyptian population and to assess the association between TPMT polymorphisms and AZA clinical efficacy and ADRs in Egyptian patients with autoimmune diseases.

Materials and Methods

Design

A prospective, observational single-center clinical trial.

Setting

Rheumatology and Rehabilitation Department, Kasr Alainy University hospital, Faculty of Medicine, Cairo University.

Patients

Patients attending Kasr Alainy Rheumatology Outpatient Clinic between December 1, 2017 and June 30, 2019 was included in the study after signing a consent form. Patients were diagnosed as having an autoimmune/chronic inflammatory disease; systemic lupus erythematosus, Behçet's disease, mixed connective tissue disease, rheumatoid arthritis, and scleroderma during the study period were included in the study after signing a consent form. TPMT genetic polymorphisms were detected for all patients and the association between the presence of polymorphisms, clinical efficacy of AZA and adverse drug reactions were determined.

Exclusion Criteria

Patients were excluded from the study if they had myelosuppression or abnormal liver function tests before receiving AZA.

DNA Isolation and Analysis of TPMT Gene Polymorphisms

A volume of 5 mL of EDTA anticoagulated whole blood was obtained from each patient, genomic DNA was then extracted from peripheral blood nucleated cells. TPMT gene polymorphisms were detected by using real time-polymerase chain reaction (RT-PCR). All samples were analyzed to detect the G238C (rs 1800462) transversion in TPMT*2, the G460A (rs 1800460), and A719G (rs 1142345) transitions in TPMT*3 alleles. Genotypes were determined by using a TaqMan^® genotyping assay.

Clinical Data Collection and Patient Follow-up

All patients’ medical files were reviewed to detect the AZA doses and its associated ADRs as well as any change in the treatment plans. Follow-up of all patients continued until the end of the study (April 30, 2020).

Myelosuppression Laboratory Values Used During Analysis

Neutropenia: ANC (absolute neutrophil count) <2000 cells/mm³

Mild neutropenia: ANC between 1000 and 1500 cells/mm³

Moderate neutropenia: ANC between 500and 1000 cells/mm³

Severe neutropenia: ANC <500 cells/mm³

Thrombocytopenia: a platelet count <100000/µL

Mild anemia: hemoglobin between 9.5 and 10.9 g/dL

Moderate anemia: hemoglobin between 8 and 9.4 g/dL

Severe anemia: hemoglobin between 6.5 and 7.9 g/dL

Life-threatening anemia: hemoglobin <6.5 g/dL

Statistical Analysis

Data were analyzed using IBM SPSS Statistics Version 22. Data were tested for normality using the Kolmogorov–Smirnov test and the Shapiro–Wilk test. Quantitative data were presented as mean and standard deviation while qualitative data were presented as number and percentage. Analysis of variance test was used to compare numerical variables between the different categories of SNPs. Chi-squared test or Fisher's exact test as appropriate were used to compare categorical variables between the different categories of SNPs. P-value set significant at 0.05 levels. All tests were two-tailed.

Ethical Approval

The study was approved by the ethical committee for grants of the British University in Egypt.

This study was conducted in accordance with the Declaration of Helsinki.

Results

This study investigated genetic polymorphism in the TPMT gene in a total of 150 patients with a mean age of 35.85 years. Table 1 shows the demographics and patients’ characteristics. The study determined the prevalence of three mutant alleles; TPMT*3 G460A (rs 1800460), TPMT*2 G238C (rs 1800462), and TPMT*3 A719G (rs 1142345). About 72% of patients were heterozygous in the TPMT*3 G460A and TPMT*3 A719G mutant alleles and 81% were wild type in the TPMT*2 G238C mutant allele as shown in Table 1. Seventy percent of patients’ diagnoses were systemic lupus erythematosus. A dose of 100 mg (1*2=one 50 mg tablet *2) was the initial dose in 94% of patients and about 75% of patients were maintained on the same dose. Abnormal liver function tests were detected in 42% of patients. Myelosuppression was represented as anemia which was detected in 63% of patients, leucopenia in 51%, and thrombocytopenia in 25% of patients. The AZA-clinical failure occurred in 50% of patients where AZA discontinuation or shift to another drug (in 45% of patients).

Table 1 Demographics and Patients Characteristics

Table 2 shows the frequencies of homozygous, heterozygous, and wild type carriers of a mutant allele of the TPMT*3 G460A. Myelosuppression was presented as anemia and leucopenia and there was a statistically significant difference between the homozygous, heterozygous and wild type carriers of the mutant allele of the TPMT*3 G460A as shown in Table 2. There was also a statistically significant difference between the three types in regard to the initial and maintenance doses, the severity of anemia as well as the treatment shift parameter.

Table 2 AZA Clinical Efficacy and Associated ADRs in Different Types of TPMT *3 G460A Mutant Allele

Table 3 shows the frequencies of homozygous, heterozygous, and wild type carriers of the mutant allele of the TPMT*2 G238C. Myelosuppression was presented as anemia and leucopenia and there was a statistically significant difference between the homozygous, heterozygous, and wild-type carriers of the mutant allele of the TPMT*2 G238C as shown in Table 3. There was also a statistically significant difference between the three types in regard to the severity of anemia as well as the treatment shift parameter.

Table 3 AZA Clinical Efficacy and Associated ADRs in Different Types of TPMT 2* G238C Mutant Allele

Table 4 shows the frequencies of homozygous, heterozygous, and wildtype carriers of the mutant allele of the TPMT*3 A719G. Abnormal liver function tests (LFTs) were presented as elevation in the aspartate transaminase (AST) and alanine transaminase (ALT) and there was a statistically significant difference between the homozygous, heterozygous and wild type carriers of the mutant allele of the TPMT*3 A719G as shown in Table 4. There was also a statistically significant difference between the three types in regard to the treatment shift parameter. Myelosuppression rates were higher in homozygous patients in the three mutant alleles, but it was statistically significant in TPMT*2 G238C while not statistically significant in TPMT*3 G460A and TPMT*3 A719G. Females had a higher risk of immunosuppression than males (p-value 0.031) as shown in Table 5.

Table 4 AZA Clinical Efficacy and Associated ADRs in Different Types of TPMT 3* A719G Mutant Allele

Table 5 Myelosuppression Rates in the Different Types of TPMT Mutant Alleles

Discussion

This current study detected the frequencies of three mutant TPMT alleles; TPMT*3 G460A, TPMT*2 G238C, and TPMT*3 A719G. About 72% of patients were heterozygous in the TPMT*3 G460A and TPMT*3 A719G mutant alleles and 81% were wildtype in the TPMT*2 G238C mutant allele. These findings are consistent with the results of many other studies conducted by Kubota and Chiba; Kumagai et al; and Hiratsuka et al, these studies showed that in Caucasians TPMT*3A (460G>A: rs1800460 and 719A>G: rs1142345) represents the most frequent allele, whereas TPMT*3C (719A>G: rs1142345) represents the most frequent allele in the Japanese population.^17–19 In this current study, TPMT*3 G460A, TPMT*2 G238C, and TPMT*3 A719G are the most common defective alleles of the TPMT gene examined. About 23% of patients were homozygous in the TPMT*3 G460A mutant allele; the rates of heterozygous and wild type were 71% and 6%, respectively. The percentages of homozygous, heterozygous and wild type in the mutant allele TPMT*2 G238C was 13, 6, and 81%, respectively, while these rates were 18, 71, and 11% in the TPMT*3 A719G mutant allele.

Our results were discordant with a study conducted on 194 Egyptian patients done by Hamdy et al that concluded that the frequency of wild type allele was 97% and 3% of patients who were heterozygous for one of the mutant alleles. TPMT*1/TPMT*3A was detected in one patient, while TPMT*1/TPMT*3C was detected in five subjects. Neither G238C (TPMT*2), G460A alone (TPMT*3B) nor the homozygous type of any mutant allele was determined in the Egyptian population. Their study also concluded that TPMT*3C is the inherited TPMT variant allele in the Egyptian population, accounting for 86% of the variant alleles detected. TPMT*3A is the second most recurrent variant and accounts for the remaining 14% of the TPMT variant alleles in Egyptians.¹⁶

In comparison with other global populations across Asia, Europe and the Americas, the gene variant that occurs most frequently in Caucasians is TPMT*3A (approximately 85%), *2, *3B and *3C occur much less frequently. In Africans and African Americans, as in Caucasians, *1 is the wild type, but the most common allele is TPMT*3C.^20,21 Regarding the Asian population, TPMT*3C is also the most common mutation reported while, TPMT*3A does not occur.^22–24 TPMT activity, in addition to nudix hydrolase 15 genetics, is a critical factor contributing to thiopurine-induced hematotoxicity.²⁵ TPMT activity differs among patients as about 87% of patients have high TPMT activity, 11.1% have intermediate activity, and 0.3% have low or absent activity.^26–28 Patients with normal TPMT activity are categorized as the wild type TPMT*1.²⁹ Patients (~1 in 178 to 1 in 3736) who are homozygous-deficient universally develop severe bone marrow suppression; an increased percentage of those who are heterozygous develop moderate-to-severe bone marrow suppression, and those who are homozygous for wild type TPMT alleles are at reduced risk of myelosuppression.¹¹ These results were consistent with our findings as 72.5% of homozygous-type carriers of the mutant allele TPMT*3 G460A developed anemia and leucopenia. Anemia and leucopenia represented 80% and 84% of homozygous-type carriers of the mutant allele TPMT*2 G238C and 59% for each of them of homozygous-type carriers of the mutant allele TPMT*3 A719G.

Myelosuppression was presented as anemia, leucopenia, or thrombocytopenia; these rates were higher in homozygous patients in the three mutant alleles, but it was statistically significant in TPMT*2 G238C while it was not statistically significant in TPMT*3 G460A and TPMT*3 A719G. This study showed ADR as well as another study that concluded that patients who inherit two nonfunctional TPMT alleles are at 100% risk for life-threatening bone marrow suppression and that heterozygous patients cannot tolerate full doses of AZA.^30,31 This current study showed that females had a higher risk of immunosuppression than males (p-value 0.031). This finding is supported by many other studies that concluded that TPMT activity increases in children compared with adults, between adults, it is elevated in males rather than in females.^32–34 During patients follow-up, AZA was discontinued in 50% of patients due to ADRs development, while treatment shift occurred in 45% of patients. Another retrospective study was conducted in the UK and included 113 patients with inflammatory bowel disease mentioned that AZA discontinuation has resulted from AZA adverse effects.³⁵ In this current study, about 94% of patients initiated on a total of 100 mg dose (<2 mg/kg) and 75% of patients could not tolerate a maintenance dose more than this dosing range. Our findings were supported by another study which concluded that there was a good clinical response in patients who received low doses of azathioprine (<2 mg/kg). Relapse or lack of response was associated with using of low-AZA dose in patients with high TPMT activity.³⁶

Conclusion

The study provided an overview of the genomic variations in the Egyptian population. The therapy should be started with a low dose of azathioprine, slowly increase the dose—under close blood counts—until an optimal effect is achieved without the occurrence of side effects.

Routine TPMT genotyping prior to the initiation of AZA therapy may be helpful for Egyptian patients in order to determine whether the drug is appropriate for each patient. Further studies should be conducted to compare the cost-effectiveness of prior screening before starting treatment and treating patients without screening in the Egyptian population. Genotyping will likely provide great pharmacologic and economic benefits in countries like Egypt.