The prevalence of polymorphisms of thiopurine s- methyltransferase gene In Iranian alopecia areata patients

Azathioprine therapy was recently used to treat dermatologic conditions such as alopecia areata (AA). Previous reports showed that thiopurine s-methyltransferase (TPMT) activities in human red blood cell are associated with a polymorphism in this gene. Therefore, patients carrying mutant allele of TPMT may show severe myelosuppression when they are treated with standard doses of Azathioprine drugs. This study aimed to evaluate the TPMT gene amongst Alopecia areata patients and healthy adult in Iranian populations. TPMT gene polymorphisms were investigated in 1285 Iranian healthy adult blood donors and 632 patients with Alopecia Areata Universalis (AU). Tetra Arms PCR, Real-Time PCR and Sequencing were used to evaluate the presence of allele-specific polymorphisms of TPMT gene (TPMT *2(c.238 GC,), TPMT *3A (c.460 GA and c.719 AG), TPMT *3B (c.460 GA), and TPMT *3C (c.719 AG). Results were shown that the TPMT*2 allele is associated with a low enzymatic activity that was detected in 22.51% (863 in 1917) of Iranian individuals. Heterozygous genotypes were in 827 (43.14%) subjects (232 AA and 595 healthy), and homozygous genotypes were in 18 (0.94%) individuals (3 AA and 15 healthy). The normal allele (wild-type) was found in 55.92% of the studied individuals (20.70% AA and 35.21% healthy). According to a higher frequency of TPMT polymorphism in Iranian population in comparison with other population, determination of TPMT genotype in may have the clinical benefit to thiopurine dosage selection and treat patients as well.


INTRODUCTION
Alopecia areata was known as hair loss of all or part of the body and classified as an autoimmune disease (Sadeghi et al., 2015;Golnoosh Taghiabadi et al., 2018). Unfortunately, several therapeutic ways which have been presented to treat alopecia areata were not curative or preventive. Also, many of therapeutic agents may have a risky efficacy of result for patients in the long-term (Alsantali, 2011). Thiopurine therapy is used to treat dermatologic conditions, rheumatic disease, inflammatory disease, autoimmune disease such as alopecia areata, organ transplant rejection, certain types of cancer such as acute lymphoblastic leukaemia (Lee et al., 2017;Murugesan et al., 2010;Caufield and Tom, 2013;Broekman et al., 2017;Liu et al., 2015). It has been proven thiopurines, 6-mercaptopurine, 6-thioguanine and azathioprine have a toxic effect on hematopoietic cells that may cause red blood cell macrocytosis, leukopenia, selective erythroid hypoplasia, severe anaemia and rarely pancytopenia (Lee et al., 2017). Thiopurines a purine analogue that intervenes the DNA synthesis of leukocytes and results in DNA damage and inhibits the proliferation and leads to cell death and myelosuppression (DiPiero and Hicks, 2015;Farfan et al., 2014) and thus decreases proliferation of cells, especially T and B lymphocytes (Alsantali, 2011;Liu et al., 2015). Metabolism of azathioprine is based on several pathways which are oxidation catalysed by xanthine oxidase (XO) and S-methylation catalysed by thiopurine methyltransferase (TPMT). This inactivates 6mercaptopurine through methylation to 6-methyl mercaptopurine (Murugesan et al., 2010;Moran et al., 2015). In alopecia areata and other skin autoimmune diseases, Azathioprine is responsible for the reduction of the number of Langerhans cells and other antigenpresenting cells in the skin (Alsantali, 2011). TPMT gene has 27kb length and is located on chromosome 6p22.3 with ten exons (Murugesan et al., 2010;Oro, 2008).
It is previously reported that TPMT activity plays a significant role in human tissue, and controlled by genetic polymorphism.
TPMT polymorphism has been recognised as a risk factor in pharmacogenetic studies and the best example for prediction of individual response to specific medicine with the use of genetic information. TPMT gene locus variation exhibit an incidence of life-threatening myelosuppression, severe toxicity due to thiopurine drugs intake (Murugesan et al., 2010). Several studies express that TPMT activity in human RBC is trimodal: -high activity, inheritance of two functional copies (90%) -intermediate activity, heterozygotes for deficient alleles (10%) -low or no detectable activity, inheritance of two nonfunctional copies (0.3%) (Murugesan et al., 2010;Caufield and Tom, 2013).
In the last group, thiopurine therapy accumulates excessive levels of active 6-mercaptopurine that turned to cytotoxic 6-thioguanine nucleotide analogues (TGNA) which may lead to hematologic toxicity, whereas heterozygous patients in risk of toxicity, too (Meijer et al., 2017;Lee et al., 2017). Extensive studies have shown that single nucleotide polymorphisms (SNPs) placed in enzymes that metabolise thiopurine drugs universally prescribed for autoimmune disease such as alopecia are prognostic factors for investigation of the toxicity and activity of TPMT level in patients with alopecia (Jimenez-Morales, 2016;Oro, 2008).

Subject selection and sampling
Subjects were included 632 patients with AA and 1285 healthy adult subjects. The age range of subjects was 40 to 50 years old and had not any history of autoimmune or allergic diseases or D3 vitamin deficiency. Also, there was no cousin marriage up to three previous generations. Subjects recruited from the different areas of Iran. K2 EDTA tube (VACUETTE® EDTA) was used to collect 2cc of the peripheral blood sample. All participants have signed a written informed consent.

DNA extraction and primer
Genomic DNA was extracted from blood samples according to protocol DNA extraction of Cinna Pure DNA (PR881612-EX6001) kit. Extracted DNA's quality was measured by both 1.5% agarose gel electrophoresis and Denovix Nanodrop device (Model Ds-11). The concentration of extracted DNA was about 250 mg/ml. Specific primers were designed using oligo 7 software and synthesised by SinaClon Company (Table1).
Three area of known TPMT gene mutations being responsible for TPMT deficiency (c.238 G›C, c.460 G›A, and c.719A›G) were determined according to the three methods Tetra Arms PCR, Real-Time PCR and Sequencing.
In brief, allele-specific PCR amplification was used to detect the c.238 G›C transversion in exon 5, c.460 G›A and c.719 A›G mutations in exon 7 and 10, respectively.

Tetra arms PCR
The final volume for all PCR reactions was 25 ml in which genomic Dezhgir et al. 69 DNA (200 to 250 ng) was amplified according to the protocol in Table 2. The reaction was used Taq DNA Polymerase Master Mix RED 2X-MgCl2; 1.5 mM ampliqon kit (#180301-50). Also, an advanced biometric thermocycler was used. The protocol of PCR amplification for three mention polymorphism included of an initial denaturation step at 95°C for 5 min followed by 32 cycles of denaturation at 95°C for 30 s, annealing at 61.7°C (c.238 G›C), 58.3°C (c.460 G›A), 55°C (c.719A›G) for 30 s and extension at 72°C for 30 s. The final extension step was 72°C for 5 min.
Electrophoresis analysed PCR products in 1.5% agarose gel and Thermo Scientific Gene Ruler 100 bp DNA Ladder. Explanation of DNA fragments was present in Figure 1.  Sequencing PCR was carried out by using an Outer primer (reverse and forward). Then PCR product was sent to SinaClon Company to sequence PCR production in both forward and reverse directions. The purpose of that confirmation of tetra arms PCR was. Sequencing results were analysed by Finch TV (Figures 2 and 3).

Real-time PCR
Polymorphisms above (TPMT*2, TPMT*3C, TPMT*3B, TPMT*3A) were assessed by real-time PCR using Corbett machine (Corbett rg6000, Australia). First, one μl of DNA template (20 ng/μl) and ten μl of a real-time PCR master mix (2xRealTime PCR Master Mix Green-no Rax Kit (#A323402-25)), as shown in Table 3, were used to perform real-time PCR. Three real-time PCR reactions should detect one sample. The tube I detected the mutation of the TPMT gene, tube II detected the wild type of TPMT gene and the tube III detected negative control. Real-time PCR was performed using the following conditions: 95°C for 5 min, followed by 45 cycles of 95°C for 30 s and 61.7°C (c.238 G›C), 58.3°C (c.460 G›A), 55°C (c.719A›G) for 30 s and 72°C for 20 s. All standards and samples were performed in triplicate. Real-time PCR results were analysed by gene runner 6000 software (Figure 4).

Statistical analysis
The data were presented as percentages and also were analysed by using SPSS version 24. 95% confidence intervals (95% CI) were estimated for all diagnostic tests. Proportions assessed categorical variables, and differences amongst the groups were compared using Fisher's exact chi-square analysis. P-values less than 0.05 were considered as statistically significant.

RESULTS
In the present study, Tetra Arms PCR and Real-Time PCR methods were used to study the polymorphisms: TPMT*2, TPMT*3C, TPMT*3B, TPMT*3A. Then DNA sequencing was carried out to ensure the accuracy of previous results. Outcomes of all used techniques were the same.
Among the 1917 Iranian participants (1285 healthy adults and 632 patients with AA), 78 were from area in the western north of Iran (4.06%) (28 AA patients and 50 controls with frequency of 35.90 and 64.10%, respectively), 88 were from the Lurs population (4.59%) (60 controls (68.18%) and 28 AA patient (31.81%)), 166 were from the Kurd population with a frequency of 8.66% (56 AA patients (33.73%) and 110 healthy adults (66.26%)), 324 (16.90%) subjects were from central and near the southern area of Iran included 213 of Esfahan, 87 of Shiraz and 24 of Kerman (220 healthy adult (67.90%) and 104 patient with AA (32.10%)), 28 were from the northern area of Iran with a frequency of 1.46% (8 AA patients (28.57%) and 20 healthy adults (71.43%)), 246 were from the southern area of Iran (12.83%) (76 AA patients (30.89%) and 170 healthy adults (69.11%)),  216 were from the eastern north of area of Iran (11.27%) (76 AA patient and 140 controls with a frequency of 35.18% and 64.81% respectively) and 771 subjects were from capital of Iran and another area close to it (589 of Tehran, 74 of Karaj, 84 of Arak, 24 of Kashan) with a frequency 40.22% (515 controls (66.80%) and 256 AA patient (33.20%)) ( Figure 5). The frequency of the TPMT*2 in the populations of the different regions of Iran are showed in Figure 6 and Table  4c.
Differences in the TPMT alleles, in the population of healthy adult and patient with alopecia areata, no significant relationship were found between them (Pvalues > 0.05) (Table 4a).
We found four new mutations not reported their relation with genetic susceptibility polymorphisms for taking Thiopurine drugs. Three of which were located in exon 5, and one other is located in exon 7. The first identified mutation was 1814381 A >G, 18143811-10 deletion GA in the non-coding area. Both of them were detected in the same subject. the other identified mutation, were 18143870 del T in exon 5 and C 18138983 T: ATT ⇾ ATC(isoleucine ====>> isoleucine) in exon 7 (Figure 3).

DISCUSSION
One of the conventional drugs prescribed for the treatment of autoimmune patients, including alopecia areata, is mercaptopurine family medicine such as azathioprine. TPMT-deficient patients are not able to treat with standard doses of mercaptopurine (6MP), and even treatment can be fatal in these patients (Lee et al., 1995;Schutz et al., 1993). Heterozygous patients have common activity, and homozygous patients have low or no activity (Rossi et al., 2001;McLeod et al., 1993;McLeod et al., 2000;Jimenez-Morales et al., 2016) (4, 34, 13 and 35).
TPMT activity in most races worldwide is known. The present study is the first assessment of TPMT allele's frequency in Iranian patients with AA population. The current research has conveyed that 43.14% of the Iranian population carringTPMT2* allele heterozygous and 0.94% carringTPMT2* allele homozygous with an overall allele frequency of TPMT allele was 22.51% in the Iranian population. That is a relatively high repartition for this polymorphic gene in Iranian compared with an array of the ethnic groups such as European (Jimenez-Morales et al., 2016), American, Latin American (Zeglam et al., 2015, Caucasians (Collie-Duguid et al., 1999) and other Asian nations (Kham et al., 2002;Lu et al., 2005;Srimartpirom et al., 2004).
In contrast to those previous reports in other national populations, TPMT*3C and TPMT*3A alleles were the most popular mutant alleles in the other studied population (Zeglam et al., 2015), they were not detected       in Iranian population. On the other hand, TPMT*2 was found to be the most prevalent mutation among Brazilians (Boson et al., 2003), Turks (Zeglam et al., 2015) and Iranians (Zeglam et al., 2015), and also in the present study, it was detected with the most common frequency. The remarkable point is that, although TPMT*2 is a rare mutant allele found in Asian populations, it was the majority proportion of our subjects carried it and this is the amount was much higher than previous studies related to Iranian population as well. This indicates the importance of TPMT testing before prescribing drug. About half of the population of Iran is susceptible to the typical dose of that drug in another hand it is toxic and would be fatal for them (44.08%). Although this study was not designed to distinguish differences in allele frequency between the different races of Iranian populations, it was determined by differences in allele frequency between the various areas of Iran.
As aforementioned, there was no significant difference between the patient and healthy subjects (P-values > 0.05). The highest frequency of mutated TPMT*2 alleles was found in the population of the capital of Iran and another area close to it (483 of 771 individuals), then the northeastern regions of Iran (104 of 216 individuals) with the frequencies 26.13 and 5.43%, respectively. It has also illustrated that the overall frequency of TPMT*2 alleles in the areas were 13.56 and 2.71% in Iranian population than 55.97% of its proportion belongs to the population of the capital of Iran and its near cities (Table  4).
In our study, the northwestern cities and southern area of Iran have the lowest share of the TPMT2* allele frequency (19 of 78 subjects with an allele frequency of 0.50% and 32 of 246 individuals with an allele frequency of 0.83%, respectively).
Via carefully examining three generations of family descent of participants in the project, it became conspicuous that most of the people were living in Tehran returned to Azeri, Tabari, Geake and Fars ethnicities. This seems to be expected owing to the high diversity of ethnic groups in the Tehran population. Given the limited access to ethnic and racial information in the project, only the ethnicities being available were examined. The results of this study showed that among the various Iranian ethnicities (Azeri, Kurd, Lurs, Baluch, Lak, Gilak, Tabari, Turkmen, Arab, Jewish, Armenian and Fars) only Azeri (223 of 378 subjects), Fars (220 0f 725 subjects), Arabs (32 of 193 individuals), Tabari (49 of 78 individuals), Gilak (53 of 85 individuals), Kurds (49 of 78 individuals) and Lurs (56 of 88 individuals) ethnicities were identifiable with a frequency of TPMT2* allele 6.51, 6.42, 0.93, 1.43, 1.55, 2.30 and 1.63%, respectively.
In this study, the frequency of allele was different among females and the male population. Although, equal populations were selected in both groups (848 females (44.24%) and 1069 males (55.76%)), the frequency of the TPMT2* heterozygous allele mutant in the male population (patients group and healthy group) was significantly higher than female groups (29.37% male and 13.77% female with a frequency allele of 14.68 and 6.88%, respectively) (CI = 95% odd ratio = 1). However, Int Res J Med Med Sci 76 the overall frequency of TPMT *2 mutant alleles of the female and male group in Iranian population were 7.88% and 9.80% respectively, and it was not significant statistically (p-values < 0.05). This demonstrates more importance to study of the TPMT gene in the both was equal (Table 4b).
Results from our study revealed that TPMT*2 was the most frequent mutant allele among Iranian population with remarkably superior frequency than other national populations and this offer that 22.50% of Iranian population might have an increased hazard of thiopurineinduced toxicity. Giving this fact that Xanthine oxidase enzyme (XO) in hematopoietic tissues is absence, TPMT seems to be only the critical enzyme causing to allay thiopurine drugs in these tissues, and thus consumption of that drug by the patients with TPMT deficiency might rapidly cause potential myelotoxicity (Zeglam et al., 2015;Srimartpirom et al., 2004). Hence determination of TPMT genotype in advance of prescribing any thiopurine dosage might have clinical benefit to treating Iranian patients as well. However, it is highly possible that other rare mutants alleles may be detected in some of the wildtype samples. Therefore, investigation of other variations in the TPMT gene would be useful.