Association between TPO gene polymorphisms with susceptibility to hyperthyroidism

Introduction and Aim: Hyperthyroidism is a disorder characterized by excessively high amounts of tri-iodothyronine and thyroxine in the bloodstream. TPO is a thyroid-specific antigen. TPO gene mutations affect thyroid hormone synthesis, causing full or partial iodide organification abnormalities. In this study, we aimed to look at the single nucleotide polymorphisms (SNPs) in the TPO gene and find possible relation of these SNPs to the development or prognosis of hyperthyroidism. Materials and Methods : Blood samples were collected from hyperthyroid patients (n=75) and healthy individuals (n=25). The concentration of triiodothyronine (T3), Tetra-iodothyronine (T4), and thyroid stimulating hormone (TSH) were estimated in all samples. The autoantibodies TPOAbs and TR-Ab levels were measured using the ELISA assay. The extracted genomic DNA from blood of participants was investigated for polymorphism in the SNPs rs1126799, rs1126797, and rs732609 within the TPO gene. Statistical methods were used to evaluate the association between the TPO SNPs studied to hyperthyroidism susceptibility. Results : No significant correlation was observed between the SNPs studied and prognosis of hyperthyroidism. However, it was found that hyperthyroid patients carrying the TPO rs1126799 T allele (CT + CC genotypes) had significantly higher serum levels of TPOAb compared to those with the TT genotype (P<0.01). Similarly, hyperthyroid patients with the TPO rs1126797 C allele (CT + TT genotypes) and TPO rs732609 A allele (CA + AA genotypes) exhibited significantly elevated levels of TPOAbs compared to individuals with the CC genotype and AA genotype, respectively (P<0.01). Conclusion: There was no correlation between the TPO rs1126799, rs1126797, and rs732609 polymorphisms with the occurrence of hyperthyroidism in the Iraqi population. However, TPO rs2071400 and rs2048722 polymorphisms were found to be correlated to serum levels of TPOAb.


INTRODUCTION
hyroid peroxidase (TPO) is a heme-binding protein primarily found on the apical membrane of thyrocytes.The activity of TPO is crucial for the process of thyroid hormonogenesis (1).Autoimmune thyroid diseases (AITDs) are a group of autoimmune disorders that affect approximately 5% of the global population.However, the prevalence of AITDs can vary among the populations associated with the levels of iodine intake (1)(2)(3)(4).Thyroid peroxidase gene mutations are inherited as autosomal recessive traits and this alteration in the structure of the enzyme leads to the production of anti-TPO autoantibodies, which can potentially contribute to the destruction of the thyroid gland (5,6) and the impairment of thyroid hormone production can occur as a result of total or partial defects in iodide organification (7,8).During the last 20 years, different loci, such as MHC, CTLA-4, PTPN22, and TSHR genes have been linked to autoimmune thyroid disease (AITD) (9,10).Several mutations have been documented in the TPO gene, which include 103 deleterious mutations, 66 missense mutations in addition to nonsense mutations, deletions, insertions and duplications (11).Human TPO gene exhibits alternative splicing, resulting in the generation of multiple TPO isoforms that may lack one or more exons.Alternative splicing of TPO gene has also been reported in other species as well (12,13).
Multiple autoimmune diseases, including but not limited to CTLA4 and PTPN22, share common susceptibility genes, suggesting the existence of overlapping genetic and molecular pathways among these conditions.This evidence indicates that autoimmune diseases may exhibit similarities in their underlying genetic and molecular mechanisms (14,15).Previous studies have revealed the existence of numerous susceptibility genes associated with autoimmune thyroid diseases (AITD).Broadly speaking, these susceptibility genes can be categorized into two main types.The first type comprises thyroidassociated genes which include: TSHR (thyroidstimulating hormone receptor) and TG (thyroglobulin).The second type consists of immunity-related genes, such as FOXP3, which play a role in immune system regulation (16), HLA-DR genes (17), and CD40 (15).This study aimed to examine the single nucleotide polymorphisms (SNPs) in the TPO gene to elucidate the potential relation between TPO gene T polymorphisms and the onset as well as the prognosis of hyperthyroidism.

MATERIALS AND METHODS
This study included 75 patients diagnosed with hyperthyroidism at the National Diabetes Center, AL-Mustansiriya University, Iraq between July and November 2021.In addition, the study also included 25 healthy volunteers who served as controls.Both genders and various age groups were represented among the patients and healthy controls.A specific questionnaire form was used to record the medical history of the patients.

Inclusion and exclusion criteria
Hyperthyroid patients were diagnosed based on early hormonal tests, specifically focusing on thyroid function, without considering the presence of other symptoms.However, hyperthyroid patients with comorbidities such as diabetes mellitus, hypertension, cardiovascular diseases, as well as pregnant women, were excluded from the study.

Measurement of thyroid hormones and autoantibodies
Blood samples were collected from all participants.Serum levels of thyroid hormones (total triiodothyronine (T3) and tetra-iodothyronine (T4) in addition to the level of thyroid stimulating hormone (TSH) were quantified using the Enzyme Linked Fluorescent Assay (ELFA) kit (Biomerx, France).Additionally, the levels of autoantibodies (TPOAbs and TR-Ab) were determined by using the Enzyme Linked ImmunoSorbent Assay (ELISA) kit (Demiditec, Germany).The assays were performed as per the manufacturer's instructions.

Extraction and genomic DNA sequence
Genomic DNA was extracted from the peripheral blood samples obtained from recruited patients by using Zymo Research Quick-DNA™ Miniprep Kit.DNA concentrations were examined by using the Nanodrop spectrophotometer.The promoter regions spanning the SNPs rs1126799, rs1126797, and rs732609 SNPs of the TPO gene were subjected to PCR amplification using precise primers as shown in Table 1.PCR cycles performed in a Thermocycler (Applied Biosystems, USA).
The volume of PCR mixture (25 μL) was composed of several components which includes: extracted DNA, MgCl2, primer dNTP and Taq DNA polymerase.The amplification process involves several serial steps including denaturation step at the temperature of 95°C for 30 seconds, annealing step at the temperature of 57-62 °C for 30 seconds, and finally the extension step at temperature of 72°C for 30 seconds, for 25 cycles.The products of PCR cycle with amplicon sizes 406, 423 and 210 bp respectively (Table 1) were subjected to direct sequencing (Macrogen Company, Korea) utilizing the Sanger sequencing method.The obtained sequences were aligned online with the corresponding TPO gene sequences as found in the National Center for Biotechnology Information (NCBI) database, using the Bioedit software.

Statistical methods
To evaluate the allele and genotype associations for each SNP, χ2 and Fisher exact tests were employed.Additionally, a χ2 test utilized each SNP in both case and control individuals.

Clinical characteristics
Hyperthyroidism is characterized by hyper metabolism and elevated serum level of T4 and T3 and low TSH level.In this study relation between hyperthyroidism and serum levels of thyroid hormones and TSH were determined.The results presented in Table 2 demonstrated that hyperthyroid patients had high significant differences in levels of free T3 and T4 compared to healthy controls (p<0.01).However, these levels still remained within the normal range.The current study also revealed a significant decrease in serum TSH levels among hyperthyroid patients (p<0.001) as described in Table 2. Furthermore, this study investigated the levels of TPOAbs among hyperthyroid patients.The results indicated a significant increase (p<0.01) in TPOAbs levels in the serum samples of hyperthyroid patients compared to healthy controls.

SNP rs1126799 T>C
The occurrence of homozygous non-polymorphic genotype (TT), heterozygous polymorphic genotype (TC) and homozygous polymorphic genotype (CC) in hyperthyroid patients were observed to be 24%, 28 % and 48% respectively (Table 3).While the frequency of these genotypes in healthy control subjects were 12%, 20% and 68 % respectively.Furthermore, the frequencies of T and C alleles among hyperthyroid patients were 38% and 62% respectively, while in healthy control allele frequencies of T and C alleles were 22% and 78% respectively.Considering T allele as a reference, the allele distribution among hyperthyroid patients and healthy was found to be non-significant.
In addition to that C and T allelic frequency in patients was 64% and 36% respectively, while in healthy control it was 76% and 24% respectively.No significant distribution was observed for alleles between hyperthyroid patients and healthy controls, with C allele as a reference allele (Table 4).

SNP rs732609 A>C
Table 5 shows results for the occurrence of homozygous non-polymorphic genotype (AA), heterozygous polymorphic genotype (AC) and homozygous polymorphic genotype (CC) in hyperthyroidism patients which is 76 %, 16% and 8% respectively.Similarly in the control group the frequency of these genotypes was observed to be 80%, 12% and 8% respectively.On the other hand, allele frequencies for A and C among hyperthyroid patients were 86% and 14% respectively, while in healthy controls the allele frequency of A and C was 84% and 16% respectively.These results showed that the distribution of A and C alleles among both hyperthyroid patients and healthy control was nonsignificant with A allele taken as a reference allele (Table 5).

Serum levels of TPOAbs among polymorphic genotypes
Results showed that the concentration of TPOAbs among the hyperthyroid, were T allele carriers (TC + CC genotypes) of SNP rs1126799 T>C (Fig. 1A), C allele carriers (CT + TT genotypes) of SNP rs1126797 C>T (Fig. 1B), and A allele carriers (AC + CC genotypes) of SNP rs732609 A>C (Fig. 1C) were significantly higher than those of wild-type genotypes (TT for rs1126799 T>C, CC for rs1126797, and AA for rs732609 respectively (p<0.01).However, it was observed that the serum levels of T4, T3, TSH were significantly different (p<0.01) in heterozygous and homozygous polymorphic genotypes than their levels in homozygous non polymorphic genotypes for the studied SNPs.

DISCUSSION
The thyroid gland plays a vital role in the synthesis of thyroid hormones (T3 and T4) by regulating the levels of TSH hormone produced by the pituitary gland.In patients with hyperthyroidism, there is often an elevation in the levels of thyroid hormones (T4 and T3) accompanied by a decrease in TSH hormone levels (18)(19)(20).Although T4 is the primary product of the thyroid gland, it is the biologically active T3 form that exerts physiological effects (21).Normally, the thyroid gland produces T4, which is then converted to T3 with the help of the TPO enzyme.However, when there is a defect in the TPO enzyme, the conversion of T4 to T3 is hindered, leading to an increase in T4 levels.The findings of this study revealed an excessive production of both T4 and T3 thyroid hormones in hyperthyroid patients which is in line with the findings by Yasaman et al., (22), who reported that TSH levels tend to decrease in hyperthyroid patients due to the negative feedback inhibition exerted by T3 and T4 on the anterior pituitary gland.
Thyroid antibodies are proteins that are produced in the blood as a response to the presence of foreign proteins (antigens).These antibodies develop when the immune system mistakenly target thyroid gland cells and tissues causing damage in the thyroid organ.The presence of these antibodies is associated with autoimmune thyroid disorders, including Graves' disease (characterized by hyperthyroidism) and Hashimoto's thyroiditis (characterized by hypothyroidism).In this study, the relation between thyroid autoantibodies was studied, results of which showed that the TPOAbs are more often raised in hyperthyroid patients which is like findings reported earlier (10,23).

TPO polymorphisms
In this study, we investigated the relation among the frequency of TPO polymorphisms and incidence of hyperthyroidism.Results showed that the frequency of the TPO rs1126799 T>C, rs1126797C>T, and rs732609 A>C were not associated with the incidence of the disease, due to non-significant difference in the dominant polymorphic alleles among both hyperthyroid patients and healthy controls, which refers that these SNPs are not a risk factor for the incidence of hyperthyroidism.

rs1126799 T>C
rs1126799 SNP in exon 15 of TPO gene was also investigated.Results showed there is no significantly association between rs1126799 TPO gene polymorphism and the incidence of hyperthyroidism due to non-significant difference in the dominant polymorphic C allele among both hyperthyroid patients and healthy controls with odd ratio 1.19 and 0.65, which mentioned that the dominant polymorphic C allele is not a risk factor for the incidence of hyperthyroidism.These findings are similar to those obtained in the Japanese population (9), but not in the Iranian population (24).

rs1126797C>T
Results of this study showed that there is no significant relation with rs1126797 TPO gene polymorphism and the development of hyperthyroidism due to nonsignificant differences in dominant polymorphic T allele among hyperthyroidism patients and healthy controls (odd ratio of 0.71).This result indicates that the dominant polymorphic T allele is not a risk factor for hyperthyroidism.However, the results of the recessive model (odd ratio=6.25) of inherited T allele were seen as a risk factor for hyperthyroidism incidence.These findings are in line with a similar work by Tomari et al., (10) who reported a lack of association between rs1126797 TPO gene polymorphism and hyperthyroidism in the Japanese population.However, this SNP was reported to be associated with the incidence of hyperthyroidism in the Egyptian population (25).

rs732609A>C
Our study found no significant relationship between rs732609 TPO gene polymorphism and the incidence of hyperthyroidism in the studied group of Iraqi patients.A non-significant difference was observed for the dominant polymorphic C allele among hyperthyroid patients and healthy controls which indicates that the dominant polymorphic allele C is not a risk factor for the incidence of hyperthyroidism.On the other hand, results indicated that the recessive model of the inherited C allele was also not a risk factor for developing hyperthyroidism, as the odd ratio was 1.3.The results of this study exhibit resemblance to the outcomes observed in the Japanese population (10), while demonstrating dissimilarity to findings in other study populations (24)(25)(26).However, our study shows the possibility of establishing an association between the rs1126799 T>C, rs1126797C>T, and rs732609A/C polymorphisms and autoimmune hyperthyroidism by examining the correlation between these genetic variations and the concentration of TPOAbs in hyperthyroid individuals of various genotypes.This also assumes significance, as previous studies have shown SNP mutations within genes to be associated with various genetic diseases (27,28).

CONCLUSION
There is no correlation between the TPO rs1126799, rs1126797, and rs732609 polymorphisms with the occurrence of hyperthyroidism in the Iraqi population.TPO rs2071400 and rs2048722 polymorphisms, on the other hand, were shown to be associated with serum TPOAb levels.

Table 1 :
The primers were used for amplification of TPO regions

Table 3 :
The genotypic and allelic frequencies of TPO rs1126799 polymorphism among hyperthyroid and healthy individuals

Table 4 :
The genotypic and allelic frequency for TPO rs1126797 polymorphism among hyperthyroid and healthy individuals

Table 5 :
The genotypic and allelic frequency for TPO rs732609 polymorphism among hyperthyroid and healthy individuals