Association of TSHR gene single nucleotide intronic polymorphism with the risk of hypothyroid and hyperthyroid disorders in Yazd province

The present study was carried out, for the first time, to evaluate the association of rs2268458 polymorphism, biochemical and environmental factors on hypothyroid and hyperthyroid disorders in thyroid patients and healthy individuals in Yazd province, Iran. In this study, blood samples were collected from a total of 100 cases, including 60 hypothyroid, 20 hyperthyroid and 20 normal individuals. DNA was extracted from blood samples and the rs2268458 single nucleotide intronic polymorphism was evaluated using Restriction Fragment Length Polymorphism PCR (RFLP-PCR). The results have shown that 59 individuals were homozygote (TT), 40 cases were heterozygote (TC) and one homozygote (CC) case. Of 59 TT homozygote cases, 25 cases were hypothyroid females and 7 hypothyroid male patients. While, heterozygote TC group consisted of 20 hypothyroid females and 7 hypothyroid male cases. Furthermore, only 1 (CC) homozygote male hypothyroid patient was observed in this study. The hyperthyroid population consisted of 7 (TT) homozygote hyperthyroid female cases, 8 (TC) heterozygote hyperthyroid female cases, 3 (TT) homozygote hyperthyroid male cases and 2 (TC) heterozygote hyperthyroid male cases. According to our study, heterozygote cases (TC) showed less severe symptoms, while homozygote cases (TT) showed no serious symptoms and the (CC) homozygote case showed severe thyroid abnormalities. So, it can be concluded that the TSHR-related rs2268458 polymorphism is associated with hypothyroidism and hyperthyroidism in the male and female populations of Yazd Province, Iran and C allele can be a risk factor for some physio-biochemical and hormonal imbalance in the thyroid disorder patients.


Materials and methods
Study population. A population-based cohort study was performed during the years 2019-2020 in Yazd, a metropolitan city of Central Iran. In short, male and female (n = 80) hypothyroid and hyperthyroid patients, and 20 healthy candidates (in total = 100 candidates) were selected and acquired the informed consent to participate in the study. The present study was approved by Human Research Ethics Committee; Academic Center for Education, Culture and Research (ACECR), Iran and the research was conducted in accordance with the Declaration of Helsinki. These patients were examined by a trained general practitioner and an endocrinologist. All data including demography, age, past history of thyroid and any other disorders, smoking history and use of medications were collected. The patients were also examined for goiter or nodules. The body mass index (BMI) was calculated by dividing weight (kg) to the height square (m 2 ).
Blood sample collection and genomic DNA extraction. Fasting blood samples were collected from hypo-and hyper-thyroid patients who matched the study criteria. Blood (5 mL) was withdrawn and transferred into plain tubes (3 mL) and EDTA tubes (2 mL). The blood samples in the plain tube were centrifuged after 30 min of sampling for serum collection, which was then stored at − 20 °C and stored for further analysis. Samples were assayed in duplicate and the mean of the paired results were analyzed. EDTA tubes were stored at − 20 °C for genomic DNA extraction. Genomic DNA was extracted and purified from whole peripheral blood samples using Blood Genomic DNA Extraction Kit (Pars Tous Biotechnology Co; Mashhad, Iran). DNA sample were stored at − 86 °C in aliquots for further analysis.
TSHR-SNP-rs2268458 analysis by PCR-RFLP. PCR amplification of the TSHR gene was carried out using PCR Master Mix (Taq DNA Polymerase Master Mix, Ampliqon A/S, Odense, Denmark). Based on the analysis of 19 , we examined TSHR-SNP-rs2268458, located in intron 1, using standard RFLP-PCR protocol 19 . Human genomic DNA (about 25 ng) was amplified by PCR machine and 415-bp products were generated, as described above. The primer sequences used for this analysis are as follows:  20,21 . Concentration and DNA purity was calculated using nanodrop with a purity range of 1.8-2.0 (ScanDrop 2 , Analytik Jena, Germany). The final amplification mixtures were then electrophoresed on 2% agarose gel for 45 min at 90 V. Visualization of the DNA fragment bands was done using Gel documentation system. Then, 8 μL of PCR products was digested for 2 h in 10 μL total volume with the restriction endonuclease Alu I (Therno Fisher Scientific Inc.) overnight according to the manufacturer's instructions. RFLP-PCR was carried out to varify the wild allele and the rs2268458 SNP variant with Alu I restriction enzyme along with a undigested DNA control. After digestion, the digested fragments were loaded onto 2% agarose gel and visualized by ethidium bromide and gel documentation to carry out genotype pattern analysis. Since Alu I digestion determines AGTT versus AGCT, this allowed the determination of each individual's hetero-or homo-genotype.
Exclusion criteria. The patients not fulfilling our study criteria and AITD subjects having other underlying diseases, thyroid cancer, renal failure, mental illness, liver failure, systemic diseases or chronic respiratory diseases were excluded from this study. Statistical analysis. The resultant data were analyzed using SPSS (version 22.0). In bivariate analysis, the numeric variables were calculated using t-test when there was a normal distribution and Mann Whitney/Kruskal Wallis test for abnormally distributed data. The bivariate analysis for categorical variables used χ 2 -squared test. For the multivariate determination, the logistic regression was used as the dependent variables were also categorical variables.

Results
The extracted DNA was clear and not fragile (Fig. 1A). PCR product of TSHR locus showed a single 162 bp band (Fig. 1B). Figure 1C shows the pattern of PCR products on agarose gel electrophoresis, related to TSHR gene    Table 1 and Fig. 3A,B, the analysis of hypothyroidism and hyperthyroidism was carried out in male and female populations, representing that the females were more likely to suffer from thyroid disorders. Moreover, hypothyroidism was predominant among study patients as compared to hyperthyroidism (p < 0.05). Table 2 shows the association of individual's age and prevalence/severity of thyroid disorders. According to our results, older study population had had higher rate of thyroid disorders (p < 0.05). While, according to Table 3, the BMI of the individual was also directly related to the thyroid dysfunction. Our results suggest that hypothyroid patients were over-weight with BMI of 25-29.9 or obese with BMI of 30 or above. While, most of the hyperthyroid patients were underweight with BMI of < 18.5.
Allele and genotype analysis. The distribution of genotype frequencies for SNP s2268458 as case-control association analysis and all has been shown in Table 4 and Fig. 4. The major allele TT genotype in s2268458 of TSHR gene was observed in 42 patients and 17 normal individuals. While, the major allele CC genotype in s2268458 was observed in only 1 patient and the normal individuals did not show this genotype. Among 100 cases, 80 cases were patients and 20 cases were normal. In total, there were 59 TT, 40 TC and 1 CC. Among TT cases, 42 of them were patients and 17 of them were normal cases. Among TC cases, 37 of them were patients and 3 of them were normal cases. There was just one CC hypothyroid male case detected in our study. According to Table 5, Among the 100 tested cases, 75 cases were female and 25 cases were male. 59 cases were TT, 40 cases were TC and one case was CC. Among TT cases, 46 cases were female and 13 cases were male. Among TC cases, 29 cases were female and 11 cases were male. Just one case was CC that was a male. According to Table 6, our analysis on 60 hypothyroid and 20 hyperthyroid cases showed that 42 cases had TT genotype, 37 cases were TC and 1 case was CC. Among TT cases, 32 cases were hypothyroid and 10 cases were hyperthyroid. Among TC cases, 27 cases were hypothyroid and 10 cases were hyperthyroid. And one CC was observed to be hypothyroid.

Discussion
During the present study, hypothyroidism was the most frequent thyroid problem among our patients but these results are not in accordance with all studies 22,23 but has a coordination with some 24 . In Iran, legislation of using iodized salt was established in 1994, however goiter and urinary iodine concentration remained elevated in many Iranian provinces. A previous population-based study assessed the prevalence of hypothyroidism in Isfahan, Iran. They found that hypothyroidism was common (12.8% of women and 4.7% of men) and probably due to autoimmunity with no correlation to iodine intake 25 . However, whether this difference is due to geographic distribution of thyroid problems or genetic or environmental difference among nations should be elucidated in well-structured ecologic studies. Present study confirmed previous reports in which female gender was described as the most frequent one among hypothyroid and hyperthyroid patients. In our study, the mean age of women with MG was lower than men 26,27 , however, these results were not statistically significant. Our study did not show gender difference in ages less than 20, however, till 60 years of age, female gender was the most frequent one among www.nature.com/scientificreports/ patients and thereafter, men were the most common presenting gender too. These findings are in accordance with previous studies 19, [28][29][30] . Since, 20-50 years is the reproductive period of women and before climacteric period, we hypothesize that the specific hormonal balance may have a role in presentation of clinical features of MG patients possibly as an exacerbating factor. Our study did not show any clinical presentation difference among MG patients with and without thyroid problem as well no para-clinical assessment EMG (electromyographic), RNS (Repetitive Nerve Stimulation), AchR-Ab (Acetylcholine Receptor Antibody)) difference was found between these two groups. Whether these findings are pure clinical finding or a consequent of not having enough study subjects remain to be elucidated by future studies with more patients. AITD is characterized by immunogenicity of the major thyroid antigens (Tg, TSHR and TPO) 29 . The study of intronic polymorphisms has been entertained because we now know that intronic DNA may be responsible for regulatory small RNAs as well as providing and/or influencing different start sites for TSHR mRNA generation 31,32 . The thyroid cells express a variety of TSHR mRNA splice variants, indicating that SNPs or small RNAs in this intronic DNA may be important in the generation of different receptor forms and/or their control. Recently, a study from Singapore demonstrated an association of a TSHR intron 1 SNP with GD. SNPs in intron 7 of the TSHR were also found to be associated with GD in Japanese, and SNPs in intron 1 of the TSHR were reported to be associated with GD in Caucasians 16 . So the result is, there is an association between rs2268458 with hypothyroidism and it has been confirmed that the allele C has a pathogenic effect on recessive, dominant and co-dominant but the biggest effect is on recessive allele and it has been perceived that the allele C is a pathogenic allele, which is usually a recessive hereditary pattern 15 . In this study, homozygote cases (TT) will not show the symptoms and they only have a 162 bp band, heterozygote cases (TC) show some of the symptoms or some light symptoms and they have 3 bands: 162, 100 and 62 but the homozygote cases (CC), show sever symptoms and they have 2 bands 100 and 62 bp which agrees www.nature.com/scientificreports/ with the Iraq's study who reported that TSHR rs2268458 polymorphisms were associated with hypothyroidism 15 and disagrees with the Lebanon's study who reported that TSHR rs2268458 polymorphisms were not associated with hypothyroidism and hyperthyroidism 26 . According to 20 , there was a correlation between relapse of Graves' disease and CC genotype of TSHR gene on the rs2268458 of intron 1 20 . In a study, related to thyroid disease association with polymorphism in Iraq, 73 cases were homozygote (TT), 20 cases were heterozygotes (TC) and 1 case was homozygote (CC) and since all the research was done on females, the homozygote (CC) case was a woman 13 but in this study, both men and females were involved and the homozygote case (CC) was a man. This shows a homozygote case (CC) can be seen in both females and men provided, both genders are studied and a large number of people are involved in the study.

Conclusion
To our knowledge, this is the very first study showing an association of TSHR gene rs2268458 polymorphism with hypothyroidism and hyperthyroidism in Iran and particularly in Yazd province. In this study, it has been shown that rs2268458 leads to a high risk in having hypothyroidism and hyperthyroidism, particularly hypothyroidism and since in yazd province some other factors such as smoking, stress, bad eating habits, not exercising and so on are high, rs2268458 is more highlighted and it leads to more number of thyroid related diseases such as hypothyroidism and hyperthyroidism. Besides, it has been confirmed that thyroid disorders are more common among females as men.