Genetic polymorphism of CTLA4 with functional thyroid tests in hypothyroid disease patients

Objective : To recognize the role of Cytotoxic T-Lymphocyte Antigen 4 CTLA4 gene polymorphism (rs231775A/G) in Hashimoto's thyroiditis (HT) patients and a study its correlation with other clinical data. Design and Methods Case-control research was conducted on 200 females, with 100 patients with Hashimoto's thyroiditis aged 18-68 years and 100 age and gender-matched healthy volunteers.


Introduction
Hashimoto's thyroiditis (HT) is an autoimmune disease, that can cause hypothyroidism in which the immune system turns against thyroid glands, in another meaning, the immune system makes antibodies that attack the thyroid follicles, which resynthesize and store thyroid hormones in a large protein called thyroglobulin these antibodies included, ant thyroglobulin (anti-TG) and anti-thyroid peroxidase (anti-TPO), which act as biochemical markers of HT illness(1) (2).Autoantibodies against TPO are found in more than 90% of HT patients, while (anti-TG) antibodies are found in approximately 80% (3).In addition, anti-TPO antibodies are found in about 10% of females, while other studies indicate that disease incidence is eight times higher in females than in males especially in elderly women, while Anti-TG antibodies are found in about 18% of elderly women (4).patients with various autoimmune disorders, such as primary adrenal hypofunction, Gravis disease, rheumatoid arthritis, pernicious anemia, gluten enteropathy, lupus disease, and type 1 diabetes, are more likely to develop Hashimoto's thyroid (5).Furthermore, many pathogeneses are linked to the interaction of hereditary and environmental variables (6).Additionally, the most prevalent cause of hypothyroidism is iodine deficiency; however, in areas where iodine usage is adequate, HT is the most common cause of hypothyroidism (7).T lymphocytes that are specific for thyroglobulin are created and sent to the thyroid glands, where they release cytokines such CTLA-4which aid in the death of thyrocytes (8).When the immune system is passively attacking and breaking down the thyroid follicles in the early stages of Hashimoto's thyroiditis, elevated levels of triiodothyronine (T3) and thyroxine (T4) are produced in the peripheral blood by destroyed thyroid glands cells, simulating a transient hyperthyroid state (9).Thyroid autoimmunity has been related to a polymorphism in the TG gene, which codes for TG forms with various immunological activities (10).Genetic susceptibility to autoimmune thyroid disease had been well investigated and verified in twin and family investigations.The formation, progression, and severity of autoimmune thyroid disease have all been related to some genes (11).HLA, CTLA-4, PTPN type-22, VDR gene, thyroglobulin gene, and cytokines such as interferon -induced helicase1 gene IFIH1, are all believed to have a role in the HT disease (12).Antigen4 is a fundamental immunosuppressive cytokine that is mostly expressed on activated T cells (13), it is a protein receptor encoded by the CTLA-4 gene, which is situated on the long arm of chromosome 2 at position 33.2 (2q33.2).It has four exons and three introns, it belongs to the immunoglobulin superfamily and encodes a protein that sends an inhibitory signal to T lymphocytes (14).The protein contains a V domain, a Tran's membrane domain, and a cytoplasmic tail.Alternative variations of transcriptional splices have been identified, each of which codes for a different isoform.The membrane-bound isoform is a disulfide-bond entangled homodimer, while the soluble isoform is a monomer.It is converted into a peptide of 233 amino acids.The CTLA-4 protein consists of a signal peptide and the main chain (the first 35 amino acids) (15).The most recent evidence of genome-wide interaction is that the CTLA-4 gene is a key factor combining the environmental with genetic factors in the pathogenesis of multiple autoimmune illnesses including systemic lupus erythematosus (SLE), Hashimoto'disese, type I diabetes, psoriasis, and vitiligo (16).

Participants
A case-control study was applied to a total number of 200 females who were classified into 100 patients with Hashimoto's thyroiditis aged 18-68 years and 100 age and gender-matched healthy volunteers.Patients were recruited from Al-Sadder medical city in Al-Najaf Governorate, Iraq.All participants underwent medical examinations to make sure they were suffering from Hashimotos thyroiditis.The subjects included in this study were selected according to inclusion and exclusion criteria, inclusion criteria involved, firstly all patients who had been previously or recently diagnosed by a physician as having autoimmune hypothyroidism (Hashimoto's thyroiditis, in addition to these patients, have all clinical signs and symptoms of Hashimoto's thyroiditis, secondly all patients have a high level of thyroid antigen tests (thyroid peroxidase and thyroglobulin).
Exclusion criteria included thyroidectomy or under radioactive iodine treatment, nonthyroidal systemic disorders including acute and chronic hepatic, renal, cardiovascular, and cerebrovascular diseases, as well as benign and malignant tumors.and patients with other autoimmune illnesses including rheumatoid arthritis, diabetes, systemic lupus erythematous (SLE), and celiac disease.

Measurements
A venous whole blood sample of five milliliters was taken from each of the participants in this study.The blood was separated into two parts and labeled as follows: 3 mL of blood was placed in a gel activator tube, then subjected to centrifuge for ten minutes at 3000 Xg and divide into small aliquots (0.5 mL) for measuring thyroid function tests, anti-TPO and anti-TG antibodies.The remaining two milliliters of whole blood were collected in K3-EDTA tubes, then DNA was extracted and the extract was kept frozen at −80 °C to be used for genotyping of CTLA4 polymorphism (rs231775A/G) by real-time polymerase chain reaction (PCR).Polymorphism within gene CTLA4 (rs231775A/G) was done by real-time PCR and allelic discrimination assay utilizing a TaqMan probe, Applied Alpha DNA (Canada).The primers, probes, and Master Mix (80×) were also supplied by Promega (USA).The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR cycles.TaqMan® probes (quencher dye) and TaqMan® MGB probes.0.2 μl Eppendorf tube in a total volume of 25mL using 2μg of genomic g DNA and a qPCR Master Mix GoTaq® Probe.The tubes were then placed in a thermal cycler, and heated at 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute.

Statistical analysis
Results were tabulated and statistically analyzed by using a personal computer using MICROSOFT EXCEL 2010 and SPSS v. 26 (SPSS Inc., Chicago, IL, USA).
Statistical analysis was done using: Descriptive: e.g.mean and standard deviation.
Analytical: that includes: Chi-Squared (χ2), t-test.A value of P less than 0.05 was considered statistically significant.

Results
This case-control study was conducted on a total number of 200 females.These included 100 patients with 100 age and gender-matched healthy subjects as a control group.Patients were (100 %) female their age ranged from 18 to 68 years with a mean of 38.40 ± 11.18) years.Control subjects were also only women their ages ranged from 18 to 68 years with a mean of 39.25 ± 12.20 years, as shown (In table 1).
The p-values of body mass index (BMI) were calculated in patients with Hashimoto's thyroiditis and control groups, it was non-significant variation between both groups in the present study (P<0.45)and the mean ± SD was (30.21±5.32,and 29.75±5.70)respectively Hashimoto's thyroiditis patients presenting with an aggressive form of hypothyroidism disease had significantly lower levels of T3, and T4, in addition to FT3 and FT4 (p˂0.000) each other when compared with control who had normal values of these hormones.
The outcomes showed a significant increase in TSH, Anti TPO, and AntiTG levels in Hashimoto's thyroiditis patients compared with healthy control as shown in In (figure 2) the frequency of the alleles of CTLA4 SNP rs231775 (A/G) has mildly different, as it shows that the G allele is (0.551 %) in groups of patients while it is ((0.522%) in the control group, which means the G allele is more frequent in patients group than a control group.
On the other hand, the A allele is (0.272%) in the patient group and (0.485%) in the control group.That means the A allele is more frequent in the control group than in the patients group.The Genotype frequencies of CTLA4 SNP rs231775 (A/G) was not consistent with Hardy-Weinberg equilibrium in control persons as seen in (table2).
In addition to, same result obtained, there were no significant variation when compared healthy control and HT patients in dominant, recessive and additive models which examined by multinomial logistic regression analysis show (Table 4) Finally, (Table 5) shows the biochemical characteristics of subjects with HT which studied according to the CTLA4 SNP rs231775 (A/G) genotype.The biochemical features of (Age, BMI, T3, T4, TSH, fT3, fT4, TPO, Anti-TG) which relative to Codominant model of CTLA4 SNP rs231775 (A/G) were studied by ANOVA test (table 5), while those of dominant model were examined by t-test (Table 6).The results in (Table 5) demonstrate a non-significant association of the Codominant model in T3, T4, TSH, fT3, fT4, TPO, and Anti-TG, the same result  studies, no correlation was identified between the +49A/G SNP of CTLA-4 and autoimmune diseases, including HT in Italy (21).and Lebanon (21).
In the current study, the outcomes show no significant differences in allele or genotype frequencies for the rs231775 SNP between Hashimotos thyroiditis patients and control subjects.In addition, there was no significant difference between cases and controls concerning CTLA4 genotype distribution with a predominance of AG and GG genotypes in studied cases.Indeed, there was a mild difference between cases and controls regarding CTLA4 alleles 53% had a G allele while 47% had an A allele.
While, in controls, 52% had a G allele while 48% of cases had an A allele.In studies conducted, results were obtained similar to those obtained in this study conducted by Mehrnaz Narooie -Nejad (22).
In contrast, another study carried out on rheumatoid arthritis disease included 1,200 samples in Chinese which identified significant associations observed between alleles of CTLA4 (rs231775A > G, P = 0.007, OR = 1.17, 95%CI = 1.04-1.30),and RA disease susceptibility (19).Demonstrating the importance of CTLA-4 in the modulation of T cell responses, as well as the breakdowns in the B7-CD28/CTLA-4 pathway may alter T cell response and affect autoimmune diseases (23).In theory, reduced expression or function of CTLA-4 could lead to autoimmune T cell proliferation and contribute to the pathogenesis of autoimmune diseases such as HT (24).
The mechanisms by which CTLA4 polymorphism contribute to HT pathogenesis remain to be explored, it is clear that the CTLA4 gene locus has a role in the susceptibility of all autoimmune thyroid diseases such as HT.Single point mutation rs231775 at the CTLA4 gene may be a potential risk factor for HT susceptibility but in more sample size.In addition, CTLA4 is a good candidate gene for autoimmune thyroid disease.Several studies carried out in different populations suggest that more than one CTLA4 polymorphisms or SNP is related to this disease, that is mean not only dose rs231775 polymorphism affect the HT susceptibility but there are many other polymorphisms more effective on the HT susceptibility (10) (25).
In conclusion, current findings identified that fact; there is no association between rs231775 SNP of the CTLA4gene and the development of HT disease in the present study population, with the limitation of the present small sample size.Finally, more new and powerful research is needed to explain the role of CTLA4gene in AITD including HT disease.

Figure 2 :
Figure 2: The Percent Frequency of A&G alleles in Two Groups According to the HWE of CTLA-4 SNP rs231775