Genotype and Phenotype Distribution in Hashimoto's Thyroiditis

Abstract

The study investigates the genotype and phenotype distribution in patients with Hashimoto's Thyroiditis (HT) and healthy controls, focusing on the association of CTLA-4 (-1661A/G) and PTPN22 (R620W) polymorphisms. Bioinformatics analysis is employed to assess the potential biological functions of these polymorphisms, including their impact on transcription factor binding sites and protein structure. The results indicate a significant association between the CTLA-4 -1661A/G polymorphism and HT, shedding light on the genetic factors contributing to this autoimmune disease.

Computational bioinformatics analysis was employed to assess the potential biological functions of these polymorphisms, focusing on their impact on transcription factor binding sites and protein structure. The results reveal a significant association between the CTLA-4 -1661A/G polymorphism and HT, providing valuable insights into the genetic factors contributing to this autoimmune disease.

Introduction

Hashimoto's Thyroiditis (HT) is a chronic autoimmune thyroid disease characterized by inflammation and destruction of the thyroid gland. While the precise etiology of HT remains elusive, it is widely accepted that both genetic and environmental factors play pivotal roles in its development and progression.

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Genetic studies have identified several polymorphisms associated with autoimmune thyroid diseases, including HT. Among these, the CTLA-4 (-1661A/G) and PTPN22 (R620W) polymorphisms have garnered significant attention due to their roles in immune regulation and potential implications in autoimmunity.

CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4) is a critical regulator of T-cell activation and immune responses. Polymorphisms in the CTLA-4 gene have been implicated in various autoimmune diseases, suggesting its importance in maintaining self-tolerance and preventing autoimmunity.

PTPN22 (Protein Tyrosine Phosphatase Non-Receptor Type 22) encodes a phosphatase that plays a crucial role in modulating T-cell receptor signaling. The R620W polymorphism in PTPN22 has been associated with multiple autoimmune diseases, highlighting its potential impact on immune dysregulation.

This study aims to investigate the genotype and phenotype distribution of HT patients and healthy controls, focusing on the CTLA-4 (-1661A/G) and PTPN22 (R620W) polymorphisms. We employ computational bioinformatics analysis to explore the potential biological functions of these polymorphisms, including their effects on transcription factor binding sites and protein structure. Understanding the genetic underpinnings of HT can provide valuable insights into its pathogenesis and may pave the way for more targeted therapeutic approaches.

Materials and Methods

The study enrolled 82 HT patients (91.5% female) and 100 healthy controls (85% female) with a mean age of 39.4±11 and 38.4±11.8 years, respectively. All participants provided informed consent, and ethical guidelines were strictly followed throughout the study.

Genomic DNA was extracted from peripheral blood samples collected from each participant. Genotyping for CTLA-4 (-1661A/G) and PTPN22 (R620W) polymorphisms was performed using polymerase chain reaction (PCR) followed by DNA sequencing. The resulting genotype and allele frequencies were determined and subjected to statistical analysis.

Results

The genotype and allele frequencies for the CTLA-4 (-1661A/G) and PTPN22 (R620W) polymorphisms are presented in Table 1.

Statistical analysis revealed no significant difference in genotype and allele frequencies for the PTPN22 (R620W) polymorphism between HT patients and healthy controls. However, for the CTLA-4 (-1661A/G) polymorphism, a positive association was observed between the heterozygous genotype (AG) and HT (P = 0.04). Additionally, genotype AA for CTLA-4 and genotype CC for PTPN22 were considered dominant, with genotype TT absent in the patient group.

Furthermore, the genotype frequencies for the PTPN22 polymorphism were in Hardy-Weinberg equilibrium (P = 0.7), indicating a stable distribution of genotypes in the study population. In contrast, the CTLA-4 genotype frequencies deviated from equilibrium (P = 0.01), suggesting a potential influence of this polymorphism on HT susceptibility.

Potential Biological Functions of CTLA-4 -1661A/G and PTPN22 R620W

The -1661A/G variant (rs4553808) in the CTLA-4 gene was subjected to in-depth bioinformatics analysis to explore its potential impact on gene regulation. This variant was predicted to be located within the core recognition sites of transcription factors C/EBPβ and GR in the CTLA-4 gene promoter region. The transition of allele A to allele G was found to result in the loss of these critical binding sites (Figure 3).

Similarly, the R620W polymorphism (rs2476601) in PTPN22 was investigated using computational tools to assess its potential biological effects. This polymorphism is located at positions 1947, 1858, and 620 of the transcript, coding sequence (CDS), and PTPN22 protein, respectively.

The physicochemical properties of PTPN22 protein were compared between the wild-type and 620R mutant phenotype, revealing significant differences (Table 2). Key parameters, such as isoelectric point, instability index, and grand average of hydropathicity, exhibited variations between the two phenotypes, suggesting potential functional consequences of the polymorphism (Table 2).

The hydrophobic score at position 620 in PTPN22 was notably different between tryptophan and arginine residues (Figure 4A). Ramachandran plot results confirmed distinct phi-psi coordinates for the mutant phenotype compared to wild types (Figure 4B). Additionally, predicted secondary structure analysis highlighted differences in residue 620, with a coil form in normal PTPN22 and a sheet form in the mutant protein (Figure 4C).

Further analysis aimed to understand the potential impact of the C1858T variation on the local RNA secondary structure of PTPN22. In silico results suggested that this SNP does not induce significant structural changes in the mRNA secondary structure (distance: 0.007, P-value: 0.8487; P-values less than 0.2 are considered significant) (Figure 5).

Moreover, a comprehensive examination of the three-dimensional structure of the mutant and normal proteins was conducted to investigate the effects of the C1858T polymorphism on PTPN22 structure and folding. Bioinformatic servers were utilized to assess the conservation of DNA sequences containing the CTLA-4- rs4553808 and PTPN22- rs2476601 polymorphism sites across multiple mammalian species (Figure 7).

Discussion

Hashimoto's Thyroiditis (HT) is a complex autoimmune disease characterized by immune-mediated destruction of the thyroid gland. Although the exact pathogenesis of HT remains incompletely understood, it is evident that both genetic and environmental factors contribute to disease susceptibility.

In this study, we investigated the genetic aspects of HT, focusing on two key polymorphisms: CTLA-4 (-1661A/G) and PTPN22 (R620W). These polymorphisms have been associated with various autoimmune diseases, prompting interest in their potential roles in HT.

Our results indicate that the CTLA-4 -1661A/G polymorphism is significantly associated with HT, with a positive correlation observed between the heterozygous genotype (AG) and disease susceptibility. This finding suggests that the presence of the G mutant allele may contribute to the development of HT. This is in line with previous studies that have reported associations between CTLA4 gene polymorphisms and various autoimmune diseases, although findings across different populations have been inconsistent.

CTLA-4 is a critical immune checkpoint receptor that regulates T-cell activation and immune responses. The loss of binding sites for transcription factors C/EBPβ and GR due to the -1661A/G polymorphism may disrupt the regulation of CTLA-4 gene expression, potentially leading to impaired self-tolerance and autoimmunity.

In contrast, the PTPN22 (R620W) polymorphism did not exhibit a significant association with HT in our study. This finding is consistent with the low frequency of the T allele in our study population, which suggests that this allele may not play a substantial role in HT susceptibility in this specific cohort.

PTPN22 encodes a phosphatase involved in T-cell receptor signaling, and the R620W polymorphism has been linked to altered immune responses in other autoimmune diseases. However, our results indicate that this particular polymorphism may not be a major contributor to HT in this population.

Further investigations into other genetic variations within these genes and the involvement of additional genes are warranted to comprehensively understand the genetic basis of HT. It is important to note that the pathogenesis of HT is likely influenced by a complex interplay of multiple genetic and environmental factors, making it a challenging and intriguing area for future research.

Conclusion

In conclusion, this study provides valuable insights into the genetic factors contributing to Hashimoto's Thyroiditis. The positive association between the CTLA-4 -1661A/G polymorphism and HT suggests a potential role for this polymorphism in the pathogenesis of the disease. Further research is needed to unravel the intricate genetic networks involved in HT and to explore the potential for targeted therapeutic interventions based on these genetic insights.