First of all, we thank the author for evaluating our article.1 As the author and we pointed out in our article, the Forkhead box P3 (FOXP3) gene is on the X chromosome. As far as we know, ten different reports in the literature have investigated the association between FOXP3 polymorphisms and the development of Graves’ disease (GD).2, 3, 4, 5, 6, 7, 8, 9, 10, 11 Seven of those reports presented the genotype and allele frequencies by pooling both sexes,2, 3, 4, 5, 6, 7, 8 and the rest stratified the participants by sex and reported the genotype and allele frequencies separately in females and males.9, 10, 11 In our article, we aimed to evaluate the frequency of FOXP3 polymorphisms in GD with or without ophthalmopathy in a Turkish population.1 Since the number of participants in each group was limited in our study and there were no corrections for the previously published articles,2, 3, 4, 5, 6, 7, 8 we did not stratify the groups by sex and used the results of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) directly to analyze genotype and allele frequencies.1 In the PCR-RFLP method, the results were shown by band patterns and those patterns do not indicate whether the sample had one or two alleles of the gene of interest. Additionally, using the same analysis method, we could compare our results with those seven reports that did not stratify the groups by sex.
However, as mentioned by the author, as males have one and females have two X chromosomes, the genotypic patterns differ between the two sexes. There are three genotypes (two homozygotes and one heterozygote) in females but only two hemizygous genotypes in males. Therefore, it will be better to stratify the groups by sex and define the genotype and allele frequencies separately in females and males for X-linked genes. Moreover, the author emphasized the importance of comparing the observed and expected genotypic values based on Hardy-Weinberg equilibrium (HWE) in his letter and explained the formula for an X-linked polymorphic locus. According to the concerns reported by the author, in this reply, we reported our additional analysis by stratifying the groups by sex as shown in Tables 1, 2, 3, and 4.
Firstly, we did the HWE analysis in both the controls and study groups, as suggested by the author. The frequencies of the observed genotypes were not significantly different from their expected frequencies based on HWE for all single nucleotide polymorphisms (SNPs) in female control groups (rs3761547, p=0.926; rs3761548, p=0.881; and rs3761549, p=0.926).
Secondly, when the groups were stratified by sex, we found that the frequency of the AC and AA genotypes of -3279 (rs3761548) and the CT genotype of -2383 (rs3761549) were significantly increased in our female study group (Table 1). Additionally, the A allele of -3279 had a significantly increased frequency both in the female (Table 1) and male study groups (Table 3) when compared separately or when compared between controls and patients regardless of sex (Table 4). The frequency of the T allele of -2383 was significantly increased both in our female study group when compared separately (Table 1) and in the whole study sample when compared regardless of sex (Table 4). However, the frequency of the T allele of -2383 was not significantly increased in our male study group when compared separately (Table 3). For polymorphism -3449 (rs3761547), allele and genotype frequency distribution were not significantly different in any comparisons between the control and study groups (Table 1, Table 3, and Table 4).
Thirdly, comparing the genotypic and allelic distributions of each of the three FOXP3 SNPs between female patients with Grave’s ophthalmopathy (GO) and GD without ophthalmopathy (non-GO) showed no statistically significant difference (Table 2).
Consequently, the results in our published article and the analysis made by stratifying the participants by sex were similar. However, as the author emphasized in his letter, polymorphic loci on X-chromosome should be analyzed differently than the loci on autosomal chromosomes. Therefore, his letter and our reply will be helpful for researchers who will investigate associations with X-linked polymorphic loci.
