It is postulated that the paternal and maternal antigens will be recognized by the immune system within the thymus, and T cells that have a high affinity for such antigens will be deleted by apoptosis [1–3]. The lack of exposure to a self-antigen in the thymus may lead to the presence of autoreactive T cells and increase the risk of autoimmunity [4]. In female mammalian cells, one of the two X-chromosomes is inactivated in early embryonic life [5]. Thus, females are mosaics for two cell populations, cells with either the paternal or the maternal X in the active form. X-chromosome choice is assumed to be random, and the result is generally 50% of cells expressing the paternal and the remaining 50% expressing the maternal genes [6]. A skewed X-chromosome inactivation (XCI) is a deviation from this ratio and is arbitrarily defined, for example, as a pattern where 80% or more of the cells inactivate the same X-chromosome [7]. This deviation may be the result of chance or genetic factors involved in the XCI or a selection process [8]. The existence of XCI in females offers a potential mechanism where by X-linked self-antigens may escape presentation in the thymus or in other peripheral sites that are involved in tolerance induction [9, 10]. This has become an attractive candidate mechanism for breakdown of self-tolerance in autoimmune diseases. An association between skewed XCI and scleroderma was recently reported [11]. A higher frequency of a skewed XCI pattern was found in patients affected with autoimmune thyroid diseases (AITDs) compared with healthy controls, indicating that skewed XCI may be associated with a predisposing factor for the development of AITDs [12–14]. It was therefore of interest to study if there is an association between skewed XCI and rheumatoid arthritis (RA) as a non-organ-specific and AITDs as an organ-specific autoimmune disease. We investigated the peripheral blood XCI patterns of 106 females affected with RA, 145 females affected with AITDs and 257 controls in the Tunisian and Turkish populations. Extremely skewed XCI was found in the blood samples of female patients affected with RA and AITDs supporting the role of skewed XCI in female predisposition to autoimmune diseases.

Extremely skewed XCI have been reported in 1 to 2% of 20 to 40 year old women, and in 2 to 4% of 55 to 72 year old women [19]. The data for RA and AITDs patients is strikingly bimodal, we plotted the distribution of the X inactivation profiles according to age. However, we did not observe a shift toward the skewed range in older patients and controls (Figures 1, 2 and 3). Characteristics of the RA and AITDs patients with skewed XCI are shown in Tables 2 and 3.

At the time of sample collection, 66 patients affected with RA were being treated with immunosuppressive therapies (methotrexate 10 to 15 mg once a week, n = 33; D-penicillamine 300 mg/day, n = 17; plaquenil 400 to 600 mg/day, n = 16). Among 76 informative patients, 46 were received immunosuppressive agents (61%). A major concern with the observed XCI patterns among RA patients was that concomitant immunosuppressive therapy could influence the results, as has been observed in feline hematopoietic cells [20]. Analysis of the data on XCI patterns according to immunosuppressive therapy did not reveal a statistically significant association between RA patients treated with methotrexate and controls (P = 0.52).

The majority of human autoimmune diseases are characterized by female predominance. RA and AITDs have a female:male ratio of approximately 3:1 and 9:1, respectively [21]. Sex hormone influences have been suggested to explain this phenomenon because the X-chromosome contains a considerable number of sex and immune-related genes such as AR, IL2 receptor gamma chain, CD40 ligand and FOXP3 [22, 23]. These genes are essential in determining sex hormone levels and, more importantly, immune tolerance [24]. The contribution of genetics to sex differences in autoimmune diseases is currently unexplored. An alternative explanation for the female predominance has been recently proposed with the finding of an enhanced skewed XCI in peripheral bloods cells of female patients with autoimmune diseases [11–14]. The present study tests the hypothesis that skewed XCI would be more prevalent in females affected with autoimmune diseases than in female control individuals. Therefore, we simultaneously examined skewed XCI in 106 patients affected with RA and 145 patients affected with AITDs. The control group consisted of 170 female age-matched healthy individuals. We have demonstrated a significantly higher prevalence of extremely skewed XCI in blood cell of females affected with RA and AITDs compared with the control group (P < 0.0001; P = 0.0015, respectively), indicating a possible role of XCI in the etiology of autoimmune diseases, and in the female preponderance of RA and AITDs.

Skewed XCI was more commonly expected in peripheral blood mononuclear cells due to the very high rate of turnover of blood cells compared with other solid tissues [25]. Then, we have examined XCI in peripheral blood mononuclear cells of patients affected with RA and AITDs, and we found a higher incidence of skewed XCI in those patients. We also tested the relationship between XCI and AITDs phenotypes (GD and HT). A skewed XCI was associated with both GD and HT (P = 0.0001 and P = 0.04). Although, our results suggest the involvement of XCI in female predisposition to RA and AITDs, this hypothesis still to be confirmed in specific tissue, because our analysis was performed in DNA from blood, and this may not be a representative tissue for all autoimmune diseases [26, 27] and there may exist locally skewed XCI in the thymus. Moreover, this study can be complicated by existing differences in peripheral blood mononuclear cells constituents in RA versus healthy controls. The XCI distribution in both Tunisian and Turkish controls (Figure 3) according to age showed that 19.5% (9 of 46) have a skewed XCI in Tunisian controls which have a mean age of 43.5 years, whereas only 8% (10 of 124) in Turkish controls with a younger mean age (35 years). This result suggests the importance of age in the difference of XCI skewing.

Our results are in agreement with those reported by Ozçelik and colleagues on 110 unrelated Turkish female AITDs patients and 160 female controls that showed a greater proportion of a skewed pattern of XCI (34%) than in controls (8%; P < 0.0001) [13]. Indeed, supporting data have been reported by Brix and colleagues, which assessed that the prevalence of skewed XCI in female twins affected with AITDs was 34% but only 11% in controls (P = 0.003) and by Yin and colleagues (P = 0.004) [12–14]. Similar positive result was described in other autoimmune diseases such as scleroderma [11]. In addition, our results are the first report that describes a significant association between extremely skewed XCI and RA. Conversely, examination of XCI pattern of 58 Caucasian female patients affected with multiple sclerosis, 46 with systemic lupus erythematosus, 18 with juvenile RA and 45 with type 1 diabetes mellitus and 30 healthy women did not reveal skewed XCI patterns [28]. Despite extensive efforts of XCI analysis in different autoimmune diseases and populations, this hypothesis remains to be confirmed because there is no apparent autoimmunity directed against protein antigens encoded on the X chromosome and the fact that, for many autoimmune diseases, we found a female predominance in inbred mice models having two identical X chromosomes and therefore no 'foreign' antigens from the XCI [29].

In humans, it was reported that XCI process was genetically controlled by genes located on X chromosome [30]. It has also been suggested that genes on the X chromosome might show linkage with AITD and RA [31, 32]. Thus, the observed association between skewed XCI and AITD and RA is not causal but could be explained by linkage disequilibrium between mutation responsible for XCI process and AITD and RA susceptibility polymorphisms. In addition, numerous environmental risk factors such as tobacco smoking, hormones, diet, drugs, toxins and/or infections are important in determining whether an individual will develop autoimmune diseases [33]. In fact, environmental agents are able to amplify autoimmunity in genetically susceptible individuals and to break tolerance in genetically resistant individuals, there by increasing the risk of developing autoimmune diseases [34]. The interaction between genetic and environmental factors remains to be achieved in order to evaluate the involvement of each component in the development of such autoimmune reactions.

We suggest a possible role of XCI mosaicism in the pathogenesis of RA and AITDs. However, the process of XCI needs to be considered as a potential factor in the predominance of females in most autoimmune diseases. It would also be of interest first to study the XCI pattern in females affected with other autoimmune diseases and second to test the XCI patterns of many cell types.