Abnormalities of the immune system or autoimmune diseases are rarely reported in patients affected by RASopathies. The current prospective study analysed immune system dysfunction and autoimmune biological markers in a cohort of 69 patients and 50 controls.
The results of the current study showed lower IgA levels in patients than in controls (p 0,00685). The worldwide prevalence of selective IgA deficiency depends on the ethnic background and it is most prevalent in Caucasians (1:600) . Most individuals are asymptomatic, but the defect may be associated with recurrent respiratory and gastrointestinal tract infections/disorders, autoimmunity and allergies.There is only one case reported in the literature of a 32-month-old female with Noonan andhypogammaglobulinemia, placed on antibiotic prophylaxis.Four of our patients with IgA deficiency also had IgG and/or IgM deficiency.Upper respiratory tract infections were recorded in 2 patients and pneumonia in 1 only.
Recently, a cohort of 42 patients with RASopathieswas evaluated for autoimmune status. Autoimmune antibodies were observed in 52% of the patients. Remarkably, three (7%) of the patients had specific gastrointestinal and liver autoantibodies without clinical findings. Six patients (14%) fulfilled the clinical criteria for autoimmune diseases [systemic lupus erythematous, polyendocrinopathy (autoimmune thyroiditis and celiac disease), primary antiphospholipid syndrome, autoimmune hepatitis, vitiligo, and autoimmune thyroiditis]. Other cases of autoimmune diseases are reported anecdotally in patients with Rasopathies[2, 30-33].
Although clinical findings suggestive for autoimmune disease were detected in only one patient, biochemical parameters showed specific alterations.
Our study has highlighted the frequent finding of positivity for thyroid autoantibodies (25%), all in conditions of euthyroidism, as already reported [34-35]. Autoimmune diseases, which are significantly increasing in the world population, currently have an estimated prevalence of around 3% in childhood and adolescence: frequent clinical pictures (constituting about 75% of total cases) include autoimmune thyroid disease (TAI), celiac disease (CD) and juvenile idiopathic arthritis (JIA). The most common form of TAI, Hashimoto's thyroiditis, shows a frequency of 1.3% in the pediatric population and mainly affects women , but it reaches 3% if the prevalence of antithyroid antibodies is evaluated which are also found more frequently in girls with goiter and after 12 years of age . However, in recent years, numerous prospective studies have demonstrated that many autoantibodies can be detected in the serum of asymptomatic or paucisymptomatic individuals who later develop an autoimmune disease. These antibodies can therefore precede the clinical symptoms of the disease by years, and could in principle be used for diagnostic and prognostic purposes, including screening studies .
Reduced CD8+ T-cells levels were also demonstrated in our patients(Figure 1).
As known, the Ras/MAPK cascade is involved in ‘immunological tolerance’, that is the ability to distinguish between self and non-self and excessive activation of MAPKs is associated with autoimmune diseases. It has been proved that a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection . Moreover, Ras/MAPK signalling is also implicated in peripheral tolerance to prevent autoimmune destruction by self-reactive T cells that escape thymic deletion. In particular, Erk MAPK pathway plays a critical role in CD8 T cell activation, proliferation, and survival . Peripheral deletion of CD8 T cells requires p38 mitogen activated protein kinase in cross-presenting dendritic cells and p38 inhibition could rescue CD8 T cells from Bim-dependent apoptosis .Activation of the MAPK kinase p38 signaling pathway had previously been reported to selectively induce apoptosis in CD8 T cells in vivo. Huang et al. demonstrated that, during immune tolerance, p38α is constitutively activated in cross-presenting Dendritic Cells (DCs) from the mesenteric lymph node, leading to the generation of induced Tregs and inhibition of Th1 T cells through a TGFβ dependent mechanism. Consequently, loss of p38α in DCs prevented induction of oral tolerance in vivo.
On the basis of these data it might be suggested that impairment of RAS-MAPK pathway alters CD8 production and function causing intolerance and cross reactivity.
In our study, CD8 deficiency is probably the result of disease-causing mutations enhancIing signal flow through RAS/MAPK pathway. Although the role of CD8+ T cells is not as well established, it is known that CD8+ T cells contribute to the induction, progression, pathogenesis and protection from many autoimmune diseases . A recently systematic review, highlighted the potential role of CD8+ T cells in different autoimmune diseases and it has been demonstrated that CD8+T cells are decreased in the peripheral blood of patients with Grave’s disease and upregulated in others autoimmune disease according to epigenetic mechanisms which participate in the activation, differentiation, and development of CD8+T cells. Although some studies have not found CD8+ T-cell deficiency in patients with autoimmune diseases or have attributed the deficiency to hormonal factors, CD8+ T-cell deficiency would appear to be a general feature of human chronic autoimmune diseases attributed to sequestration of CD8+T cells in the target organ. CD8+ T-cell deficiency also occurs in healthy blood relatives of patients with these diseases. It is proposed that this deficiency is genetically determined and underlies the development of chronic autoimmune diseases .
We hypothesized that reduced CD8+ T-cells levels demonstrated in our patients as a consequence of the alteration RAS/MAPK pathway might predispose to autoimmune disorders patients affected by RASopathies and therefore could be the first detectable sign of possible emergence of autoimmune disease.
The increase ofinflammatory moleculeslevels, in ours patients,might instead suggest the presence of a state of chronic low-grade inflammation.On the other hand, cytokines including proinflammatory cytokines (IL-1, TNFα, IFN, IL-2, IL-6, IL-12) and consequently anti-inflammatory cytokines (IL-10, IL-11, IL-13, IL-1ra) are important players in the pathogenesis of autoimmune disease through multiple ways, such as regulating inflammation and angiogenesis [47-48].
For example, ours patients showed high levels of IL-4 and it is well known the B-cell stimulatory and Th2 promoting properties of IL-4 in the development of autoantibodies and autoantibody mediated diseases.Even more important, IL-6 is a critical cytokine that mediates numerous inflammatory and immunomodulatory pathways. In this regard, dysregulated and persistent IL-6 production results in severe inflammatory and autoimmune disorders .There are also in support for susceptibility to autoimmune phenomena, increased of IL-7 that abrogates suppressive activity of human
regulatory T cells and allows expansion of alloreactive and autoreactive T cells  and increased of IL-15 implicated in the pathogenesis of several immune diseases . Interestingly, IP-10 is also significantly higher in patients than in controls. IP-10 and its receptor, CXCR3, appear to contribute to the pathogenesis of many autoimmune diseases and high levels of circulating IP-10, have been shown in patients with autoimmune thyroiditis also relating to a stronger and more aggressive inflammatory response in the thyroid[53-54].
Likely, as a counter-regulatory action, we demonstrated IL-2 and IL-10 increased, the first performs an essential function in the control immune responses and maintain self-tolerance  and the secondis another key mediator of the anti-inflammatory response  (Saraiva et al 2020). It might be suggested that the increase of inflammatory molecules levels with a state of chronic low-grade inflammation represents the underlying pathological mechanism driving immune and metabolic pathways involved in autoimmune diseases.
In conclusion, in light of our data we can underline thatthe altered regulation of RAS/MAPK pathway that characterizes the cohort of patients could be responsible of the altered function of the immune system. In particular, the results suggested a major tendency to autoimmune phenomena than to an immunodeficiency as demonstrated by the finding of circulating autoantibodies, low levels of CD8 T cells and high levels of inflammatory cytokines. So, these evidences may be the first markers of the possible evolution in overt autoimmune disease.
Limits of study
A limitation of our study is the lack of a comprehensive study in the literature that has determined the exact frequency of the autoimmune markers or autoimmune diseases in the general population which allows comparison with RASopathy patients. In addition, we have not considered a control group for the detection of autoantibodies.
Moreover, the average age of our patients is relatively low, which probably limits the diagnosis of autoimmune disorders that have a later onset.