Non-celiac gluten sensitivity (NCGS) is a condition characterized by gastrointestinal and extraintestinal symptoms which are triggered by gluten ingestion in the absence of celiac disease (CD) and wheat allergy. In the last years studies suggested that wheat components other than gluten can be responsible of symptom's triggering, thus the term "non-celiac wheat sensitivity" (NCWS) has been proposed as a more appropriate label. To date, different pathogenetic mechanisms have been proposed, but no conclusive data have been reported; among these, some study groups a possible role of innate immunity and of Natural Killer (NK) cells. KIR (Killer Immunoglobulin-like Receptors) regulate the activation of NK cells through their interaction with Human Leucocyte Antigens (HLA). Both KIR and HLA loci are highly polymorphic, and, in the case of KIR, two main haplotypes have been identified: A and B. Haplotype A is the simplest and correlates mainly with NK inhibition, while haplotype B has a variable number of genes, most of which activate NK cells. The investigators hypothesis is that the genetic variants of KIR, which define the haplotype "inhibitor" or "activator", can affect the development and the course of NCWS too. Thus, the researchers aimed to:1. Identify putative KIR genetic variants in NCWS patients (50 subjects) respect to celiac disease patients (50 subjects) and blood donors (50 subjects); 2. Evaluate the possible association of KIR genetic variants with specific clinical manifestations of patients with NCWS.

Non-celiac gluten sensitivity (NCGS) is a condition characterized by gastrointestinal and extraintestinal symptoms which are triggered by gluten ingestion in the absence of celiac disease (CD) and wheat allergy. Despite the great interest in NCGS, much remains unknown about its pathogenesis. Some studies seem to suggest that wheat components other than gluten (i.e. amylase/trypsin inhibitors, ATIs) can be responsible of symptom's triggering, and therefore the term "non-celiac wheat sensitivity" (NCWS) has been proposed as a more appropriate label. NCWS pathogenesis has been attributed to very different mechanisms: innate or adaptive immunity, incomplete digestion and/or absorption of fermentable oligosaccharides and disaccharides, monosaccharides, and polyols, and, finally, psychological effect. Although NCWS might be considered in its clinical features like CD, to date, no data are available about genes that confer a higher genetic predisposition to the disease. It is known that, in contrast to CD, patients with NCWS do not have a characteristic Human Leucocyte Antigens (HLA) genotype/phenotype, even if HLA DQ2/DQ8 alleles are present in 40-50% of these patients, a value higher than that of the general population (30%). Many studies ascertained the importance of innate immunity and of Natural Killer (NK) cells in the pathogenesis of NCWS. KIRs (Killer Immunoglobulin-like Receptors) regulate the activation of NK cells through their interaction with HLA. Both KIR and HLA loci are highly polymorphic, and, in the case of KIR, two main haplotypes have been identified: A and B. Haplotype A is the simplest and correlates mainly with NK inhibition, while haplotype B has a variable number of genes, most of which activate NK cells. More in general, several studies have shown that haplotypes containing predominantly activator genes confer protection against viral infections and susceptibility to the development of autoimmune and neoplastic diseases. In the context of CD, NK cells are one of the main components of innate immunity and are involved in the destruction of epithelial cells and their cytotoxic activity is closely related to the function of KIRs. The investigators hypothesis is that the genetic variants of KIR, which define the haplotype "inhibitor" or "activator", can affect the development and the course of NCWS too. Thus, the researchers aimed to: 1. Identify putative KIR genetic variants in NCWS patients (50 subjects) respect to celiac disease patients (50 subjects) and blood donors (50 subjects); 2. Evaluate the possible association of KIR genetic variants with specific clinical manifestations of patients with NCWS.

1. Inclusion Criteria for NCWS patients

   • age >18 and <65 years
   • subjects with gluten/wheat-dependent symptoms, both intestinal and extra-intestinal
   • negativity of anti-deamidated gliadin protein (DGP) immunoglobulins (Ig) class A (IgA) and IgG, anti-tissue transglutaminase (tTG) IgA and IgG, Endomysium antibody (EMA)
   • absence of villous atrophy at the duodenal level, documented in all patients with HLA DQ2 and/or DQ8 (therefore, regardless of the negativity of CD-specific serum antibodies), evaluated when patients had a minimum intake of 100 grams of pasta and/or bread a day, for at least 45 days
   • absence of wheat allergy (negative prick-test and/or specific serum IgE assay for wheat, gluten and gliadin)
   • resolution of symptoms with a strict standard elimination diet, i.e., "oligoantigenic" (without wheat, cow's milk, egg, tomato and chocolate, and other foods self-reported by the patient as causing symptoms), for at least 4 weeks, followed by the reappearance of the same after a Double-Blind Placebo-Controlled Challenge (DBPCC) with gluten/wheat
   • complete medical records
   • duration of follow-up greater than 12 months after initial diagnosis and at least 2 outpatient visits during the follow-up period.

2. Exclusion Criteria for NCWS patients

   • age <18 and >65 years
   • alcohol and/or drug abuse
   • self-exclusion of gluten/wheat from the diet and refusal to reintroduce it, for diagnostic purposes, before entering the study
   • treatment with steroids and/or non-steroidal anti-inflammatory drugs (NSAIDs) in the 2 weeks prior to performing duodenal biopsies
   • positivity of EMA in the culture medium of duodenal biopsies, even in the presence of a normal villus/crypt ratio in the duodenal mucosa
   • incomplete medical records
   • lack of clinical follow-up of at least 12 months from diagnosis
   • pregnancy
   • diagnosis of chronic inflammatory bowel disease or other organic pathology affecting the digestive system, diseases of the nervous system, major psychiatric disorders, infectious diseases, immunological deficits, and impairments that limit physical activity.

3. Criteria for inclusion of patients with CD

   • age >18 and <65 years
   • subjects with gluten/wheat-dependent symptoms, both intestinal and extraintestinal, who meet the diagnostic criteria of CD reported in the current guidelines ("four out of five rule"): 1) typical intestinal and extraintestinal signs and symptoms of CD; 2) antibody positivity (both immunoglobulin (Ig) A class anti-tTG and EMA in IgA-sufficient or IgG class anti-tTG and EMA in IgA-deficient subjects); 3) HLA-DQ2 and/or -DQ8 positivity; 4) intestinal damage (demonstrated by histology on duodenal biopsies according to the Marsh classification); 5) clinical response to gluten-free diet (GFD) (e.g., resolution of intestinal and/or extra-intestinal symptoms).

4. Exclusion criteria for patients with CD

   • age <18 and >65 years
   • alcohol and/or drug abuse
   • incomplete medical records
   • lack of clinical follow-up of at least 12 months from diagnosis
   • pregnancy
   • diagnosis of chronic inflammatory bowel disease or other organic pathology affecting the digestive system, diseases of the nervous system, major psychiatric disorders, infectious diseases, immunological deficits and impairments that limit physical activity.

50 NCWS patients, 50 CD patients, and 50 healthy blood donors.