In recent years, a new gluten- or wheat-related disease has emerged, a condition labelled "non-celiac gluten sensitivity" (NCGS) or "non-celiac wheat sensitivity" (NCWS). This is very often a self-reported condition, since patients refer to intestinal [mainly irritable bowel syndrome (IBS)-like] and/or extra-intestinal symptoms (i.e. fatigue, headache, anemia) caused by gluten or wheat ingestion, even though they do not suffer from celiac disease (CD) or wheat allergy (WA).

Among the extra-intestinal symptoms, several studies have shown, in patients with NCWS, the presence of anemia, generally mild, often with iron or folate deficiency characteristics, but no research has ever been planned with the specific intention of analyze this particular aspect of the disease.

Therefore, the aim of the present multicentric research was to analyze, both retrospectively and prospectively, the laboratory data of NCWS patients, compared to CD and IBS controls, to identify: a) the presence, severity and morphologic characteristic of anemia; 2) possible pathogenic mechanisms.

In recent years, a new gluten- or wheat-related disease has emerged, a condition labelled "non-celiac gluten sensitivity" (NCGS) or "non-celiac wheat sensitivity" (NCWS). This is very often a self-reported condition, since patients refer to intestinal [mainly irritable bowel syndrome (IBS)-like] and/or extra-intestinal symptoms (i.e. fatigue, headache, anemia) caused by gluten or wheat ingestion, even though they do not suffer from celiac disease (CD) or wheat allergy (WA).

There are conflicting data about the real mechanisms which induce symptoms in NCGS/NCWS patients after wheat ingestion. Some authors suggested a prevalent role for Fermentable Oligosaccharides-Disaccharides-Monosaccharides and Polyols (FODMAPs), rather than gluten in determining the symptoms. Other studies underlined the activation of mechanisms of both innate and acquired immunity in NCWS patients after wheat ingestion.

Given the lack of a diagnostic biomarker, NCGS/NCWS mostly remains a diagnosis of exclusion, especially respect to CD and WA, so a confirmatory test is required. The "Salerno criteria" suggested the double-blind, placebo-controlled (DBPC), cross-over, gluten/wheat challenge as the gold standard test to discriminate true NCGS/NCWS patients.

By definition, NCGS/NCWS symptoms generally occur after the ingestion of gluten/wheat, disappear within a few days of a gluten-free diet (GFD) and quickly reappear when gluten/wheat is, voluntarily or accidentally, reintroduced. However, GDF is very difficult and onerous from a social (presence of gluten in many industrial food products and "contamination", both domestic and extra-domestic), psychological (e.g. for adolescents, exclusion from the "peer group", with difficulty in accepting the diagnosis) and economic point of view.

Among the extra-intestinal symptoms, several studies have shown, in patients with NCWS, the presence of anemia, generally mild, often with iron or folate deficiency characteristics, but no research has ever been planned with the specific intention of analyze this particular aspect of the disease.

Therefore, the aim of the present multicentric research was to analyze, both retrospectively and prospectively, the laboratory data of NCWS patients, compared to CD and IBS controls, to identify: a) the presence, severity and morphologic characteristic of anemia; 2) possible pathogenic mechanisms, with particular attention to iron, vitamin B12 and folate metabolism, thyroid hormones, and autoimmune gastric involvement.

Inclusion Criteria:

1. To diagnose NCWS the recently proposed criteria will be adopted. All the patients will meet the following criteria:

   - negative serum anti-transglutaminase (anti-tTG) and anti-endomysium (EmA) immunoglobulin (Ig)A and IgG antibodies, absence of intestinal villous atrophy, negative IgE-mediated immune-allergy tests to wheat (skin prick tests and/or serum specific IgE detection), resolution of the IBS symptoms on standard elimination diet, excluding wheat, cow's milk, egg, tomato, chocolate, and other self-reported food(s) causing symptoms, symptom reappearance on double-blind placebo-controlled (DBPC) wheat challenge. As the investigators previously described in other studies, DBPC cow's milk protein challenge and other "open" food challenges will be performed too.

2. To diagnose CD the standard criteria will be adopted. All the patients will meet the following criteria:

   - positive serum anti-transglutaminase (anti-tTG) and anti-endomysium (EmA) immunoglobulin (Ig)A and IgG antibodies presence of intestinal villous atrophy.

3. To diagnose IBS the standard Rome II (for retrospective patients) and Rome III (for prospective patients) Criteria will be adopted. None of these subjects improved on an elimination diet without wheat, cow's milk, egg, tomato, or chocolate.

Exclusion Criteria:

For NCWS diagnosis it will be evaluated the following exclusion criteria:

- positive EmA in the culture medium of the duodenal biopsies, also in the case of normal villi/crypts ratio in the duodenal mucosa, self-exclusion of wheat from the diet and refusal to reintroduce it before entering the study, other previously diagnosed gastrointestinal disorders, other previously diagnosed gynaecological disorders, nervous system disease and/or major psychiatric disorder, physical impairment limiting physical activity.

We will enroll NCWS patients, both with retrospective and prospective methods. As controls groups, we will enroll CD and IBS patients, both with retrospective and prospective methods.