The inclusion/exclusion criteria used to select the study population have been previously validated in other retrospective studies. Additional exclusion criteria related to steatosis and other liver diseases and specifically required for this study were adopted.

Inclusion criteria for Non-Celiac Wheat Sensitivity (NCWS) patients

• age >18 and <65 years;
• subjects with wheat-dependent symptoms, both gastrointestinal and extra-intestinal;
• negativity of IgA and IgG anti-deamidated gliadin peptide (DPG) antibodies, immunoglobulin (Ig)A and IgG anti-tissue transglutaminase (tTG) antibodies, and anti-endomysial antibodies (EMA);
• absence of duodenal villous atrophy, documented in all patients carrying the human leukocyte antigen (HLA) DQ2 and/or DQ8 haplotypes (therefore regardless of the negativity of celiac disease (CeD)-specific serum antibodies), evaluated when the patients had consumed a minimum of 100g of pasta and/or bread a day, for at least 45 days;
• absence of IgE-mediated wheat allergy (WA): negative skin prick-test and/or specific serum IgE assay for wheat, gluten and gliadin);
• resolution of symptoms on a strict standard elimination diet (i.e. extended oligoantigenic, excluding wheat, cow's milk, egg, tomato and chocolate and other foods self-reported by the patient as causing symptoms), followed for at least 4 weeks, and the recurrence of the same symptoms after double-blind placebo-controlled challenge (DBPCC) with wheat (for further details see below);
• complete medical records;
• duration of follow-up longer than 12 months after initial diagnosis, with at least 2 outpatient visits during the follow-up period.

Inclusion criteria for Irritable Bowel Syndrome/Functional Dyspepsia (IBS/FD) and other functional gastrointestinal disorders unrelated to NCWS or other food allergies/intolerances patients

• age >18 and <65 years;
• subjects diagnosed with IBS/FD and other functional gastrointestinal disorders, according to the Rome IV classification, 1 who did not specifically report symptoms/signs, whether gastrointestinal or extra-intestinal, following ingestion of wheat or other foods and who did not respond to gluten-free diet (GFD).

Inclusion criteria for Celiac Disease (CeD) patients

• age >18 and <65 years;
• subjects with gastrointestinal and extra-intestinal wheat-dependent symptoms that meet the diagnostic criteria of CeD 2: positivity of anti-tTG IgA and/or IgG antibodies and evidence of villous atrophy, according to the Marsh-Oberhuber classification, demonstrated by histology on duodenal biopsy;
• clinical response to the GFD: resolution of gastrointestinal and/or extra-intestinal symptoms.

Exclusion criteria for all the patients enrolled in the study

• self-exclusion of wheat from the diet and refusal to reintroduce it for diagnostic purposes, before entering the study;
• drug abuse;
• treatment with steroids and/or non-steroidal anti-inflammatory drugs in the 2 weeks before duodenal biopsy;
• pregnancy or breastfeeding;
• diagnosis of chronic inflammatory bowel disease or other organic pathologies affecting the digestive system (e.g., wheat allergy, microscopic colitis, diverticulitis, segmental colitis associated with diverticulosis, etc.), neurological diseases, major psychiatric disorders, infectious diseases, immunological deficiencies, and impairments limiting physical activity;
• incomplete medical records;
• lack of clinical follow-up for at least 12 months after diagnosis with >2 outpatient visits during the follow-up period.

Additional exclusion criteria related to liver steatosis and other liver diseases

• absence of abdominal ultrasound (US) imaging performed before diagnosis (i.e. before starting the wheat-free/gluten-free diet in NCWS and CeD patients, and before any lifestyle modifications and/or drug/prebiotic/probiotic intake in IBS/FD patients);
• incomplete clinical records, lacking the data considered for the present study;
• chronic alcohol intake (>30 g/day for men and >20 g/day for women);
• chronic hepatotropic virus infections [hepatitis B virus (HBV) and hepatitis C virus (HCV)];
• autoimmune liver diseases;
• congenital metabolic liver diseases (e.g. alpha-1 antitrypsin deficiency, hemochromatosis, Wilson's disease, porphyria, other storage diseases, etc.);
• chronic long-term treatment with drugs associated with both macrovesicular (glucocorticoids, estrogens, tamoxifen, amiodarone, methotrexate, and 5-fluorouracil) and microvesicular (glucocorticoids, valproic acid, tetracycline, and zidovudine) steatosis.