Over 50% of non-celiac wheat sensitivity (NCWS) patients are HLA DQ2/DQ8 positive and often have a Celiac Disease (CD) family history. Studies have identified a subgroup of NCWS patients whose clinical and immunological features are closer to CD than to irritable bowel syndrome/functional dyspepsia (IBS/FD) ('inflammatory subgroup'). The investigators hypothesized that among CD patient's relatives, there might be a high number of NCWS subjects, who hypothetically belong to the 'inflammatory subgroup'. Therefore, the aim of this multi-step project is to identify the prevalence of both self-reported NCWS and IBS/FD not related to wheat ingestion among CD patient's relatives (parents, grandparents, siblings and sons).

Hypothesis, Rationale and Aims Over 50% of non-celiac wheat sensitivity (NCWS) patients are HLA DQ2/DQ8 positive and often have a Celiac Disease (CD) family history. Studies have identified a subgroup of NCWS patients whose clinical and immunological features are closer to CD than to irritable bowel syndrome/functional dyspepsia (IBS/FD) ('inflammatory subgroup'). The investigators hypothesized that among CD patient's relatives, there might be a high number of NCWS subjects, who hypothetically belong to the 'inflammatory subgroup'.

To the best of investigators' knowledge, very few studies have tried to identify NCWS, or even IBS/FD not related to wheat ingestion prevalence among CD patient's relatives, nor have studied their clinical, immunological, intestinal permeability (IP) and gut microbiota (GM) features, or the changes induced by a wheat-free diet (WFD). More specifically, in 1996, Troncone et al. showed high lymphocyte counts (i.e. total intraepithelial lymphocyte number and/or numbers of lamina propria and intraepithelial CD3+ and γδ+ cells) after rectal gluten instillation in siblings of CD children, revealing a gluten sensitization not restricted to HLA. Esteve M et al. analyzed the duodenal histological pattern and clinical features of 110 HLA-DQ2 positive CD patient's first-degree relatives, proving that 22.2% of them had some evidence of 'gluten-sensitive enteropathy'; subjects with Marsh I and Marsh II-III lesions were significantly more often symptomatic (56.3% and 53.8%, respectively) than patients with normal mucosa (Marsh 0 21.1%, p=0.002). Another Spanish study group analyzed the prevalence of gastrointestinal symptoms and the influence of gluten intake in 139 CD patient's first-degree relatives, finding that 57.6% had gastrointestinal symptoms [bloating (16.5%), constipation (15.1%), diarrhea (14.4%), and abdominal pain (5.8%)] and 32.7% (n. 37/113) had a Marsh I degree at duodenal histology examination. At baseline, 45.7% of the participants reported symptoms (evaluated with CD-specific symptom index), which reduced to 24.5% (i.e. reduction ≥20%) during a 4-week gluten-free diet, going-back to 38.1% during a gluten-overload diet (i.e. 15g of gluten supplement to their normal diet containing gluten). Moreover, Araya M et al. reported that among 166 CD patient's first-degree relatives, the most spontaneously declared being asymptomatic, but detailed questioning revealed that 60.7% experienced symptoms, which had not been investigated.

Therefore, the aim of this multi-step project is to identify the prevalence of both self-reported NCWS and IBS/FD not related to wheat ingestion among CD patient's relatives (parents, grandparents, siblings and sons).

Inclusion criteria

• CD patient's relatives
• >18 years old
• reporting IBS/FD-like and extraintestinal (EI) symptoms

Exclusion criteria

• self-exclusion of wheat from the diet and refuse to reintroduce it for diagnostic purposes;
• drug and/or alcohol (>30 g/day for men and >20 g/day for women) abuse;
• treatment with steroids and/or non-steroidal anti-inflammatory drugs in the 2 weeks before duodenal biopsy;
• pregnancy or breastfeeding;
• Helicobacter pylori and other bacterial and/or parasitic infections;
• diagnosis of chronic inflammatory bowel disease or other organic pathologies affecting the digestive system [e.g., IgE-mediated Wheat Allergy (WA), microscopic colitis, diverticulitis, segmental colitis associated with diverticulosis, etc.], neurological diseases, major psychiatric disorders, infectious diseases, immunological deficiencies, and impairments limiting physical activity;
• patients undergoing radiotherapy and chemotherapy.

Diagnostic criteria of NCWS and IBS/FD not related to wheat ingestion

1. NCWS

   • subjects with wheat-dependent symptoms, both gastrointestinal and extraintestinal;
   • negativity of anti-deamidated gliadin peptide (DGP) immunoglobulin (Ig)A and IgG antibodies, anti-tissue transglutaminase (tTG) IgA and IgG antibodies, and anti-endomysial antibodies (EMA);
   • absence of duodenal villous atrophy in all patients carrying the human leukocyte antigen (HLA) DQ2 and/or DQ8 haplotypes (therefore regardless of the negativity of CD-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 WA: negative skin prick test and/or specific serum immunoglobulin E (IgE) assay for wheat, gluten and gliadin;
   • resolution of symptoms on a strict WFD.

2. IBS/FD not related to wheat intake - subjects diagnosed with IBS/FD, according to the Rome IV classification, who did not specifically report gastrointestinal or EI symptoms/signs following ingestion of wheat and who did not respond to a WFD.

Research plan, Experimental design and Methodologies The study consists of 2 work packages (WP) that will be carried out in collaboration between the partners and according to a prospective study design.

WP1: Identification of CD patient's first-degree relatives with IBS/FD-like and extraintestinal (EI) symptoms

CD patient's relatives (>18 years old, non-pregnant, non-breastfeeding) will fill out the following online questionnaires (T0):

• Demographic/anthropometric features [Sex, Age, Ethnicity, Height, Weight, Body Mass Index (BMI)]
• Self-perceived food intolerance (including NCWS)
• Gastrointestinal Symptom Rating Scale (GSRS)
• Bristol Stool Scale
• IBS-Symptom Severity Scale (IBS-SSS)
• Extraintestinal Symptom Rating Scale (ESRS)
• IBS-Quality of Life (IBS-QoL). All patients complaining of IBS/FD-like and EI symptoms will undergo HLA DQ2/DQ8 and CD serological screening (DGP IgA and IgG antibodies, tTG class IgA and IgG antibodies, EMA). Symptomatic patients with CD suspicion (HLA DQ2/DQ8 positivity, regardless of serology results) will undergo duodenal biopsy (examined according to Marsh-Oberhuber classification). Other organic diseases will be excluded according to the clinician's choice, according to the National Health Systems (NHS) screening programs for symptomatic CD relatives. At the end of all required examinations, we will identify a subgroup of patients suffering from IBS/FD-like and EI symptoms not related to other organic diseases (T1).

WP2: Identification of CD patient's relatives with potential NCWS vs IBS/FD not related to wheat intake with 6-week long strict WFD Patients, identified at T1, will undergo a 6-week long strict WFD (preliminary dietician consul will be granted to all subjects), at the end of which (T2) they will fill-out an online form including a WFD adherence questionnaire (i.e. modified 'Biagi' score) and the questionnaires reported in WP1 (except the self-perceived food intolerance one). Patients reporting a reduction ≥30% in at least one questionnaire after WFD will be identified as self-reported NCWS, all the others will be identified as IBS/FD not related to wheat intake.

Power Calculation The sample size is difficult to determine as very low data are known about NCWS/IBS/FD prevalence among CD patient's relatives. According to previous studies, up to 50-60% CD patient's relatives complain symptoms. In addition, some studies, assessing NCWS immunological pattern, obtained statistical significance with almost 35 patients.

According to the multicenter design of this study, the investigators planned to screen CD patient relatives as much as possible (at least 600 subjects), with esteemed identification of 200-300 subjects with IBS/FD-like and/or EI symptoms (WP1). It is not possible to establish a priori how many patients will be identified as affected by self-reported NCWS and, therefore, it is not possible to indicate how many will access the subsequent phases of the study (WP2).

Potential pitfalls and caveats, and alternative approaches WP1, which consists of a simple screening survey and subsequent clinical assessment of CD patients' relatives, should be carried out without pitfalls, mostly following the NHS recommendations for CD early screening.

Usually, patients who self-report NCWS easily agree to start a period of WFD. A 6-week long WFD challenge represents a sufficient period to evaluate the evolution of symptoms in these patients, without risking determining nutritional deficiencies that could alter the patient's state of health.

Expected results, and Impact This multicenter study will define exactly, for the first time, the prevalence of self-reported NCWS and IBS/FD not related to wheat intake among CD patient's relatives. In addition, a screening carried out on these subjects will allow both to early identify subjects with CD, and to direct subjects affected by NCWS or IBS/FD not related to wheat intake towards a more rapid diagnosis, consequently reducing direct and indirect costs for NHS, and an adequate therapeutic approach, consistently improving their QoL.

• Clinical benefits: thanks to this project, which starts with a general screening of CD patient's first-degree relatives, physicians will both early diagnose new CD patients and identify and treat those subjects who, although not celiac, suffer from NCWS or IBS/FD not related to wheat intake. Moreover, the early identification of new CD subjects will be useful to prevent possible long-term consequences, such as malnutrition, osteoporosis, and neoplastic diseases development.
• Research benefits: this study will add a piece to the knowledge about CD, NCWS, and IBS/FD not related to wheat ingestion, and defining, for the first time, the prevalence of the last 2 conditions in CD patient's familiar.
• Social benefits: the early diagnosis of CD, NCWS and IBS/FD not related to wheat intake will improve health status (considering physical, psychological and social issues) of recruited subjects.
• Economic benefits: the early diagnosis of CD, NCWS and IBS/FD not related to wheat intake would determine a reduction in the number of medical visits and examinations, as well as of lost workdays, with substantial economic savings for the NHS and improvement of patient's QoL. In addition, in new CD subjects, the early diagnosis will also reduce the costs related to the development of CD long-term complications.

Inclusion criteria

• CD patient's relatives
• >18 years old
• reporting IBS/FD-like and extraintestinal (EI) symptoms

Exclusion criteria

• self-exclusion of wheat from the diet and refuse to reintroduce it for diagnostic purposes;
• drug and/or alcohol (>30 g/day for men and >20 g/day for women) 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., IgE-mediated Wheat Allergy (WA), microscopic colitis, diverticulitis, segmental colitis associated with diverticulosis, etc.], neurological diseases, major psychiatric disorders, infectious diseases, immunological deficiencies, and impairments limiting physical activity.