Coeliac disease BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000536 on 15 December 2020. Downloaded from Characterisation of gene and pathway expression in stabilised blood from children with coeliac disease Hanna Gustafsson Bragde ,1,2 Ulf Jansson,3 Mats Fredrikson,2 Ewa Grodzinsky,2 Jan Söderman1,2 To cite: Bragde HG, ABSTRACT Summary box Jansson U, Fredrikson M, Introduction A coeliac disease (CD) diagnosis is et al. Characterisation of likely in children with levels of tissue transglutaminase What is already known about this subject? gene and pathway expression autoantibodies (anti- TG2) >10 times the upper reference in stabilised blood from Paediatric coeliac disease (CD) diagnosis still re- value, whereas children with lower anti- TG2 levels need ► children with coeliac quires sampling and evaluation of small intestinal an intestinal biopsy to confirm or rule out CD.A blood disease. BMJ Open Gastro biopsies in many cases. sample is easier to obtain than an intestinal biopsy 2020;7:e000536. doi:10.1136/ Gene expression in small intestinal biopsies differs sample, and stabilised blood is suitable for routine ► bmjgast-2020-000536 between patients with active CD and patients with Universitets Bibliotek. Protected by copyright. diagnostics because transcript levels are preserved at non- CD. ► Additional material is sampling. Therefore, we investigated gene expression Blood is generally a more accessible sampling ma- published online only. To view in stabilised whole blood to explore the possibility of ► terial compared with small intestinal biopsies. please visit the journal online gene expression- based diagnostics for the diagnosis and (http:// dx. doi. org/ 10. 1136/ Stabilised blood is suitable for routine gene follow- up of CD. ► bmjgast- 2020- 000536). expression- based diagnostics because transcript Design We performed RNA sequencing of stabilised levels are preserved at sampling. whole blood from active CD cases (n=10), non- CD cases Received 4 September 2020 (n=10), and treated CD cases on a gluten- free diet (n=10) What are the new findings? Revised 5 November 2020 to identify diagnostic CD biomarkers and pathways Accepted 21 November 2020 ► Stabilised whole blood is not a suitable sample for involved in CD pathogenesis. clinical diagnostics of CD based on single genes. Results No single gene was differentially expressed ► Pathways of potential interest in CD have been between the sample groups. However, by using gene set identified. enrichment analysis (GSEA), significantly differentially expressed pathways were identified in active CD, and How might it impact on clinical practice in the these pathways involved the inflammatory response, foreseeable future? negative regulation of viral replication, translation, as well ► The results provide a potential starting point for the as cell proliferation, differentiation, migration, and survival. development of pathway- focused gene expression The results indicate that there are differences in pathway panels for CD. http://bmjopengastro.bmj.com/ regulation in CD, which could be used for diagnostic ► These findings add to the knowledge of CD purposes. Comparison between GSEA results based pathogenesis. on stabilised blood with GSEA results based on small intestinal biopsies revealed that type I interferon response, © Author(s) (or their defence response to virus, and negative regulation of viral symptomatic children with high levels (>10 employer(s)) 2020. Re- use replication were identified as pathways common to both times the upper reference value, URV) of permitted under CC BY- NC. No tissues. tissue transglutaminase autoantibodies (anti- commercial re- use. See rights Conclusions Stabilised whole blood is not a suitable 1 and permissions. Published TG2) if additional requirements are met. In sample for clinical diagnostics of CD based on single symptomatic children with anti- TG2 levels by BMJ. genes. However, diagnostics based on a pathway- focused 1 that are 1–10 times the URV, an intestinal on December 21, 2020 at Linkopings Laboratory Medicine, Region gene expression panel may be feasible, but requires Jönköping County, Jönköping, further investigation. biopsy is needed to confirm or rule out CD Sweden as a diagnosis. If the biopsy sample is Marsh 2 Department of Biomedical and grade 0 or 1, the diagnosis is unclear and Clinical Sciences, Linköping INTRODUCTION 1 University, Linköping, Sweden further investigations are recommended. 3Department of Paediatrics, Small intestinal biopsies have long been an In symptomatic IgA- competent patients with Region Jönköping County, essential part of coeliac disease (CD) diag- anti- TG2 <1 times the URV, a CD diagnosis Jönköping, Sweden nostics, but based on the recommendations is less likely, but not ruled out.1 Additional from the European Society for Paediatric biomarkers would aid in diagnosing CD; we Correspondence to Dr Hanna Gustafsson Bragde; Gastroenterology, Hepatology, and Nutri- previously investigated gene expression in hanna. gustafsson. bragde@ tion in 2012, the intestinal biopsy may small intestinal biopsies by RNA sequencing rjl. se be excluded from the diagnostic flow for and found differences in gene expression Bragde HG, et al. BMJ Open Gastro 2020;7:e000536. doi:10.1136/bmjgast-2020-000536 1 Open access BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000536 on 15 December 2020. Downloaded from in active CD cases versus non- CD cases that were detect- Study groups able also in CD cases with no or low-grade intestinal inju- Two study groups (table 1) were defined based on subjects ries (Marsh 0–1).2 However, blood is a more accessible on a gluten- containing diet: (1) group CDM3 with a CD sampling material than small intestinal biopsies that can diagnosis (histopathologic assessment Marsh 3B–3C and be sampled repeatedly to both diagnose and follow- up anti- TG2 >7 U/mL), (2) group M0 cleared from CD with patients with CD. RNA sequencing of CD4+ T cells (Marsh 0 and anti- TG2 ≤7 U/mL). A third study group and whole genome microarray transcriptome analysis of (table 1) was defined based on CD subjects at follow-up peripheral blood mononuclear cells from mainly adult on a GFD: (3) Group CDM0 (Marsh 0 and anti- TG2 CD cases have identified genes and pathways of interest ≤7 U/mL). Whole blood RNA samples were sequenced in CD.3–5 Because stabilised whole blood preserves tran- from all study subjects in all groups. Additionally, RNA script levels during collection, transport, and storage,6 samples from small intestinal biopsies from subjects in the use of stabilised whole blood would facilitate the groups M0 and CDM3 were sequenced. These subjects incorporation of gene expression-based diagnostics in were included as subgroups in a previous study involving clinical practice. We performed RNA sequencing of stabi- RNA sequencing of small intestinal biopsies.2 Based on lised whole blood from paediatric patients with active the previous analysis, one subject in group M0 was more CD, non- CD, and treated CD on a gluten- free diet (GFD) similar to Marsh 3 subjects, and therefore, that subject to identify potential CD diagnostic biomarkers and path- was excluded from all analyses. ways involved in CD pathogenesis. Sample collection and processing Sera were collected using blood tubes containing polymer METHODS gel and clot activator (Becton, Dickinson and Company, Universitets Bibliotek. Protected by copyright. Study subjects Franklin Lakes, New Jersey, USA). Serum levels of anti- Children and adolescents (<18 years of age) were TG2 and IgG antibodies against deamidated gliadin referred to Ryhov County Hospital in Jönköping, were determined using EliA-kits from Thermo Fisher Sweden with suspected CD or were followed-up after Scientific (Waltham, Massachusetts, USA) according to a period on a GFD to verify mucosal recovery. The Bragde et al.7 A cut- off of 7 U/mL was used. patients included in this study were enrolled during Blood for RNA isolation was collected in Tempus Blood the years 2009–2014, where biopsy samplings on GFD RNA tubes (Life Technologies, Carlsbad, California, were still performed as a part of the standard diagnostic USA), kept overnight at 4°C, and then stored at −20°C process for CD at the hospital. Most patients were until RNA isolation. Total RNA from stabilised blood was referred for small intestinal biopsy due to elevated anti- purified using the Tempus Spin RNA Isolation Reagent TG2 regardless of symptoms. Some children with anti- kit (Life Technologies) according to the manufactur- TG2 levels below the URV on a gluten- containing diet er’s instructions. RNA concentrations were determined (with or without selective IgA deficiency) were referred using the Qubit 2.0 Fluorometer and the Qubit RNA for biopsy to exclude CD. The patients provided written BR Assay Kit (Thermo Fisher Scientific). RNA integrity consent before blood and duodenal biopsy specimens was assessed using the Agilent 2100 Bioanalyzer with the were collected. Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa http://bmjopengastro.bmj.com/ Table 1 Descriptive statistics of the study groups No. of Age at biopsy Gender Marsh grade cases (years)* M/F (span) Anti- TG2*† (U/mL) Anti- DG*‡ (U/mL) HLA- DQ2.5cis§ M0 9 8.4 (2.2–17) 4/5 M0 0.29 (<0.10–1.3) 0.57 (<0.40–1.6) 67%, 22%, 11% CDM0 10 8.8 (3.0–17) 2/8 M0 2.3 (0.10–7.0) 2.5 (0.50–5.8) 10%, 70%, 20% CDM3 10 8.6 (2.3–16) 4/6 M3B–3C 600 (36–2858) 151 (9.0–781) 10%, 80%, 10% on December 21, 2020 at Linkopings Subjects in the M0 and CDM3 groups consumed a gluten- containing diet. Study subjects in group M0 did not receive a coeliac disease (CD) diagnosis, whereas a CD diagnosis was confirmed in study subjects in group CDM3. Group CDM0 contained subjects with a CD diagnosis who were followed- up after a period on a gluten- free diet. *Mean (min–max) †Levels of IgA autoantibodies against tissue transglutaminase (anti-TG2) in sera.