Genetic and Inflammatory Biomarkers Classify Small Intestine Inflammation in Asymptomatic First-degree Relatives of Patients With Crohn’s Disease

doi:10.1016/j.cgh.2019.05.061

Clinical Gastroenterology and Hepatology

Volume 18, Issue 4, April 2020, Pages 908-916.e13

https://doi.org/10.1016/j.cgh.2019.05.061 Get rights and content

Background & Aims

Relatives of individuals with Crohn's disease (CD) carry CD-associated genetic variants and are often exposed to environmental factors that increase their risk for this disease. We aimed to estimate the utility of genotype, smoking status, family history, and biomarkers can calculate risk in asymptomatic first-degree relatives of patients with CD.

Methods

We recruited 480 healthy first-degree relatives (full siblings, offspring or parents) of patients with CD through the Guy’s and St Thomas’ NHS Foundation Trust and from members of Crohn’s and Colitis, United Kingdom. DNA samples were genotyped using the Immunochip. We calculated a risk score for 454 participants, based on 72 genetic variants associated with CD, family history, and smoking history. Participants were assigned to highest and lowest risk score quartiles. We assessed pre-symptomatic inflammation by capsule endoscopy and measured 22 markers of inflammation in stool and serum samples (reference standard). Two machine-learning classifiers (elastic net and random forest) were used to assess the ability of the risk factors and biomarkers to identify participants with small intestinal inflammation in the same dataset.

Results

The machine-learning classifiers identified participants with pre-symptomatic intestinal inflammation: elastic net (area under the curve, 0.80; 95% CI, 0.62–0.98) and random forest (area under the curve, 0.87; 95% CI, 0.75–1.00). The elastic net method identified 3 variables that can be used to calculate odds for intestinal inflammation: combined family history of CD (odds ratio, 1.31), genetic risk score (odds ratio, 1.14), and fecal calprotectin (odds ratio, 1.04). These same 3 variables were among the 5 factors associated with intestinal inflammation in the random forest model.

Conclusion

Using machine learning classifiers, we found that genetic variants associated with CD, family history, and fecal calprotectin together identify individuals with pre-symptomatic intestinal inflammation who are therefore at risk for CD. A tool for detecting people at risk for CD before they develop symptoms would help identify the individuals most likely to benefit from early intervention.

Section snippets

Participants

We recruited 480 healthy FDRs (full siblings, offspring, or parents) through CD patients attending the IBD service at Guy’s & St Thomas’ NHS Foundation Trust (GSTT) and from members of Crohn’s & Colitis UK (a charity supporting patients with IBD). CD diagnosis in the probands was confirmed by their gastroenterologist or general practitioner. FDRs provided information regarding family history of IBD, medical history (including gastrointestinal symptoms), smoking status, medications, allergies,

Calculating the High- and Low-Risk Quartiles

The relative risk of CD in the 454 asymptomatic FDRs generated from 72 genetic risk markers and smoking status (Figure 1) ranged from 0.03 to 38.3 and was significantly higher than expected in the general population (Supplementary Figure 1) (P = .013). Demographic information for each risk quartile is shown in Supplementary Table 2. FDRs in the highest-risk and lowest-risk quartiles (n = 228) were invited to participate in phase 2 of the study (Figure 1); of these 81 were unable to attend for

Discussion

We found that the combination of CD family history, genetic risk, and level of FC was a good predictor of SI inflammation in our cohort of FDRs. Both machine-learning classifiers performed similarly, with significant prediction of SI inflammation, and both put CD family history, genetic risk, and FC as top predictors, which increases confidence in the model’s utility. CD family history was the strongest predictor in the elastic net model and ranked fifth most important predictor in the random

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Second, our analyses are based on biomarkers that may have inherited limitations according to the assay or approach; LMR does not capture colonic permeability, yet it is associated with risk of future onset of CD.15 Also, CRP and FCP may miss low-grade small bowel inflammation,33–35 and even when FCP is elevated, it is not always associated with intestinal inflammation as measured using video capsule endoscopy;36,37 in spite of this, FCP is a predictor of future onset of CD in unaffected FDRs.15,16 Regardless, we show that despite correcting for these biomarkers, we still see a robust effect of high AS on future CD onset, suggesting that the high AS signal is not simply a reflection of elevated LMR, CRP, FCP, or CD-PRS.
Altered host immune reactivity to microbial antigens is hypothesized to trigger the onset of Crohn’s disease (CD). We aimed to assess whether increased serum anti-microbial antibody response in asymptomatic first-degree relatives (FDRs) of CD patients is an independent risk factor for future CD development.
We measured host serum antibody response to 6 microbial antigens at enrollment (Prometheus enzyme-linked immunosorbent assay test: anti-Saccharomyces cerevisiae antibodies immunoglobulin A/immunoglobulin G, anti-OmpC, anti-A4-Fla2, anti-FlaX, anti-CBir1) and derived the sum of positive antibodies (AS). We used samples at enrollment of prospectively followed healthy FDRs from a nested case-control cohort of the Crohn’s and Colitis Canada Genetics Environment Microbial Project. Those who later developed CD (n = 77) were matched 1:4 by age, sex, follow-up duration, and geographic location with control FDRs remaining healthy (n = 307). To address our research aims, we fitted a multivariable conditional logistic regression model and performed causal mediation analysis.
High baseline AS (≥2) (43% of cases, 11% of controls) was associated with higher risk of developing CD (adjusted odds ratio, 6.5; 95% confidence interval, 3.4–12.7; P < .001). Importantly, this association remained significant when adjusted for markers of gut barrier function, fecal calprotectin, C-reactive protein, and CD-polygenic risk score, and in subjects recruited more than 3 years before diagnosis. Causal mediation analysis showed that the effect of high AS on future CD development is partially mediated (42%) via preclinical gut inflammation.
Our results suggest that increased anti-microbial antibody responses are associated with risk of future development of CD, independent of biomarkers of abnormal gut barrier function, subclinical inflammation, and CD-related genetic risks. This suggests that anti-microbial antibody responses are an early predisease event in the development of CD.
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Increased Intestinal Permeability Is Associated With Later Development of Crohn's Disease
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It remains possible that some individuals may have early presymptomatic CD; however, this proportion is likely small. A study of 147 asymptomatic FDRs of patients with CD who underwent video capsule endoscopy showed that only 1 individual had marked inflammation typical of CD.30 Indeed, previous studies that followed asymptomatic patients with isolated terminal ileitis that was found in screening colonoscopies showed that fewer than 1.1% to 4.6% develop CD during a median follow-up of 29 to 54 months.31,32
Increased intestinal permeability has been associated with Crohn’s disease (CD), but it is not clear whether it is a cause or result of the disease. We performed a prospective study to determine whether increased intestinal permeability is associated with future development of CD.
We assessed the intestinal permeability, measured by the urinary fractional excretion of lactulose-to-mannitol ratio (LMR) at recruitment in 1420 asymptomatic first-degree relatives (6–35 years old) of patients with CD (collected from 2008 through 2015). Participants were then followed up for a diagnosis of CD from 2008 to 2017, with a median follow-up time of 7.8 years. We analyzed data from 50 participants who developed CD after a median of 2.7 years during the study period, along with 1370 individuals who remained asymptomatic until October 2017. We used the Cox proportional hazards model to evaluate time-related risk of CD based on the baseline LMR.
An abnormal LMR (>0.03) was associated with a diagnosis of CD during the follow-up period (hazard ratio, 3.03; 95% CI, 1.64–5.63; P = 3.97 × 10^–4). This association remained significant even when the test was performed more than 3 years before the diagnosis of CD (hazard ratio, 1.62; 95% CI, 1.051–2.50; P = .029).
Increased intestinal permeability is associated with later development of CD; these findings support a model in which altered intestinal barrier function contributes to pathogenesis. Abnormal gut barrier function might serve as a biomarker for risk of CD onset.
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The full results of the study are eagerly awaited. Most recently, Taylor et al. tried to further stratify this at-risk population and assess the predictive value of clinical features (family history and smoking), genetics, and biomarkers, in accurately detecting the presence of small bowel lesions, in the unaffected FDRS of patients with CD [17]. According to the study results, those with higher polygenic risk score, positive family history and elevated FC are more likely to present asymptomatic small bowel lesions, and therefore have, hypothetically, a higher risk to develop CD in the future [17].
There is growing evidence that inflammatory bowel disease has a preclinical phase that precedes disease symptoms, during which immune system activation has already occurred and inflammatory pathways have been primed, setting the stage for disease to expand and leading up to clinical diagnosis. Gaining insight to this pre-diagnosis period could improve our knowledge about disease pathogenesis and potentially result in the detection of biomarkers that could predict disease development. The ultimate goal of such research is to identify a population at risk for developing IBD, so preventive strategies could be implemented and disease prevention be achieved.
Deciphering the different phases of preclinical inflammatory bowel disease
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: Conflicts of interest The authors disclose no conflicts.

: Funding This study was undertaken as part of a Guy's and St Thomas' Charity funded clinical research fellowship (R080522). The authors also acknowledge support from the National Institute for Health Research, Guy's and St Thomas' Biomedical Research Centre, London, United Kingdom, and at South London and Maudsley NHS Foundation Trust, in partnership with King's College London.

^a: Authors share co-first authorship.

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Original ArticleAlimentary TractGenetic and Inflammatory Biomarkers Classify Small Intestine Inflammation in Asymptomatic First-degree Relatives of Patients With Crohn’s Disease

Background & Aims

Methods

Results

Conclusion

Section snippets

Participants

Calculating the High- and Low-Risk Quartiles

Discussion

Gastroenterology

Lancet

Mayo Clin Proc

Dig Liver Dis

Am J Hum Genet

Clin Gastroenterol Hepatol

Family and twin studies in inflammatory bowel disease

World J Gastroenterol

Genome-wide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease

Nat Genet

Heritability in inflammatory bowel disease: from the first twin study to genome-wide association studies

Inflamm Bowel Dis

IBD genetic risk profile in healthy first-degree relatives of Crohn’s disease patients

J Crohns Colitis

Epidemiology and risk factors for IBD

Nat Rev Gastroenterol Hepatol

Family studies in Crohn’s disease: new horizons in understanding disease pathogenesis, risk and prevention—Figure 1

Gut

Tissue studies in screened first-degree relatives reveal a distinct Crohnʼs disease phenotype

Inflamm Bowel Dis

220 Asymptomatic first degree relatives of Crohn’s patients display endoscopic small intestinal lesions independent of their gut permeability status

Gastroenterology

Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci

Nat Genet

Promise and pitfalls of the Immunochip

Arthritis Res Ther

A population-based case-control study of CARD15 and other risk factors in Crohn’s disease and ulcerative colitis

Am J Gastroenterol

Original Article
Alimentary Tract
Genetic and Inflammatory Biomarkers Classify Small Intestine Inflammation in Asymptomatic First-degree Relatives of Patients With Crohn’s Disease