Exome sequencing in patient-parent trios suggests new candidate genes for early-onset primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a rare bile duct disease strongly associated with inflammatory bowel disease (IBD). Whole-exome sequencing (WES) has contributed to understanding the molecular basis of very early-onset IBD, but rare protein-altering genetic variants have not been identified for early-onset PSC. We performed WES in patients diagnosed with PSC ≤ 12 years to investigate the contribution of rare genetic variants to early-onset PSC. METHODS: In this multicentre study, WES was performed on 87 DNA samples from 29 patient-parent trios with early-onset PSC. We selected rare (minor allele frequency < 2%) coding and splice-site variants that matched recessive (homozygous and compound heterozygous variants) and dominant (de novo) inheritance in the index patients. Variant pathogenicity was predicted by an in-house developed algorithm (GAVIN), and PSC-relevant variants were selected using gene expression data and gene function. RESULTS: In 22 of 29 trios we identified at least 1 possibly pathogenic variant. We prioritized 36 genes, harbouring a total of 54 variants with predicted pathogenic effects. In 18 genes, we identified 36 compound heterozygous variants, whereas in the other 18 genes we identified 18 de novo variants. Twelve of 36 candidate risk genes are known to play a role in transmembrane transport, adaptive and innate immunity, and epithelial barrier function. CONCLUSIONS: The 36 candidate genes for early-onset PSC need further verification in other patient cohorts and evaluation of gene function before a causal role can be attributed to its variants.

Time-to-reach Target Calprotectin Level in Newly Diagnosed Patients With Inflammatory Bowel Disease.

OBJECTIVES: Treatment targets in inflammatory bowel disease (IBD) move away from controlling symptoms towards complete recovery of the intestinal mucosa. Currently, the most frequently used noninvasive surrogate marker of mucosal healing is a faecal calprotectin concentration in the target range. This study tested if there was a relation between time-to-reach target calprotectin and first flare. METHODS: We prospectively included new-onset IBD patients ages 17 and younger in a cloud-based registry (FastForwardCare) and followed them for at least 52 weeks. They were treated according to Dutch national guidelines that advocate a step-up approach. Time-to-reach target was defined as the first calprotectin measurement below 250 µg/g after the start of induction therapy. Time-to-first flare was the time from the first calprotectin measurement below 250 µg/g until reappearance of symptoms with calprotectin values above 250 µg/g. RESULTS: We included 76 patients (luminal Crohn disease [CD] 43); ulcerative colitis [UC] 33). Median age at diagnosis was, respectively 14.5 and 14.1 years. Median time-to-reach target calprotectin was 37 weeks in CD and 11 weeks in UC patients (Log-rank test, P = 0.001). Once the calprotectin target was reached, time-to-first flare was significantly longer in CD than in UC patients (Log-rank test, P = 0.001). CD patients with time-to-reach target calprotectin ≤12 weeks after conventional induction therapy (ie, exclusive enteral nutrition or steroids) had a more favorable disease course in the first year than those with time-to-reach target calprotectin >12 weeks (Log-rank test, P = 0.057). In UC patients, time-to-reach target calprotectin ≤12 weeks is not associated with a favorable disease course in the first year. CONCLUSIONS: The findings of this prospective registry suggest that a quick response to conventional therapy predicts a favorable disease course in new-onset paediatric CD, but not in UC. The concept "time-to-reach target calprotectin level" rationalizes the indefinite term "response to treatment" and is well suited for studying treatment effectiveness in real-world practices.

Calprotectin instability may lead to undertreatment in children with IBD.

BACKGROUND: Treatment decisions in children with inflammatory bowel disease (IBD) are increasingly based on longitudinal tracking of faecal calprotectin concentrations, but there is little known about the stability of this protein in stool. METHODS: We stored aliquots of homogenised stool at room temperature and at 4°C, and measured the calprotectin concentration for 6 consecutive days with three different assays. In addition, we assessed calprotectin stability in assay-specific extraction buffers kept at room temperature. RESULTS: After 6 days of storage at room temperature, mean percentage change from baseline calprotectin concentrations in stool and extraction buffer was 35% and 46%, respectively. The stability of calprotectin was significantly better preserved in samples stored at 4°C (p=0.0066 and 0.0011, respectively). CONCLUSIONS: Calprotectin is not stable at room temperature. Children with IBD and their caretakers may be falsely reassured by low calprotectin values. The best advisable standard for preanalytical calprotectin handling is refrigeration of the stool sample until delivery at the hospital laboratory.

No association between abdominal pain and Dientamoeba in Dutch and Belgian children.

OBJECTIVE: To study the association between Dientamoebafragilis colonisation and faecal calprotectin to see whether the parasite is a harmless commensal or a gut pathogen. DESIGN: Cross-sectional study of previously collected stool samples. SETTING AND PATIENTS: Two hundred stool samples originated from children aged 5-19 years with chronic abdominal pain and diarrhoea, who were seen in paediatric clinics in the Netherlands and Belgium and in whom somatic gastrointestinal disorders were excluded. Another 122 samples came from a healthy community-based reference population of the same age. All stool samples were analysed with real-time PCR for the detection of D. fragilis and with an ELISA for calprotectin-a biomarker of gastrointestinal inflammation. MAIN OUTCOME MEASURES: Prevalence of D. fragilis colonisation and results of stool calprotectin testing. RESULTS: D. fragilis was detected in 45% (95% CI 38% to 51%) of patients and in 71% (95% CI 63% to 79%) of healthy children. Median (IQR) concentrations of calprotectin in patients and healthy children with a positive PCR result were not different from those with a negative PCR result (40 (40-55) µg/g vs 40 (40-75) µg/g, respectively). CONCLUSION: Since D. fragilis colonisation is most prevalent in healthy children and is not associated with an increase in faecal calprotectin concentration, our data do not support the inference that D. fragilis is a pathogenic parasite. Routinely testing for D. fragilis in children with chronic abdominal pain should therefore be discouraged.

Head-to-head comparison of three stool calprotectin tests for home use.

OBJECTIVE: Treatment decisions in inflammatory bowel diseases are increasingly based on longitudinal tracking of calprotectin results. Many hospital laboratories measure calprotectin levels in sent-in stool samples with an enzyme-linked immunosorbent assay (ELISA). Several manufacturers introduced a lateral flow-based test with software application that turns a smartphone camera into a reader for quantitative measurements. We compared three home tests (IBDoc, QuantonCal and CalproSmart) and companion ELISA tests (fCAL, IDK-Calprotectin and Calprotectin-ALP) to see if measurement pairs agreed sufficiently. DESIGN: A method comparison study was conducted with stool samples from patients with active or quiescent inflammatory bowel disease. Medical students without any specific laboratory training carried out the home tests with two iOS (iPhone 6 and 7) and two Android devices (Samsung Galaxy S6 and Motorola Moto G5 Plus). Two experienced laboratory technicians measured the calprotectin concentration with the ELISA method. Primary outcome was test agreement (defined as percentage of paired measurements within predefined limits of difference). Secondary outcome included reading error rate (RER) per smartphone type. RESULTS: We performed 1440 smartphone readings and 120 ELISA tests. In the low calprotectin range (≤500 µg/g) IBDoc, QuantOnCal and CalproSmart showed 87%, 82% and 76% agreement with their companion ELISAs. In the high range (>500 µg/g) the agreement was 37%, 19% and 37%, respectively. CalproSmart and QuantOnCal had significantly higher RERs than IBDoc (respectively 5.8% and 4.8%, versus 1.9%). Forty-three percent of reading errors was on the Motorola device, in particular with the QuantOnCal application. CONCLUSIONS: All three calprotectin home tests and companion ELISAs agreed sufficiently when concentrations are ≤500 µg/g. To minimize wrongful interpretation of calprotectin changes over time it is essential to always use the home test and companion ELISA of one and the same manufacturer. Manufacturers should explicitly evaluate and report the suitability of commonly used smartphones for quantitative calprotectin readings.

Methotrexate for maintaining remission in paediatric Crohn's patients with prior failure or intolerance to thiopurines: a multicenter cohort study.

BACKGROUND AND AIMS: Methotrexate [MTX] is an immunomodulating drug that can be used to maintain remission in patients with Crohn's disease [CD], but data on efficacy and tolerability in children and teenagers are scarce. We evaluated the long-term efficacy and tolerability of MTX monotherapy after thiopurine therapy in paediatric CD patients. METHODS: A multicenter cohort of paediatric MTX users who stopped thiopurines due to ineffectiveness or intolerance between 2002 and 2012 were included and followed for at least 12 months. Relapse-free use was defined as steroid and biologics-free clinical remission after the introduction of MTX, and included intentional discontinuation of successful therapy before the end of the observation period. RESULTS: A total of 113 patients with CD in remission were followed while on MTX monotherapy, of whom 75 [66%] had failed on thiopurines and 38 [34%] had stopped thiopurines due to side effects. Median age at the introduction of MTX was 14 years [range 7 to 17], and 93% used the subcutaneous route. Kaplan-Meier analysis showed that 52% of the study cohort were still in steroid- and biologics-free remission after 12 months of MTX monotherapy, with a difference that did not reach significance between thiopurine-intolerant and thiopurine-failing patients [p = 0.21, log-rank test]. CONCLUSIONS: The findings of this cohort study suggest that MTX is an effective immunomodulator to maintain remission after stopping thiopurines. MTX maintenance should be considered before stepping up to anti-tumor necrosis factor alpha therapy. MTX is probably somewhat more effective in patients who stopped thiopurines due to side effects than in those who failed on thiopurines.