Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract and can occur at any age. Crohn’s disease (CD), ulcerative colitis (UC) and indeterminated colitis are defined according to the clinical, endoscopic, radiological and histopathological features. IBD is a multifactorial disease caused by genetic, bacterial, nutritional, and immunological factors. There is clear evidence for a strong genetic influence in the pathogenesis of IBD, especially in CD, coming from twin studies [1] with disease concordance up to 50% for monozygotic CD twins. In addition, pediatric-onset CD is characterized by distinct phenotypic differences compared to adult-onset suggesting that a genetic susceptibility may play an important role [2]. Polymorphisms in the leucine-rich repeats (LRR) region of the NOD2/CARD15 gene were identified as independent risk factors for CD in Caucasians [3, 4], which varies depending on the number of mutations from a 2 to 4-fold increased risk with 1 of the 3 major alleles to 40-fold increase in homozygous or compound heterozygous carriers [5]. The three major polymorphisms in the coding region of NOD2/CARD15, associated with CD, are p.R702W, p.G908R, and p.L1007fs. The NOD2 gene is involved in the innate immune response by recognizing muramyl dipeptide of intracellular bacterial lipopolysaccharides and activating nuclear factor κB pathways [6]. NOD2 is mainly expressed in Paneth cells in the small bowel and is involved in the innate immune response to bacterial pathogens [7]. Thus variant NOD2 alleles are associated with reduced defensin expression in response to bacteria [8]. Children have shorter environmental exposure and therefore genetic factors such as NOD2 variants may have a greater influence in the etiology and severity of CD [9]. Disease behaviour may change over time, thus long-term follow-up data on pediatric-onset disease are needed [10]. Indeed, the few studies analyzing genotype-phenotype in pediatric-onset CD with longer observation time revealed associations of NOD2 variants with disease severity and risk for surgery [11, 12].

Children and adolescents suffering from IBD are at risk of developing osteoporosis. The prevalence of severe demineralization in children and in adults ranges from 4.35% to 42% [13]. The pathogenesis of osteoporosis in CD is not completely understood. Bone mineral density (BMD) is influenced by several factors including chronic or recurrent corticosteroid use, malnutrition or malabsorption, low body mass index (BMI), hypogonadism, immobilisation, vitamin D deficiency [14]. Together these factors result in low turnover bone loss. However, also inflammation may significantly contribute to a high-turnover mineral bone loss in CD [14–16]. Osteoclastogenesis and bone resorption is driven by inflammatory mediators such as tumor necrosis factor (TNF) α, Interleukin (IL)-1β and Il-6 in synergy with receptor activator of nuclear factor kappa B ligand (RANKL). RANKL is released by activated T-cells and binds RANK on osteoclastic precursors and thereby induce and activate osteoclasts [17]. Thus a chronic inflammatory state, such as the gut in CD patients, in addition affects bone remodelling and contributes to high turn-over mineral bone loss. The influence of NOD2 genotype on osteoporosis in CD has not been studied yet.

We therefore wished to investigate the potential relationship between the three major NOD2/CARD15 variants in a well characterized, long term follow up dataset of Crohn patients with pediatric- and adult-onset and inflammatory phenotype, response to therapy and lower bone mineral density.

Evaluation of disease behaviour by genotype is complex and may change throughout the course of disease. Dissecting pediatric- and adult-onset CD represents a model to identify further associations between NOD2 gene variants and phenotypes and may help in selecting individual treatment regimes for patients with CD. We here demonstrate greater influence of NOD2 variants in pediatric-onset CD patients towards higher disease activity, risk for underweight, osteoporosis and more intensive immunosuppressive therapy. This is the first study identifying an influence of NOD2 variant alleles in patients with CD on osteoporosis and an age of onset dependent risk for steroid dependent or refractory course, long-term use of immunosuppressive drugs.

In this retrospective single center study in a tertiary center we report a high prevalence of CD patients carrying at least one NOD2 variant allele with 44% in the pediatric- and 42% in adult-onset CD similar to other European pediatric multicenter cohort studies with a prevalence of 35 up to 45.6% [12, 23, 24]. In addition, the presence of two NOD2 mutant alleles was found more frequent in the pediatric-onset CD patients as reported by others [23, 25] and may predict complicated disease [26].

Growth retardation is a frequent specific feature of pediatric-onset CD [27], which manifest as poor weight gain or short stature and may be the first presenting symptom. We observed a trend in CD patients with NOD2 variants towards underweight at diagnosis in our pediatric-onset cohort analogous to other studies [28–30] describing lower weight and a tendency to growth retardation more or less pronounced in their pediatric cohorts. In addition, we found also significant ongoing underweight in the course of disease in patients with NOD2 variant alleles. Growth failure seems to be associated with disease severity [29] and high levels of TNFα in pediatric IBD [31]. Indeed, the average disease activity in pediatric-onset CD patients carrying at least one NOD2 variant allele was significantly higher and associated with chronic active or moderate to severe clinical course of disease. However, other studies did not found an influence of NOD2 variants towards higher disease activity [25, 29]. This is explicable by the fact, that in most instances disease activity was educed from hospitalization, need for immunosuppressive drugs, surgery or occurrence of complications but not by PCDAI at onset and during course of disease [25, 28, 29, 32]. The PCDAI-score was only used by one other pediatric study with 65 CD patients, which described an influence of NOD2 variants towards a higher PCDAI [30]. Besides actual history and laboratory values (e.g. haematocrit, erythrocyte sedimentation rate and albumin) the PCDAI score includes also weight loss, growth delay, perirectal disease and extraintestinal manifestation. Therefore a high PCDAI score in our pediatric-onset CD patients with NOD2 variants reflects a more severe disease behaviour indicated by underweight, stricturing and penetrating behaviour. Moreover, an aggressive disease manifestation in pediatric-onset CD patients with NOD2 variants leading to complications and surgical resection is described by Lacher et al. [12] and Kugathasan et al. [32, 33].

The prevalence of osteoporosis ranges from 7 to 30% and osteopenia from 22 to 70% in pediatric IBD studies [13, 34, 35]. In fact, 30% of our patients with pediatric-onset CD have osteoporosis and 31% osteopenia, some even before corticosteroid treatment confirming data from Walther [13] and Harpavat et al. [36]. Possible risk factors for pathologic bone density in CD are disease activity, lifetime steroid dosage over 10 g, CD, multiple bowel resections, young age and low body mass index according to a large epidemiological study [37]. We also observed low BMI and a higher disease activity associated with reduced bone density in pediatric-onset CD. Similar Paganelli et al. described an inversely correlation of volumetric BMD with disease activity measured by PCDAI and IL-6 [38]. Other risk factors such as ileal localization and bowel resection did not play a role in our pediatric-onset cohort. Lifetime corticosteroid treatment in children with IBD seems to be less important as previously believed, as children with a chronic non-inflammatory type of disease such as steroid sensitive nephritic syndrome (SSNS) receiving glucocorticoids chronically do have a better bone mineral density than children with CD [39]. Certainly osteoporosis was associated with a steroid refractory or dependent course of disease in the pediatric-onset group. In fact the glucocorticoid dosage in the treatment of pediatric CD may be not as high as in previous older studies; children with CD often receive enteral nutrition therapy and there are more patients with steroid dependent or refractory course of disease in the low BMD group actually switched earlier to a long-term immunosuppressive therapy including azathioprine, methotrexate and anti-TNF-α antibody treatment. Thus the inflammatory state in CD might basically contribute to high-turn over bone mineral loss. It is also known that NOD2 variant alleles induce an inflammatory response e.g. via NF-кB activation [40]. Thus some cytokines with osteolytic activity such as TNFα, IL-1β, IL-6, IFNγ, RANK, RANKL and osteoprotegrin may also be elevated. IL-1β for example stimulates osteoclast activity and enhances mineral bone loss. In fact carriers of IL1β -511b allele have significant lower BMD [41]. Interestingly, CD patients carrying NOD2 variant alleles have an increased risk towards osteoporosis. This is more pronounced in pediatric-onset CD, especially in patients carrying homozygous NOD2 variant alleles. However, we could not prove our hypothesis that osteoporosis is significantly more frequent in pediatric-onset CD patients carrying any NOD2 variant allele may be due to a lack of power. Taken together, these data support screening of osteoporosis in pediatric onset CD, especially in children with NOD2 variants, low BMI and high PCDAI.

We recently reported that CD patients with NOD2 carrier status were more refractory for steroid treatment but responded well to immunomodulators such as AZA/6-MP [42]. This was in contrast to another study that could not find an association between NOD2 carrier status or age of onset of disease and response to steroids [43]. We speculated that treatment response might vary analyzing pediatric and adult CD patient study cohorts regarding NOD2 genotype. To corroborate this hypothesis we now included a pediatric CD cohort and reclassified all CD patients in our adult cohort according to the onset of disease in pediatric and adult onset. Using this approach we found a significant association for steroid failure in CD patients with NOD2 variant alleles and younger age of onset in contrast to Weiss et al. [43]. In addition, the need for long-term immunosuppressive therapy and second line therapy with anti-TNF-alpha agents was also only associated with younger age of onset and the presence of any NOD2 variant allele. Although a positive response to the anti-TNFα agent infliximab was found to be associated with a higher systemic inflammation [44], the NOD2 genotype was not predictive of treatment outcome in their cohort [45]. The diverging results regarding our study cohort might be due to the subgroup analysis of pediatric-onset CD. The fact that we did not investigate treatment outcome is another disadvantage of this study and its retrospective approach.

In summary, dissecting a large unselected group of patients with long-term CD duration in pediatric- and adult-onset of disease revealed a more pronounced difference between NOD2 genotype and phenotype in the pediatric-onset group. There was an association of NOD2 gene variants towards a more affected phenotype with underweight, higher disease activity, steroid failure and the need for long-term and intensive immunosuppressive therapy. Thus pediatric-onset CD and NOD2 variant alleles are risk factors frequently associated with chronic and moderate to severe course of disease. In addition, the prevalence of low bone mineral density is considerably high in pediatric Crohn patients with high average PCDAI, low BMI, steroid failure and NOD2 variant alleles.

The major goal in the treatment of CD is to change the fatal course of chronic active disease especially in children and adolescents in order to improve growth and prevent osteoporosis. Until now testing for NOD2 variants is not part of the diagnostic work-up in pediatric-onset CD. We conclude from our study, that genetic testing for NOD2 variant alleles is useful in pediatric-onset CD as it may identify patients who are at risk for a more affected phenotype. Given the beneficial effect of TNF-α blockade on bone formation and on CD activity this may be an alternative early treatment strategy in children [5, 46].