Candidate Genes Involved in Beneficial or Adverse Responses to Commonly Eaten Brassica Vegetables in a New Zealand Crohn’s Disease Cohort

Crohn’s disease (CD) is one of the two manifestations of inflammatory bowel disease. Particular foods are thought with CD to exacerbate their illness. Vegetables, especially Brassicaceae, are often shunned by people with CD because of the negative effects they are alleged to have on their symptoms. Brassicaceae supply key nutrients which are necessary to meet recommended daily intakes. We sought to identify the candidate genes involved in the beneficial or adverse effects of Brassicaceae most commonly eaten, as reported by the New Zealand adults from the “Genes and Diet in Inflammatory Bowel disease Study” based in Auckland. An analysis of associations between the single nucleotide polymorphisms (SNPs) and the beneficial or adverse effects of the ten most commonly eaten Brassicaceae was carried out. A total of 37 SNPs were significantly associated with beneficial effects (p = 0.00097 to 0.0497) and 64 SNPs were identified with adverse effects (p = 0.0000751 to 0.049). After correcting for multiple testing, rs7515322 (DIO1) and rs9469220 (HLA) remained significant. Our findings show that the tolerance of some varieties of Brassicaceae may be shown by analysis of a person’s genotype.


Introduction
Crohn's disease (CD) is one of two commonly identified inflammatory bowel diseases (IBD) the other being ulcerative colitis (UC).Both disorders are inflammatory and people experience phases of remission and deterioration.
The incidence of CD seems to be increasing and it appears that New Zealand (NZ) in 2006 had the highest rate at 16.5/10 5 [1].Increases are observed particularly in the western world, and research has shown that the environment plays a considerable role [2].It has been observed that individuals from Bangladesh (where the incidence of IBD is very low) on shifting to the United Kingdom, develop a very high incidence of IBD within a generation [3].Nutrition is thought to play a key role and is considered to be an important environmental factor influencing the development of CD and its symptoms [4], through the nutrigenomic and epigenetic modification of the susceptibility genes [5].
Vegetables, especially Brassicaceae, are often shunned by people with CD because of the negative effects they are alleged to have on their symptoms [6,7].However, Brassicaceae supply key nutrients.Some varieties of Brassicaceae also appear to be well tolerated by people with CD [8].Several studies show that Brassicaceae contain many significant nutrients: fibre, the antioxidants vitamin A and C, the vitamin folate and vitamin K and the minerals such as potassium, calcium, selenium and zinc as well as the numerous phytochemicals which have key roles in maintaining health [9][10][11][12].They help improve immunity as well as contributing to anti-inflammatory and anti-cancer activities of the body [13][14][15][16][17][18][19][20][21].The intake of sufficient amounts of these nutrients is important for people with CD.These nutrients are necessary to meet the daily intakes as recommended in the Nutrient Reference Values for Australia and New Zealand [22].
By studying the interaction of different Brassicaceae varieties with single nucleotide polymorphisms (SNPs) in people with CD, it may be possible to uncover a genetic basis for individual tolerances.This could lead to more specific nutrition advice with respect to these important vegetables, and enhance the opportunities for CD patients to avail themselves of the benefits of Brassicaceae.The aim of this study was therefore to identify the candidate genes involved in the beneficial or adverse effects of the Brassicaceae most commonly eaten as reported by NZ adults from the "Genes and Diet in IBD Study" [4], based in Auckland.

Brassicaceae Selection
The Brassicaceae analysed for tolerability were those reported to be consumed by the subjects in the "Genes and Diet in IBD Study" based in Auckland NZ.These Brassicaceae were: broccoli, cabbage, cauliflower, Chinese greens, rocket (arugula), watercress, horseradish, mustard sauce, mustard powder and wasabi.

Tolerability of Brassicaceae
The tolerability of the Brassicaceae was ascertained from secondary analysis of the information gained from the responses to the dietary questionnaire.This questionnaire was based on Joachim's methodology [23] and feedback from a group of patients with CD [4].Questions in the survey were asked (and scored on a five-point scale on whether the person's IBD condition became either: definitely better, (++) probably better (+), had no effect (=), probably worse (−), and definitely worse (− − in response to the listed food.This allowed information to be collected on how food affected their disease symptoms.This takes into account that many foods are never consumed because they are not considered palatable irrespective of the effects they are perceived to have on their symptoms of CD. Two scales were used: the percentage of beneficial effects (definitely better and probably better) and adverse effects (definitely worse and probably worse).The responses to "makes no difference" were omitted.A nutritionist or registered nurse checked out the responses with respondents when there were any queries [4].The dietary questionnaire also included open ended questions.These were also evaluated for additional information on foods which may affect symptoms associated with CD.

Study Population
Study participants were from the main North Island centre, Auckland and other major North Island centres.They were enrolled as part of a population based study in the IBD project whose purpose was to determine the genetic and environmental factors of CD aetiology.These people were enlisted from gastroenterology clinics or by their response from advertising in the media between May 2005 and April 2009.A total of 339 Caucasian subjects gave their informed consent to take part.The nutritional questionnaire was completed by 290 and up to 323 were genotyped (Table 1).Consent was obtained before the collection of peripheral blood for DNA extraction and genotyping.The questionnaires were taken away for participants to complete and on their return scrutinised for accuracy and completion and where necessary subjects were contacted again for clarification.All of the participants were selected on the basis of self-disclosed Caucasian ancestry only [4].Clinical data (age, IBD diagnosis and the most recent Montreal classification of CD location [24]) were extracted from the clinical questionnaire and patient medical notes, which were supplied by the diagnosing gastroenterologist in the primary "Genes and Diet in IBD Study".Ethical approval was given by the NZ Multi-region Human Ethics Committee (MEC04/12/11).

Genotyping
SNP data with respect to the beneficial or adverse effects of selected Brassicaceae were taken from the original "Genes and Diet in IBD Study" based in Auckland, NZ.Gene polymorphisms in this study were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.The SNPs identified for each Brassicaceae variety were corrected for multiple testing using a false discovery rate [25].R was used for statistical analyses.A p-value was considered significant if it was less than 0.05 [26].

Results
Table 2 shows the candidate genes.Table 3 shows the SNPs that were significantly associated with beneficial effects while Table 4 shows the significant SNPs which were associated with adverse effects on the symptoms of people with CD.Table S1 shows the Brassicacea frequencies [n(%)] by SNPS.A total of 37 variants were identified as having beneficial effects (p values from 0.00097 to 0.0497) and 64 SNPs with adverse effects (0.0000751 to 0.049).After correcting for multiple testing, two SNPs in two genes remained significant with adverse reactions to Brassicaceae.The rs7515322 variant in DIO1 was associated with Broccoli, and the rs9469220 variant in HLA was associated with rocket.These p-values were marked with an asterisk (*) (Table 4).

Discussion
On looking more closely at the candidate SNPs associated with CD that were tested for beneficial or adverse effects with the ingestion of selected Brassicaceae, we noted, as one would expect with an inflammatory disorder like Crohn's disease that several of the genes associated with these SNPs were involved in functions relating to immunity.AGT12 (rs26532) and AGT16L1 (rs10210302) are necessary for autophagy; CSFIR (rs2282804), governs macrophages, DEFA6 (rs712276) relates to the Paneth cells and its role in defence and HLA (rs9469220) a major histocompatibility complex, also involves the immune system.The ICAM1gene, (rs1799969) is expressed on the cells of the immune system and the endothelium [27].IL12B, (rs6887695), has also been associated with another immune disorder, asthma [28].IRGM, (rs4958847) is a gene associated with the autophagy pathway in CD [29].IL23R, (rs11209026 and rs7517847) is another gene recognised as being involved in the adaptive immunity pathway as is JAK2 (rs10758669) which is also essential for signalling events in innate immunity [28].PTPN2 (rs2542151) controls a range of cellular processes e.g., cell growth and TLR9 (rs5743836) has a major role in both innate and adaptive immunity [28].
It is curious to note that six of the candidate genes identified have SNPS that are linked with both adverse and beneficial effects.The SNP rs7629936 (CDH29) is associated with adverse effects for cauliflower and beneficial effects for cabbage.The SNP rs2250114 (FHIT) is associated with adverse effects to Chinese greens and wasabi but beneficial effects for mustard powder and mustard sauce.The SNP rs3792796 (GPX3) is associated with adverse effects to mustard powder with beneficial effects to cauliflower and broccoli.The SNP rs2066844 (NOD2) is associated with adverse effects to cauliflower and mustard sauce and beneficial effects to mustard powder.The SNP rs5859 (SEP15) is associated with adverse effects to cabbage and watercress but beneficial effects to mustard powder.The SNP rs9881860 (USP4) is associated with adverse effects to broccoli and Chinese greens and beneficial effects to mustard sauce.
These differences in tolerance may relate to the different composition of each Brassicaceae.Brassicaceae differ in their nutritional components especially with respect to their phytochemical composition [8].They are also unique in that they contain the phytochemicals known as glucosinolates.Up to two hundred different glucosinolates have been recorded [39].These have been intensely researched because of their links with cancer reduction and properties associated with destroying or inhibiting the growth of bacteria, fungi and nematodes [40].Each plant species of the Brassicaceae family may also contain up to four different classes of glucosinolates.The most common glucosinolates in cabbage are for example glucobrassicin, glucoraphanin, glucotropaeolin and sinigrin whereas in broccoli the abundant glucosinolates are glucoraphanin, glucobrassicin, gluconapin, and progoitrin [41].Rocket's predominate glucosinolates are glucoraphanin, DMB-GLS and glucoerucin [42].
When the vegetables are cooked, the glucosinolates reach the large intestine intact and the microbiota release the isothiocyanates and other metabolites with diverse outcomes [43].The different combinations of glucosinolates in combination with particular SNPs may contribute to dissimilar outcomes produced by the microbiota.People with Crohn's disease also have a microbiota community that differs from those with a normal gut [44].So these differences too in the microbiota communities will also play a part in the difference in response.Escherichia coli are bacteria which have been identified as having increased numbers in those with Crohn's disease [45][46][47][48].They have also been associated with formation of granuloma (a common feature of Crohn's disease) when internalised by macrophages in vitro [49].Escherichia coli movement across Membranous cells (M-cells) has been shown in vitro to be reduced by soluble plant fibres such as broccoli [50].[M-cells are part of the epithelium on the lymphoid follicles of the large intestine and act as a portal by which microorganisms can gain entry] [51].The ingestion of broccoli has also been shown in the IBD mouse model mdr1a(−/−) to lower inflammation in the large bowel through changes in the microbiotametabolism [52].However, from the analysis provided in this paper, this response maybe modulated in a different way in humans depending on the particular SNP they have.For example the SNP rs9469220 variant in HLA and the SNP rs7515322 in DIO1 which remained significant after correcting for multiple testing.
The rs9469220 variant in HLA was associated with an adverse reaction to the Brassicaceae rocket.HLA (human leucocyte antigen) genes are located in the region of MHCII in chromosome 6.This is very close to the HLA complexP5 (HCP5) which is associated with susceptibility to autoimmune diseases [53].Variations in the HLA region have been associated with an inflammatory colonic phenotype [54][55][56].HLA contains 4 classes and this SNP is with the class II MHC subgroup DO alpha.Both Class I and II HLA genes are necessary for normal lymphocyte performance [56].This subgroup is associated with extracellular proteins that regulate peptide loading with antigens [57,58].Class II molecules engage with CD4 T cells.These activate an immune response which may involve inflammation with the enlisting of phagocytes or activate B cells engaging antibodies in the immune response [59,60].The polymorphism rs9469220 (identified in this study with an adverse response to rocket) has been linked to total IgE levels [61,62].IgE has a pivotal role in type I sensitivity associated with allergic forms of asthma, rhinitis, urticaria and dermatitis [63].IgE is also recognized as providing immunity to parasites [64].The fact that the SNP rs9469220, in people with CD in this study is significantly associated with an adverse reaction to the Brassicaceae rocket would suggest that ingestion of this food engages the immune response in a type I sensitivity reaction.Hence an exacerbation of symptoms of CD occurs.
The other SNP which remained significant after correcting for multiple testing, was SNP (rs7515322) in DIO1.DIO1 is part of the family of selenoenzymes which are important signalling molecules which activate or deactivate thyroid hormones, so play a key role in thyroid metabolism.As thyroid hormones are key to the development and metabolism of most tissues, these selenoproteins have a significant contribution to make.The DIO1 gene is located in chromosome 1p33-p32 [28].It is described as a thioredoxin fold integral membrane protein found in the plasma membrane.Transcripts of it have been identified in the intestines, thyroid, gonads, pituitary gland and placenta [65].DIO1 has a number of roles.It supplies a large portion of circulating plasma T 3 , it also acts as a scavenging enzyme removing inactive iodothyronines and recycling iodine, as well as playing a part in thyronamine biosynthesis [66][67][68].Studies have also shown DIO activity associated with local inflammation and tumoural tissues [69,70].DIO1 as a selenoprotein has also been discovered to be protective against iodine deficiency when its activity is reduced with selenium deficiency.This was observed in regions of endemic goitre, when selenium was given to people with iodine deficiency with a consequent lowering of thyroid function [71].Selenium levels have been reported as being significantly lower in New Zealand people with Crohn's disease [72].People in New Zealand with Crohn's disease and low selenium levels and with this SNP rs7515322 may have also a heightened adverse response to Broccoli.
However, another mechanism may be involved.Critically ill people have been shown to have lower DIO1 hepatic activity, and this has been thought to be modulated through Cytokines e.g., IL1, IL6 and TNFα [73].Cytokines may also decrease the function of DIO1and IL-1β has been shown to inhibit DIO1 activities in hepatocarcinoma cell lines [74].IL-1β is one of the cytokines increased in patients with Crohn's disease [75] and associated with inflammation in this disorder.This SNP rs9469220 could link to an adverse response through progoitrin one of the most abundant glucosinolates in broccoli [41].Progoitrin after ingestion is converted to goitrin by the activity in bacteria [76] and goitrin is known to decrease thyroid hormone production [77].
In addition, many adults in NZ are in a state of mild iodine deficiency with the adult population having a medium iodine concentration of 53 µg/L [78].Schneider et al. in their murine study showed that DIO1 may enable the major thyroid hormone T 3 to be released into the circulatory system when an animal was iodine deficient [66].Thyrocytes also react quickly to iodine deficiency by increasing the release of angiogenic signals.This is a VEGF-A dependent process [79].VEGF production has been shown to be significantly increased with active Crohn's disease [80].
Those people with CD having this SNP rs7515322 may with the ingestion of broccoli experience a greater degree of deficiency in iodine.This combined with a decrease in DIO1 activity from the influence of IL-1beta activity associated with CD (DIO1 normally increases with iodine deficiency) as well as an increased VEGF levels with the release of angiogenic signals with thyrocyte production, may exacerbate their symptoms of CD.
When investigating individual SNPs from this analysis of a New Zealand population with CD, it is interesting to note the two SNPs which are significantly associated with adverse effects (after correcting for multiple testing) to the two Brassicaceae-broccoli and rocket.Studying a wider range of Brassicaceae would increase the possibility of the identification of more significant SNPs and improve the range of Brassicaceae that would be available to choose from.If people want to avoid the long and tedious trial and error approach to working out what they can eat, or be more discerning about advice given to avoid many of the Brassicaceae as in the FODMAP diet [81] knowledge of their SNP profile and how it interacts with Brassicaceae would be helpful.It would enable people to select Brassicaceae more appropriately and maximise on their nutritional benefits.

Conclusions
It is now possible to identify two specific SNPs rs7515322 (DIO1), and rs9469220 (HLA) associated with the two forms of Brassicaceae broccoli and rocket respectively, with regard to having an adverse effect on the symptoms of people with CD.Further research is required to substantiate our findings and to conclusively determine the nature of the observed association of an adverse effect with the two forms of Brassicaceae broccoli and rocket and the two SNPs rs7515322 (DIO1), and rs9469220 (HLA) respectively, with CD.This study indicates that the tolerance of some varieties of Brassicaceae may be predicted by analysis of a person's genotype.This information is a step towards enabling those with CD to select appropriate Brassicaceae and be exposed to key nutrients.This raises the possibility that with suitable nutrition, there is a real prospect of a significant improvement in symptoms associated with this disorder.

Table 1 .
Summary of sample sizes and clinical data for those with Crohn's disease (CD).

Table 2 .
CD related single nucleotide polymorphisms (SNPs) for association with Brassicaceae.

Table 3 .
Results of SNPs from the candidate genes associated with beneficial effects of Brassicaceae; showing p-values only; significant p values highlighted in italic and bold; n/r: no response.

Table 4 .
Results of SNPs from candidate genes associated with adverse effects of Brassicaceae; showing p-values only; significant p values highlighted in italic and bold.(* Remained significant after applying multiple testing correction using false discovery rate).