Polymorphisms in the Mannose-Binding Lectin Gene are Associated with Defective Mannose-Binding Lectin Functional Activity in Crohn's Disease Patients

Abstract

Mannose-binding lectin, together with mannose-associated serine proteases, activates the lectin pathway of the complement system and subsequent inflammatory mechanisms. An association between mannose-binding lectin deficiency and anti-Saccharomyces cerevisiae antibody levels is observed in Crohn's disease and this deficiency is frequently associated with a severe Crohn's disease phenotype. In the present study, we assessed the relationship between serum concentrations of mannose-binding lectin, mannose-binding lectin functional activity, MBL2 and NOD2 polymorphisms, anti-S. cerevisiae antibody levels and clinical Crohn's disease phenotype in 69 Crohn's disease patients and 30 age- and sex-matched healthy controls. The results show that the MBL2 variant rs5030737 at codon 52 was associated with a low level of mannose-binding lectin and impaired mannose-binding lectin-mannose-associated serine protease (MBL-MASP) functional activity in Crohn's disease patients. This MBL2 variant was also associated with a higher level of anti-S. cerevisiae antibodies. In addition, the NOD2 variant rs2066844, which is associated with susceptibility to Crohn's disease, was significantly correlated with an impairment in MBL-MASP functional activity. These results provide evidence that Crohn's disease patients have an impairment in MBL-MASP functional activity and that this defect is associated with MBL2 and NOD2 variants.

Polymorphisms in the Mannose-Binding Lectin Gene are Associated with Defective Mannose-Binding Lectin Functional Activity in Crohns Disease Patients

OPEN

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Laura Choteau,,, FrancisVasseur, Frederic Lepretre, Martin Figeac, Corine Gower-Rousseau,, Laurent Dubuquoy,, Daniel Poulain,,,

Jean-Frederic Colombel, Boualem Sendid,, & Samir Jawhara,,

Mannose-binding lectin, together with mannose-associated serine proteases, activates the lectin Saccharomyces cerevisiae antibody levels is mannose-binding lectin, mannose-binding lectin functional activity, and polymorphisms, anti-S. cerevisiae variant was also associated with a higher level of anti-S. cerevisiae the Crohns disease patients have an impairment in MBL-MASP functional activity and that this defect is associated with and

Inammatory bowel disease is a chronic inammatory disease of the gastrointestinal tract which includes Crohns disease and ulcerative colitis1. Although the aetiology of irritable bowel disease is unclear several studies have showed that genetic susceptibility, the microbiota, environment and immune system are all involved in its pathogenesis24. Studies in twins have provided the best evidence for genetic predisposition to irritable bowel disease5. Relatives of patients with Crohns disease have a higher risk of developing irritable bowel disease than those of patients with ulcerative colitis5. In addition to the clinical characteristics of irritable bowel disease such as patient age at diagnosis, disease location and disease behaviour, serological markers, in particular anti-Saccharomyces cerevisiae antibodies, can improve the accuracy of diagnosis of irritable bowel disease. Anti-S. cerevisiae antibodies have the highest sensitivity as serological markers of Crohns disease6. Plevy et al. showed that incorporating a combination of serological, genetic and inammatory markers into a diagnostic algorithm improved the accuracy of diagnosis of irritable bowel disease7. The genetic association of NOD2/CARD15 with Crohns disease has established a critical link between innate immune cells, the intestinal epithelium and development of the disease8. In addition, mannose-binding lectin is another critical protein, which has a major role in the innate

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immune defence against pathogens and promotes intestinal homeostasis9,10. This lectin circulates in the blood as a complex with mannose-associated serine proteases. The mannose-binding lectinmannose-associated serine protease (MBL-MASP) complex activates the lectin complement pathway aer recognition of microorganisms through the carbohydrate recognition domain11,12. The carbohydrate recognition domain of mannose-binding lectin senses polysaccharide patterns such as D-mannose, L-fucose and N-acetylglucosamine on several clinically relevant pathogens13. This results in activation of MASP-1 and MASP-2 leading to the complement cascade and to pathogen neutralisation. It has been shown that MASP-1 is the main activator of MASP-214,15. Both MASP-1 and MASP-2 can form co-complexes with mannose-binding lectin and are crucial to lectin pathway activation. They are also able to cleave prothrombin, promoting clot formation1618.

It has been reported that single nucleotide polymorphisms located within the promoter region and exon 1 of the MBL2 gene are correlated with mannose-binding lectin serum levels and, consequently, are associated with a higher risk of developing infectious disease. Several studies have shown an association between mutations in the MBL2 gene and Crohns disease9,19. Seibold et al. suggested that an impaired innate immune system dened by mannose-binding lectin deciency may lead to increased reactivity to mannan antigens in the microbial cell wall and that this enhanced mannan exposure contributes to the generation of anti-S. cerevisiae antibodies in patients with Crohns disease20,21. Uemura et al. showed that mannose-binding lectin is expressed in epithelial cells in the murine small intestine22. Recently, we showed that mannose-binding lectin is produced locally by human colon epithelial cells in response to intestinal inammation where it plays a crucial role in the mucosal antifungal defence and intestinal homeostasis10. Although the association between mannose-binding lectin serum concentrations and MBL2 gene mutations in Crohns disease patients has been studied previously, the functional activity of the MBL-MASP complex has not yet been investigated in any clinical cohort of Crohns disease patients.

The present study aimed to investigate the relationship between mannose-binding lectin serum concentrations, mannose-binding lectin functional activity, MBL2, MASP1, MASP2 and NOD2 variants, anti-S. cerevisiae antibody levels and clinical Crohns disease phenotype in a cohort of Crohns disease patients in comparison with healthy subjects.

Results

Association between mannose-binding lectin serum concentrations and clinical phenotype of Serum concentrations of mannose-binding lectin were not statistically dierent between Crohns disease patients and healthy controls although a slightly elevated mannose-binding lectin level was observed in Crohns disease patients (P= 0.8). The concentration of mannose-binding lectin was not associated to the clinical phenotype of the disease (B1, B2, or B3) (Fig.1A,B).

To assess whether mannose-binding lectin is able to bind to mannose-associated serine proteases and then to activate the complement system in the serum of Crohns disease patients we explored the functional activity of the MBL-MASP complex. The functional activity assay was based on the ability of mannose-binding lectin to bind to S. cerevisiae mannan and the ability of mannose-associated serine proteases to cleave the uoregenic substrate of thrombin.

The functional activity of the MBL-MASP complex was measured in 69 Crohns disease and 30 healthy control sera. No functional activity of the MBL-MASP complex was detected in either Crohns disease patients or healthy control subjects when the mannose-binding lectin level was <500 ng/mL (Fig.1C). Furthermore, no signicant dierence in functional activity of the MBL-MASP complex was observed between healthy controls and Crohns disease patients. Increased functional activity of the MBL-MASP complex was correlated with the mannose-binding lectin serum level in both healthy controls (P < 0.0001, r = 0.8, Fig.1D) and Crohns disease patients (P<0.0001, r= 0.75, Fig.1E), particularly when the mannose-binding lectin serum concentrations were >500ng/mL. This suggests that the serum concentration of mannose-binding lectin has an important impact on mannose-binding lectin functional activity.

Association between high anti-S. cerevisiae Anti-S. cerevisiae antibody levels were signicantly higher in Crohns disease patients compared to healthy controls (P< 0.0001) (Fig.2A). Furthermore, anti-S. cerevisiae antibody levels were signicantly elevated in Crohns disease patients with the B2 phenotype compared to patients with the B1 phenotype (P < 0.01) and there was also a tendency towards elevated anti-S. cerevisiae antibody levels in Crohns disease patients with the B3 phenotype (P = 0.0516) (Fig.2B). Mannose-binding lectin levels were inversely correlated with anti-S. cerevisiae antibody levels in Crohns disease patients with severe clinical phenotypes (P<0.015, r=0.72) (Fig.2C).

Relationship between the concentrations, MBL-MASP functional activity and anti-S. cerevisiae antibody levels in Crohns To explore whether MBL2 polymorphisms are associated with susceptibility to Crohns disease and to its clinical and serological manifestations, the MBL2 gene and its promoter were genotyped in 69 Crohns disease patients and 30 healthy controls. Two polymorphisms of the MBL2 gene were identied: rs930508 and rs1800450, which were associated with signicant mannose-binding lectin deciency (P<0.01 and P< 0.0001, respectively; Fig.3A,B). The polymorphism rs5030737 (codon 52) was associated with a decrease in mannose-binding lectin serum levels (P< 0.0001) and a low level of MBL-MASP functional activity (P<0.05) in Crohns disease patients (Fig.3C,D). In addition, the polymorphism rs5030737 was associated with signicantly increased levels of anti-S. cerevisiae antibodies (P < 0.01) in Crohns disease patients (Fig.3E). In terms of the association between mannose-binding lectin polymorphisms and clinical phenotype of Crohns disease, the polymorphism rs5030737 was more common in Crohns disease patients with B2 and B3 phenotypes than in those

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Figure 1. Relationship between functional activity of the MBL-MASP complex and mannose-binding lectin concentration. (A) Mannose-binding lectin level was determined in 30 healthy control subjects and 69 Crohns disease patients. There was no signicant dierence between the two groups. (B) No signicant dierence was found between the concentration of mannose-binding lectin and clinical phenotype of Crohns disease. Scatter plots of these data with the median line are shown. Mannose-binding lectin concentration was determined in duplicate for each sample. (C) Functional activity of the MBL-MASP complex was determined in 30 healthy controls (open dot) and 69 Crohns disease patients (black dot). Data are the mean SD of two independent experiments. (D,E) Correlation between functional activity of the MBL-MASP complex and mannose-binding lectin concentration in 30 healthy controls (P<0.0001, R= 0.8) and 69 Crohns disease patients (P<0.0001, R= 0.75). AU, Arbitrary units.

with B1 (Table1). 13% of Crohns disease patients had heterozygous mutations for the rs5030737 variant, which represent 77.7% for B2 and B3 clinical phenotypes vs. 22.3% for B1.

Association between the

To investigate an additional genetic marker for Crohns disease, the NOD2 gene was genotyped in 69 Crohns disease patients and 30 healthy controls. A signicant association was found between the rs2066847 polymorphism and Crohns disease (P= 0.0177) and there was a tendency for the rs2066844 polymorphism to be associated with the disease (P = 0.0518). In terms of the association between clinical phenotype and the NOD2 polymorphism, the rs2066847 variant was found in 15 Crohns disease patients with clinical phenotypes B1 (33.3%), B2 (40.1%) and B3 (26.6%), respectively, carrying the heterozygous mutation C_C (Table2) and in two Crohns disease

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Figure 2. Anti-S. cerevisiae antibody levels in healthy control subjects and Crohns disease patients. (A) Anti-S. cerevisiae antibody level was increased in Crohns disease patients when compared to healthy controls (P< 0.0001). Scatter plots of these data with the median line are shown. (B) Crohns disease patients with the clinical phenotype B2 had higher anti-S. cerevisiae antibody levels than those with B1 (P<0.01)and there was a tendency for Crohns disease patients with B3 to have higher levels than patients withB1 (P=0.0516). (C) Correlation between anti-S. cerevisiae antibody levels and mannose-binding lectin concentrations in Crohns disease patients with B3 (P<0.015, r=0.72). The results are expressed in arbitrary units (AU).

patients carrying the homozygous mutation (both B3). The NOD2 variant rs2066844 was found in 19 Crohns disease patients (heterozygous mutations C_T) with B1 (73.3%), B2 (15.8%) and B3 (10.5%), respectively (Table2).

While the NOD2 variant rs2066844 was not associated with mannose-binding lectin serum levels, the polymorphism was associated with functional activity of the MBL-MASP complex (Fig.3F). Signicantly lower functional activity of the MBL-MASP complex was observed in Crohns disease patients carrying the NOD2 variant rs2066844 (R702W) compared to those with the NOD2 wild-type (P< 0.05) (Fig.3F). In addition to genotyping the MBL2 and NOD2 genes, the MASP1 gene was also genotyped in this study. No association was found between MASP1 and Crohns disease (Table3).

Discussion

The MBL-MASP complex is an activator of the lectin pathway of the complement system and subsequent inammatory mechanisms23. In the present study, mannose-binding lectin serum levels did not vary signicantly between Crohns disease patients and healthy controls. In addition, mannose-binding lectin serum levels were not associated with the clinical phenotype of Crohns disease. These data are consistent with previous clinical studies that show the absence of mannose-binding lectin level changes in Crohns disease patients24,25. Experimental studies showed that MASP-1 and MASP-2 are involved in blood coagulation12,26,27. Hajela et al. showed that soluble

native human MASP-1 has a thrombin-like substrate specicity, cleaving and activating the coagulation proteins Factor XIII and brinogen12,18. In addition, MASP-2 is capable of generating thrombin via prothrombin17.

In the present study, we explored the functional activity of the MBL-MASP complex using a coagulation protease substrate. The test was based on the ability of mannose-binding lectin to bind to S. cerevisiae mannan through its carbohydrate recognition domain and the ability of the mannose-associated serine protease-associated collagen-like domain of mannose-binding lectin to cleave the uorogenic protease substrate27. We observed a signicant correlation between mannose-binding lectin serum concentrations and functional activity of the MBL-MASP complex in both Crohns disease patients and healthy controls. However, a mannose-binding lectin concentration of <500 ng/mL was associated with an impairment in MBL-MASP functional activity and the absence of enzymatic activity in serum samples from Crohns disease patients. To corroborate the functional

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Figure 3. Association between MBL2 and NOD2 polymorphisms, mannose-binding lectin concentration, functional activity of the MBL-MASP complex and anti-S. cerevisiae antibody levels in Crohns disease patients (AC). Mannose-binding lectin concentration was signicantly associated with rs930508 (wild-type C/C heterozygous C/G or homozygous G/G; P< 0.01), rs1800450 (wild-type C/C, heterozygote C/T; P<0.001) and rs5030737 (wild-type G/G, heterozygote G/A; P<0.0001) of MBL2 polymorphisms in Crohns disease patients. (D) Functional activity of the MBL-MASP complex was signicantly associated with the rs5030737 MBL2 polymorphism in Crohns disease patients (wild-type G/G, heterozygote G/A; P<0.05). (E) Relationship between the rs5030737 MBL2 polymorphism and anti-S. cerevisiae antibody levels in Crohns disease patients. Anti-S. cerevisiae antibody level was signicantly higher in heterozygous Crohns disease patients (G/A; n=9) than in wild-type patients (G/G; n= 60) for the rs5030737 MBL2 variant (P<0.01). (F) Association between functional activity of the MBL-MASP complex and the NOD2 polymorphism in Crohns disease patients. Functional activity of the MBL-MASP1 complex was signicantly higher in heterozygous Crohns disease patients (C/T; n= 20) when compared to wild-type patients (C/C; n= 50) for the rs2066844 NOD2 variant (P<0.05).

activity observed in the uorogenic thrombin assay, two other assays were performed. The rst was based on cleavage of complement C4 protein to C4b fragments and the second on the activation of platelets that had been exposed to the MBL-MASP complex trapped on mannan-coated plates. In these assays, we found a signicant correlation between mannose-binding lectin concentrations and functional activity of the MBL-MASP complex in both Crohns disease patients and healthy controls. These results are consistent with those from the uorogenic thrombin assay (Supplementary data).

Anti-S. cerevisiae antibodies are important serological markers that can help to dierentiate Crohns disease from ulcerative colitis6. In the present study, anti-S. cerevisiae antibody levels were signicantly elevated in

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Mann-Whitney

P (=0.05) Number (%) 60 (87%) 9 (13%)

Behaviour, n (%)B1 35 (58.3%) 2 (22%)

B2 12 (20%) 4 (44.4%)

B3 11 (18.3%) 3 (33.3%)

IBDU 2 (3.3%)

ASCA level (AU) 56.55 110.4 <0.05 MBL level (ng/mL) 3211.9 366.4 <0.0001 MBL-MASP activity

(%EF/ng of MBL) 0.35 0.13 <0.05

Table 1. Evaluation of mannose-binding lectin phenotype and genotype in relation to the Crohns disease phenotype. IBDU: inammatory bowel disease not identied; AU: arbitrary unit; ASCA: anti-Saccharomyces cerevisiae antibodies; MBL: mannose-binding lectin; MASP: mannose-associated serine protease; EF: emitted uorescence.

MBL2 rs5030737

Mann-Whitney

P (=0.05) Number (%) 50 (72.5%) 19 (27.5%)

Behaviour

B1 23 (46%) 14 (73.3%)

B2 13 (26%) 3 (15.8%)

B3 12 (24%) 2 (10.5%)

IBDU 2 (4%)

ASCA level (AU) 62.8 66.7 ns MBL level (ng/mL) 3046.5 2299.46 ns MBL-MASP activity

(%EF/ng of MBL) 0.38 0.16 p<0.05

Table 2. Relationship between the rs2066844 NOD2 polymorphism and clinical phenotype of Crohns disease. IBDU: inammatory bowel disease not identied; AU: arbitrary unit; ASCA: anti-Saccharomyces cerevisiae antibodies; MBL: mannose-binding lectin; MASP: mannose-associated serine protease; EF: emitted uorescence.

Crohns disease patients with stricture formation and penetrating disease complications indicating that higher anti-S. cerevisiae antibody levels could be a predictive marker of Crohns disease severity. This observation is consistent with clinical studies showing that high levels of anti-S. cerevisiae antibodies are associated with a complicated clinical phenotype of Crohns disease and the need for surgery28,29.

In the present study, the analysis of MBL2 polymorphisms revealed an association between three variants, rs930508, rs1800450 and rs5030737, and a reduction in mannose-binding lectin serum levels in Crohns disease patients. In addition, both homozygous and heterozygous MBL2 mutations were associated with a decrease in mannose-binding lectin concentrations. Swale et al. showed that the presence of these variants (rs1800450 and rs5030737) was the major contributing factor for lower mannose-binding lectin concentrations30. However, we found that although the MBL2 variant rs5030737 was associated with a low level of MBL-MASP functional activity, this variant was related to high anti-S. cerevisiae antibody levels in Crohns disease patients. These data corroborate previous observations, which showed that patients with low serum mannose-binding lectin or mannose-binding lectin deciency were more oen anti-S. cerevisiae antibody-positive than patients with normal levels of mannose-binding lectin20,31. The variant rs5030737 was found to be related to Crohns disease in a paediatric cohort19. In addition, Schoepfer et al. showed that a low mannose-binding lectin serum level was highly associated with complicated Crohns disease31. Altogether, our data emphasise the role of the rs5030737 variant in MBL-MASP functional activity and suggest that this variant aects the binding of mannose-associated serine proteases to the collagen-like domain of mannose-binding lectin. This may alter the innate immune response and increase the risk of developing a complicated Crohns disease phenotype.

The NOD2 gene is involved in the innate immune response and is highly associated with Crohns disease32.

Crohns disease-associated NOD2 polymorphisms exhibit a reduced capacity to activate NF- following muramyl dipeptide stimulation, suggesting that the loss of NOD2 activation promotes Crohns disease33. NOD2

polymorphisms, rs2066844 (R702W) and rs2066845 (G908R and rs2066847 (l1007fs), are the most common genetic variants associated with an increased risk of Crohns disease34. In addition, these variants alter the structure of either the leucine-rich repeat domain of the protein or the adjacent region3. In the present study, we observed that Crohns disease patients carrying the NOD2 rs2066844 variant had low functional activity of the MBL-MASP complex, suggesting that both the NOD2 rs2066844 variant and impairment of the functional

Wild-type

(G/G)

Wild-type

(C/C)

Heterozygote

(G/A)

NOD2 rs2066844

Heterozygote

(C/T)

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Crohns disease patients vs. healthy controls

Wild-type Heterozygous Homozygous Ref Muted

European allele

frequency Crohns disease

association P

MBL-MASP

activity P

SNP

MASP1

rs850312 C/C: 56.5

vs. 54 C/T: 27.5 vs. 38 T/T: 16 vs. 8 C: 66 T: 34

rs72549154 C/C: 94

vs. 88 C/A: 6 vs. 12 C: 97 A: 3

rs3774268 A/A: 64

vs. 78 G/A: 32 vs. 19 G/G: 4 vs. 4 A: 15 G: 85

rs34090319 G/G: 48

vs. 50 G/GG: 46 vs. 42 GG/GG: 6 vs. 8

rs78008995 G/G: 87

vs. 88 G/T: 13 vs. 8 T/T: 0 vs. 4

rs72549251 T/T: 97

vs. 92 T/C: 3 vs. 8

rs16861895 C/C: 69

vs. 35 C/G: 28 vs. 61 G/G: 3 vs. 4

rs16861896 G/G: 69

vs. 35 G/A: 28 vs. 61 A/A: 3 vs. 4

rs72549254 G/G: 69

vs. 35 G/A: 28 vs. 61 A/A: 3 vs. 4

NOD2

rs2066844 (R702W) C/C: 72

vs. 92 C/T: 28 vs. 8 C: 95 T: 5 0.0518 <0.05

rs2066845 (G908R) G/G: 88

vs. 88 G/C: 12 vs. 12 G: 99 C: 1

rs2066847 (l1007fs) C/C: 75

vs. 96 C/CC: 22 vs. 4 CC/CC: 3 vs. 0 0.0177

rs2076753 G/G: 35

vs. 50 G/T: 36 vs. 46 T/T: 29 vs. 4 0.0119

rs2067085 C/C: 55

vs. 35 C/G: 32 vs. 54 G/G: 13 vs. 11 C: 57 G: 43

rs2066842 C/C: 33

vs. 58 C/T: 38 vs. 38 T/T: 29 vs. 4 C: 76 T: 24 0.002

rs2066843 C/C: 33

vs. 58 C/T: 38 vs. 38 T/T: 29 vs. 4 C: 76 T: 24 0.002

rs1861759 T/T: 56

vs. 31 T/G: 30 vs. 54 G/G: 13 vs. 15 T: 58 G: 42

rs5743291 G/G: 91

vs. 85 G/A: 7 vs. 15 A/A: 1 vs. 0 G: 90 A: 10

rs1077861 A/A: 55

vs. 4 T/A: 33 vs. 69 T/T: 12 vs. 27

Table 3. Frequency of MASP1 and NOD2 polymorphisms in 69 Crohns disease patients and 30 healthy controls. SNP: single nucleotide polymorphism; MBL: mannose-binding lectin; MASP: mannose-associated serine protease.

activity of the MBL-MASP complex could aect the innate immune response against pathogens, leading to an increased risk of Crohns disease35,36.

In conclusion, although mannose-binding lectin serum levels did not vary signicantly between Crohns disease patients and healthy controls, we selected 69 Crohns disease patients for a preliminary polymorphism analysis who reected the characteristics of the original cohort in terms of the diversity of clinical phenotypes, age and sex. This made it possible to explore multiple genetic targets with a more focused approach (Supplementary data). The number of patients with the B3 phenotype was very low in our initial cohort (n= 40). We intend to increase our statistical power by including new Crohns disease patients with the B3 phenotype. Overall, our ndings provide evidence that Crohns disease patients with severe clinical phenotypes have an impairment of MBL-MASP functional activity and that this defect is associated with MBL2 and NOD2 variants. This study will enable us to determine the relationship between MBL2 and NOD2 in Crohns disease and the way in which each aect the other by studying the signalling pathways.

Methods

The Crohns disease patients included in this study had previously been included in the MINOTOR cohort study at Lille University Hospital. At inclusion, the patients underwent a thorough clinical and laboratory examination. The diagnosis of Crohns disease was based on standard endoscopic, histological and radiographic ndings37. The clinical phenotype of Crohns disease was determined according to the Montreal classication on the basis of age at onset (A), disease location (L) and behaviour (B)38. B1 corresponds to non-ste-nosing, non-penetrating disease, B2 to stenosing behaviour and B3 to penetrating behaviour. Sixty-nine Crohns

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Crohns disease

patients (n=69)

Mean age of onset (years) 23 Female/male 42/27 Montreal classication

16-years (A1) 17 (24.6%) 1640-years (A2) 48 (69.6%)

>40-years (A3) 3 (4.3%)

Terminal ileum (L1) 15 (21.7%) Colon (L2) 14 (20.3%) Ileocolon (L3) 34 (49.3%) Non-stricturing/non-penetrating (B1) 37 (53.6%) Stricturing (B2) 16 (23.2%) Penetrating (B3) 14 (20.3%) Perianal lesions 23 (33.3%) Surgery 42 (60.9%) Predisposed subjects 14 (40.6%)

Table 4. Clinical characteristics of the Crohns disease patients. Values shown are n, or n (%). A: age; L: disease location; B: behaviour.

disease patients (42 females/27 males; age at diagnosis: 1050 years; B1 =37, B2=16, B3=14, unclassied=6) were included in the cohort. Thirty healthy control subjects (14 females/16 males; age 1940 years) were also included. All healthy controls were free of symptoms and had a normal clinical examination. Details of the clinical phenotype of the Crohns disease patients including: age at diagnosis (A1: <16 years, A2: 1740 years, A3: >40 years), disease location (L1: ileal, L2: colonic, L3: ileocolonic) and behaviour (B1: non-stricturing, non-penetrating, B2: structuring, B3, penetrating) are shown in Table4.

All subjects were informed about the study and gave their written consent to participate. The study protocol was reviewed and approved by the Ethics Committees of Lille University Hospital (CP 05/86). The study was conducted according to the principles expressed in the Declaration of Helsinki.

Measurement of mannose-binding lectin and anti-S. cerevisiae

Mannose-binding lectin concentrations were measured by enzyme-linked immunosorbent assay according to the manufacturers instructions (BioPorto, Denmark) and are expressed as ng/mL. A concentration of <500ng/mL was considered to represent mannose-binding lectin deciency, low levels were 5001000ng/mL, normal levels were 10004000ng/ml and a high mannose-binding lectin level was >4000ng/mL.

Anti-S. cerevisiae antibodies were also detected by enzyme-linked immunosorbent assay (IBDX gASCA; Glycominds, Israel)39. Briey, 50L of 1:100 diluted serum was added to the coated wells. Absorbance was read at 450 nm (reference lter, 620 nm) in a microplate reader (Bio-Rad) aer addition of tetramethylbenzydine40.

Results are expressed as arbitrary units (AU). Crohns disease patients were declared anti-S. cerevisiae antibody-positive when serum levels were >50AU.

Activity of the MBL-MASP complex in serum was determined using a modied version of a method described previously27. This assay is based on the thrombin-like activity of mannose-associated serine protease to cleave thrombin substrate. Briey, 96-well plates (Nunc-Immuno, Maxisorp, Germany) were coated with 50 l/well of S. cerevisiae mannan (1 mg/mL). Aer incubation for 24h at 4C, the plates were washed twice with wash buer (20mM HEPES, 140 NaCl, 0.1% Tween, pH 7.4) and then incubated for 4h at 4C with 200L of blocking buer (20mM HEPES, 140mM NaCl, 5 mM EDTA, pH 7.4). Aer several washes, 50 L of serum sample was mixed with 50 L of dilution buer (HEPES 40 mM, NaCl 2 M, CaCl2 10 mM, pH 7.4) and then added to each well for 1 h at 4 C. Aer several washes, uorogenic thrombin substrate (VPR-AFC; Sigma, SCP0216) was added to each well and the plate was incubated at 37C for 1h in a spectrouorometer to measure the uorescence released by cleavage of the thrombin substrate every minute (excitation 395 nm, emission 500 nm). The results are expressed as a percentage of emitted uorescence (EF)/ng mannose-binding lectin. All samples were tested in duplicate. The increased uorogenic signal was directly proportional to the mannose-associated serine protease activity (Supplemental data). Aer optimisation of various parameters, in particular the duration of measuring MBL-MASP activity, there was a plateau of activity at 1h and no further variation in enzymatic activity could be detected (Supplemental data).

DNA extractions were performed as described previously10. DNA was extracted using a commercial kit according to the manufacturers instructions (Kit Nucleon BACC3; GE Healthcare). AmpliSeq libraries were prepared using an ion AmpliSeq library kit 2.0 and ion AmpliSeq custom panel (Life Technologies). AmpliSeq technologies were used to design a custom NGS library including 110 amplicons in two pools, covering all targets of interest (19.87kb covered at 99.42%). The targets of interest were the MBL2, NOD2 and MASP1 genes. The design is available in the supplementary data. 10ng of each DNA sample was used as a template to prepare the library. Quality control of all libraries was performed with an Agilent bioanalyser using high sensitivity

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chips. Template dilutions were calculated aer library concentrations were normalized to ~100pM using an ion library equalizer kit (Life Technologies). Library templates were amplied clonally using an ion one touch 2,

following the manufacturers protocol. Recovered template-positive ion sphere particles were subjected to enrichment according to the manufacturers instructions. Samples were subjected to the ion PGM 200 sequencing v2 protocol using ion 318 v2 chips (Life Technologies). Thirty-two barcoded samples were loaded per chip to ensure an average depth of 1500. For data analysis, alignment of the sequences to the human genome build 19 reference genome and base calling were performed using Torrent Suite soware. Identication of variants was performed with an ion torrent variant caller and coverage analysis was generated using coverage analysis plugins (Life Technologies). Allelic frequencies and their association with anti-S. cerevisiae antibody and mannose-binding lectin levels were determined with Haploview soware41.

Statistical analysis was performed using Prism 4.0 from GraphPad and XLSTAT. Data were analysed using either the Kruskal-Wallis or Mann-Whitney U test to compare pairs of groups and the Chi2-test for comparison of two independent groups with categorical data. Dierences were considered significant when the P value was as follows: P < 0.05; P < 0.01; P < 0.001. Fitting the Hardy-Weinberg equilibrium, allelic frequencies and their association with Crohns disease or anti-S. cerevisiae antibody levels were determined with Haploview soware.

References

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Acknowledgements

The authors thank Ms. Shhrazade Sebda and Ms. Nadine Franois for their excellent technical assistance. The authors would like to thank the digestScience Foundation for their support. This work was funded by the FP7 Health 260338 ALLFUN project Fungi in the setting of inammation, allergy and auto-immune diseases: translating basic science into clinical practices.

Author Contributions

L.C. developed the test, collected and tested the patients sera, prepared the diagrams and contributed to the interpretation of the results. F.V. contributed to the interpretation/presentation of the data and to the statistical analysis. F.L. and M.F. contributed to the mannose-binding lectin genotyping and the interpretation of the results. L.D. and D.P. contributed to the interpretation/presentation of the data. C.G.-R. and J.-F.C. were the principal investigators of the Minotor cohort study and J.-F.C. was the reference physician throughout the study. B.S. contributed to the interpretation/presentation of the data and to the statistical analysis. S.J. designed and managed the study, managed the completion of the data and the writing of the manuscript.

Supplementary information accompanies this paper at http://www.nature.com/srep

Competing nancial interests: The authors declare no competing nancial interests.

How to cite this article: Choteau, L. et al. Polymorphisms in the Mannose Binding Lectin Gene are Associated with Defective Mannose Binding Lectin Functional Activity in Crohns Disease Patients. Sci. Rep. 6, 29636; doi: 10.1038/srep29636 (2016).

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