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ORIGINAL ARTICLE Fucosyltransferase 2 (FUT2) non-secretor status and blood group B are associated with elevated serum lipase activity in asymptomatic subjects, and an increased risk for chronic pancreatitis: a genetic association study Frank Ulrich Weiss,1 Claudia Schurmann,2,3 Annett Guenther,1 Florian Ernst,2 Alexander Teumer,2 Julia Mayerle,1 Peter Simon,1 Henry Völzke,4 Dörte Radke,4 Andreas Greinacher,5 Jens-Peter Kuehn,6 Martin Zenker,7 Uwe Völker,2 Georg Homuth,2 Markus M Lerch1

▸ Additional material is ABSTRACT published online only. To view Objective Serum lipase activities above the threefold Significance of this study please visit the journal online (http://dx.doi.org/10.1136/ upper reference limit indicate acute pancreatitis. We gutjnl-2014-306930). investigated whether high lipase activity—within the reference range and in the absence of pancreatitis—are What is already known on this subject? For numbered affiliations see ▸ The definition of acute pancreatitis includes end of article. associated with genetic single nucleotide polymorphisms (SNP), and whether these identified SNPs are also characteristic clinical symptoms in combination Correspondence to associated with clinical pancreatitis. with elevated serum enzyme activities (amylase Professor Markus M Lerch, or lipase) above three times the upper Methods Genome-wide association studies (GWAS) on 1 Department of Medicine A, ‘ ’ ‘ reference limit. Elevated lipase levels below University Medicine Greifswald, phenotypes serum lipase activity and high serum lipase activity’ were conducted including 3966 German this cut-off are not considered diagnostic for Fleischmannstrasse 41, pancreatitis, and can be observed in a number 17475 Greifswald, Germany; volunteers from the population-based Study-of-Health-in- [email protected] of non-pancreatic conditions. Pomerania (SHIP). Lead SNPs associated on a genome- ▸ wide significance level were replicated in two cohorts, No serum biomarker presently exists for chronic FUW and CS are equally pancreatitis contributing first authors. 1444 blood donors and 1042 pancreatitis patients. GH and MML are equally Results Initial discovery GWAS detected SNPs within or What are the new findings? contributing senior authors. near genes encoding the ABO blood group specifying ▸ In asymptomatic volunteers from a transferases A/B (ABO), Fucosyltransferase-2 (FUT2), and population-based study (N=3966) and healthy Received 4 February 2014 fi Revised 20 June 2014 Chymotrypsinogen-B2 (CTRB2), to be signi cantly blood donors (N=1444), genetic polymorphisms Accepted 23 June 2014 associated with lipase activity levels in asymptomatic that determine the so-called subjects. Replication analyses in blood donors confirmed Fucosyltransferase-2 non-secretor status and the association of FUT-2 non-secretor status (OR=1.49; the ABO blood-type B, were significantly p=0.012) and ABO blood-type-B (OR=2.48; associated with increased serum lipase −8 p=7.29×10 ) with high lipase activity levels. In activities. pancreatitis patients, significant associations were found ▸ This study is the first to identify Lewis and AB0 −4 for FUT-2 non-secretor status (OR=1.53; p=8.56×10 ) blood group systems as disease- relevant for −4 and ABO-B (OR=1.69, p=1.0×10 ) with chronic the development of pancreatitis. FUT2 pancreatitis, but not with acute pancreatitis. Conversely, non-secretors and blood group B carriers carriers of blood group O were less frequently affected by displayed 1.53-fold and 1.69-fold odds of −05) chronic pancreatitis (OR=0.62; p=1.22×10 and less developing chronic pancreatitis, whereas in likely to have high lipase activity levels (OR=0.59; blood type 0 carriers, the odds were clearly −05 p=8.14×10 ). reduced (0.62-fold). Conclusions These are the first results indicating that ▸ FUT2 non-secretor status and blood group ABO blood type-B as well as FUT2 non-secretor status are represent genetic cofactors in the development common population-wide risk factors for developing of alcoholic as well as idiopathic chronic chronic pancreatitis. They also imply that, even within the pancreatitis, but apparently do not influence reference range, elevated lipase activities may indicate the risk for acute pancreatitis. subclinical pancreatic injury in asymptomatic subjects.

To cite: Weiss FU, Schurmann C, Guenther A, et al. Gut Published Online pancreatic duct system and into the duodenum. First: [please include Day INTRODUCTION Here, it has a major role in breaking down dietary Month Year] doi:10.1136/ Human pancreatic lipase (PNLIP, OMIM 246600) lipids such as triglycerides, oils and fats. In healthy gutjnl-2014-306930 is secreted by the exocrine pancreas through the individuals, only little lipase activity is found in the

CopyrightWeiss FU, et Article al. Gut 2014; author0:1–11. doi:10.1136/gutjnl-2014-306930(or their employer) 2014. Produced by BMJ Publishing Group Ltd (& BSG) under licence.1 Downloaded from gut.bmj.com on September 29, 2014 - Published by group.bmj.com

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4308 volunteer participants (2008 males, aged 20–81 years) Significance of this study with German citizenship, European ancestry and principal resi- dence in the study area. Participants with a history of pancrea- titis or renal insufficiency, due to estimated glomerular filtration How might it impact on clinical practice in the rate (eGFR CKD-EPI) <30 mL/min per 1.73 m2)13 were foreseeable future? excluded from further analysis. For our study population of ▸ Blood group B (12–15% in a Western population) and FUT2 3966 participants, serum lipase activity was determined, and non-secretor status (20%) are common genetic traits genome-wide SNP-typing was performed. Approval was associated with serum lipase activities and the risk of obtained by the local ethic committee, and informed consent developing chronic pancreatitis. was given by all participants. ▸ Future studies investigating genetic or environmental risk factors for pancreatitis will have to correct for these factors Blood donors and pancreatitis patients in their cohorts because they vary greatly between The replication cohort consisted of 1500 blood donors (18–68 populations (eg, blood group B among Kashmiris 32%, years, mean age 32.4 years, 58.2% male) who were recruited Norwegians 8%). for the study in 2010 at University Medicine Greifswald. ▸ Elevated serum lipase activities in asymptomatic subjects Written consent and information on alcohol and tobacco con- may indicate, even when within the reference range, sumption, as well as a history of pancreatic disease was subclinical pancreatic injury. recorded. Subjects with a self-reported history of pancreatitis (N=9) were excluded from further analysis. To test the disease relevance of the identified SNPs we performed a second replica- blood, the source of which is either basolateral exocytosis of tion in 1042 German pancreatitis patients (5–97 years, mean 2 pancreatic secretory enzymes , or physiological turnover of age 46.6 years, 62.3% male) that had been enrolled in an obser- 3 acinar cells. vational study (ProZyt) between 2003 and 2011. This cohort – Almost a century ago it was recognised that high serum levels included patients with AP (n=505) and CP ( n=537).14 16 All of pancreatic secretory enzymes (initially amylase and later individuals gave their written informed consent for participation 4 lipase) indicate acute injury of the pancreas. Today, all current and genotyping, and the local ethics committee approved the guidelines require for the diagnosis of acute pancreatitis (AP), in study protocol. Definition for AP was abdominal pain in com- addition to characteristic abdominal symptoms, an elevation of bination with a greater than three-fold elevation of serum amylase or lipase of three times the upper limit of normal or amylase or lipase activity. Diagnosis of CP included the presence 15 higher. Conversely, for the diagnosis of chronic pancreatitis of unequivocal morphological evidence on computed tomog- (CP) no such serum marker exists, and neither elevated nor raphy (CT) and/or endoscopic retrograde cholangio pancrreato- fi reduced lipase activity levels are of suf cient diagnostic accuracy graphy (ERCP) and was labelled idiopathic (ICP (idiopathic 67 to be of clinical use. High subthreshold serum lipase activity chronic pancreatitis); n=229) in subjects in whom no known is common in patients with critical illness of any kind, cholecyst- risk factors, including gallstones, hyperlipidaemia or hypercal- itis, gastric ulcer disease, bowel obstruction, viral gastroenteritis caemia could be identified. Alcoholic aetiology (ACP (alcoholic and renal failure, so that an underlying pancreatic disorder chronic pancreatitis); n=214) was assumed in subjects with a 8 could so far not safely be assumed in symptomatic patients. history of regular alcohol consumption (more than two drinks In a previous population-based study, subthreshold elevated or 20 g/day). serum lipase activities were found to be associated with renal impairment, advanced age, or the use of various medications Phenotype determination that modulate the immune system,9 all of which can occasion- Serum lipase activities in the SHIP-cohort were measured using ally be associated with subclinical pancreatic damage, or overt a turbidometric assay on a Hitachi 717 analyser (Roche pancreatic disease.6 However, lipase elevations in this range are Diagnostics GmbH, Mannheim, Germany). The ‘serum lipase not generally regarded as diagnostic for pancreatic disease.10 activity’ phenotype in the continuous association analysis refers The question of whether or not a genetic predisposition that to the square root of serum lipase activity, and the ‘high serum involves increased lipase serum activities in asymptomatic sub- lipase’ phenotype in the dichotomous analysis was defined as jects represents a risk factor for either pancreatitis or pancreatic binary variable comparing samples with the highest 25% of cancer has remained unresolved.11 To test this hypothesis we serum lipase activity versus the remaining 75%. Serum lipase have performed genome-wide association studies (GWAS) on activity could be analysed in 1444 blood donors using the the phenotypes ‘serum lipase activity’ and ‘high serum lipase ADVIA 1800 Siemens Lipase (LIP), colorimetric test (Siemens, activity’ analysing asymptomatic volunteers of the population- Germany) according to the manufacturer’s protocol, and were based Study-of-Health-in-Pomerania (SHIP). The results indicate in the range between 0.21–3.05 μmol/sl (Median 0.45). Again, that ABO-blood group B and FUT2 non-secretor status are asso- the ‘high serum lipase activity’ phenotype was defined as binary ciated with asymptomatic hyperlipasemia and also increase the variable comparing samples with the highest 25% of serum risk of developing CP. lipase activities (>0.55 mmol/sl) versus the remaining 75%.

MATERIAL AND METHODS Genome-wide SNP genotyping SHIP study population and sample selection SHIP samples were genotyped using the Affymetrix Human SHIP is a population-based study conducted in West Pomerania, SNP Array 6.0 as previously described.12 17 Imputation was the northeastern part of Germany, and its details have been pre- carried out using the HapMap v22 reference panel. For our ana- viously reported.12 From 212 000 inhabitants of this area, a lysis, we excluded all SNPs with a Hardy–Weinberg equilibrium random sample was drawn via the residence registration office p value <0.001, a minor allele frequency (MAF) <0.01 and an where all residents are registered. The SHIP-0 cohort included imputation quality score <0.4.

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Genotyping of blood donor and pancreatitis cohorts activity’ and ‘high serum lipase activity’. The GWAS identified Genotyping was performed using specific TaqMan assays three and two loci that exhibited genome-wide significant asso- (Applied Biosystems) for rs632111, rs8176693, rs889512, ciations for ‘serum lipase activity’ and ‘high serum lipase activ- rs17303392 and rs601338. In some individuals, also the ABO ity’, respectively (figure 1A,B, see online supplementary figure blood group was analysed or verified by genotyping analysis of 1A,B and table 1). The ABO locus at chromosome 9q34 exhib- rs8176719, rs8176747, rs8176750. The genotypes were ana- ited the strongest association with ‘serum lipase activity’ − lysed using TaqMan 7500 RT-System (Applied Biosystems). (p=1.29×10 30) as well as with ‘high serum lipase activity’ − Samples were, in part, verified and analysed by (p=1.28×10 22) where the minor allele of the common lead PCR-amplification with specific oligonucleotide primers fol- SNP of both analyses, rs8176693_T, was associated with lowed by Sanger sequencing using the BigDye V.3.1 terminator increased lipase activity (figure 2A, B). The second locus is − sequencing kit and a 3130xl genetic analyser (Applied located in the FUT2 region at 19q13 (p=6.15×10 9 and − Biosystems). All SNPs were in Hardy–Weinberg equilibrium (p 5.72×10 9 for ‘serum lipase activity’ and ‘high serum lipase value >0.001) and had a MAF >0.01. activity’, respectively; figure 2C, D). As for the first locus, the minor allele of the common lead SNP, rs632111_G, was asso- ciated with increased lipase activities. The lead SNP rs889512 Statistical analysis of the third locus CTRB2 at 16q23 was only genome-wide sig- Because lipase activity values (and corresponding residuals after nificantly associated with the ‘serum lipase activity’ phenotype adjusting for covariates age, sex, smoking and alcohol) were − (p=1.91×10 8; figure 2E, F). In this case, the minor allele right-skewed, we performed a square-root transformation of the rs889512_G was associated with decreased lipase activity. lipase measurements to normalise the distribution and to reduce false positive results. To maximise the statistical power, serum lipase activity levels were analysed as a continuous outcome Replication analysis using linear regression. As a complement, and to consider pos- Serum lipase activity was determined in 1444 local blood sible non-linear effects of the allele counts on serum lipase activ- donors who were also individually genotyped for the lead SNPs ity levels, we also performed logistic regression defining the of the discovery-stage GWAS. Association analyses revealed that outcome according to the phenotype ‘high serum lipase activ- again the ABO locus exhibited the strongest association with the ity’. Linear regression is normally used to establish a relation- − ‘serum lipase activity’ phenotype (p=2.92×10 19) as well as ship between dependent and independent variables, whereas − with the ‘high serum lipase activity’ phenotype (p=5.19×10 9; logistic regression can ascertain the probability of an event table 2). Significant association of the FUT2 and CTRB2 loci (‘high lipase’). − with both phenotypes was also demonstrated (p=2.05×10 6 For a total 3966 SHIP-0 samples and 1444 blood donors, − − and 9.94×10 6 for ‘serum lipase activity’ and p=1.98×10 4 SNP associations were tested using linear regression on the − and 3.31×10 4 for ‘high serum lipase activity’). In all cases, the square root transformed serum lipase levels in mmol/s/l for observed effect directions were the same as in the discovery the ‘serum lipase activity’ phenotype, and logistic regression for GWAS. These results confirmed the significant association of the ‘high serum lipase activity’ phenotype assuming additive polymorphisms within the ABO, FUT2 and CTRB2 loci with genetic effects. Adjustment for age, gender, smoking and serum lipase activity in asymptomatic subjects. alcohol was performed. Furthermore, adjustment was per- formed as follows: in SHIP-0 for the smoking status (never- smoker, ex-smoker and current smoker) and the amount of − Analysis of the correlation between ABO locus association alcohol usage in g day 1, in the replication cohort for the life- and ABO blood group time tobacco exposure in pack years, and the Alcohol Use The GWAS lead SNP rs8176693 is located in the first intron of Disorders Identification Test—C (AUDIT-C) score as a measure- ABO, which raised the question whether it is associated with a ment for the alcohol usage. For the discovery-stage GWAS in − distinct ABO blood group. As blood groups were not deter- SHIP-0, associations with a p value below 5×10 8 were consid- mined in SHIP, we analysed the correlation between rs8176693 ered genome-wide significant.18 The estimated genomic control and ABO blood group subtypes in our blood donor replication was low for ‘serum lipase activity’ and ‘high serum lipase activ- cohort (n=1491) where blood groups were typed serologically ity’ (λ =1.0185 and λ =0.9952, respectively), suggesting GC GC and verified, in specific cases, using TaqMan or sequencing ana- little residual confounding due to population stratification. lysis of ABO (see online supplementary table S1). We found Genomic control was applied for the ‘serum lipase activity’ ana- complete co-occurrence of rs8176693_T and blood group B, lysis only. To identify possible independent associations per with a single exception in one individual, in which locus, lead SNPs were selected by the PLINK clumping algo- rs8176693_T was associated with blood group A /0. Obviously rithm (R2 > 0.01, 1 Mb distance19;) based on genotype data of 1 the minor allele of rs8176693 belongs to the ABO haplotype the HapMapII r28 CEU samples, but no additional hits were that determines blood group B. A high linkage disequilibrium found. Associations were considered replicated in the blood (LD) is indicated by a 2.5-fold over-representation of individuals donor cohort with a p value below 0.05/4=0.0125 − with blood group B (p=7.3×10 08) in the blood-donor sub- (Bonferroni-correction for 4 tests); χ2 tests were performed group exhibiting the 25% highest lipase activities (figure 3). By with help of the interactive calculation tool for χ2 tests of contrast, blood group 0 was significantly under-represented goodness-of-fit and independence, available at (http://quantpsy. − (OR=0.59; p=8.1×10 05) in this subgroup, and carriers of org/chisq/chisq.htm). blood group A were equally frequent in both groups. After stratification for blood groups, we could confirm that in the RESULTS blood donor cohort, the mean lipase activity was highest in car- Discovery-stage GWAS riers of the blood groups B and AB. These results indicate sig- In the discovery-stage GWAS, 3966 individuals were analysed nificant associations of blood groups B and 0 with high and low for associations of SNPs with the phenotypes ‘serum lipase serum lipase activities, respectively.

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Figure 1 Manhattan plot showing significance of association for all SNPs with a minor allele frequency >1% in the genome-wide association analysis. The ‘serum lipase activity’ phenotype (A) refers to the square root of serum lipase concentrations and the ‘high serum lipase’ phenotype (B) was defined as binary variable comparing samples with the highest 25% of serum lipase concentrations versus the remaining 75%. SNPs are plotted on the x-axis according to their chromosomal position against the association with the phenotype (shown as -log10 p value) on the y-axis. The grey − horizontal line indicates the threshold for genome-wide significance (p=5×10 8).

Table 1 Lead SNP associations from the discovery-stage Study-of-Health-in-Pomerania-Genome-wide association studies (GWAS) on the phenotypes ‘Serum lipase activity’ and ‘High serum lipase activity’ Chr Locus lead SNP p Value Effect OR SE Minor allele Major allele MAF HWE pval N Nearest gene ‘Serum lipase activity’ phenotype 9 rs8176693 1.29×10−30 0.139 − 0.012 T C 0.1 0.851 3966 ABO (intronic) 19 rs632111 6.15×10−9 0.04 − 0.007 G A 0.43 0.041 3966 FUT2 (3‘-UTR) 16 rs889512 1.91×10−8 −0.063 − 0.011 G C 0.11 0.575 3966 CTRB2 (5‘ near gene) ‘High serum lipase activity’ phenotype 9 rs8176693 1.28×10−22 0.819 2.27 0.084 T C 0.1 0.851 3966 ABO (intronic) 19 rs632111 5.72×10−9 0.306 1.36 0.053 G A 0.43 0.041 3966 FUT2 (3‘-UTR) − 16 rs889512 6.41×10 4 −0.322 0.72 0.094 G C 0.11 0.575 3966 CTRB2 (5‘ near gene) Chr, chromosome; Effect, effect strength (β) and direction of minor SNP allele; HWE pval, Hardy–Weinberg p value; MAF, minor allele frequency; N, sample size; P, p value of association between locus lead SNP and phenotype; genomic control was applied for the ‘serum lipase activity’ analysis only.

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Figure 2 Regional plots of the three loci associated with ‘serum lipase activity’ and ‘high serum lipase activity’. Shown are the ABO region (A,B), the FUT2 region (C,D) and the CTRB2 region (E,F), displaying the results for ‘serum lipase activity’ on the left and for ‘high serum lipase activity’ on the right, respectively. The region’s lead SNP from the ‘serum lipase activity’ Genome-wide association studies is coloured in purple while other SNPs in the region are given as colour-coded smaller circles. Red indicates high correlation with the top SNP (R2>0.8); orange indicates moderate correlation (0.6

Analysis of the correlation between FUT2 locus association independent of the ABO blood type. Twenty percent of indivi- and secretor status duals with European ancestry are ‘non-secretors’ because they Fucosyltransferase 2 (FUT2) determines an individual’s aibility to represent homozygous carriers of the FUT2 W134X allele (SNP secret ABO blood type antigens into body fluids. This so-called rs601338_A) resulting in a FUT2 null phenotype.20 The observa- secretor (Se) or a non-secretor status (se) is completely tion that the GWAS lead SNP rs632111 is located in the

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Table 2 Replication of the discovery Genome-wide association studies (GWAS) lead SNPs in a validation cohort of 1444 local blood donors Locus Chr lead SNP p Value Effect OR SE Minor allele Major allele MAF HWE pval N Nearest gene ‘Serum lipase activity’ phenotype − 9 rs8176693 2.92×10 19 0.0613 – 0.007 T C 0.09 0.743 1444 ABO (intronic) − 19 rs632111 2.05×10 6 0.019 – 0.004 G A 0.43 0.555 1444 FUT2 (3‘-UTR) − 16 rs889512 9.94×10 6 −0.028 – 0.006 G C 0.11 1 1444 CTRB2 (5‘ near gene) ‘High serum lipase activity’ phenotype − 9 rs8176693 5.19×10 9 0.849 2.34 0.145 T C 0.09 0.743 1444 ABO (intronic) − 19 rs632111 1.98×10 4 0.346 1.41 0.093 G A 0.43 0.555 1444 FUT2 (3‘-UTR) − 16 rs889512 3.31×10 4 −0.594 0.55 0.165 G C 0.11 1 1444 CTRB2 (5‘ near gene) Linear regression analysis was performed after adjustment for age, sex, alcohol consumption and smoking. Chr, chromosome; Effect, effect strength (β) and direction of minor SNP allele; HWE pval: Hardy–Weinberg p value; MAF, minor allele frequency of the SNP in the analysed sample; N, sample size; P, p value of association between locus lead SNP and phenotype.

30-untranslated region (30-UTR) of FUT2 raised the question patients with acute and CP. Genotype distributions for all SNPs whether it is also associated with the non-secretor (W134X) were in Hardy–Weinberg equilibrium (see online supplementary allele. Therefore, rs601338 was additionally genotyped in the table S3). In addition, ABO blood groups, if not available from 1444 individuals of our blood donor replication cohort (table 3, medical records, were also typed (see online supplementary table see online supplementary table S3). The corresponding R2-value S2). The obtained results were compared with those determined was 0.88, indeed demonstrating a shared underlying haplotype in the blood donor replication cohort. These analyses revealed an for rs601338_A and rs632111_G. Similar results (R2=0.87) over-representation of blood group B prevalence in patients with were obtained for the pancreatitis patients cohort subsequently CP (OR=1.69, p=0.0001) but not in those with AP (figure 4). analysed (N=973, see below). In accordance with these results, This association was found in CP of either idiopathic (ICP) or we identified a significant overpresentation of the non-secretor alcoholic (ACP) aetiology (OR=1.57, p=0.0189 and OR=1.65, − rs601338_A allele (OR=1.42; p=1.88×10 4) in the blood- p=0.0097). Further nominal significant associations were found donor subgroup exhibiting 25% highest lipase activities (table 3). between A and AB blood groups and ICP and AP, respectively Homozygous carriers of the rs601338_A allele (non-secretors) (OR=1.36; p=0.032 and OR=1.71; p=0.008). Carriers of were significantly over-represented (OR=1.49; p=0.012) in this blood group O tended to be less frequent among pancreatitis group. patients, and their proportion was significantly decreased in − patients with ICP (OR=0.51, p=2.51×10 05). Replication of associations in pancreatitis patients We further identified a significant allele imbalance for the two In order to test whether the loci identified as associated with FUT2 SNPs typed in the pancreatitis cohort (table 4). increased serum lipase activities are also associated with an Homozygosity for the minor alleles (table 4, Recessive Model) increased risk for pancreatitis, we typed the FUT2 locus lead SNP of rs632111 (GG) as well as of rs601338 (AA, non-secretors) − rs632111, the linked W134X SNP rs601338, and the CTRB2 was significantly associated with CP (OR=1.58, p=2.4×10 4 locus lead SNP rs889512 in a further cohort consisting of and OR=1.57, p=0.001) but not with AP. Interestingly, FUT2

Figure 3 Association of blood group and serum lipase activity in the blood donor replication cohort. ABO blood groups were typed in 1444 donors by serological tests. Blood group prevalences and mean lipase activities (mmol/sl) were calculated for the blood donor cohort as well as for subcohorts of high lipase (25%) and low lipase (75%) phenotypes (A). A forest plot illustrates the different ORs (and 95% CI) of specific blood groups in the 25% of blood donors with highest lipase activities. Results of Pearson’s chi-square tests are also shown (B).

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Table 3 Association of rs601338 (W134X SNP) and secretor status with lipase activity in blood donor cohort p Value Effect OR SE Minor allele Major allele MAF HWE pval N Nearest gene (A) ‘Serum lipase activity’ phenotype 2.47×10−6 0.019 – 0.004 A G 0.403 0.307 1444 FUT2 (3‘-UTR) ‘High serum lipase activity’ phenotype 1.88×10−4 0.349 1.42 0.094 A G 0.403 0.307 1444 FUT2 (3‘-UTR)

75% of sample with low 25% of sample with high Blood Donor Cohort serum lipase activity serum lipase activity Phenotyp (Genotype) N=1444 N=1098 N=346 OR (95% CI) p Value (B) Secretor (GG or GA) 1214 (84.1) 938 (85.4) 276 (79.8) 1.49 (1.08 to 2.05) 0.0121 Non-secretor (AA) 230 (15.9) 160 (14.6) 70 (20.2) FUT2 secretor status was analysed in 1444 blood donors by genotyping,the W134X SNP rs601338_A using TaqMan analysis and genomic sequencing. (A) Linear regression analysis was performed after adjustment for age, sex, alcohol consumption, and smoking. Effect, effect strength (beta) and direction of minor SNP allele; HWE pval, Hardy–Weinberg p value; MAF, Minor allele frequency of the SNP in the analysed sample; n: sample size. (B) Pearson’s χ2 test was calculated for secretor/non-secretor phenotypes in groups of 25% highest vs 75% lowest serum lipase activities. p, p value of association between locus lead SNP and phenotype. non-secretors are over-represented in idiopathic (OR1.7, in the blood donor replication cohort, this was found to be the p=0.003) but not in alcoholic CP. Taken together, these results case in the pancreatitis cohort, where carriers of blood groups suggest that the FUT2 and ABO glycosyltransferases, both AB and O less frequently and more frequently, respectively, cooperating in the synthesis of Lewis antigens Leb and LeY, con- represented non-secretors. Subsequently, we analysed whether tribute to the risk of developing pancreatitis. The CTRB2 locus blood group B and non-secretor status (se/se) represent inde- lead SNPs rs889512 was not significantly associated with pan- pendent or combined/synergistic risk factors for CP.As shown in creatitis of any aetiology in the analysed patient cohort. table 5 no change in combined risk was detected in the pancrea- titis cohort, neither in ICP nor in ACP. However, it has to be Co-occurrence of blood group B and non-secretor considered that the corresponding double genotype/phenotype phenotype (se/se-blood plus group B) is quite rare, as B represents a low- While the non-secretor phenotype (se/se) was not significantly frequency blood group in the general population. Therefore, the different distributed between the individual ABO blood groups statistical power of this analysis is rather limited.

Figure 4 Association results of blood groups in pancreatitis cohort versus healthy blood donors. ABO blood groups were typed by TaqMan analysis or serologic testing in 1042 pancreatitis patient and compared with 1444 blood donors. Pie charts (A) show the blood group distribution in BD, blood donor cohort; Pan, total pancreatitis cohort; CP, chronic pancreatitis; AP, acute pancreatitis; ICP, idiopathic chronic pancreatitis; ACP, alcoholic chronic pancreatitis. Pearson’s χ2 tests were calculated of blood type frequencies in patient cohorts vs. blood donors (B). PVAL, p value (χ2 test), 95% CI, 95% CI of OR, grey shaded cells are nominal significant (PVAL<0.05) and red shaded cells are significant after Bonferroni correction for 15 tests (PVAL<0.05/15 = 0.003).

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blood group frequencies between different populations were Table 4 Association results of Genome-wide association studies identified. This study is the first to identify components of the (GWAS) top SNPs in the pancreatitis cohort vs healthy blood donors Lewis and ABO blood group systems as genetic risk factors for Additive genetic model Recessive genetic model the development of CP. Our findings suggest that non-secretor Model PVAL OR 95% CI PVAL OR 95% CI status and blood group B both predispose to this chronic disease in the absence of other known precipitating risk factors. While rs632111 (FUT2 GWAS lead SNP) blood group B was associated with an increased disease risk, Pan vs BD 0.054 1.12 1.00 to 1.26 0.046 1.23 1.00 to 1.51 − group O was significantly under-represented and appears to CP vs BD 0.003 1.24 1.08 to 1.44 2.4×10 4 1.58 1.24 to2.02 have a protective effect. In the initial GWAS and the replication AP vs BD 0.999 1 0.86 to 1.16 0.469 0.9 0.68 to 1.19 study, we identified FUT2 non-secretor status and the blood ICP vs BD 0.017 1.27 1.04 to 1.55 0.001 1.72 1.25 to 2.38 group B as being significantly associated with elevated serum ACP vs BD 0.497 1.08 0.87 to 1.34 0.276 1.23 0.85 to1.79 lipase activity in asymptomatic volunteers. The fact that pancre- rs601338 (W134X SNP) atic injury is associated with increased serum lipase activity has Pan vs BD 0.047 1.12 1.00 to 1.26 0.031 1.26 1.02 to 1.56 long been established for AP.In CP,on the other hand, increased CP vs BD 0.015 1.2 1.03 to 1.38 0.001 1.57 1.22 to 2.02 activities due to acute inflammatory episodes, as well as AP vs BD 0.501 1.05 0.91 to 1.22 0.839 0.97 0.73 to 1.29 decreased lipase levels due to reduced exocrine tissue have been ICP vs BD 0.081 1.2 0.98 to 1.46 0.003 1.7 1.21 to 2.37 observed, and serum lipase measurements were, therefore, con- ACP vs BD 0.592 1.06 0.85 to 1.32 0.168 1.32 0.90 to 1.93 sidered to be of little diagnostic value. Elevated lipase activity in rs889512 (CTRB2 locus lead SNP) asymptomatic subjects has been correlated in previous studies Pan vs BD 0.809 0.9 0.81 to 1.18 0.974 0.99 0.46 to 2.12 with increased serum creatinine levels, advanced age, use of ster- CP vs BD 0.830 0.98 0.77 to 1.23 0.647 1.23 0.51 to 2.99 oids or antihypertensive medication as well as self-reported CP, AP vs BD 0.868 0.98 0.77 to 1.24 0.564 0.73 0.25 to 2.19 but was not considered to be a risk factor for CP in itself. The ICP vs BD 0.757 1.05 0.77 to 1.44 0.720 0.77 0.18 to 3.36 current data raise the possibility that hyperlipasemia—even ACP vs BD 0.073 0.71 0.48 to 1.05 0.902 0.91 0.21 to 3.98 below the 3× upper reference limit and, therefore, within the ACP, alcoholic chronic pancreatitis; AP, acute pancreatitis; BD, blood donor cohort; range defined as normal—may be a marker of subclinical and CP, chronic pancreatitis; ICP, Idiopathic chronic pancreatitis; Pan, total pancreatitis cohort; PVAL, p value (χ2 test); 95% CI, 95% CI of OR, grey shaded cells are nominal continuous pancreatic injury in asymptomatic subjects. The significant (PVAL<0.05) yellow shaded cells are significant after Bonferroni correction common denominator for the observed clear associations of the for 15 tests (PVAL<0.05/15=0.003) and red shaded cells are significant after ABO FUT2 Bonferroni correction for 30 tests (PVAL<0.05/30=0.0017). Genotyping of SNPs was and loci with high lipase activity as well as with pan- performed by TaqMan analysis or genomic sequencing. creatitis might be that, on a population-based level rather than an individual level, high lipase activity indicates subclinical pan- creatic damage. An alternative explanation is that a common underlying mechanism causes pancreatitis and elevated serum DISCUSSION lipase activity, and that this mechanism involves specific glycosy- Relationships between blood groups and disease phenotypes lation patterns mediated by the haplotypes underlying blood have been debated for almost a century, when variations in group B and the non-secretor status. Under the latter scenario,

Table 5 Association results (A) and comparison (B) of extreme groups in the pancreatitis cohort vs healthy blood donors BD Pan CP AP ICP ACP (A) Blood group B (G1) vs remaining (G0) G1 180 171 100 71 40 39 G0 1311 871 437 434 189 175 Non-secretor (G1) vs secretor (G0) G1 233 186 113 73 54 38 G0 1258 795 389 406 172 156 Blood group B and non-secretor (G1) vs remaining (G0) G1 30 25 17 8 6 6 G0 1461 956 485 471 220 188

Blood group B and non-secretor vs Blood group B vs non-B Non-secretor vs secretor remaining

PVAL OR (95% CI) PVAL OR (95% CI) PVAL OR (95% CI) (B) Pan vs BD 0.0023 1.43 (1.14 to 1.79) 0.0352 1.26 (1.02 to 1.56) 0.4562 1.27 (0.74 to 2.18) − − CP vs BD 2.2×10 4 1.67 (1.28 to 2.18) 7.2×10 7 1.57 (1.22 to 2.02) 0.1129 1.71 (0.93 to 3.12) AP vs BD 0.2774 1.19 (0.89 to 1.6) 0.8958 0.97 (0.73 to 1.29) 0.7776 0.83 (0.38 to 1.82) ICP vs BD 0.0300 1.54 (1.06 to 2.24) 0.0026 1.70 (1.21 to 2.379) 0.7044 1.33 (0.55 to 3.23) ACP vs BD 0.0161 1.26 (1.11 to 2.37) 0.1907 1.32 (0.9 to 1.93) 0.4744 1.55 (0.64 to 3.78) ACP, alcoholic chronic pancreatitis; AP, acute pancreatitis; BD, blood donor cohort; CP, chronic pancreatitis; ICP, idiopathic chronic pancreatitis; Pan, total pancreatitis cohort; PVAL, p value (χ2 test). 95% CI, 95% CI of OR, grey shaded cells are nominal significant (PVAL<0.05) and red shaded cells are significant after Bonferroni correction for 15 tests (PVAL<0.05/15 = 0.003). Genotyping of SNPs was performed by TaqMan analysis or genomic sequencing.

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Pancreas serum lipase activity would simply represent a surrogate marker 2.42.29 Strikingly, according to the HapMap (release 22) data- for other, not yet identified disease-relevant proteins involved in base, the ABO lead SNP rs8176693 identified in our study to be pancreatitis pathogenesis. associated with ‘serum lipase activity’ as well as with ‘high The third locus that was significantly associated with high serum lipase activity’ is in perfect LD (D0 value: 1.0) with lipase activity in the population-based GWAS, CTRB2, was not rs505922, the lead SNP of the initial GWAS on pancreatic associated with pancreatitis in the patient cohort. There are, cancer.24 The minor alleles of both SNPs were associated with again, different explanations for this finding, the most simple of increased serum lipase activity and increased risk for pancreatic which is that the association of this locus with pancreatitis cancer, respectively, indicating a shared haplotype that appar- simply is much weaker than the associations between pancrea- ently also carries the polymorphisms specifying blood group titis and the ABO and FUT2 loci. In this case, a larger patient B. Indeed, the D0 values between blood group B defining SNPs cohort would be necessary to reach a comparable level of statis- rs8176743, rs8176746 and rs8176747 on the one hand, and tical significance for CTRB2. rs8176693 on the other hand also consistently amounted to A, B and H antigens, the antigenic determinants of the ABO 1.0. The corresponding D0 values between these three ABO-B blood group system, are formed by the sequential addition of SNPs and rs505922 were 1.0, 1.0, and 0.56. Interestingly, while specific monosaccharides to the carbohydrate side chains of gly- the r2 values between the three ABO-B SNPs and rs8176693 colipids and glycoproteins. These specific glycans decorate the were also consistently high (1.0, 1.0, 0.69), this was not the surface of erythrocytes. The biosynthesis of A and B antigens case between the ABO-B SNPs and rs505922 (0.16, 0.16, involves glycosyltransferases that catalyse the addition of 0.073) as well as between rs505922 and rs8176693 (0.16). This N-acetyl-D-galactosamine or D-galactose to core H antigens. reflects the higher MAF (according to HapMap data) of the A single base pair deletion resulting in the inactivation of pancreatic cancer risk lead SNP rs505922 (0.363) as compared these glycosyltransferases is the reason why in individuals with to the ABO-B SNPs (0.08, 0.075, 0.123) and the lead SNP blood group O the H antigen remains unmodified. The rs8176693 (0.083) in individuals with European ancestry. We common denominator of the H antigen, the Fucα1.2-glycan therefore hypothesise that rs505922 represents a phylogenetic structure, is formed by specific α (l.2) fucosyltransferases which older SNP which has already been present on an ancestral play a regulatory role in the tissue expression of the ABH anti- haplotype and is only associated with increased pancreatic gens. In the bone marrow (from where erythrocytes originate) cancer risk via its LD with the more recently arisen haplotype fucosylation of ABH antigens is performed by the common that is associated with blood group B and increased serum lipase H-(fucosyl)-transferase (FUT1). However, ABH antigens are not activity. According to this model, the increased pancreatic confined to erythrocytes but are expressed on other tissue cells, cancer risk would be caused by the presence of the latter haplo- body fluids or along the gastrointestinal mucosal lining in indivi- type detected in our study. Inactivation of the ABO-encoded duals of ‘positive secretor status’ (secretor phenotype, Se).21 blood group specifying glycosyltransferases mediates a protect- This is because Se individuals carry at least one functional allele ive effect against pancreatic cancer and, according to the results (Se/Se or Se/se) for the Secretor-(fucosyl)-transferase (FUT2), of our study, mediates a decreased risk for developing CP.Today, which produces the Fucα1.2-glycan structure in secreted body we have only a limited understanding of the initiating molecular fluids, such as saliva, tears, breast milk, gastric juice and gastro- events in pancreatic cancer and the potential role of ABH blood intestinal mucus secretions. Individuals who are ‘secretors’ groups in pancreatic tumorigenesis. It is, however, well estab- express blood group O antigens (Lewis b (Leb) and H antigens), lished that persisting CP increases the risk of developing pancre- which can be further converted by the ABO glycosyltransferases atic cancer.31 32 One possible mechanism explaining our into A or B antigens, respectively.22 By contrast, individuals of findings is a 25% increased level of von-Willebrandt-Factor non-secretor phenotype, Se0, lack the secretor-transferase (VWF) in individuals who have a blood group other than O.33 altogether, and synthesise the shorter Lewis a antigen (Lea).20 The glycosylation of VWF by ABO A and B glycosyltransferases Approximately 20% of the world population are non-secretors, affects circulating VWF levels and function through several but geographical and ethnical differences do exist.20 mechanisms.34 These may include the rate of VWF synthesis The functional significance of ABO antigen expression and secretion, its proteolysis by metalloprotease ADAMTS13, outside the blood has not been defined, but several studies have and the modulation of VWF clearance.35 VWF is a negative found correlations between disease susceptibilities and ABO modulator of angiogenesis, and displays proinflammatory activ- blood group or secretor status. One long-established association ity which could promote a chronic inflammatory state. is that between blood group O and peptic ulcer disease.23 Reilly A recent GWAS demonstrated that the FUT2 non-secretor et al recently reported an association between ABO polymorph- status is a risk factor for Crohn’s disease,36 but apparently is no isms and myocardial infarction in patients with coronary artery risk modifier in the association of ABO antigens with pancreatic disease. The ABO association was attributable to the glycotrans- cancer.37 Being a non-secretor has several potential health disad- ferase deficiency specified by the ABO blood group O genotype vantages. Secreting blood-type antigen into saliva, mucus, and previously proposed to protect against myocardial infarction.24 other body fluids may add a degree of protection against Of particular importance in the context of our study are the environmental factors, particularly with respect to micro- recently reported associations between ABO blood group and organisms. Secretors have a better capacity to stabilise the – the risk and outcome of pancreatic cancer.25 27 Cohort studies blood-type-dependent intestinal bacterial ecosystem. A part of in independent populations confirmed a significant higher risk the intestinal microbiome that is considered beneficial (probio- of developing sporadic pancreatic cancer in carriers of the blood tics) preferentially feed on the glycosylation products that define group B. Carriers of blood groups A and AB exhibited an inter- the blood type system. Bacteria in the digestive tract of secretors mediate risk, and carriers of blood group O demonstrated the may benefit from a constant nutritional supply by the secretion – lowest risk.28 30 Compared with the OO genotype, addition of of blood group antigens in the mucus that lines the intestinal each non-O allele increased the pancreatic cancer risk. Subjects mucosa. The composition of this commensal flora is a contribut- with AO and AA genotype had ORs of 1.33 and 1.61, while ing mechanism of the ABO and Lewis blood group-dependent individuals with BO and BB genotypes had ORs of 1.45 and risk for inflammatory bowel disease,36 various gastrointestinal

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Fucosyltransferase 2 (FUT2) non-secretor status and blood group B are associated with elevated serum lipase activity in asymptomatic subjects, and an increased risk for chronic pancreatitis: a genetic association study

Frank Ulrich Weiss, Claudia Schurmann, Annett Guenther, et al.

Gut published online July 15, 2014 doi: 10.1136/gutjnl-2014-306930

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