Contribution of MDR1 gene polymorphisms on IBD predisposition and response to glucocorticoids in IBD in a Chinese population

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This article is protected by copyrig1h t. All rights reserved.

Contribution of MDR1 gene polymorphisms on IBD

predisposition and response to glucocorticoids in IBD in Chinese

population1

Short title: MDR1 SNPs and IBD in Chinese population

Qing Fan YANG*1

, Bai Li CHEN1, Qing Sen ZHANG

1, Zhen Hua ZHU

1 , Bin HU

2 ,

Yao HE1

, Xiang GAO1, Yi Ming WANG

2, Pin Jin HU

1, Min Hu CHEN

1, Zhi Rong

ZENG#1

1: Department of Gastroenterology, the First Affiliated Hospital, Sun

Yat-sen University, Guangzhou, P.R. China

2: Department of Medical Genetics, Zhongshan School of Medicine and

Center for Genome Research, Sun Yat-Sen University, Guangzhou, China

#: The corresponding author of this study

Correspondence: Zhirong Zeng, Department of Gastroenterology, the

First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan II road,

Guangzhou, P.R. China, 510080. Tel: +86 020 8733 2741. Fax: +86 020

8733 2741. E-mail: [email protected]

The abstract of this study has been accepted as oral presentation in the 1st Annual

meeting of Asian Organization for Crohn's & Colitis held on 13th to 14th June 2013,

Tokyo, Japan and also accepted as poster presentation in the Asian Pacific Digestive

Week 2013/World Congress of Gastroenterology, 21-24 September 2013, Shanghai,

China.

This article has been accepted for publication and undergone full peer review but has not been through

the copyediting, typesetting, pagination and proofreading process, which may lead to differences

between this version and the Version of Record. Please cite this article as doi:

10.1111/1751-2980.12205

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Abstract

Background: Inflammatory bowel diseases (IBD) are chronic diseases of

unknown etiology, in which genetic factors contribute to the pathogenesis.

The cornerstone of conventional treatment is glucocorticoid (GCs) whose

sensitivity varies from patient to patient. Genes such as Multidrug

resistance 1 (MDR1), NACHT leucine-rich-repeat protein 1 (NALP1),

Glucocorticoid receptor (GR) and its co-chaperone FKBP5 participate in

the anti-inflammatory mechanism of GCs. Variations of these genes are

related to GCs response，and MDR1 polymorphisms are also associated

with the susceptibility to IBD in Caucasians. However, whether similar

relationships exist in Chinese population remains unclear. Thus, the aims

of this research were to investigate the polymorphisms of these genes

influence the response to GCs in Chinese IBD patients and the

relationships between MDR1 and IBD susceptibility.

Methods: 8 Single-nucleotide polymorphisms (SNPs) were selected and

genotyped in 156 IBD patients treated with GCs and 223 healthy controls

by MALDI-TOF MS assay. Patients were defined as GCs responders,

dependants or resistants after one year follow up.

Results: The CC genotypes of rs1128503 and rs1045642 in MDR1 gene

were more frequent in GC dependants compared with the responsive Acc

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patients of Crohn’s Disease (CD) (OR 6.583, 95%CI 1.760-24.628,

P=0.019 and OR 3.873, 95%CI 1.578-9.506, P=0.009, respectively).

The G allele of MDR1 rs2032582 was less frequent among CD cases than

in controls (OR 0.668, 95%CI 0.484-0.921, P=0.014). G allele carriers

were also less likely to develop non-stricturing and non-penetrating CD

(OR 0.661, 95% CI 0.462-0. 946, P=0.023) and ileocolonic CD (OR

0.669, 95%CI 0.472-0.948, P=0.024). There was no significant finding in

Ulcerative Colitis ( UC).

Conclusion：Polymorphisms of MDR1 associated with GCs response and

the predisposition to CD in Chinese population. More studies are needed

to elucidate the functions of MDR1 polymorphisms in IBD and their role

as genetic markers for GCs response.

Key words: Inflammatory bowel disease; Single Nucleotide

Polymorphism; Glucocorticoids; Clinical phenotypes;

Introduction

Inflammatory bowel diseases (IBD), including Crohn’s disease (CD)

and Ulcerative colitis (UC), are a collection of chronic inflammatory

diseases characterized by destructive relapsing inflammation of the

gastrointestinal tract. During the last few decades, the incidence of IBD Acc

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has been increasing in previously low morbidity areas, including China [1]

.

Glucocorticoids (GCs) are the important and effective treatment of

moderate to severe IBD, while the sensitivity to which varies between

individuals. However, it also brings a lot of side effects, ranging from

mild to life-threatening adversary events [2]

. According to an investigation

of our center, 20-30% of IBD patients in our country were GCs dependent,

2-9% were resistant and approximately 15% of CD patients required

surgery within one year when the treatment with GCs failed [3]

. Thus it is

useful and attractive to search for biomarkers which enable the prediction

of GCs response to avoid non-effective administering and insensitive IBD

patients.

Recent studies from some populations have confirmed that single

nucleotide polymorphisms (SNPs) of MDR1 (drug transporter

P-glycoprotein encoding gene), NR3C1 (glucocorticoid receptor ɑ and ß

encoding gene), FKBP5 (encoding FK506-binding protein, the

co-chaperone of glucocorticoid receptor), and the pro-inflammatory

cytokine activator NALP1 (NACHT domain, LRR domain, and pyrin

domain-containing protein 1) are associated with the steroid response in

IBD patients [3,4,5,6]

, Hence it is safe to infer that they are potential

genetic predictors of GCs responsiveness in IBD. Mechanism studies

have revealed that the weakened function of glucocorticoid receptor ɑ

(GR-ɑ), abnormal activity of FKBP5, overexpression of P-glycoprotein Acc

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(p-gp), and constitutive activation of some inflammatory factors such as

IL-1ß, regulated by NALP1, may inhibit the anti-inflammatory function

of glucocorticoid. It has indicated close connections between these genes

and GCs response[8,9,10]

. Since the relationship between genes mentioned

above and GCs therapeutic effect in Chinese population remains unclear,

we selected 8 SNPs from these genes to investigate whether they are

associated with the sensitivity to steroid in IBD patients in our population.

As mentioned before, SNPs of MDR1 gene has a relationship with the

susceptibility to IBD in some populations [11, 12]

, so we also assessed the

role of these SNPs in the predisposition to IBD and in affecting clinical

phenotypes of IBD in Chinese population.

MATERIALS AND METHODS

Patients and controls

156 IBD patients (CD: n=117, UC: n=39) were included from our IBD

outpatient Clinic and 223 healthy individuals were included as control

from general physical examination in our hospital Patients enrolled were

diagnosed clinically between 2005 and 2010 according to previously

established international diagnosis criteria [13]

and followed up for at

least 1 year to confirm diagnosis. This study included all consecutive

patients who used steroid for the first time. They were prescribed with oral

GCs for at least 30 days and had a minimum of one year follow-up. Acc

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Patients were initially administered prednisone about 40-60mg/d, and the

doses were subsequently tapered every one or two weeks. We separated

patients into different groups according to the effect of the first course GC

therapy. Patients who maintained complete or partial remission after GCs

withdrawal were defined as GC responders; while patients who were

either i) unable to reduce steroids below the equivalent of prednisolone 10

mg/day within 3 months of starting steroids, without recurrent active

disease, or ii) who have a relapse within 3 months of stopping steroids

were defined as GC dependants; GC resistants were patients who did not

respond after short time therapy (30 days) of GCs [14]

.Clinical

classification of all patients was assessed according to Montreal

classification of IBD [15]

. All subjects were given written informed consent

and this study was approved by the ethic committee of the First Affiliated

Hospital of Sun Yat-sen University.

Genotyping of polymorphism

Blood samples of IBD patients and healthy controls were collected.

Genomic DNA was extracted from peripheral blood leukocytes using the

TIANamp Blood DNA kit (Tiangen Biotech, Beijing, China). The

polymorphisms were genotyped by Beijing Genomics Institute (BGI,

Shenzhen, China) using MALDI-TOF MS assay (MassArray™,

Sequenom, Inc., San Diego, CA, USA). Primers for Polymerase chain

reaction (PCR) were designed (Table-1). Details are available from the Acc

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authors on request. Twenty randomly selected DNA samples were

sequenced to validate the genotyping results by MALDI-TOF MS.

Results of the MALDI-TOF MS method corresponded with the results of

sequencing.

Statistical analysis

All the analyses were carried out by SPSS 13.0 software (SPSS Inc.,

Chicago, Ill., USA). Hardy-Weinberg equilibrium was assessed using the

X2 test. Single factor analysis and subsequent logistic regression model

were used to analyze relationship between genotype and GCs response as

well as relationships between genotypes and disease phenotypes.

Pearson-X2 test was used to analyze differences in allele and genotype

distribution between cases and controls. Odds ratio (OR) and 95%

confidence interval (CI) were carried out by adjusting age and sex. The

criterion for significant difference was two-side probability <0.05.

RESULTS

Demographic characteristics of study participants

A total of 156 IBD patients (97 males and 59 females) and 223

controls (129 males and 94 females) were genotyped for selected SNPs.

The average age of the case and control groups was 32.0±6.8 and

30.9±13.6, respectively. There was no significant difference in sex and

age between cases and controls (P>0.05). All genotypes of the selected Acc

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SNPs were consistent with Hardy-Weinberg equilibrium (P>0.05) in both

case and control groups except that SNP rs1045642 was disequilibrium in

the control group (p=0.04) and hence was excluded when performing

genotype and disease susceptibility analyses. Among the included 117

CD patients, 83 patients were GC responders（83/117, 70.9%）, 29 GC

dependants（29/117, 24.8%）, and 5（5/117, 4.2%） were considered

resistants. Among the 39 UC patients, we identified 22 GC responders

（22/39, 56.4%）, 17 GC dependants（17/29, 43.6%）, and none of them

considered as resistant. Detailed demographic characteristics of the CD

and UC patients are summarized in Table 2 and 3.

Frequency distribution of the selected SNPs between steroid respond

and dependent IBD patients

The number of resistant patients is insufficient (only 5), so these

patients were not included in the statistical analysis. The genotype

frequency of polymorphism rs1128503 (C1236T) and rs1045642

(C3435T) in the MDR1 gene distributed significantly differently between

GCs respond and dependent IBD patients (P=0.027 and 0.023

respectively, Table 4). These SNPs were further found only associated

with the steroid effect of CD patients but not with UC patients (Table 5).

As reported in Table 5, genotype frequency distribution of SNP

rs1128503 (C1236T) and rs1045642 (C3435T) were obviously different Acc

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between the responders and dependants of CD patients. The wild

genotype CC of rs1128503 (C1236T) and rs1045642 (C3435T) were both

more frequent in GCs dependants with CD than in the responders

[adjusted P=0.019 and 0.009，OR(95%CI) 6.583 (1.760-24.628) and

3.873 (1.578-9.506) , respectively], so CC genotype of the two SNPs may

be risk factors for GCs dependence. We further performed haplotype

analysis and found the frequencies of C3435/C1236/T2677 and

T3435/T1236/T2677 haplotypes of the three SNPs was much different

between steroid responders and dependants with CD. Haplotype analysis

revealed that carriers of C3435/C1236/T2677 haplotype had a

significantly higher risk of having CD (p=0.004), while carriers of

T3435/T1236/T2677 had a significantly reduced risk of having CD

(P=0.006, OR (95%CI) 0.383(0.189-0.775)). No significant association

was found between haplotypes of MDR1 SNPs and UC. Besides, the

other 6 SNPs and their haplotypes analysis failed to show any

significant association with GCs effects (P >0.05).

Frequency distribution of the selected polymorphisms in IBD

patients and controls

The secondary aim of our research was to assess whether SNPs of

MDR1 contribute to IBD susceptibility and phenotypes by case-control

study. However, since rs1045642 was not in Hardy–Weinberg equilibrium

in the control group, we did not analyze it. As shown in Table-6, the two Acc

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polymorphisms selected from MDR1 gene were distributed significantly

differently between IBD cases and controls (P<0.05). In further separate

analyzing, we found the mutant genotypes of rs2032582 (G2677T/A) was

more frequent among CD cases in comparison to controls, which

suggested that the G allele of MDR1 rs2032582 (G2677T/A) (OR: 0.668,

95%CI 0.484-0.921, P=0.014) might reduce the risk of CD, while there

was no difference of the genotypes distributions between UC patients and

controls.

Genotype-phenotype analysis

Significant associations were found between MDR1 rs2032582

(G2677T/A) and disease phenotypes (Table 7). The wild allele G of

MDR1 rs2032582 (G2677T/A) was protective for ileocolonic CD (OR:

0.669, 95% CI 0.472-0.948, P= 0.024). MDR1 rs2032582 (G2677T/A)

also had a relationship with CD behavior. Patients who carried G allele of

MDR1rs2032582 had a 0.661-fold less risk of developing non-stricturing

and non-penetrating CD (OR: 0.661, 95%CI 0.462-0. 946, P=0.023). No

significant association had been observed between phenotypes of IBD

and the other SNPs.

Discussion

GCs play a pivotal role in inducing remission of IBD by activating the

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anti-inflammatory reaction in target cells. Once diffusing into cells, GCs

binds to its receptor GR-ɑ and subsequently transported into the nucleus

together to inhibit the transcriptional regulation of many

pro-inflammatory cytokines and finally reduced their production[8]

. The

aberrant activity of GR, for example, declined expression of GR-ɑ or

increased expression of GR-ß which doesn’t bind to GCs will undermine

the mediation function of GCs [16]

. Moreover, the inactivated GR-ɑ is

bound to the heat-shock protein (HSP) complex and immunophilin

FK506-binding proteins FKBP5. Once after binding to GCs, GR-ɑ

becomes activated, and being released from HSP and FKBP5. The

increasing expression of these chaperones such as FKBP5 can modify

GR-ɑ activity by reducing its affinity to GCs, which also lead to

weakened GCs function[17]

. On the other hand, some other molecules also

modulate GCs function. For example, the MDR1 coding protein

P-glycoprotein (P-gp) is a drug transporter which promotes the outflow of

glucocorticoid from target cells. In that case, the overexpression of P-gp

may hinder the effect of GCs by reducing the concentration of GCs in the

cytoplasm. In addition, pro-inflammatory factors, such as TNF-ɑ, IL-1ß

will interference the activity of GCs[18]

; Some of these pro-inflammatory

cytokines must be activated by caspase-1 which needs the help of NALP1

to convert it from inactive precursor [8, 9]

. Therefore, NALP1 participates

in the mediating of GCs function indirectly by affecting the activation of Acc

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pro-inflammatory factors. On the genetic aspect, some researchers found

SNPs of MDR1, NR3C1, FKBP5 and NALP1 also associated with GCs

responsiveness [3, 4, 5, 6]

, which strengthens the role of these genes in GCs

function meditation. In this study, we mainly tried to identify the

relationships between SNPs of these genes and GCs therapy response in

our Chinese population.

Our results suggested SNPs of NR3C1, FKBP5 and NALP1 did not

relate to the GCs therapy response in IBD patients in Chinese population.

The mutated genotype of NR3C1 rs41423247 (OR 0.15, 95% CI 0.03 to

0.68, p=0.0075) was reported to be in association with GC

hypersensitivity in IBD patients in Italian population [3, 4]

. NR3C1

polymorphism rs7701443 (OR 3.43; 95% CI: 1.79–6.57; P= 0.042) and

rs860457 (OR 3.43; 95% CI: 1.79–6.57; P< 0.001) were also related at

the allelic level to corticosteroid resistance in pediatric-onset CD in

Canada population [5]

. The variation of FKBP5 rs4713916 significantly

associated with resistance to GC treatment in CD in Italian population

(responder 17% versus resistant 35%; P=0.0043) [6]

.Furthermore, NALP1

involved in some autoimmune disease [7]

and the variant allele of its

polymorphism rs12150220 was less frequent in the GC responsive IBD

patients in Italian population(P=0.02)[4]

. But here we found these SNPs

don’t affect GCs therapy responsiveness in Chinese patients with IBD,

which may be due to two reasons. Firstly, as the contribution of genetic Acc

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polymorphism differs significantly between ethnicities, it could partially

explain our result. Secondly, former studies suggested some SNPs were

related to GCs resistance [5,6], while we did not analyze the relationship

between GCs resistance and SNPs because it enrolledonly 5 patients.

The exclusion of this group from statistics inevitably risked selection

bias. As mentioned before, 2-9% of IBD patients who used steroid in our

country were GCs resistant [3]

, which was much lower than the dependent

or response rate. Moreover, we only enrolled patients who used steroid

for the first time, which further reduced the number of resistant patients

included. Because of these limitations, it calls for further studies with

larger sample size to further elucidate the relationship between genotype

and phenotype

Despite the fact that, there was no interaction found between SNPs

and NR3C1, FKBP5, NALP1 and GCs efficacy, we did find two SNPs

from MDR1 gene which were positively associated with GCs effect in

IBD patients. The wild genotype CC of rs1128503 (C1236T) and

rs1045642 (C3435T) might lead to the lower sensitivity to GCs and

should be regarded as risk factors for the GCs dependent in CD in

Chinese population. In the previous studies, Krupoves A et al collected

260 pediatric CD patients in Canada population and found rs1045642

(C3435T) did not correlate to GCs sensitivity [19]

. This contrast result

reminded us the influence of polymorphisms might vary depending on the Acc

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age, ethnicity and environment. In mechanism researches, no report is

about how SNP C1236T impact on MDR1 or P-gp functions so far.

Nevertheless, S. Hoffmeyer et al observed a significant correlation of

polymorphism rs1045642 (C3435T) with expression levels and function

of MDR1. The homozygous CC carriers had two-fold higher MDR1 and

P-gp expression levels [20]

. In consistent with S. Hoffmeyer et al, Hitzl

M et al found individuals of the 3435 CC genotype had significantly

higher MDR1 activity measured by rhodamine efflux in CD56+ natural

killer cells than those of TT genotype [21]

.These studies indicated that

C3435T CC genotype might cause more GCs transporting out of the

cytoplasm and GCs less responsiveness, which were in accord to our

results. On the contrary, some other studies found the function of MDR1

and P-gp were independent of rs1045642 (C3435T) [22, 23]

. Though the

associations of these SNPs with GCs effect are contradictory in different

populations and the mechanism is still unclear, our result strongly

demonstrated that MDR1 SNP played a critical role of in GCs effect in

Chinese IBD patients.

MDR1 gene is located in chromosome 7q21.1, which has been

identified as a locus of susceptibility for IBD [24]

, thus we also evaluated

the relationship between MDR1 polymorphisms and IBD in this research.

We discovered that the G allele of MDR1 rs2032582 (G2677T/A) was

less frequent among CD cases in comparison to controls (OR: 0.668, Acc

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95%CI 0.484-0.921, P=0.014), Furthermore, the genotype-phenotype

analysis revealed the G allele of MDR1 rs2032582 (G2677T/A) was

protective from ileocolonic CD and non-stricturing and non-penetrating

CD. However, no significant association between the other MDR1

polymorphisms and the susceptibility to IBD was found, nor was there

any other association with subphenotypes of IBD. In consistent with our

result, Brant et al [25]

found rs2032582 (G2677T/A) had an obviously

association with CD in North American cohort. Whereas Potocnik et al [26]

and Huebner C [27] found a connection with UC and no association was

observed in studies of Ho et al [28]

, Ardizzone S [29]

and Palmieri et al [30]

.

Because of Hardy–Weinberg disequilibrium, we did not analyze MDR1

C3435T association with IBD in Chinese population, though a lot of

studies from different populations found obviously interactions between

MDR1 C3435T and UC or CD [12]

. Heterozygous carriers for the variants

C1236T showed a lower risk of developing ulcerative colitis (odds ratio

0.63, 95% confidence interval 0.42-0.93, P = 0.03; ) as compared with

homozygotes[31]

, while we did not find any association between C1236T

and UC, which might be partially attributed to the small size of the

involved UC patients. P-gp expresses in the epithelial cells and plays a

role in decreasing the absorption of endogenous and exogenous toxins in

the GI tract [32]

. The mechanism is unknown how the G allele of SNP

rs2032582 (G2677T/A) plays a protective role in IBD; Kim RB et al [33] A

ccep

ted

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found nonsynonymous SNP rs2032582 could alter transport function of

P-gp; The G allele of SNP rs2032582 enhanced the transport ability of

P-gp; So SNP rs2032582 (G2677T/A) may affect the development of IBD

by influencing P-gp function.

In conclusion, for the first time we demonstrated MDR1

polymorphisms C1236T and C3435T were associated with GCs

sensitivity in Chinese patients with CD; the G2677T/A polymorphism

correlated to susceptibility to CD and phenotypes of CD. Therefore

MDR1 polymorphism may be a potential genetic marker of GCs response

in IBD patients in Chinese population. Studies with more individuals are

required to further identify the findings Especially in UC patients, the

study should be repeated again. In this study, we had only enrolled 39 UC

patients which was partially due to the relatively low prevalence of IBD

and a lower using rate of steroid in UC than CD. Moreover, functional

studies are also needed to assess the role of these SNPs in modulating

GCs therapy responsiveness and the pathogenesis of IBD.

Acknowledgement

This project was sponsored by Natural Science Foundation of Guangdong

Province, China (No.10151008901000223) and Clinical Key Technology

Program Grant from Ministry of Health of China (No.020426902). Acc

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Contribution of authors and Conflict of interest

Prof. Zingrong Zeng, Minhu Chen and Pinjin Hu helped to design the

project; Prof. Yiming Wang and Bin Hu provided a good lab for us to

perform the experiment; Prof. Xiang Gao and Yao He helped us to collect

subjects’ sample and to inform the patients of this subjects; Doctor

Qingsen Zhang and Zhenhua Zhu helped to analyze the results and

perform part of the experiment. Prof. Baili Chen and doctor Qingfan Yang

participated in all parts of this experiment and wrote the manuscript.

There is no conflict of interest among authors.

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25. Brant SR, Panhuysen CI, Nicolae D, et al. MDR1 Ala893 polymorphism is associated with

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26. Potocnik U, Ferkolj I, Glavac D, et al. Polymorphisms in multidrug resistance 1 (MDR1)

gene are associated with refractory Crohn disease and ulcerative colitis. Genes Immun. 2004

Nov; 5(7):530-9.

27. Huebner C, Browning BL, Petermann I,et al. Genetic analysis of MDR1 and inflammatory

bowel disease reveals protective effect of heterozygous variants for ulcerative colitis.

Inflamm Bowel Dis. 2009 Dec; 15(12):1784-93.

28. Ho GT, Nimmo ER, Tenesa A, et al. Allelic variations of the multidrug resistance gene

determine susceptibility and disease behavior in ulcerative colitis. Gastroenterology. 2005

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susceptibility to inflammatory bowel disease. Inflamm Bowel Dis. 2007 May; 13(5):516-23.

30. Palmieri O, Latiano A, Valvano R, et al. Multidrug resistance 1 gene polymorphisms are not

associated with inflammatory bowel disease and response to therapy in Italian patients.

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Financial Support

The project was supported by research grants of Natural science

foundation of Guangdong province in China (Program number

S2013010016835)

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Table-1 Selected SNPs and their primers

Genes and SNPs Primers Type

MDR1

rs2032582

(G2267T/A)

1st-PCR Primer 5’ACGTTGGATGGAAAATGTTGTCTGGACAAGC3’

2nd-PCR Primer 5’ACGTTGGATGCATATTTAGTTTGACTCACC3’ CNS

rs1128503 (C1236T) 1st-PCR Primer 5’ACGTTGGATGGTTTTTTTCTCACTCGTCCTG3’

2nd-PCR Primer 5’ACGTTGGATGTCTGCCCACTCTGCACCTT3’

CS

rs1045642 (C3435T) 1st-PCR Primer 5’ ACGTTGGATGTATGGAGACAACAGCCGGGT 3’

2nd-PCR Primer 5’ ACGTTGGATGAAGGCATGTATGTTGGCCTC 3’

CS

NR3C1

rs41423247

1st-PCR Primer 5’ACGTTGGATGACCATGTTGACACCAATTCC3’

2nd-PCR Primer 5’ACGTTGGATGACAGGGTTCTTGCCATAAAG3’

NC

rs7701443 1st-PCR Primer 5’ ACGTTGGATGTGTCTCCATTTCCTCCAGAG 3’

2nd-PCR Primer 5’ ACGTTGGATGTGTTAGGCCCCAGTATAAGG 3’

NC

rs860457 1st-PCR Primer 5’ACGTTGGATGAGGTGACCTTCCTCTTGTTC3’

2nd-PCR Primer 5’ACGTTGGATGTAGGTTGCAGAGTCAGTCAC3’

NC

FKBP5

rs4713916 1st-PCR Primer 5’ACGTTGGATGTATCTGGCAACCCTAACCTC3’

2nd-PCR Primer 5’ACGTTGGATGCCTAACGAGATAGTGAGGAG3’

NC

NALP1

rs12150220 1st-PCR Primer 5’ACGTTGGATGCTTGGAGACTCATGGTCTGG3’

2nd-PCR Primer 5’ACGTTGGATGTTTCATTCCCCCCAGAAATC3’

CNS

Abbreviation: CNS, coding nonsynonymous; CS, coding synonymous; NC, noncoding;

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Table-2 Demographic characteristic of CD Patients（n=117）

Responders

（n=83）

Dependants

(n=29)

Resisters

(n=5)

Gender

Male

Female

50（60.2%）

33（39.8%）

18（62.1%）

11（37.9%）

3

2

Age at onset

X±s

25.0±11.8

35.7±12.2

Age at diagnosis

＜17

17-40

＞40

13(15.7%)

59(71.1%)

11(13.3%)

2(6.9%)

24(82.8%)

3(10.3%)

Family history

Yes

No

1（1.2%）

82（98.8%）

1（3.4%）

28（96.6%）

Smoking

Yes

No

Not available

14（16.9%）

66（79.5%）

3(3.6%)

1（3.4%）

28（96.6%）

Appendectomy

Yes

No

Not available

8（9.6%）

74 （ 89.2% ）

1（1.2%）

2（6.9%）

26（89.7%）

1（3.4%）

Location

L1

L2

L3

L4

7（8.4%）

8（9.6%）

63（75.9%）

5（6.0%）

1（3.4%）

7（24.1%）

19（65.5%）

2（6.9%）

Behavior

B1

B2

B3

56（67.5%）

15（18.1%）

12（14.5%）

23（79.3%）

5（17.2%）

1（3.4%）

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Abbreviation: L1: Terminal ileum; L2: colon; L3: ileocolon, L4: upper GI; B1: non-stricturing,

non-penetrating; B2: structuring; B3: penetrating

Table-3 Demographic characteristic of UC Patients（n=39）

Corticosteroid responders

（n=22）

Corticosteroid dependants

(n=17)

Corticosteroid resisters

(n=0)

Gender

Male

Female

16（72.7%）

6（27.3%）

10（58.8%）

7（41.2%）

Age at onset

X±s

36.1±15.2

27.6±9.0

Age at diagnosis

X±s

38.0±14.8

29.8±9.7

Family history

Yes

No

0（0%）

22（100%）

0（0%）

17（100%）

Smoking

Yes

No

Not available

3（13.6%）

16（72.7%）

3(13.6%)

3（11.6%）

13（76.5%）

1 (5.9%)

Appendectomy

Yes

No

Not available

14（63.6%）

0（0%）

8（36.4%）

2（11.8%）

14（82.4%）

1（5.9%）

Location

E1

E2

E3

10（45.5%）

1(4.5%)

11(50%)

1(5.9%)

3(17.6%)

13(76.5%)

Abbreviation: E1: ulcerative proctitis; E2: left-sided UC; E3: extensive UC.

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Table-4 Comparison of the SNP frequency distribution between steroid

respond and dependent IBD patients

SNP Corticosteroid responders VS. dependants

P-value

MDR1

rs2032582

rs 1128503

rs1045642

0.294

0.027

0.023

NR3C1

rs41423247

rs7701443

rs860457

0.685

0.804

0.724

KBP5

rs4713916 0.666

NALP1

rs12150220

0.101

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Table-5 SNP frequency in the steroid dependent or respond patients

with CD

SNP Corticosteroid responders Corticosteroid dependants Ajusted P-value OR (95%CI)

Genotypes Genotypes

WT (%) HET (%) MUT (%) WT (%) HET (%) MUT (%) ( H+M vs W)

MDR1

rs2032582 GG 17(21) GT 32(39.5) TT 21(25.9) 11(40.7) GT 7(25.9) TT 3(11.1) 0.177

TA 11(13.6) AA 0 TA 5(18.5) AA 1(3.7)

rs 1128503 4(4.8) 35(42.2) 44(53) 7(25.0) 12(42.9) 9(32.1) 0.019 6.583 (1.760-24.628)

rs1045642 21(25.6) 45(54.9) 16(19.5) 16(57.1) 9(32.2) 3(10.7) 0.009 3.873 (1.578-9.506)

NR3C1

rs41423247 49（59） 31（37.3） 3（3.7） 15(51.7) 13(44.8) 1(3.5) 0.714

rs7701443 29(34.9) 40(48.2) 14(16.9) 10(34.5) 14(48.3) 5(17.2) 0.841

rs860457 71(85.5) 11(13.3) 1(1.2) 24(82.8) 5(17.2) 0 0.974

KBP5

rs4713916 47(56.6) 27(32.6) 9(10.8) 16(59.2) 9(33.3) 2(7.4) 0.941

ALP1

rs12150220 78（96） 5（4） 0 29(100) 0 0 0.999

Abbreviation: WT, wild type homozygote; HET, heterozygote; MUT, mutant homozygote.

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Table-6 SNP frequency distribution among CD, UC and Controls

SNP CD

n (%)

UC

n (%)

Control

n (%)

IBD (UC+CD) vs Control

Wt+Het vs Mut

P-value

CD vs Control

Wt+Het vs Mut

Adjusted P-value

UC vs Control

Wt+Het vs Mut

Adjusted P-value

MDR1

rs2032582

WT(%) 28(25.9) 10(25.6) 86(38.9) 0.006 0.014 0.205

HET(%) 55(50.9) 23(59.0) 112(50.7) OR=0.668 (95%CI 0.484-0.921)

MUT(%) 25(23.1) 6(15.4) 23(10.4)

rs 1128503

WT(%) 11(9.9) 13(33.3) 35(15.7) 0.058 0.137 0.128

HET(%) 47(42.3) 24(61.5) 101(45.3)

MUT(%) 53(47.7) 2(5.1) 87(39.0)

rs1045642

WT(%) 37(33.6) 15(38.5) 90(40.4)

HET(%) 54(49.1) 19(48.7) 114(51.1)

MUT(%) 19(17.3) 5(12.8) 19(8.5)

NS: No Significance; WT, wild type homozygote; HET, heterozygote; MUT, mutant homozygote.

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Table-7 Correlation between MDR1 Polymorphisms and selected

phenotypic characteristics in CD patients

Disease

phenotypes

rs2032582

Genotypes

P-val

ue

OR(95%CI)

WT(%) HET(%) MUT(%) (WT+HET

vs MUT)

Location

L1 1（12.5） 6（75.0） 1（12.5） 0.746 -----

L2 5 (38.5) 5(38.5) 3(23.1) 0.944 -----

L3 18(22.5) 42(52.5) 20(25) 0.024 0.669

(0.472-0.948)

L4 4(57.1) 2(28.6) 1(14.3) 0.873 -----

Behavior

B1 21(27.3) 38(48.1) 18(22.8) 0.023 0.661

(0.462-0.946 )

B2 3(16.7) 10(55.6) 5(27.8) 0.128 -----

B3 4(30.8) 7(53.8) 2(15.4) 0.699 -----

Abbreviation: L1: Terminal ileum; L2: colon; L3: ileocolon, L4: upper GI; B1: non-stricturing,

non-penetrating; B2: structuring; B3: penetrating; WT, wild type homozygote; HET, heterozygote;

MUT, mutant homozygote.

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Documents

Contribution of MDR1 gene polymorphisms on IBD predisposition and response to glucocorticoids in IBD in a Chinese population