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: zengzhirong@vip.163.com

    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 responseand 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 Crohns 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).

    ConclusionPolymorphisms 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 Crohns 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 and

    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

    (GR-), 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-1, 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). 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 responders83/117, 70.9%, 29 GC

    dependants29/117, 24.8%, and 55/117, 4.2% were considered

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

    22/39, 56.4%, 17 GC dependants17/29, 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 0.009OR(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 HardyWeinberg 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

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

    binds to its receptor GR- 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- or

    increased expression of GR- which doesnt bind to GCs will undermine

    the mediation function of GCs [16]

    . Moreover, the inactivated GR- is

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

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

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

    increasing expression of these chaperones such as FKBP5 can modify

    GR- 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-, IL-1

    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...

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