Polymorphisms in RYBP and AOAH Genes Are Associated with Chronic Rhinosinusitis in a Chinese Population: A Replication Study

Background The development of CRS is believed to be the result of combined interactions between the genetic background of the affected subject and environmental factors. Objectives To replicate and extend our recent findings from genetic association studies in chronic rhinosinusitis (CRS) performed in a Canadian Caucasian population in a Chinese population. Methods In a case-control replication study, DNA samples were obtained from CRS with (n = 306; CRSwNP) and without (n = 332; CRSsNP) nasal polyps, and controls (n = 315) in a Chinese population. A total of forty-nine single nucleotide polymorphisms (SNPs) selected from previous identified SNPs associated with CRS in Canadian population, and SNPs from the CHB HapMap dataset were individually genotyped. Results We identified two SNPs respectively in RYBP (rs4532099, p = 2.15E–06, OR = 2.59) and AOAH (rs4504543, p = 0.0001152, OR = 0.58) significantly associated with whole CRS cohort. Subgroup analysis for the presence of nasal polyps (CRSwNP and CRSsNP) displayed significant association in CRSwNP cohorts regarding to one SNP in RYBP (P = 3.24E–006, OR = 2.76). Evidence of association in the CRSsNP groups in terms of 2 SNPs (AOAH_rs4504543 and RYBP_rs4532099) was detected as well. Stratifying analysis by gender demonstrated that none of the selected SNPs were associated with CRSwNP as well as CRSsNP. Meanwhile 3 SNPs (IL1A_rs17561, P = 0.005778; IL1A_rs1800587, P = 0.009561; IRAK4_rs4251513, P = 0.03837) were associated with serum total IgE level. Conclusions These genes are biologically plausible, with roles in regulation of transcription (RYBP) and inflammatory response (AOAH). The present data suggests the potential common genetic basis in the development of CRS in Chinese and Caucasian population.


Introduction
Chronic rhinosinusitis (CRS) is a common inflammatory disorder of the sinus and paranasal sinus mucosa with a highly heterogeneous pathogenesis. Because of its negative impact on patients' quality of life and the concomitant increasing social economic burden, CRS has become a global health problem [1]. The development of CRS is believed to be the result of combinations between the genetic background of the affected subject and environmental factors [1,2]. However, there is still no clear answer as to their exact contributions to the process and the mechanisms of pathogenesis of CRS.
A genetic basis to sinusitis is strongly suggested by clinical and experimental evidence [3][4][5][6][7][8][9][10]. In humans, genetic disorder such as cystic fibrosis and primary ciliary dyskinesia [11] are frequently associated with development of sinusitis in knockout mouse models, deletion of several genes leads to development of sinusitis.
Given the evidence above, genetic backgroung plays potencial roles in the development of CRS and we hypothesized genes which were demontrated as susceptible genes for CRS in Caucasian population also exerted effects in Chinese cohort. Therefore, the aim of this study was to replicate polymorphisms in the genes performed in Canadian Caucasian population previously are associated with the Chinese population. A population-based case-control association analysis was used to assess the risk of CRS conferred by SNPs in the candidate genes in our Han Chinese cohort.

Study Subjects
306 CRS with nasal polyps (CRSwNP subjects) (180 males and 126 females) and 332 individuals affected with CRS without nasal polyps (CRSsNP) (190 males and 142 females) were prospectively recruited from the rhinology ward of Beijing Tongren Hospital between February 2008 to July 2009. A total of 315 healthy controls, of which 146 (46.3%) were female, were recruited as well. All the subjects were of Chinese Han ethnic origin and all from the north region of China. The study was approved by the Beijing Tongren Hospital Ethics Committee, and written informed consent was obtained from all participants.
Diagnosis of CRSwNP and CRSsNP was based on American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) 2004 guidelines [17], based on assement by a single ENT doctor specialized in sinus diseases. All the CRS cases recruited in present study were unresponsive to all forms of medical therapies such as topical or intranasal corticosteroid and long term-low dose antibiotic or presented persistent signs/symptoms of CRS despite previous endoscopic sinus surgery (ESS). Patients were interviewed by trained personnel, and a standardized questionnaire was used to obtain items including demographic variables and personal and familial antecedents of allergies. Patients also underwent a standard set of laboratory tests that included measurements of total IgE to assess allergic status. Controls were recruited following two strategies: either spouses or non-blood relatives living in the same household and individuals recruited from a geographic area similar to that of CRS patients. The only attempt at matching subjects and control is their geographical location to minimize differences secondary to differences in potential environmental exposures. Nevertheless, a standardized questionnaire assessing age, sex and ethnic origin (but not smoking, history of atopy or physician diagnosed asthma) was obtained for controls. Moreover, all the controls showed negative of serum phadiatop determination.

SNP Selection
A total of forty-one single nucleotide polymorphisms (SNPs) selected from previous identified associated with CRS in Canadian population [3,[5][6][7]9,10,16,18,19] were choose for genotying (Table 1). In addition, SNPs in IRAK4 gene from the CHB HapMap dataset were also individually genotyped. Briefly, the International Haplotype Mapping (HapMap) (www.hapmap.org) SNP databases were used to select SNPs in the IRAK4 gene region. The screened region was extended 10 kilobases upstream of the annotated transcription start site and downstream at the end of the last IRAK4 exon. The SNPs were selected to extract the most genetic information based on CHB haplotype data using the HAPMAP database (Hapmap Data Rel 27 Phase II+III, Feb09) [20]. From this dataset, 34 SNPs in IRAK4 gene region were selected using a pairwise tagging algorithm implemented in Haploview version 4.1 program [21]. In addition, when we set Hardy-Weinberg p value cutoff, minor allele frequency and r2 thresholds at 0.01, 0.05 and 0.8, respectively, the LD pattern for IRAK4 gene in our population showed strong LD in several groups of SNPs, indicating that the SNPs in each group represent a common region ( Figure 1). Consequently, we choose 10 SNPs including rs4251513, rs1461567, rs3794262, rs4251481, rs4251540, rs4251569, rs6582484, rs4251431, rs1870765 and rs12302873 to represent the entire 34 loci for eventual genotyping and the former two SNPs were composed in the selected SNPs from the previous identified associated with CRS in Canadian population. Therefore, 49 SNPs constituted the selection set to be genotyped in our patient and controls eventually.

Genotyping
DNA was collected in EDTA-treated tubes and isolated from peripheral blood leukocytes, using the DNA Isolation Kit for Mammalian Blood (Roche, Indianapolis, USA). Isolated DNA from blood was stored at 4uC prior to use. To reduce genotyping cost, the majority of the selected SNPs were genotyped by the MassArray system (Sequenom) with primers and probes (Table 2) as described. One SNP (rs12302873) which was evaluated by preliminary test unsuitable to be genotyped through MassArray approach were identified by direct sequencing of PCR products of genomic DNA (Table 3). Genotyping was performed without knowledge of the case or control status. A 10% random sample was tested in duplicate by different persons, and the reproducibility was 100%.

Statistical Analyses
PLINK program version v1.02 was used to determine association. The association test is based on comparing allele frequencies between cases and controls using Chi-squared tests (x2). We estimated odds ratios (OR) and 95% confidence intervals (95% CI) for the effect of polymorphisms on CRS risk. A corrected p-value of ,0.05 was considered statistically significant. Bonferroni correction over the tested SNPs was performed for multiple adjustments. Subanalysis restricted to the presence of nasal polyps was also performed to examine whether the effect of observed associations within the population differed within the subgroups. Associations between genotype and IgE levels for all patients were assessed using an Anova test, which was performed in the R statistics software version 2.3.1. Haploview 4.1 software was used to generate the linkage disequilibrium (LD) plot. Table 4 provides a summary of the demographic characteristics of the study population. Age and gender were all well-balanced between cases and controls. The cohort of 306 CRSwNP patients had a mean age of 43 years and consisted of slightly more men (58.8%) than women (41.2%), while the 332 CRSsNP individuals

Association Analysis
Allele frequencies for all 49 SNPs were calculated and the significant associations between alleles and CRS phenotype were shown in Table 5. The significant associations (P , 0.05) only existed among the genes coding RYBP (rs4532099), AOAH (rs4504543) and IRAK4 (rs1461567, rs4251559 and rs3794262) genes at 5 loci. Table 5 shows odds ratios for risk allele and the corresponding P values. Two SNPs respectively in RYBP (rs4532099, P = 2.15E-06, OR = 2.59) and AOAH (rs4504543, P = 0.0001152, OR = 0.58) remained significant following application of the Bonferroni correction for multiple testing for 49 simultaneous tests (P,0.001).
In order to verify potential association between total serum IgE levels and the selected SNPs, a quantitative trait analysis was performed. As presented in Table 7, 3 SNPs (IL1A_rs17561, P = 0.005778; IL1A_rs1800587, P = 0.009561; IR-AK4_rs4251513, P = 0.03837) were associated with serum total IgE level.

Discussion
In this study, we replicate a number of genes in CRS in previously identified in Caucasians in a Han Chinese population. Genes associated with CRS and the Caucasian population has not yet being replicated in the Chinese population and the replication suggests a common basis.
The genes identified are of potential important biological significance. RYBP is a regulator of transcription [12]. ILIRL1 is associated with Toll-like receptor (TLR) signaling regulation [14]. SNPs in the IL1RL1 gene have previously been shown to affect  serum level of eosinophilia and IgE in other models [5]. IRAK4 also is implicated as a signaling intermediate in the TLR signaling pathway [16,22], and SNPs in the IRAK4 gene have been documented to have a functional impact, within genotype specific effect on serum IgE level [6,23]. Importance of TLR signaling is suggested by their role in detecting and regulating responses to gram-positive and gramnegative bacteria. Importance of IRAK-4 is suggested by the description of an enhanced susceptibility to infection with grampositive bacteria in IRAK-4 deficient children. Previous work from the Desrosiers group has identified altered function of the TLR signaling system as key to the pathogenesis of CRS. Using complex-model analysis of pooling-based genome wide association testing on the Canadian population with CRS, they identified the polymorphisms at multiple levels of the TLR signaling cascade all confer an increased risk in CRS [2,16]. Functional support for this concept has been provided by in vitro model of epithelial cell culture documenting reduced response to TLR agonists in epithelial cells from CRS patients [24].
The identification of the AOAH gene is also of potential significant interest and suggests a novel mechanism for the development of CRS, again implicating an innate immune signaling, but in a novel fashion. AOAH is responsible for degrading lipopolysaccharide (LPS), and dysfunctional AOAH gene function leads to decreased LPS degradation with unopposed continued LPS stimulation via a TLR-4 dependant mechanism. In AOAH knockout mouse models, persistent inflammation following LPS stimulation is observed [25][26][27]. Supporting a role in airway disease, the AOAH gene has previously been implicated in a genome wide scan for asthma [13]. Corresponding to the above literature, here we presented that rs4504543 in the AOAH gene played a protective role (OR = 0.58) in CRS with a strong P value (P = 0.0001152). Moreover, the AOAH_ rs4504543 loci was also revealed as a protective factor (OR = 0.30) in CRSsNP group with a stronger P value (P = 8.11-011), indicating that AOAH gene might exert a crucial protective role in the development of CRS.
RYBP is a zinc finger protein with an essential role during embryonic development, which binds transcriptional factors, polycomb products, and mediators of apoptosis, suggesting roles in apparently,unrelated functions. Gene products of the RYBP gene inhibit ubiquitination and subsequent degradation of TP53, and, by interacting with MDM2, play a role in regulating transcription of TP53 target genes and promoting apoptosis. Recent findings have also suggested that RYBP may also play a role in epigenetic regulation [28], and contribute to defense against retroviruses [29]. It is thus possible that polymorphisms in the RYBP gene may be implicated in CRS by dysregulating TP53 activity in TP53 or in its target genes via alteration of RYBP gene products or by altering binding at regulatory binding sites in the RYBP gene promotor area, which contains binding sites for the following transcription factors (YY1, IRF-1, C/EBPA, GATA-1, POU2F1). This may contribute to the inflammation observed in CRS, promoting epithelial dysfunction with secondary bacterial colonization. In contrast to AOAH gene, polymorphisms in the RYBP gene was exhibited here for the first time as a significantly risk factor of CRS and either of the subgroups (CRSsNP and CRSwNP) with high OR values (OR CRS = 2.59; OR CRSsNP = 2.76; OR CRSwNP = 2.45), suggesting the variation of rs4532099 in RYBP could increase the risk of CRS development.
IL1RL1 gene is involved in regulation of TLR signaling and has recently also been implicated as the receptor for IL-33. Its role in  Taken overall, all of the replicated genes have disparate functions, but evidence supports that dysfunctions in each these genes may conceivably contribute to development of CRS, underlining the concept that CRS represents a common morphological appearance of clinical disease as an endpoint of multiple unique pathogenic mechasnisms.
Our study has obvious limitations. First and most noticeable is the small group size of the sample used. Nevertheless, given the limited number of genes genotyped in this replication study, feet corrected P value remains significant and, in the case of the RYBP and AOAH genes, is highly significant with a high odds ratio. A second consideration is the ethnic variability in SNP frequency known for these genes. It is clear that in this study, we have replicated SNPs associated in a Caucasian population, and have not performed extensive fine mapping studies of the gene which might identify other risk SNPs. As shown in this example however, when gene coverage is adjusted to better reflect tagging SNP selection for the CHB data set, we are able to identify significant polymorphisms within the IRAK4 gene.
We replicate several genes which were proved to be associated with CRS in Caucasian population in a Chinese population, suggesting a common basis to the development of the disease in both population types. Of interest is the potential implication of inflammatory pathways, suggesting dysfunction in TLR signaling as a critical element in chronic rhinosinusitis. While these studies have significant limitations, they nevertheless offer a basis for further exploration of a role for immune signaling and response to bacteria in further studies of CRS.