Analysis of Clinical Indexes and RUNX3,TBKBP1,PPARGC1B Polymorphisms in Chinese Han Patients with Ankylosing Spondylitis

Abstract

Background: Ankylosing spondylitis (AS) is a genetically determined disease. Runt-related transcription factor 3 (RUNX3), tumor necrosis factor family member-associated NF-κB activator binding kinase 1 binding protein (TBKBP1), and peroxisome proliferator-activated receptor-gamma coactivator 1 beta (PPARGC1B) have recently been found to be associated with susceptibility to AS in patients of Western European descent. We hypothesize that these three genes may be related to clinical outcomes of Chinese Han AS patients. Methods: Blood samples were drawn from 396 HLA-B27-positive Chinese Han AS patients. Clinical indexes were scored for each patient, including the Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and modified Stoke Ankylosing Spondylitis Spine Score (mSASSS), which measure patients' function of daily life and severity of AS. Twelve tagSNPs were selected from these three genes and genotyped. We analyzed the clinical indexes in different genotyped patients to investigate the relationship between severity of AS and different genotypes. Results: The rs11249215 SNP in RUNX3 and the rs7379457 and rs32579 SNPs in PPARGC1B significantly affect the BASFI score in patients. The rs11249215, rs7551188, and rs1395621 SNPs in RUNX3 significantly affect the BASDAI scores. The two selected single nucleotide polymorphisms (SNPs) in TBKBP1 show no relationship with the clinical outcomes. None of the 12 SNPs is related to mSASSS. In conclusion, RUNX3 is related to both the severity of AS and the function of daily life. PPARGC1B is related to the function of daily life.

Introduction

Ankylosing spondylitis (AS) is a chronic inflammatory disorder characterized by inflammation in the spine and sacroiliac joints causing initial bone and joint erosion and subsequent ankylosis (Brown et al., 2002). Most of the patients develop first symptoms of AS younger than 30 years of age (Braun and Sieper, 2007). Scientists found that significant radiographic progression occurs in the first 10 years of disease, and structural damage at initial presentation is the best predictor of further damage (Carette et al., 1983; Gran and Skomsvoll, 1997; van der Heijde, 2004).

AS patients' disease severity is largely genetically determined (Hamersma et al., 2001). In genome-wide association studies, rs11249215 in runt-related transcription factor 3 (RUNX3), rs8070463 in tumor necrosis factor family member-associated NF-κB activator binding kinase 1 binding protein (TBKBP1), and rs11959820 in peroxisome proliferator-activated receptor-gamma coactivator 1 beta (PPARGC1B) are related to AS susceptibility in patients of Western European descent (WTCCC and TAST, 2007, 2011; Sirota et al., 2009). We hypothesize that the RUNX3, TBKBP1, and PPARGC1B genes may predict the severity of AS.

We use three common indexes to describe the clinical outcomes of each patient. These indexes are BASFI, BASDAI, and mSASSS. The Bath Ankylosing Spondylitis Functional Index (BASFI) is used to evaluate the patients' function of daily life. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) is used to evaluate the severity of AS. The modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) is used to evaluate the structural damage to the patients' spine from AS.

Materials and Methods

Study population

In this work, 396 HLA-B27-positive Chinese Han AS patients were recruited, and they have been treated by nonsteroidal anti-inflammatory drug routinely; no other treatments were used for patients. Among the AS patients, there were 345 males (87.1%) and 51 females (12.9%); the average age was 29.6 years (range 16-60 years). The average duration since AS diagnosis was 11.5 years (range 8-18 years) (Table 1). The diagnosis of AS has been made by experienced rheumatologists; all diagnoses satisfied the modified New York criteria (van der Linden et al., 1984). Subjects with rheumatoid arthritis, psoriasis, inflammatory bowel disease, or other autoimmune diseases were excluded from both the AS and the control groups.

Table 1.

Demographic Data of Ankylosing Spondylitis Patients and Controls

Sex
Male	345 (87.1%)
Female	51 (12.9%)
Age	29.6±8.5
Duration of diagnosis	11.5±2.1
BASFI	3.97±1.49
BASDAI	3.95±1.05
mSASSS	12.6±13.4

Numerical values presented as mean±standard deviation.

BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; mSASSS, modified Stoke Ankylosing Spondylitis Spine Score.

Clinical indexes

The BASFI and BASDAI were administered to the patients and the controls using questionnaires; these indexes are the most widely used tools for the assessment of AS functional status and disease activity (Calin et al., 1994; Garrett et al., 1994). The mSASSS is a scoring system for quantification of chronic spinal changes (Baraliakos et al., 2009). Standard anteroposterior and lateral radiographs of the cervical and lumbar spine were obtained for each patient and control, and the lateral view was used to derive a mSASSS for each patient (Wanders et al., 2004; Creemers et al., 2005; Sieper et al., 2009). Three of the authors separately assigned the mSASSS, and we used the average.

Single nucleotide polymorphism selection

The single nucleotide polymorphisms (SNPs) in this study include five in RUNX3, two in TBKBP1, and five in PPARGC1B. These three genes localize to chromosome, 1, 17, and 5, respectively. The selected SNPs serve as a multimarker tagging algorithm with criteria of r² more than 0.8 and for all SNPs with minor allele frequency more than 5% from the Han Chinese in Beijing.

Population in the HapMap database

Haploview 4.2 software (Broad Institute, Cambridge, Massachusetts, USA) is used to select the tagSNPs. Figure 1 shows the positions of each tagSNP. The rs11249215 SNP is located in the promoter of RUNX3. The rs8070463 SNP is located in the promoter of TBKBP1. The rs7379457 SNP is located in the promoter of PPARGC1B. Other SNPs are located in the introns. Most SNPs in TBKBP1 are in high linkage disequilibrium (LD), hence only two tagSNPs are selected.

FIG. 1.

Positions of each selected tagSNP on the genes. The rs11249215 SNP is in the promoter of runt-related transcription factor 3 (RUNX3). The rs8070463 SNP is in the promoter of tumor necrosis factor family member-associated NF-κB activator binding kinase 1 binding protein (TBKBP1). The rs7379457 SNP is in the promoter of peroxisome proliferator-activated receptor-gamma coactivator 1 beta (PPARGC1B). Other SNPs are all in introns. SNP, single nucleotide polymorphism.

DNA extraction and genotyping analysis

DNA was isolated from 2 mL whole blood samples using the AxyPrep Blood Genomic DNA Miniprep kit (Axygen Biosciences, Union City, CA). Detection of the SNPs was performed by MassARRAY system (Sequenom, San Diego, CA). The chip-based matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry technology was used in this procedure (Tost et al., 2005). Most of the SNPs were successfully genotyped. The rs109077 SNP was 98.5% genotyped in the case group and 97.5% in the control group. The rs4648884 SNP was 98.5% genotyped in the case group. The rs9438876 was 90.9% genotyped in the case group and 95.5% in the control group. In the other SNPs, more than 99.5% was genotyped in both case and control groups.

Statistical analyses

The Hardy-Weinberg equilibrium was tested for all 12 tagSNPs. All the clinical indexes are summarized in Table 2 (RUNX3), Table 3 (TBKBP1), and Table 4 (PPARGC1B). The Levene's test was used to test homogeneity of variance. A p-value less than 0.05 indicates heterogeneity of variance; therefore, we used the Kruskal-Wallis test, a nonparametric test. A p-value more than 0.05 indicates homogeneity of variance; we additionally used the analysis of variance (ANOVA) test. A p-value in these two kinds of analyses of less than 0.05 was considered significantly related to severity of AS. Statistical analyses were carried out with the SPSS v.17.0 software package (IBM, Armonk, NY).

Table 2.

Clinical Indexes About Severity of Ankylosing Spondylitis in RUNX3

rs11249215	Freq. ^a	BASFI	BASDAI	mSASSS	Duration of disease
Levene's test^b		4.758E-4^c	0.153	0.040	0.012
GG^d	64	4.059±0.184^e	3.844±0.129	14.156±1.738	11.609±0.263
AG	184	3.760±0.108	3.769±0.076	11.441±1.020	11.478±0.154
AA	146	4.207±0.122	4.205±0.085	13.397±1.151	11.479±0.174
Kruskal-Wallis test		0.061	<0.001 ^*, ^f	0.188	0.549
ANOVA			0.001 ^*, ^g
rs7551188
Levene's test		0.026	0.138	0.005	0.168
TT	86	4.151±0.159	4.000±0.111	12.302±1.499	11.360±0.226
CT	200	3.760±0.104	3.770±0.073	11.650±0.983	11.490±0.148
CC	110	4.222±0.141	4.255±0.098	14.564±1.325	11.627±0.200
Kruskal-Wallis test		0.005 ^*	<0.001 ^*	0.538	0.801
ANOVA			4.038E-4 ^*
rs1395621
Levene's test		0.169	0.210	0.128	0.030
AA	58	4.147±0.192	4.033±0.134	11.633±1.797	11.683±0.271
AG	189	3.836±0.108	3.777±0.076	12.000±1.015	11.441±0.153
GG	149	4.077±0.122	4.149±0.085	13.757±1.144	11.500±0.173
Kruskal-Wallis test		0.145	0.002 ^*	0.343	0.736
ANOVA		0.209	0.004 ^*	0.437
rs4648884
Levene's test		0.345	0.685	0.003	0.224
CC	62	4.265±0.189	4.194±0.133	13.806±1.770	11.790±0.267
CT	196	3.911±0.106	3.867±0.075	11.480±0.996	11.459±0.150
TT	136	3.944±0.128	3.971±0.090	13.735±1.195	11.426±0.180
Kruskal-Wallis test		0.131	0.090	0.459	0.712
ANOVA		0.252	0.100		0.492
rs9438876
Levene's test		0.094	0.100	2.304E-5	0.906
GG	43	3.833±0.229	3.762±0.164	8.333±2.141	11.762±0.329
AG	129	4.034±0.130	3.938±0.093	13.446±1.217	11.546±0.187
AA	188	3.895±0.108	4.011±0.077	12.809±1.012	11.351±0.155
Kruskal-Wallis test		0.890	0.205	0.729	0.793
ANOVA		0.653	0.379		0.463

Frequencies of each genotype.

Levene's test is used to test homogeneity of variance. p-Value less than 0.05 indicates heterogeneity of variance; therefore, we use Kruskal-Wallis test, a nonparametric test. Otherwise, we use ANOVA.

p-Value for Levene's test.

All clinical indexes are analyzed due to the genotype of each SNP.

The mean±deviation of each index is calculated for different genotypes.

p-Value for Kruskal-Wallis test.

p-Value for ANOVA.

p-Value is less than 0.05; it is written in bold so it is more visible.

ANOVA, analysis of variance; RUNX3, runt-related transcription factor 3; SNP, single nucleotide polymorphism.

Table 3.

Clinical Indexes About Severity of Ankylosing Spondylitis in TBKBP1

rs8070463	Freq. ^a	BASFI	BASDAI	mSASSS	Duration of disease
Levene's test^b		0.125^c	0.202	2.856E-4	0.007
CC^d	72	3.651±0.172^e	3.784±0.122	9.595±1.617	11.216±0.244
CT	198	4.082±0.105	4.010±0.075	13.374±0.988	11.707±0.149
TT	124	4.008±0.134	3.967±0.095	13.246±1.259	11.336±0.190
Kruskal-Wallis test		0.306^f	0.438	0.062	0.086
ANOVA		0.101^g	0.283
rs4439799
Levene's test		0.163	0.308	0.040	0.077
TT	60	3.673±0.192	3.867±0.136	10.667±1.802	11.217±0.270
CT	182	4.038±0.110	3.945±0.078	13.110±1.035	11.769±0.155
CC	152	4.026±0.121	4.000±0.085	12.816±1.132	11.289±0.170
Kruskal-Wallis test		0.207	0.892	0.328	0.019 ^*
ANOVA		0.226	0.699		0.060

Frequencies of each genotype.

p-Value for Levene's test.

All clinical indexes are analyzed due to the genotype of each SNP.

The mean±deviation of each index is calculated for different genotypes.

p-Value for Kruskal-Wallis test.

p-Value for ANOVA.

p-Value is less than 0.05; it is written in bold so it is more visible.

TBKBP1, tumor necrosis factor family member-associated NF-κB activator binding kinase 1 binding protein.

Table 4.

Clinical Indexes About Severity of Ankylosing Spondylitis in PPARGC1B

rs7379457	Freq. ^a	BASFI	BASDAI	mSASSS	Duration of disease
Levene's test^b		0.009^c	0.111	2.482E-6	0.042
TT^d	8	3.000±0.521^e	3.500±0.370	7.750±4.887	11.125±0.743
CT	50	4.492±0.209	4.240±0.148	17.840±1.955	11.800±0.297
CC	336	3.919±0.080	3.923±0.057	11.982±0.754	11.464±0.115
Kruskal-Wallis test		0.004 ^*	0.106^f	0.086	0.430
ANOVA			0.063^g
rs1422429
Levene's test		0.402	0.383	0.007	0.329
CC	44	4.155±0.224	4.000±0.159	15.545±2.102	11.614±0.317
CT	194	4.062±0.107	3.979±0.076	12.299±1.001	11.572±0.151
TT	156	3.824±0.119	3.910±0.084	12.205±1.117	11.378±0.168
Kruskal-Wallis test		0.109	0.966	0.446	0.883
ANOVA		0.253	0.792		0.440
rs109077
Levene's test		0.457	0.845	0.010	0.357
GG	34	3.572±0.248	3.667±0.175	9.444±2.324	11.444±0.350
GT	188	4.024±0.108	3.947±0.076	13.116±1.012	11.511±0.152
TT	168	4.001±0.1115	4.024±0.081	12.750±1.076	11.488±0.162
Kruskal-Wallis test		0.092	0.202	0.683	0.752
ANOVA		0.236	0.179		0.984
rs32582
Levene's test		0.126	0.982	0.028	0.457
TT	8	4.500±0.525	3.750±0.370	16.000±4.923	12.125±0.742
GT	124	3.855±0.133	3.871±0.094	11.484±1.251	11.468±0.189
GG	264	4.013±0.091	4.000±0.064	13.023±0.857	11.496±0.129
Kruskal-Wallis test		0.832	0.523	0.912	0.576
ANOVA		0.373	0.452		0.691
rs32579
Levene's test		0.033	0.318	5.070E-5	0.944
AA	38	3.563±0.241	3.632±0.170	9.368±2.254	11.237±0.341
AG	172	4.127±0.113	4.023±0.080	14.244±1.059	11.634±0.160
GG	184	3.925±0.109	3.957±0.077	11.783±1.024	11.429±0.155
Kruskal-Wallis test		0.049 ^*	0.183	0.774	0.755
ANOVA			0.114		0.473

Frequencies of each genotype.

p-Value for Levene's test.

All clinical indexes are analyzed due to the genotype of each SNP.

The mean±deviation of each index is calculated for different genotypes.

p-Value for Kruskal-Wallis test.

p-Value for ANOVA.

p-Value is less than 0.05; it is written in bold so it is more visible.

PPARGC1B, peroxisome proliferator-activated receptor-gamma coactivator 1 beta.

Ethics statement

The blood samples of both AS patients and controls used in this study were part of samples taken for diagnostic tests. During the collection and use of DNA samples, clinical data guidelines, regulations of the local Ethics Committee, and the Helsinki Declaration in 1975 were followed. Written informed consents were obtained from all the patients and subjects (or their parents in the case of two patients younger than 18 years). The study procedure was approved by our Institutional Review Board.

Results

The rs11249215 SNP in RUNX3 is related to BASDAI (p=0.001), with the BASDAI in the AA genotype (4.205±0.085) higher than in other genotype patients. The rs7551188 SNP in RUNX3 is related to BASFI (p=0.005), with the BASFI in the CC genotype higher (4.222±0.141) than in other genotype patients; this SNP is also related to BASDAI (p=4.038×10⁻⁴), with the BASDAI in the CC genotype higher (4.255±0.098) than in other genotype patients. The rs1395621 SNP in RUNX3 is related to BASDAI (p=0.004), with BASDAI in the GG genotype higher (4.149±0.085) than in other genotype patients (Table 2).

None of the SNPs selected in TBKBP1 is related to clinical indexes (Table 3).

The rs7379457 SNP in PPARGC1B is related to BASFI (p=0.004), with the BASFI in the CT genotype (4.492±0.209) higher than in other genotype patients. The rs32579 SNP in PPARGC1B is related to BASFI (p=0.049), with the BASFI in the AG genotype higher (4.127±0.113) than in other genotype patients (Table 4).

In conclusion, RUNX3 is related to both the severity of AS and the function of daily life. PPARGC1B is related to the function of daily life.

Discussion

Reveille found the pathogenesis of AS remains poorly understood, but genetic factors play a significant role (Reveille et al., 2001). Changes in the spine involve syndesmophytes forming bony ankylosis of adjacent vertebrae or ankylosis of small vertebral joints. Some classification systems exist based on clinical and radiographic criteria (Taylor et al., 1998; Braun et al., 2002). Most of our patients present with mild symptoms and do well with chronic medical treatments; however, some patients have more severe manifestations that require surgery within the first 10 years of diagnosis. We aimed to find genetic markers that are associated with the severity of AS and analyzed the most commonly used clinical indexes BASFI, BASDAI, and mSASSS, which describe the function status, activity of AS, and structure damage, respectively.

The RUNX3 gene encodes for RUNX3, and it localizes to chromosome 1p36.11. RUNX3 is a downstream target of the transforming growth factor-β (TGF-β) pathway, which is considered a tumor suppressor pathway, as components are frequently altered in cancers, especially those of the gastrointestinal tract (Derynck et al., 2001). RUNX3 is inactivated in gastric cancer by hemizygous deletion, promoter hypermethylation, histone modification, and protein mislocalization, suggesting a tumor-suppressive role of RUNX3 in this malignancy (Li et al., 2002, 2005; Fujii et al., 2008). Since the discovery of the potential role of RUNX3 in the initiation and the progression of gastric cancer, RUNX3 has been found to be involved in the development of a variety of cancers, including colon, liver, lung, and breast cancer (Goel et al., 2004; Li et al., 2004; Lau et al., 2006; Subramaniam et al., 2009). RUNX3 knockout mice spontaneously develop inflammatory bowel disease characterized by leukocyte infiltration, mucosal hyperplasia, formation of lymphoid clusters, and increased production of IgA. RUNX3 belongs to the runt domain family of transcription factors, which are key regulators of lineage-specific gene expression and more recently found to be linked to human autoimmunity (Alarcón-Riquelme, 2004). When RUNX3 is suppressed in human T cells, either through gene inactivation or with small interference RNA, Foxp3 expression is reduced, which in turn disrupts the recognition of regulatory T cells. As with other autoimmune diseases, AS patients exhibit an imbalance of CCR4+CCR6+ helper T cells and regulatory T cells (Klunker et al., 2009; Wu et al., 2011). RUNX3 is highly expressed in dendritic cells, where it functions as a component of the TGF-β signaling cascade (Fainaru et al., 2004). It is obvious that RUNX3 is not only a tumor suppressor but also plays an important role in autoimmune diseases and inflammations. In this work, the rs11249215 SNP in RUNX3 affected patients' BASFI. The rs11249215, rs7551188, and rs1395621 SNPs in RUNX3 affected patients' BASDAI. Figure 2 shows LD of the three genes and the left part is RUNX3. The rs7551188 is in high LD with rs1395621, which means these two SNPs have the same trend in genotype distribution. RUNX3 is related to both the activity of AS and the function of daily life. Because previous studies have all focused on inflammatory pathways, we conclude that the RUNX3 can influence the AS severity due to its effect on the inflammatory process.

FIG. 2.

Linkage disequilibrium (LD) map comparing all ankylosing spondylitis (AS) patients and controls. Darker color indicates higher LD, lighter color indicates less LD. Numbers in the squares indicate correlation coefficient (R²) value. The left part of the figure contains five SNPs (from rs9438876 to rs11249215). They are from RUNX3. The middle part of the figure contains two SNPs (rs8070463 and rs4439799). They are from TBKBP1. The right part of the figure contains four SNPs (from rs7379457 to rs32579). They are from PPARGC1B.

TBKBP1 is an adaptor protein that binds to TBK1, also known as sintbad. The precise function of TBKBP1 in the process of TBK1 activation has not been defined fully, but there is evidence that the adaptor proteins link the kinases to the upstream signaling pathways, possibly by interaction with TRAF3 (Unterholzner et al., 2011). Ishii et al. performed bone marrow transfer experiments, which revealed that TBK1-mediated signaling in hematopoietic cells is critical for the induction of antigen-specific B and CD4+ T cells, whereas in nonhematopoietic cells, TBK1 was required for CD8+ T-cell induction. These data suggest that TBK1 is a key signaling molecule for DNA-vaccine-induced immunogenicity, in addition to being part of the classic NF-κB inflammatory pathway (Ishii et al., 2008). Acute or chronic inflammation is an important feature of AS, with the degree of inflammation correlated to severity of disease (Reveille et al., 2001). Our data showed that the TBKBP1 SNPs examined did not affect patients' clinical indices. We conclude that the pathogenesis of AS is not strictly through inflammatory pathways, and that further research is necessary to study the role TBKBP1 plays in AS.

The human PPARGC1B gene, encoding PGC-1β, localizes to chromosome 5q32, a region that shows linkage to type 2 diabetes (Vionnet et al., 2000). Wirtenberger and colleagues found PGC-1β to be associated with familial breast cancer (Wirtenberger et al., 2006). The mechanism that PPARGC1B relates to these diseases is still being investigated. Numerous publications indicate that PPARGC1B plays a critical role in regulating the multiple aspects of energy metabolism, including mitochondrial biogenesis, thermogenesis, gluconeogenesis, and fatty acid β-oxidation (Puigserver et al., 1998; Lehman et al., 2000; Vega et al., 2000; Herzig et al., 2001; Yoon et al., 2001). Oxidative phosphorylation (OXPHOS) dysfunction plays a critical pathogenic role in several human diseases (Dimauro and Schon, 2008). Srivastava found that PGC-1β overexpression can lead to a marked improvement in OXPHOS defects caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) (Srivastava et al., 2009). Ishii found PGC-1β accelerates osteoclastic bone resorption through the coordination of mitochondrial biogenesis and the cell differentiation program (Ishii et al., 2009). Our data showed that the rs7379457 and rs32579 SNPs in PPARGC1B affected patients' functional status (BASFI). The right part of Figure 2 shows LD of PPARGC1B and the rs7379457 and rs32579 SNPs are in 100% LD, suggesting that the two SNPs affected BASFI equally.

None of the 12 SNPs affected patients' mSASSS, proxy for spinal structure damage. The recruited patients had duration of disease of 10-12 years, which helps us to investigate the speed of disease progression. The duration of disease showed no significant difference in any of the SNP groups. In conclusion, RUNX3 is related to both the activity of AS and the function of daily life. PPARGC1B is related to the function of daily life.

Our findings can provide context for better understanding of the genetic and molecular pathogenesis of AS. The specific SNPs in these genes can be used to guide genetic analysis and counseling, medical and surgical treatment options, and ultimate prognosis of severity of AS. Further studies are needed to elucidate the molecular roles these genes play in AS.

Footnotes

Acknowledgments

The authors wish to thank all the patients and families who participated in this study, and all the clinical doctors who helped them in the Chinese PLA General Hospital.

Author Disclosure Statement

No competing financial interests exist.

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