Differences in the expression of variants across ethnic groups in the systemic lupus erythematosus (SLE) patients have been well documented. However, the genetic architecture in the Thai population has not been thoroughly examined. In this study, we carried out genome-wide association study (GWAS) in the Thai population.
Methods
Two GWAS cohorts were independently collected and genotyped: discovery dataset (487 SLE cases and 1606 healthy controls) and replication dataset (405 SLE cases and 1590 unrelated disease controls). Data were imputed to the density of the 1000 Genomes Project Phase 3. Association studies were performed based on different genetic models, and pathway enrichment analysis was further examined. In addition, the performance of disease risk estimation for individuals in Thai GWAS was assessed based on the polygenic risk score (PRS) model trained by other Asian populations.
Results
Previous findings on SLE susceptible alleles were well replicated in the two GWAS. The SNPs on HLA class II (rs9270970, A>G, OR = 1.82, p value = 3.61E−26), STAT4 (rs7582694, C>G, OR = 1.57, p value = 8.21E−16), GTF2I (rs73366469, A>G, OR = 1.73, p value = 2.42E−11), and FAM167A-BLK allele (rs13277113, A>G, OR = 0.68, p value = 1.58E−09) were significantly associated with SLE in Thai population. Meta-analysis of the two GWAS identified a novel locus at the FBN2 that was specifically associated with SLE in the Thai population (rs74989671, A>G, OR = 1.54, p value = 1.61E−08). Functional analysis showed that rs74989671 resided in a peak of H3K36me3 derived from CD14+ monocytes and H3K4me1 from T lymphocytes. In addition, we showed that the PRS model trained from the Chinese population could be applied in individuals of Thai ancestry, with the area under the receiver-operator curve (AUC) achieving 0.76 for this predictor.
Conclusions
We demonstrated the genetic architecture of SLE in the Thai population and identified a novel locus associated with SLE. Also, our study suggested a potential use of the PRS model from the Chinese population to estimate the disease risk for individuals of Thai ancestry.
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Abkürzungen
GWAS
Genome-wide association study
SLE
Systemic lupus erythematosus
SNP
Single nucleotide polymorphism
HLA
Human leukocyte antigen
MHC
Major histocompatibility
CHR
Chromosome
MAF
Minor allele frequency
OR
Odds ratio
CI
Confidence interval
bp
Base pair
BCR
B cell receptor
TCR
T cell receptor
IL
Interleukin
PD
Program cell death
MDA
Melanoma differentiation-associated gene 5
Background
The systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by loss of tolerance to self-antigens, inappropriate immune activation, and inflammation [1]. The severity is various depending on affected organs [2]. Genetic susceptibility has been widely accepted as one of the critical factors driving disease development [2]. Recently, the genetic architecture of SLE has been examined worldwide [3]. Using GWAS, more than 90 loci have been found associated with SLE across at least four ethnic groups, including Han Chinese, European, North America, and Africa [4, 5]. The strongest signal was identified at the HLA class II allele, which replicated in all of the different populations [4]. These findings indicate critical biological mechanisms underlying the disease, which will be the candidate in further functional studies [6].
However, heterogeneity of disease between different ethnicities drives a question of whether genetic background in different ancestries could affect the clinical manifestations. It is known that Asian SLE patients have higher disease severity compared to Europeans [2]. However, only a few studies on SLE associations that were based on candidate genes were performed in the Thai population [7‐11]. In this study, we conducted GWAS using the SLE samples collected from two tertiary referral hospitals in Thailand. We aim to replicate known SLE-associated variants in the Thai population and identify novel SNPs associated with SLE.
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Methods
Sample collection and preparation
We calculated the power of our study by using an online tool called Genetic association study (GAS) Power Calculator [12]. With 800 cases and 1600 controls at 5E−08 significant level, we obtained 0.934 expected power for the study. The EDTA blood samples from SLE patients (n = 487) were collected at King Chulalongkorn Memorial Hospital as cases for the discovery phase. All procedures were approved by the ethical committee from the Faculty of Medicine, Chulalongkorn University (COA no. 923/2017). For the replication cohort, the samples (n = 405) were collected from the Rheumatology clinic, Ramathibodi Hospital, with ethical approval from the Faculty of Medicine, Mahidol University (MURA no. 2015/731, EC no. 590223, Protocol-ID 12-58-12). All patients were carefully recruited regarding the criteria from the American College of Rheumatology (ACR) [13]. Patients’ demographic data from both datasets have been summarized in Table 1. For healthy controls (n = 1606) and unrelated disease controls including breast cancer, periodontitis, tuberculosis, drug-induced liver injury, epileptic encephalopathy, dengue hemorrhagic fever, thalassemia, and cardiomyopathy (n = 1590), data were provided from the Department of Medical Science, Ministry of Public Health, Thailand.
Table 1
SLE patients’ characteristics of both observatory and replication datasets
Patients’ characteristics
Clinical cases
Observatory cohort n = 455a
Replication cohort n = 371a
n
(%)
n
(%)
Age of onset (mean ± SD)
30.38
± 13.68
30.39
± 11.43
Sex
Female
425
(93.41%)b
337
(90.84%)c
Male
26
(5.71%)b
27
(7.28%)c
Clinical aspects
Hemologic disorders
243
(53.41%)b
136
(36.66%)c
Neurological disorders
62
(13.63%)b
33
(8.89%)c
Ulcer
115
(25.27%)b
52
(14.02%)c
Discoid rash
161
(35.38%)b
49
(13.21%)c
Malar rash
142
(31.21%)b
82
(22%)c
Arthritis
133
(29.23%)b
148
(39.89%)c
Renal disorders
284
(62.42%)b
149
(40.16%)c
ANA
350
(76.92%)b
214
(57.68%)c
aThe sample number after quality control processes
bThe percentages of unknown clinical data (n/a) in the observatory dataset are listed here. Sex = 0.88%, hematologic disorder = 1.76%, neurological disorder = 2.20%, ulcer = 4.18%, discoid rash = 3.96%, malar rash = 5.71%, arthritis = 4.18%, renal disorders = 1.76%, and ANA = 9.89%
cThe percentages of unknown clinical data (n/a) in the replication dataset are listed here. Sex = 0.00%, hematologic disorder = 36.93%, neurological disorder = 37.2%, ulcer = 37.4%, discoid rash = 37.2%, malar rash = 37.47%, arthritis = 37.2%, renal disorders = 37.74%, and ANA = 36.93%
DNA extraction
Buffy coats were extracted using the QIAGEN® EZ1® DNA blood kit (QIAGEN GmbH, Hilden, Germany). We used 200 μl of a buffy coat as recommended by the manufacturer’s instruction. Buffy coat samples were transferred into tube or sample cartridge for EZ1 Advanced XL (QIAGEN GmbH, Hilden, Germany) and extracted using EZ1® Advanced XL DNA Buffy coat protocol. From this protocol, DNA was eluted at 200 μl. DNA was diluted and quantitated using Qubit™ dsDNA BR Assay Kit according to the manufacturing protocol (Invitrogen, Thermo Fisher Scientific, MA, USA).
Genotyping and quality control
Genotyping was performed using Infinium Asian Screening Array-24 v1.0 BeadChip with 659,184 SNPs (Illumina, San Diego, CA, USA) at the Department of Medical Sciences (DMSC, Ministry of Public Health, Thailand) based on the protocol recommended by the manufacturer. The Genome Studio data analysis software v2011.1 (Illumina, San Diego, CA, USA) was used for calling genotypes. Samples and SNP markers were tested for quality control (QC) using PLINK genomic analysis software (v1.90b5.4) [14]. Individuals with autosomal genotype call rate ≤ 0.98, gender inconsistency based on heterozygosity rate of X chromosome (maleTh = 0.8, femaleTh = 0.2), and genome-wide estimates of identity-by-descent (pihat) ≥ 0.185 (3rd generation) were excluded from analysis. SNPs with more than 5% missing genotyping rate or significant deviation of Hardy-Weinberg equilibrium (p value ≤ 1 × 10−8) were also removed. After quality control (QC), we obtained a dataset of 2041 individuals with 421,909 variants for the discovery phase and 1955 individuals with 446,139 variants for replication. The flow diagram of the analysis process is shown in Fig. 1a.
×
GWAS data imputation
Pre-phasing was performed using SHAPEIT [16]. After that, genotype data for individuals was imputed to the density of the 1000 Genomes Project Phase 3 reference using IMPUTE2 [17]. After all the QC processing, 6,657,806 were left for further analysis. The processed data were publicly available at http://2anp.2.vu/GWAS_SLE_Thailand.
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Association study, meta-analysis, and statistical analysis
The association studies were conducted by using SNPTEST [18], and the factored spectrally transformed linear mixed models (FaST-LMM v.0.2.32) program [19]. The results from FaST-LMM were analyzed and visualized by RStudio to obtain genomic inflation factor (λ), quantile-quantile plot, and Manhattan plot [20]. The SNPs with p value ≤ 1 × 10−5 were plotted to obtain the regional plot by using LocusZoom [21]. Haplotype block and linkage disequilibrium structure were analyzed by Haploview software version 4.2 [22]. The characterized SLE susceptible loci were downloaded from a previous study [23] and GWAS catalogue (the NHGRI-EBI catalogue of published genome-wide association studies). Meta-analysis was studied based on the inverse variant strategy in the METAL program [24]. The genetic inheritance pattern was calculated from the frequency of different genotyping on risk alleles using R-Bioconductor. Simultaneously, functional annotation was predicted by using SNPnexus, which applied data from the Reactome database [25]. The histone markers and transcription factor binding sites were predicted from an online tool called HaploReg V4.1 [26].
Polygenic risk score calculation
Lassosum [27] was used to calculate PRS for individuals. The summary statistics for SLE association in East Asians [28], involving 2618 cases and 7446 controls with Chinese ancestry, were used to train the model. The area under the ROC curve (AUC) was calculated using R package pROC [29].
Results
Known SLE associations found in the Thai population
In the discovery dataset, the association studies were initially performed using healthy controls (n = 1606) and SLE patients (n = 487) collected from King Chulalongkorn Memorial Hospital. Regarding the result, we found that variants at the HLA class II regions were strongly associated with SLE (p value < 5E−08). Similarly, GWAS from 405 SLE cases and 1590 non-immune-mediated disease controls found variants at the HLA class II regions reached the genome-wide significant threshold (p value < 5E−08). Our findings were consistent with previous reports in other ethnic groups [30]. Inflation factors from both datasets were calculated as reported in Supplementary figure 1.
Subsequently, a meta-analysis of the two Thai GWAS was carried out, and we systematically examined associations across the 90 known SLE-associated loci, which were collected from the GWAS catalogue (https://www.ebi.ac.uk/gwas/) and previous review articles [23]. Of these loci, the HLA-DQA1, HLA-DRB1, STAT4, FAM167A-BLK, and GTF2I loci have reached the genome-wide significant threshold (p value < 5E−08; Fig. 1b, Table 2) in Thai population, and the variants at the PROS1C1, NOTCH4, HCP5, C6orf10, TAP2, TNFSF4, RasGRP3, TERT, TNPO3-IRF5, CXCR5, GPR19, SLC15A4, and ITGAM loci showed suggestive evidence of associations with SLE (p value < 5E−05, Supplementary Table 1). These loci have been found in several ancestries, including Han Chinese, Korean, North American, European, African, and Hispanic populations [31, 32].
Table 2
List of significant variants at individual locus from the meta-analysis
HAPa
dbSNPb
CHRc
BPd
RAe
MAF affected
MAF unaffected
Locus
Locus upstream
Locus downstream
Discovery dataset
Replication dataset
Meta-analysis
phetf
OR (95% CI)
p
OR (95% CI)
p
OR
p
q32.3
rs7574865
2
191,099,907
A
0.47
0.36
STAT4
1.54 (1.33–1.79)
1.45E−08
1.61 (1.37–1.89)
7.45E−09
1.57
8.218E−16
0.69
q23.3
rs74989671
5
128,398,268
G
0.16
0.11
FBN2
1.52 (1.24–1.86)
4.31E−05
1.58 (1.26–1.98)
7.61E−05
1.54
1.611E−08
0.81
p21.32
rs9270970
6
32,605,797
G
0.42
0.30
HLA-DRB1
HLA-DQA1
2.02 (1.73–2.35)
8.71E−20
1.63 (1.39–1.93)
4.15E−09
1.83
3.617E−26
0.07
q11.23
rs73366469
7
74,619,286
G
0.14
0.09
RP5-1186P10.2
GTF2I
1.8 (1.45–2.24)
1.09E−07
1.65 (1.3–2.1)
2.84E−05
1.73
2.42E−11
0.61
p23.1
rs13277113
8
11,491,677
G
0.26
0.32
FAM167A
BLK
0.64 (0.54–0.76)
2.16E−07
0.74 (0.61–0.88)
8.76E−04
0.68
1.58E−09
0.27
q24.33
rs1385374
12
128,816,149
A
0.20
0.15
SLC15A4
1.54 (1.28–1.85)
5.76E−06
1.37 (1.12–1.69)
2.36E−03
1.46
7.62E−08
0.43
p11.2
rs1143679
16
31,265,490
A
0.07
0.03
ITGAM
1.67 (1.21–2.28)
1.39E−03
2.27 (1.6–3.23)
2.55E−06
1.91
5.81E-08
0.2
aHaplotype
bdbSNP from single nucleotide polymorphisms database (NCBI)
cChromosome
dPosition
eRisk alleles
fp value of heterogeneity
We noticed that some of the previously characterized nonsynonymous polymorphisms also showed certain evidence of association (p value < 0.05) in Thai population, such as rs11235604 (ATG16L2, R58W), rs13306575 (NCF2, R395W), rs1990760 (IFIH1, A946T), rs3734266 (UHRF1BP1, Q454L), rs2841280 (PLD4, E27Q), and rs2230926 (TNFAIP3, F127S). Details of these associations are summarized in Table 3. All significant variants were calculated for Hardy-Weinberg equilibrium, as reported in Supplementary Table 2.
Table 3
List of known SLE susceptible SNPs in Thai SLE patients
dbSNPa
CHRb
BPc
RAd
Locus
Annotation
MAF affected
MAF unaffected
OR
SE
p
rs35426045
1
161,649,724
A
FCGR2B
Intergenic
0.80
0.75
1.38
0.09
1.83E−04
rs1234315
1
173,209,324
A
TNFSF4
Intergenic
0.53
0.46
1.27
0.07
1.02E−06
rs2205960
1
173,191,475
T
TNFSF4
Intergenic
0.27
0.22
1.26
0.08
2.37E−03
rs34889541
1
198,594,769
A
ATP6V1G3,
Intergenic
0.10
0.13
0.75
0.11
7.66E−03
rs1418190
1
173,361,979
T
LOC100506023
ncRNA_intronic
0.59
0.56
1.18
0.07
1.55E−02
rs13306575
1
183,563,302
A
NCF2
Nonsynonymous
0.11
0.08
1.48
0.09
1.73E−02
rs13385731
2
33,701,890
C
RASGRP3
Intronic
0.13
0.17
0.70
0.09
1.71E−05
rs6705628
2
74,208,362
T
DGUOK-AS1
ncRNA_exonic
0.11
0.13
0.79
0.10
1.83E−02
rs1990760
2
163,124,051
T
IFIH1
Missense
0.23
0.21
1.17
0.08
4.93E−02
rs10936599
3
169,492,101
T
MYNN
Synonymous SNV
0.52
0.56
0.84
0.07
6.95E−03
rs564799
3
159,728,987
T
IL12A
ncRNA_intronic
0.12
0.14
0.80
0.10
1.97E−02
rs10028805
4
102,737,250
A
BANK1
Intronic
0.45
0.49
0.87
0.07
4.08E−02
rs7726159
5
1,282,319
A
TERT
Intron
0.43
0.40
1.25
0.07
5.00E−05
rs2736100
5
1,286,401
C
TERT
Intron
0.51
0.43
1.25
0.07
4.67E−05
rs10036748
5
150,458,146
T
TNIP1
Intronic
0.66
0.61
1.16
0.07
3.04E−02
rs2431697
5
159,879,978
C
PTTG1; MIR146A
Intergenic
0.07
0.09
0.77
0.13
3.36E−02
rs548234
6
106,568,034
T
PRDM1; ATG5
Intergenic
0.67
0.72
0.81
0.07
2.21E−03
rs2230926
6
138,196,066
G
TNFAIP3
Missense
0.04
0.03
1.49
0.18
2.92E−02
rs3734266
6
34,823,187
C
UHRF1BP1
Intronic
0.21
0.19
1.18
0.08
4.68E−02
rs4728142
7
128,573,967
A
KCP; IRF5
Intergenic
0.19
0.13
1.61
0.09
1.34E−07
rs729302
7
128,568,960
C
KCP; IRF5
Intergenic
0.25
0.30
0.77
0.07
3.32E−04
rs12531711
7
128,617,466
G
IRF5; TNPO3
Intron
0.03
0.01
2.03
0.25
4.27E−03
rs4917014
7
50,305,863
G
C7orf72; IKZF1
Intergenic
0.15
0.17
0.81
0.09
1.84E−02
rs7097397
10
50,025,396
A
WDFY4
Missense
0.59
0.64
0.78
0.07
3.84E−04
rs4948496
10
63,805,617
C
ARID5B
Intronic
0.66
0.62
1.17
0.07
2.19E−02
rs1128334
11
128,328,959
T
ETS1
UTR3
0.35
0.28
1.36
0.07
1.50E−05
rs2732552
11
35,084,592
C
PDHX
Intergenic
0.78
0.75
1.18
0.08
3.04E−02
rs11235604
11
72,533,536
T
ATG16L2
Missense
0.04
0.05
0.70
0.17
3.93E−02
rs1385374
12
129,300,694
T
SLC15A4
Intronic
0.21
0.15
1.46
0.09
7.62E−08
rs10845606
12
12,834,894
A
GPR19
Intronic
0.32
0.37
0.75
0.07
3.19E−06
rs2841280
14
105,393,556
C
PLD4
Nonsynonymous
0.52
0.45
1.91
0.07
5.81E−08
rs1143679
16
31,276,811
A
ITGAM
Missense
0.07
0.04
1.71
0.14
6.18E−08
rs11860650
16
31,315,385
A
ITGAM
Intronic
0.09
0.07
1.74
0.10
4.64E−03
rs1170426
16
68,603,798
T
ZFP90
Intronic
0.69
0.73
0.82
0.07
5.91E−03
rs7444
22
21,976,934
C
UBE2L3
UTR3
0.64
0.60
1.17
0.07
1.81E−02
rs463426
22
21,809,185
C
HIC2; TMEM191C
Intergenic
0.38
0.40
0.85
0.08
4.50E−02
adbSNP from single nucleotides polymorphisms database (NCBI)
bChromosome
cPosition
dRisk alleles
Identification of novel loci associated with SLE
Excluding the variants at the known SLE-associated loci, we discovered a novel variant on FNB2 (rs74989671, OR = 1.54, p value = 1.61E−08) specifically associated with SLE in Thai population (Figs. 1b and 2a, Table 2) when comparing the association in Europeans (OR = 0.998, p value = 0.979) and in Chinese populations (OR = 0.982, p value = 0.692) [28]. Further analyses based on different genetic inheritance models suggested that the disease risk was associated with the copy number of risk alleles that the individuals carried (additive model) (Table 4). Three SNPs on FBN2 loci (rs74989671, rs35983844, rs6595836) showed linkage disequilibrium (LD r2 = 0.82) (Fig. 2b, Supplementary Table 1). Of these variants, rs74989671 was found to locate within the peak of H3K36me3 derived from CD14-positive monocytes and H3K4me1 (associated with active enhancers) derived from the primary T cells (Fig. 2c).
Table 4
Analyses based on different inheritance models on the FBN2 locus
Locus SNPs
Model
Genotypes or alleles
SLE n
Control n
OR
95% CI
p
FBN2
Codominant
GG
21
26
1.75
0.93–3.27
7.96E−02
rs74989671
Dominant
AG
235
334
1.53
1.25–1.86
2.38E−05
AA
562
1219
ref
ref
ref
AG+GG
256
360
1.54
1.27–1.87
8.83E−06
AA
562
1219
ref
ref
ref
Recessive
GG
21
26
1.57
0.84–2.93
0.161
AG + AA
797
1553
ref
ref
ref
Allelic
A
277
386
ref
ref
ref
G
1359
2772
1.38
1.17–1.64
1.31E−04
FBN2
Codominant
GG
655
1366
0.72
0.23–2.47
5.80E−01
rs76835745
Dominant
AG
162
212
1.15
0.36–4
1.00
AA
6
9
ref
ref
ref
GG+GA
817
1578
0.78
0.25–2.66
0.60
AA
6
9
ref
ref
ref
Recessive
GG
655
1366
0.63
0.5–0.79
5.43E−05
AA+GA
168
221
ref
ref
ref
Allelic
A
174
230
ref
ref
ref
G
1472
2944
12.34
10.6–14.4
2.20E−16
×
In addition, we found variants at the ATP6V1B1, MIR4472-2, MYO5C, ADCY5, and DGKG, showing suggestive evidence of associations with SLE in Thai population (p value < 5E−05) (Supplementary Figure 2, Supplementary Table 1). Though these polymorphisms are likely to associate with Thai SLE patients, an independent GWAS dataset of SLE patients with Thai background is needed for further validation.
In silico functional annotation of SLE-associated variants in Thai population
To understand the biological meaning underlying the SLE-associated loci in the Thai population, we performed the pathway analysis using the SNPnexus program [25]. Variants with p value < 5E−05 were involved in this study. Notably, we found that 50% of all variants were located within the coding region, by which 10% is nonsynonymous polymorphisms. Pathway analysis results revealed that SLE-associated variants were highly enriched in the regulation of interferon signaling, PD-1 signaling, MHC-class II antigen presentation, TCR/BCR signaling, cytokine signaling, TNF signaling, NOTCH4 signaling, calcium-activated potassium channels, and cell-cell junction organization pathways. Furthermore, we found that extracellular matrix organization was significant in our results (Fig. 3). It indicated that Thai SLE patients might have a higher risk of fibrosis-associated inflammation.
×
Anzeige
Polygenic risk score prediction for the individuals
To apply the GWAS result to predict the Thai SLE outcome, we also tested the hypothesis of whether the PRS models trained by individuals with Chinese ancestry could be applied for Thai SLE patients. We calculated PRS for individuals in the Thai GWAS, based on the training data from the Chinese population (2618 cases and 7446 controls) [28]. Significantly, the PRS for SLE cases were higher than controls (mean difference = 0.89; p value = 2.2E−16; Fig. 4a), and the area under the receiver-operator curve (AUC) achieved 0.76 for this predictor. This analysis indicated the potential application for the PRS in the Thai population, based on the results from other Asian populations. Regarding the analysis, this might be a clue for predicting an outcome of SLE clinical characteristics in Thai SLE patients, and it is a good source for further genetic analysis to identify actual SLE pathogenesis in the different ancestry.
×
Discussion
The present study is the first largest GWAS cohort conducted among Thai SLE patients. The highest significant association in the region of HLA class II was consistent with previous reports from other ethnic groups [30]. Since there are vast differences of HLA class II allele frequency among populations and sophisticated genetic structure, the study of the specific HLA class II haplotype is needed. Currently, there were two publications reported about specific HLA haplotypes in Thai SLE patients. First, the fine genetic mapping of the HLA allele from SLE patients in the northern part of Thailand has identified the association of HLA-DRB5*01:01 and HLA-DRB1*16:02 [34]. Secondly, HLA haplotype analysis found HLA-DRB1*15:02 and HLA-DQ*05:01 associated with Thai SLE patients [35]. Further study on the HLA class II allele using whole-genome sequencing or exome sequencing might be helpful to specify the impact of the HLA class II allele on Thai SLE patients.
Apart from the HLA class II alleles, our study found variants in STAT4, GTF2I, and BLK regions. For BLK locus, this gene encoded for Src-tyrosine kinase, which is an important signaling molecule under B cell development [36]. This gene showed protein-protein interaction with BANK1 (B cell-specific cytoplasmic protein involved in B cell receptor signaling) and might plausibly involve in dysregulation of the B cell receptor, which is a common feature found in SLE patients [37]. For STAT4 and GTF2I alleles, these genes are encoded for the transcription factors that mediate many immune-related genes and inflammatory cytokine transcription machinery. Both BLK and STAT4 loci have been reported as SLE susceptible alleles in Thai SLE patients recently [7], whereas GTF2I locus has firstly identified in our study. Interestingly, the variants on STAT4 and GTF2I loci were correlated with lupus nephritis (LN) in the various SLE ancestries [32]. The GTF2I allele was likely to be specific in Asian background, mainly Han Chinese [38].
Our analysis found several LN-susceptible loci such as IRF5 [39, 40], ITGAM [9, 41], IKZF1 [42], and TNFSF4 [43]. While IKZF1 is a co-transcription factor with STAT-4 mediated Th1 lymphocyte differentiation and interferon pathways [44], the TNFSF4 locus, also called OX40L, encoded for the TNF superfamily ligand, which actively stimulates CD4+ T cell activation [43]. Study in the Finnish and Swedish SLE patients found the correlation of ITGAM with cutaneous discoid lupus erythematosus (DLE) and LN as well as Ro/SSA auto-antibody positive [45]. Not only LN, but we also found several loci that have been verified in the specific sub-phenotype of SLE patients. For example, our result found a variant on ETS1, which previously showed association with juvenile SLE, as well as a variant on RasGRP3, which was involved in malar rash or discoid rash [42]. The recent SLE susceptible loci identified in the cardiac manifestation of neonatal lupus, NOTCH4, was found in our results [46].
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Note that we found some of the known SNPs which are nonsynonymous variants such as NCF2 [47], IFIH1 [48], TNFAIP3 [49], UHRF1BP1 [50], ATG16L2 [51], and PLD4 [52]. A few pieces of evidence have revealed the impact of those variants on various pathways including neutrophil extracellular traps (NETs) formation [53], sensor molecule to detect viral genome inside cells [54], a negative regulator for NF-kB transcription factors [55], and a negative regulator of cell growth [56]. These pathways resembled with our functional enrichment pathways analysis. Interestingly, our results found extracellular matrix organization (ECM) pathways associated with Thai SLE patients. Previously, single-cell transcriptome analysis in non-responder LN patients highlighted the upregulated genes in the ECM pathway correlated with treatment failure [57]. The ECM reflected the active fibrotic process, which was a marker of poor prognosis LN [58]. Remarkably, the prevalence of severe LN was high in the South East Asian ethnic included Thai [59]. As regards our SLE patients’ demographic data, we found that the frequency of clinical phenotype was roughly similar to other ethnic [60]. The LN has the highest abundance found among Thai SLE patients. Thus, our results supported that genetic background was a pivotal factor driving a severe LN among Thai SLE patients. Taken together, these pieces of evidence could justify the link between genetic variants and clinical involvement in Thai SLE patients.
The study of known SNPs showed most of the polymorphisms resembled with previous reports in Thais, such as ARID5B, TNFSF4, BANK1, TNFAIP3, CXCR5 SLC15A, ITGAM, WDFY4, ETS1, and BLK [7‐11]. It confirmed that our analysis processes were reliable. Noticeably, the allele frequency of ITGAM was higher among Thai SLE when compared to Chinese Hong Kong [9], but has no association with Japanese and Korean background [61]. Thus, this implies the specificity of these variants to the Thai SLE patients. Although we did not recognize polymorphisms on chromosome 11q23.3 (rs11603023 on PHLDB1 and rs638893 on DDX6), which has been identified in the Thais’ SLE, our meta-analysis enhanced signal from rs10845606 on GPR19 allele which does not correlate with Thai SLE patients previously [8].
It is noteworthy that meta-analysis in the Thai population discovered novel SLE susceptible variants on FBN2. The FBN2 allele is located on a chromosome 5 encoded protein called fibrillin-2 [62]. Fibrillins-2 is one of the glycoprotein components incorporated extracellularly on microfibrils and is essential in bone, muscle, and extracellular matrix formation [63]. It is well known that mutation of FBN2 leads to dominant heritable connective tissue disorders [64]. Importantly, a recent review article has gained insight on fibrillin-2 as a critical mediator that binds to transforming growth factor-beta (TGF-β) during extracellular matrix formation [65]. The TLR9/TGF-β1/PDGF-β pathway was excessively activated in peripheral mononuclear cells isolated from LN patients [66]. Besides, the upregulation of FBN2 correlated with fibrosis prevalence in the spontaneous LN developed mouse model (SWR X NZB1 F1) [67]. Although the function of FBN2 in SLE is unclear, collective evidence led us to hypothesize that this variant might drive either fibrosis-associated inflammation or inflammatory induction during disease pathogenesis. Due to whole-genome sequencing data in the Thai population is lacking, further study using FBN2 target sequencing, whole-genome sequencing, and variant functional characterization in a large cohort is needed. This knowledge could be useful to identify rare coding variants and genetic propensity eliciting SLE pathogenesis in Thais.
Note that some of the variants were previously characterized in other autoimmune diseases, including rheumatoid arthritis and primary Sjögren syndrome (pSS). It, therefore, indicates the sharing of underlying genetic factors between autoimmune disease. However, predisposing factors which could affect clinical manifestation driving different autoimmune disease outcome has not been elucidated yet. Recently, the GRS (genetic risk score) has been widely adopted to predict disease outcomes from genetic variants [68]. The previous studies in SLE showed that overall mortality was higher in the striking GRS SLE patients; also, the high cumulative genetic risk could predict the specific organ damages such as proliferative nephritis and cardiovascular disease [69]. Our study showed a high sensitivity for using polygenic risk scored as a marker for SLE disease development in the Thai population. It is exciting for further study to calculate the genetic risk score and specific clinical manifestation among Thai SLE patients.
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Conclusions
In conclusion, our study reported susceptible loci of SLE patients in Thai ancestry, which were variants on the HLA class II allele, STAT4, GTF2I, and BLK. Additionally, we confirmed those variants which had been reported previously in the Thai populations, which were ARID5B, TNFSF4, BANK1, TNFAIP3, CXCR5 SLC15A, ITGAM, WDFY4, and ETS1. Interestingly, we identified novel variants associated with the Thai SLE patients, which were on the FNB2 allele. Summary loci associated with the Thai SLE were seen in Fig. 4b. Functional annotation analysis highlighted extracellular matrix organization pathways specific to the Thai population. The PRS using GWAS data is useful for SLE prediction with sensitivity and specificity of more than 70%. Further whole-genome sequencing study with a large sample size might help to validate our results. Finally, our finding provides the necessary genetic background susceptible to SLE disease, expanding the number of molecular targets for treatment options.
The technical support group from the Department of Medical Sciences is gratefully acknowledged. We would like to thank the valuable suggestion from Associate Professor Wanling Yang from the Department of Paediatrics and Adolescent Medicine, Queen Mary Hospital, Li Ka Shing Faculty of Medicine, and Centre for Genomic Sciences, Li Ka Shing Faculty of Medicine, and The University of Hong Kong.
Ethics approval and consent to participate
All the procedures were followed in compliance with the principles of the Declaration of Helsinki, and informed consent was obtained from all participants. The study and the consent procedures were reviewed and approved by the local institutional review board including the ethical committee from the Faculty of Medicine, Chulalongkorn University (COA no.923/2017), and the Faculty of Medicine, Mahidol University (MURA no. 2015/731, EC no. 590223, Protocol-ID 12-58-12).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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