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Microrna-Mediated Regulation Explains
Allelic Risk of TLR7 Variant Predisposing to
Systemic Lupus Erythematosis
Disclosure
All authors have no competing financial
relationships to disclose.
Yun Deng, Jian Zhao, Jennifer M. Grossman and Betty P. Tsao
Division of Rheumatology,
UCLA David Geffen School of Medicine
Identification of TLR7 3’UTR SNP Associated with
SLE Susceptibility in 9,274 Eastern Asians
Introduction
 SLE: Excessive innate immune activation involving
TLR7/8/9 and IFN-α
an important pathogenic mechanism in the disease
 Increased Tlr7 Gene Dosage
(Shen N, Fu Q, Deng Y, et al, PNAS, 2010;107:15838-15843)
Autoimmunity In Lupus Mice
Pisitkun P, et al. Science. 2006;
Deane JA, et al. Immunity. 2007;
Barrat FJ, et al. Eur J Immunol . 2007.
 No Strong Evidence For Common Copy Number Variations
At Human TLR7-TLR8 Locus:
1. Real time PCR;
2. Genomic Southern and pulse field gel electrophoresis;
3. Reports from customized CGH arrays;
4. Database of Genomic Variants.
Explore Functional TLR7 Single Nucleotide Polymorphisms
Confirmation of the Association Between Rs3853839 and
SLE Susceptibility in 13,339 non-Asian Ancestries
Meta-analysis Showing Robust Association of rs3853839
with SLE in 22,613 individuals of Four Ancestries
(Deng Y, Zhao J, et al, PLoS Genetics, 2013;9:e1003336)
(Deng Y, Zhao J, et al, PLoS Genetics, 2013;9(2):e1003336)
rs3853839
Trans-ethnic meta analysis
Frequency
3,936 SLE vs. 3,491 Ctrls
All
1,679 SLE vs. 1,934 Ctrls
P meta
OR (95%CI)
Combined EA+AA+HS
7.5E-11
1.24 (1.18-1.34)
Combined EA+AA+HS+AS
2.0E-19
1.25 (1.20-1.32)
Sex EthnicityCase/Control Test Allele Case Control
P
OR (95%CI)
EA
3,936/3,491
G
20.3%
17.2%
6.5E-06 1.23 (1.13-1.35)
AA
1,679/1,934
G
19.8%
16.7%
1.1E-03 1.24 (1.09-1.41)
HS
1,492/807
G
44.8%
37.3%
7.5E-04 1.26 (1.10-1.43)
AS*
4,334/4,940
G
81.0%
77.0%
6.5E-10 1.27 (1.17-1.36)
1,492 SLE vs. 807 Ctrls
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Risk Allele of rs3853839
TLR7 expression
Risk Allele of rs3853839
mRNA Level in PBMCs:
Slow degradation
TLR7 expression
Protein Level in PBMCs:
Reporter assay:
Xhol
Renilla
SV40
promoter
Notl
TLR7 3’UTR
Firefly
rs3853839 G/C
RNA-containing
autoantigens
IRF7/IRF5
TLR7
IFN-α
Explore whether differential miRNA binding could explain
allelic risk of TLR7 for SLE.
IFN-α inducible genes:
MX1, Ly6E, IFIT1, IFIT3
Risk Allele of rs3853839
Risk Allele of rs3853839
TLR7 expression
IFN response
Reduced modulation
by miRNA(s)
TLR7 expression
Only miR-3148 levels show an inverse
correlation with TLR7 mRNA levels in PBMCs
Co-transfection of each miRNA with TLR7 3’UTR reporter vector into HEK 293 cells
SLE
Control
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Overexpression of miR-3148 in PBMCs leads
to reduction in TLR7 mRNA expression
PBMCs from
healthy donors
(n=15)
miR-3148 mimic
Non-target mimic
24h
miR-3148 inhibitor
Non-target inhibitor
PBMCs stimulated
with TLR7 agonist
24h
Overexpression of miR-3148 in PBMCs results
in a trend of decreased IFN-α production
IFN-α production
IFN-α inducible
genes mRNA level
TLR7 mRNA level
mimic
mimic NC
inhibitor NC
No miRNA transfection
NC
inhibitor NC
No miRNA transfection
miR-3148
miR-3148
Conclusion
 The
TLR7 3’UTR SNP (rs3853839) was strongly associated with SLE
susceptibility in populations of multiple ancestries.
 Among
the 6 miRNAs predicted to target the sequences at/around
rs3853839 at TLR7 3’UTR, miR-3148 and miR-2278 showed reduced
modulation on the risk allele of rs3853839 which may confer decreased
degradation of TLR7 transcripts, resulting in elevated TLR7 expression.
 miR-3148 levels in PBMCs showed an inverse correlation with TLR7 mRNA
Acknowledgement
UCLA
Betty P. Tsao
Yun Deng
Jian Zhao
Daisuke Sakurai
Erika Magdangal
Jennifer M. Grossman
Rita M. Cantor
Bevra H. Hahn
levels. Overexpression of miR-3148 reduced the endogenous expression of
TLR7 in PBMCs, resulting in a trend of decreased downstream production of
type I IFNs; whereas inhibition of miR-3148 has an opposite effect.
Thank You !
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