Transcript Whole Genome Linkage Disequilibrium Mapping with SNPs to

大規模連鎖不平衡マッピングによる
関節リウマチ関連遺伝子解析
山田 亮
理化学研究所
遺伝子多型研究センター
関節リウマチ関連遺伝子研究チーム
平成16年9月18日
Today’s contents
1. 自己免疫疾患関連遺伝子の同定
2. SNPによる大規模LDマッピング
3. 関節リウマチとPADI、抗シトルリン化ペプ
チド抗体
4. PADI4多型の同定
Genetic predisposition in autoimmunities
Wandstrat A. and Wakeland E. Nat Immunol 2(9) 802,2001
HLA and autoimmunities
Overlaps of associated genes
Genetics and Genetic Analysis of
Rheumatoid Arthritis
• Twin and family studies
– Relative risk to monozygotic twin ( λMZ )
• 12~62
– Relative risk to siblings (λsib)
• 2~17
– HLA locus explains 1/3-1/2 of total genetic
components.
– There are multiple non-HLA genes.
• Multiple linkage studies
• Many candidate-approach studies
Genetic analysis of common diseases
• Hypothesis-free whole-genome approach
– In order to identify novel pathologic
mechanisms
Large-scale case-control screening with SNPs
Two Ways of Whole Genome Approach
Map-based Approach
2
1
6
3
7
5
4
Gene D
Gene A
Gene B
Gene C
Gene-based Approach
12 3
8 910
567
4
Gene D
Gene A
Gene B
Gene C
8
9 10
Whole Genome Survey with SNPs by
Linkage Disequilibrium Mapping
• Prospects
– Map-based approach
• Markers are evenly distributed throughout the genome
• No. of SNPs to cover the whole genome: 500,000 –
1,000,000
– Gene-based approach
• Markers are distributed in gene-containing regions
• No. of SNPs to cover the whole genome: 50,000 –
100,000
D. Botstein & N. Risch Nat Genet 33 Suppl,228-237(2003)
Public gene database
Build (NCBI)
No. mRNA
No. genes
27
37823
37486
28
51689
51258
29
46438
45753
30
35273
33809
31
27634
25336
32
34839
32208
33
When completion of human
genome sequence was
announced.
37498
34731
34
25334
22193
Adopted SNPs from JSNP db
~Gene-based approach~
• 105,123 SNPs
– In 16,676 genes
• 5.6 SNPs per a gene in average
Gene structure
5flank
Exon
Intron
3flank
Inter-gene
Undetermined
4022
18305
68542
2076
10024
2154
4%
17%
65%
2%
10%
2%
Case-control association tests for
screening the whole genome
• Two steps
(1) 94 cases vs. 658 controls
(2) 846 cases vs. 658 controls
• 1,165 /105,123 SNPs passed an initial step (1)
• 50 / 1,165 SNPs passed the second step (2)
in progress
• 50 SNPs are located in 25 LD blocks
Identification of SLC22A4 and
RUNX1 as RA-associated genes
RA or Crohn disease-associated SNPs in Ch5q31
Cytokine cluster
Chromosome 5q31
IL3, IL4, IL5, IL9, IL13, CSF2, IRF1 and TCF7 are in the region
• Recessive association
• Relative Risk = 2
The SNP changes affinity of RUNX1
10kb
Genes
RIL
SLC22A4
SLC22A5
IRF1
Exons
SNPs
RA-associated SNP
1
2 3
4
Exons
SNPs
……CAGGTTATGTGG C/T GAAGGATAAG……
RUNX1 binding site
5
6
78
9
10
The SNPs associated with RA or Crohn
disease
10kb
Genes
RIL
SLC22A4
SLC22A5
Exons
RA
Crohn
SNPs
RUNX1 binding site
L503F
Heat shock elementbinding site
IRF1
SLC22A4 and RUNX1
• SLC22A4
– Organic cation transporter
– Unknown physiologic function
– In 5q31 cytokine cluster linked with Crohn disease and
asthma/atopy
• RUNX1
– Hematologic transcriptional factor
– Responsible for leukemia
– Links with autoimmune diseases (SLE, psoriasis)
Tokuhiro S et al. Nat Genet 35, 341-348 (2003)
RA, SLE and Psoriasis-associated SNPs disrupt RUNX1 binding motif
3 genes with RUNX1 binding sequence
SLE
Psoriasis
RA
Table
Disease
Gene
Cytoband
Function of gene
RUNX1 binding motif
Location
Length evaluated
with reporter assay
SLE
PDCD1
2q37.3
An immunoreceptor
with tyrosine-based
inhibitory motif
TGCg/aGT
Intron 4
Not Done
TGTg/aGT
Intergene
102 bp
TGTGGt/c
Intron1
24 bp
SLC9A3R1
Psoriasis
17q24-q25
NAT9
Rheumatoid
arthritis
SLC22A4
Regulatory
molecule of
functional
membrane
N-acetyltransferase
5q31
Organic cation
transporter
Identification of RA-susceptible
variant in PADI4
関節リウマチ診断における、最も信頼できる自己抗体
抗シトルリン化ペプチド抗体（抗CCP抗体)
感度
特異度
PPV
リ ウマ トイド因
子陽性
54%
89%
85.70%
抗シ トルリ ン化
ペプチド抗体陽
性
38%
98.70%
97.40%
リ ウマ トイド因
子&抗シ トルリ
ン化ペプチド抗
体陽性
30%
100%
100%
Citrulline
• Citrulline：
– One of native amino acids, but not among 20
coding amino acids
– Free citrulline and peptidyl citrulline
• Hereditary citrullinemia (a metabolic disorder)
– Unclear physiologic function of peptidyl
citrullination
Peptidyl citrullination
Arginine
Citrulline
NH2
C=NH2 +
NH
CH2
CH2
CH2
HCNH3+
COO-
NH2
C=O
NH
CH2
PADIs
CH2
CH2
HCNH3+
COO-
Loss of ionic NH2+ of
Arg residue
Effects on intra- and intermolecular interactions
Detection of citrullinated filaggrin
by SDS-PAGE and Western blotting
Augmentation of T-cell response
by citrullinated peptides
J.Hill E Cairns et al.
J Immunol 2003
HLA class II genes and aCCP
• Carrier of SE DRB1 was associated with
aCCP positivity
SE status OR(95% CI) for aCCP positivity
SE/SE
13.3 (4.6-40.4)
SE/x
3.3 (1.8-6.0)
x/x
1
SE: Shared epitope
P
<0.001
<0.001
F van Gaalen R de Vries et al.
Arthritis & Rheum 2004
Molecular modification as
peptidyl-citrullination alters
tertially structure of self-peptides.
Production of anti-citrullinated
antibodies under the influence of
HLA molecules ?
Chromosome 1
PADI cluster
Result of 1st step screening by case-control association
Results of Affected Sib-pair
Linkage Analyses
Association Plots in the PADI Cluster
PADI4
Exons
-log10(P)＝５
Two main transcript variants in PADI4
663 amino acids, 4 SNPs with 3 amino acid substitutions
Gly 55 Ser, Val 82 Ala, Gly 112 Ala
RA-susceptible haplotype
25％ in population
RA-non-susceptible haplotype
60％ in population
●
RR of Individuals with two copies of susceptible types is 2.
Fraction
RA patients with 2 copies of susceptible type were more
frequently to be positive for anti-citrullinated filaggrin
antibody
100
90
80
70
60
50
40
30
20
10
0
P-0.04
Positive
Negative
2 copies
1 copy
0 copy
Number of copies of RA-susceptible allele
Following UK study on PADI
variants(1)
Haplotype frequency
Haplotype frequency in
British and Japanese
100%
Others
80%
60%
haplotype 2
40%
RA-susceptible
haplotype
haplotype 1
20%
0%
Japanese
British
Ethnic groups
UK study on PADI variants(2)
Japan
UK
Case
Control
Case
Control
N
830
736
839
481
Susceptible
haplotype
frequency
0.32
0.25
0.32
0.30
Protective
haplotype
frequency
0.52
0.60
0.56
0.59
Hypothetical mechanism of RA-susceptible variant
Summary for PADI4
• Hypothesis-free approach identified an RAassociated gene that was functionally
strongly relevant to RA.
• Functional variants of PADI4 were
consisted of SNPs.
• Many issues are still present to understand
PADI and citrullination in RA pathogenesis.
Acknowledgment
• Lab. Rheumatic Diseases
–
–
–
–
–
–
–
–
–
–
Dr. K. Yamamoto
Dr. A. Suzuki
Dr. X.Chang
Dr. Y. Kochi
Mr. S. Tokuhiro
Ms. R. Kawaida
Ms. K. Kobayashi
Ms. M. Ohtake
Ms. E. Kannno
Ms. K. Komakine
• SRC, RIKEN
–
–
–
–
Dr. A. Sekine
Dr. T. Tsunoda
Dr. Y. Nakamura
Mr. H. Kawakami
• Clinical Institutes
– Dr. T. Sawada
– And many other
collaborators
http://134.160.84.101/ra/