Toll-like receptor 9 gene mutations and polymorphisms in Japanese ulcerative colitis patients

Table 1 Basic subject data (mean ± SD) n (%)

	UC (n = 48)	Controls (n = 47)
Age (yr)	43.49 ± 15.48	41.21 ± 15.36
Sex (M/F)	21 (43.8)/27 (56.2)	21 (44.7)/26 (55.3)
Age at diagnosis (yr)	35.07 ± 14.39
Disease duration (yr)	8.42 ± 8.49
Familial disease	0 (0)	0 (0)
Disease location
Proctitis	3 (6.25)
Left colitis	27 (56.3)
Right colitis	1 (2.1)
Pancolitis	17 (35.4)
Disease severity
Light	17 (35.4)
Mild	18 (37.5)
Severe	13 (27.1)
History of colectomy	5 (10.4)
History of immunosuppressant	6 (12.5)
History of colon cancer	3 (6.3)

UC: Ulcerative colitis.

Table 2 Primers used in polymerase chain reaction for genotyping Toll-like receptor 9 polymorphisms and three single nucleotide polymorphisms

Fragment (product)	Forward	Reverse
TLR9 polymorphisms
-2109 to -1293 (817 bp)	5'-CCAAGGGACTCTGGGAAAG-3'	5'-CATGTCACCCTCTCAACAGGG-3'
-1626 to -1095 (532 bp)	5'-CAGCCTTCACTCAGAAATACCC-3'	5'-GGCCAACAAGGCCCTATG-3'
-1296 to -553 (662 bp)	5'-CATGGGAGCAGAGACATAATG-3'	5'-GCCAGGGTGTAGCTTGA-3'
-795 to -134 (662 bp)	5'-GAGTCTCCTCACCTAGATCAG-3'	5'-TATACCAGCCTAGTAGC-3'
79 to 962 (884 bp)	5'-CTGCAAGCAACAGTGACGG-3'	5'-AGCTTTCACTTAACCAATCCC-3'
-393 to 1698 (2092 bp)	5'-TACCCGCTACTGGTGCTATC-3'	5'-TGGCAGAGTCTAGCATCAGG-3'
996 to 1687 (692 bp)	5'-CTGGTTCTGAAGCCTAATTC-3'	5'-AGCATGAGGATGTTGGTATGG-3'
976 to 1899 (924 bp)	5'-CTGGATTCTAGGTCTCAGTCC-3'	5'-CTGGATTCTAGGTCTCAGTCC-3'
1540 to 2368 (829 bp)	5'-CCTGCCACATGACCATCGAG-3'	5'-CCTGCCACATGACCATCGAG-3'
1812 to 2520 (709 bp)	5'-AACCTCACCCACCTGTCAC-3'	5'-AACCTCACCCACCTGTCAC-3'
2407 to 3350 (944 bp)	5'-TGCAGATGAACTTCATCAACC-3'	5'-GCTGTTGCAGCTGACATC-3'
3267 to 4057 (791 bp)	5'-CAGGAAACCAGCTGAAGG-3'	5'-CAGGAAACCAGCTGAAGG-3'
3867 to 4701 (835 bp)	5'-GACTGGGTGTACAACGAGCTT-3'	5'-TTCTGCATGGGAAAGGTAGG-3'
SNP
-1486	5'-CAGCCTTCACTCAGAAATACCC-3'	5'-GGCCAACAAGGCCCTATG-3'
1174	5'-CTGGTTCTGAAGCCTAATTC-3'	5'-AGCATGAGGATGTTGGTATGG-3'
2848	5'-TGCAGATGAACTTCATCAACC-3'	5'-GCTGTTGCAGCTGACATC-3'

TLR9: Toll-like receptor 9; SNP: Single nucleotide polymorphism.

Table 3 Allele frequencies in ulcerative colitis cases and disease-free controls for Toll-like receptor 9 single nucleotide polymorphisms at -1486, 1174, 2848

SNP1	Allele	UC (n = 48)	Controls (n = 47)	dbSNP2	JSNP3
-1486	T/C	0.344/0.656a	0.532/0.468	0.647/0.353	ND
1174	A/G	0.344/0.656a	0.532/0.468	0.512/0.488	0.517/0.483
2848	G/A	0.354/0.646a	0.543/0.457	0.481/0.519	0.517/0.483
					aP = 0.013

¹Positions were calculated taking the A of the Toll-like receptor 9 (TLR9) ATG start codon as position 1 based on Genbank Accession No. NM_017442;

²dbSNP public database: http://www.ncbi.nlm.nih.gov/SNP.

³JSNP is a database of Japanese SNPs: http://snp.ims.u-tokyo.ac.jp/index_ja.html;

^aP value by Fisher’s Exact test, cases vs controls. SNP: Single nucleotide polymorphisms; UC: Ulcerative colitis; ND: No data.

Table 4 Genotype frequencies in ulcerative colitis cases and disease-free controls for Toll-like receptor 9 single nucleotide polymorphisms at -1486, 1174, 2848 n (%)

Genotype at TLR9		UC (n = 48)	Controls (n = 47)
-1486	CC	20 (41.7)a	10 (21.3)
	CT	23 (47.9)	24 (51.1)
	TT	5 (10.4)a	13 (27.7)
1174	GG	20 (41.7)a	10 (21.3)
	GA	23 (47.9)	24 (51.1)
	AA	5 (10.4)a	13 (27.7)
2848	AA	19 (39.6)a	9 (19.1)
	AG	24 (51.1)	25 (53.2)
	GG	5 (10.4)a	13 (27.7)
			aP < 0.05

^aP value by Fisher’s exact test, ulcerative colitis (UC) cases vs controls. TLR9: Toll-like receptor 9.

Table 5 Haplotype frequencies in ulcerative colitis cases and disease-free controls for Toll-like receptor 9 single nucleotide polymorphisms at -1486, 1174, 2848 n (%)

Haplotype			UC (n = 96)	Controls (n = 94)
-1486	1174	2848
C	G	A	62 (64.6)a	43 (45.7)
T	A	G	33 (34.4)a	50 (53.2)
C	G	G	1 (1.04)	1 (1.06)
				aP < 0.05

^aP value by Fisher’s exact test, ulcerative colitis (UC) cases vs controls.

Table 6 Diplotype frequencies in ulcerative colitis cases and disease-free controls for Toll-like receptor 9 single nucleotide polymorphisms at -1486, 1174, 2848 n (%)

Diplotype			UC (n = 48)	Controls (n = 47)
-1486	1174	2848
CC	GG	AA	19 (39.6)a	9 (19.1)
CC	GG	AG	1 (2.1)	1 (2.1)
CT	GA	AG	23 (47.9)	24 (51.1)
TT	AA	GG	5 (10.4)a	13 (27.7)
				aP < 0.05

^aP value by Fisher’s exact test, ulcerative colitis (UC) cases vs controls.

Table 7 Linkage disequilibrium among single nucleotide polymorphisms of Toll-like receptor 9 in ulcerative colitis cases and disease-free controls

SNPs		UC (n = 48)		Controls (n = 47)
		|D’|	r2	|D’|	r2
-1486 T/C	1174 A/G	1	1	1	1
-1486 T/C	2848 G/A	1	0.9522	1	0.9581
1174 A/G	2848 G/A	1	0.9522	1	0.9581

Lewontin’s D’ (|D’|) and the linkage disequilibrium coefficient r² between each pair of single nucleotide polymorphisms (SNPs) were examined by the expectation-maximization algorithm of SNPAlyze software. UC: Ulcerative colitis.