Clinical significance of NOD2/CARD15 and Toll-like receptor 4 gene single nucleotide polymorphisms in inflammatory bowel disease

Table 1 Primers sequences and restriction enzymes used for genotyping TLR4 and NOD2/CARD15

Gene locus	SNPs		Sequence	Restriction enzyme
NOD2/CARD15	R702W	For	5’ TTCAGATCACAGCAGCCTTC 3’	MspI
		Rev	5’ CCCACACTGCAAAATGTCAAC 3’
	G908R	For	5’ AGCCACTGAAAACTCTTGG 3’	HhaI
		Rev	5’ TCTTCACCTGATCTCCCCAA 3’
	L1007finsC	For	5’ CCTGCAGTCTCTTTAACTGG 3’	NlaIV
		Rev	5’ CTTACCAGACTTCCAGGATG 3’
TLR4	D299G	For	5’ TTAGAAATGAAGGAAACTTGGAAAAG 3’	BsaBI
		Rev	5’ TTTGTCAAACAATTAAATAAGTGATTAATA 3’
	T399I	For	5’ GGTTGCTGTTCTCAAAGTGATTTTGGGAGAA 3’	HinfI
		Rev	5’ CCTGAAGACTGGAGAGTGAGTTAAATGCT 3’

Table 2 NOD2/CARD15 and TLR4 SNPs allele frequencies of CD patients vs control group and UC patients

Polymorphisms of NOD2/CARD15 and TLR4 genes	CD (n = 133)	Allele frequency (%)	Controls (n = 103)	Allele frequency (%)	1P	OR (95% CI)	UC (n = 45)	Allele frequency (%)	2P	OR (95% CI)
R702W
Wild-type	107 (80.4%)	9.8	98 (95.1%)	2.4	0.001	4.09 (1.5-11.9)	43 (95.5%)	2.3	0.03	4.49 (1.02-19.8)
Heterozygous	26 (19.6%)		5 (4.9%)				2 (4.5%)
G908R
Wild-type	120 (90.2%)	4.5	94 (91.2%)	4.3	NS	1.01 (0.3-3.5)	41 (91.1%)	4.4	NS	0.90 (0.28-2.92)
Heterozygous	13 (9.8%)		9 (8.8%)				4 (8.9%)
L1007finsC
Wild-type	107 (80.4%)	9.8	97 (94.1%)	2.9	0.002	3.92 (1.55-9.95)	43 (95.5%)	2.3	0.01	5.22 (1.19-22.98)
Heterozygous	26 (19.6%)		6 (5.9%)				2 (4.5%)
D299G
Wild-type	123 (92.5%)	3.7	95 (92.2%)	3.9%	NS	0.96 (0.37-2.54)	42 (93.3%)	3.4	NS	0.87 (0.23-3.35)
Heterozygous	10 (7.5%)		8 (7.8%)				3 (6.7 %)
T399I
Wild-type	125 (94%)	3.0	97 (94.1%)	2.9%	NS	1.15 (0.28-4.64)	42 (93.3%)	3.4	NS	1.11 (0.28-4.40)
Heterozygous	8 (6.0%)		6 (5.9%)				3 (6.7%)

No patients homozygous for NOD2/CARD15 gene R702W, G908R and L1007finsC SNPs were found in this study population; No patients homozygous for TLR4 gene D299G and T399I SNPs were found in this study population.

¹CD patients vs control group;

²CD patients vs UC patients. NS: No significance.

Table 3 Genotype-phenotype correlations in CD patients

Total CD patients (n = 133)	CARD15 no risk alleles (n = 68, 51.1%)	R702W 1 risk allele (n = 26, 19.6%)	G908R 1 risk allele (n = 13, 9.7%)	L1007finsC 1 risk allele (n = 26, 19.6%)	CARD15 at least 1 risk allele (n = 65, 48.8%)	CARD15 compound heterozygous (n = 48, 36.1%)
Age (mean ± SD)	41.5 ± 11.2	41.2 ± 11.9	43.02 ± 10.9	42.0 ± 12.8	42.3 ± 12.1	42.7 ± 11.9
Sex (m/f, 70/63)	32/36	13/13	8/5	10/16	30/35	25/23
Localization (%)
Ileum (n = 61)	25 (36.8)	11 (42.3)	4 (30.8)	21 (80.7)	38 (58.5)	30 (62.5)
Ileo-colon (n = 39)	22 (32.3)	10 (38.5)	4 (30.8)	3 (11.5)	15 (23.0)	10 (20.8)
Colon (n = 30)	18 (26.5)	5 (19.5)	5 (38.4)	2 (7.8)	12 (18.5)	8 (16.7)
Upper GI (n = 3)	3 (4.4%)				0	0
P		> 0.051	> 0.051	0.0011	0.042	0.033
Disease type (%)
Inflammatory (n = 37)	14 (20.6)	7 (27.0)	6 (46.0)	10 (38.5)	23 (35.4)	8 (16.6)
Stenosing (n = 56)	32 (47.0)	11 (42.3)	7 (54.0)	6 (23.0)	24 (37.0)	34 (70.8)
Fistulizing (n = 40)	22 (32.4)	8 (30.7)	0	10 (38.5)	18 (27.6)	6 (12.6)
P		> 0.051	0.031	> 0.051	> 0.052	0.023
Resective Surgery (%)	18 (26.4)	9 (34.6)	9 (69.2)	14 (53.9)	32 (49.2)	31 (64.6)
P		> 0.051	0.0031	0.011	0.012	0.0003
Extraintestinal manifestations (n = 18, %)	10 (55.5)	3 (11.5)	1 (7.7)	4 (15.3)	8 (13.3)	3 (6.2)

No patients homozygous for R702W, G908R and L1007finsC SNPs were found in this study population.

¹CD patients no risk allele vs CD patients with risk allele;

²CARD15 at least 1 risk allele vs CD patients no risk allele;

³CARD15 compound heterozygous vs CD patients no risk allele.