Single nucleotide polymorphism in the tumor necrosis factor-alpha gene affects inflammatory bowel diseases risk

Table 1 Summary of clinical and demographic data on Caucasian IBD patients genotyped for at least one TNF-α polymorphism n (%)

	CD	UC	IC
Gender	388	405	27
Female	249 (64.2)	214 (52.8)	15 (55.6)
Male	139 (35.8)	191 (47.2)	12 (44.4)
Age at first diagnosis
Below 17	39 (10.0)	26 (6.4)	0
Between 17 and 40	199 (51.3)	184 (45.4)	15 (55.6)
Above 40	150 (38.7)	195 (48.2)	12 (44.4)
CD location
Ileal	125 (32.2)
Colonic	169 (43.6)
Ileocolonic	90 (23.2)
Unknown (U + UN)	4 (1.0)
UC location
Proctitis		140 (34.6)	3 (11.1)
Left colon		107 (26.4)	5 (18.5)
Pancolitis		154 (38.0)	19 (70.4)
Unknown		4 (1.0)	0
Behaviour
Non-structuring, non-penetrating perianal disease: With	47 (21.5)
Without	172 (78.5)
Structuring perianal disease: With	46 (38.0)
Without	75 (62.0)
Penetrating perianal disease: With	17 (35.4)
Without	31 (64.6)
Any relative with IBD: Yes (n = 143)	74 (19.1)	65 (16.1)	5 (18.5)
Bowel resection: Yes (n = 214)	142 (36.6)	70 (17.3)	2 (7.4)
Smoker at diagnosis: Yes (n = 147)	97 (25.7)	49 (12.3)	2 (7.7)
Ever used immunomo- dulators: Yes (n = 296)	203 (52.3)	86 (21.2)	8 (29.6)
Extra intestinal manifestations: Yes (n = 142)	75 (19.3)	64 (15.8)	3 (11.1

Table 2 Primer and probe sequences for custom made TaqMan SNP genotyping assay for TNF-α -857C/T, TNF-α -308G/A and TNF-α -238G/A

TaqMan SNP genotyping assay	DNA sequence
TNF-α_-857C/T _forward primer	5’-GGGCTATGGAAGTCGAGTATGG -3’
TNF-α_-857C/T _reverse primer	5’-GTCCTGGAGGCTCTTTCACT -3’
TNF-α_-857C/T_VIC probe	5’-CCCTGTCTTCGTTAAGG -3’
TNF-α_-857C/T _FAM probe	5’-CCTGTCTTCATTAAGG -3’
TNF-α_-308G/A_forward primer	5’-CCAAAAGAAATGGAGGCAATAGGTT -3’
TNF-α_-308G/A _reverse primer	5’-GGACCCTGGAGGCTGAAC -3’
TNF-α_-308G/A _VIC probe	5’-CCCGTCCCCATGCC -3’
TNF-α_-308G/A _FAM probe	5’-CCCGTCCTCATGCC -3’
TNF-α_-238G/A_forward primer	5’-CAGTCAGTGGCCCAGAAGAC -3’
TNF-α_-238G/A _reverse primer	5’-CCCTCACACTCCCCATCCT -3’
TNF-α_-238G/A _VIC probe	5’-CTCGGAATCGGAGCAG -3’
TNF-α_-238G/A _FAM probe	5’-CTCGGAATCAGAGCAG -3’

Table 3 Genotype and allele counts for TNF-α variants in New Zealand IBD patients and Caucasians

SNP	Controls	CD	UC	CD + UC	IC
TNF-α -238 G/A
AA	0	2 (0.5)	2 (0.5)	4 (0.5)	0
AG	50 (12.1)	47 (12.1)	39 (9.6)	86 (10.9)	2 (7.4)
GG	365 (87.9)	338 (87.3)	364 (89.9)	702 (88.6)	25 (92.6)
Genotype P		0.51	0.19	0.37
HWE P	0.38	0.68	0.31	0.35	1
A	50 (6.0)	51 (6.6)	43 (5.3)	94 (5.9)	2 (3.7)
G	780 (94.0)	723 (93.4)	767 (94.7)	1490 (94.1)	52 (96.3)
OR (95%CI)		1.10 (0.72, 1.68)	0.87 (0.56, 1.36)	0.98 (0.68, 1.42)
Allelic P		0.64	0.53	0.93
TNF-α -308 G/A
AA	10 (2.4)	16 (4.1)	23 (5.7)	39 (4.9)	1 (4.0)
AG	123 (29.7)	112 (28.9)	122 (30.3)	234 (29.6)	4 (16.0)
GG	282 (67.9)	260 (67.0)	258 (64.0)	518 (65.5)	20 (80.0)
Genotype P		0.41	0.049	0.1
HWE P	0.49	0.4	0.1	0.07	0.29
A	143 (17.2)	144 (18.6)	168 (20.8)	312 (19.7)	6 (12.0)
G	687 (82.8)	632 (81.4)	638 (79.2)	1270 (80.3)	44 (88.0)
OR (95% CI)		1.09 (0.84, 1.42)	1.27 (0.98, 1.63)	1.18 (0.94, 1.48)
Allelic P		0.49	0.06	0.14
TNF-α -857C/T
CC	359 (88.2)	332 (86.0)	358 (88.4)	690 (87.2)	21 (77.8)
CT	45 (11.1)	51 (13.2)	41 (10.1)	92 (11.6)	6 (22.2)
TT	3 (0.7)	3 (0.8)	6 (1.5)	9 (1.1)	0
Genotype P		0.61	0.57	0.85
HWE P	0.2	0.45	0.004	0.01	1
C	763 (93.7)	715 (92.6)	757 (93.5)	1472 (93.0)	48 (88.9)
T	51 (6.3)	57 (7.4)	53 (6.5)	110 (7.0)	6 (11.1)
OR (95% CI)		0.84 (0.56,1.26)	0.95 (0.63,1.45)	0.89 (0.63,1.28)
Allelic P		0.38	0.82	0.52

Table 4 Allelic odds ratios and 95% confidence intervals for comparison of TNF-α variants with IBD status in New Zealand IBD patients and Caucasians

	TNF-α_361525 (-238 G/A)				TNF-α_1799724 (-857C/T)				TNF-α_1800629 (-308 G/A)
	CD		UC		CD		UC		Crohn's		UC
	OR	95% CI	OR	95% CI	OR	95% CI	OR	95% CI	OR	95% CI	OR	95% CI
Female	1.00	0.59-1.69	0.64	0.35-1.18	1.06	0.65-1.74	1.07	0.64-1.79	1.01	0.72-1.41	1.19	0.85-1.67
Male	1.29	0.69-2.41	1.15	0.63-2.08	1.08	0.62-1.89	1.46	0.84-2.53	1.26	0.85-1.87	1.34	0.93-1.93
Age at first diagnosis
0-16 yr	1.30	0.54-3.14	0.31	0.04-2.29	1.67	0.51-5.48	0.8	0.28-2.31	1.66	0.97-2.85	1.60	0.83-3.07
17-40 yr	0.92	0.55-1.54	0.8	0.46-1.39	0.85	0.53-1.36	0.75	0.47-1.20	1.01	0.74-1.39	1.23	0.90-1.68
> 40 yr	1.30	0.78-2.17	1.02	0.62-1.69	0.72	0.44-1.18	1.31	0.76-2.25	1.08	0.77-1.52	1.26	0.93-1.71
CD location
Ileal	0.93	0.51-1.71			0.98	0.55-1.75			1.00	0.69-1.45
Colonic	1.09	0.65-1.83			1.00	0.59-1.69			1.25	0.91-1.72
Ileocolonic	1.44	0.79-2.63			0.56	0.32-0.97			0.92	0.59-1.42
UC location
Proctitis			1.07	0.61-1.87			0.68	0.41-1.12			0.90	0.62-1.30
Left colon			0.76	0.38-1.52			1.36	0.68-2.73			0.97	0.65-1.45
Pancolitis			0.74	0.40-1.36			1.08	0.62-1.88			1.91	1.41-2.60
CD Behaviour
Inflammatory	1.27	0.81-2.00			1.05	0.64-1.71			1.19	0.89-1.60
Structuring	1.19	0.67-2.10			0.66	0.39-1.11			1.01	0.69-1.47
Penetrating	0.16	0.02-1.17			0.65	0.31-1.37			0.89	0.50-1.59
Ileal/Structuring	1.34	0.70-2.58			0.95	0.47-1.92			1.04	0.66-1.64
Colonic/Inflammatory	1.29	0.77-2.17			1.06	0.60-1.87			1.29	0.92-1.80
	TNF-α_361525 (-238 G/A)				TNF-α_1799724 (-857C/T)				TNF-α_1800629 (-308 G/A)
Any relative with IBD	1.15	0.57-2.32	0.89	0.39-2.01	1.02	0.49-2.12	1.02	0.47-2.20	1.49	0.98-2.27	0.98	0.60-1.60
Bowel resection	1.12	0.65-1.93	0.7	0.29-1.66	0.59	0.37-0.95	0.97	0.47-2.02	0.93	0.65-1.34	1.57	1.02-2.41
Smoker at diagnosis	1.04	0.54-1.99	0.66	0.23-1.87	1.00	0.52-1.91	0.48	0.25-0.94	0.84	0.54-1.30	1.08	0.63-1.86
Ever used immunomodulators	1.07	0.66-1.75	0.66	0.29-1.48	0.93	0.57-1.51	0.75	0.41-1.39	1.09	0.80-1.48	1.46	0.98-2.17
Any EIMs	1.11	0.55-2.24	0.5	0.18-1.41	1.60	0.67-3.80	2.07	0.74-5.83	1.46	0.96-2.22	1.28	0.81-2.03

Table 5 Comparison of TNF-α variants with IBD status between Males and Females in New Zealand IBD patients and Caucasians

	Gender	n	TNF-α-238		TNF-α-857		TNF-α-308
			OR (95% CI)	P	OR(95% CI)	P	OR (95% CI)	P
CD vs Control	F	248/239	1.31 (0.99-1.75)	0.06	1.30 (0.98-1.73)	0.07	1.32 (0.99-1.75)	0.056
	M	139/176	1.00		1.00		1.00
UC vs Control	F	214/239	0.83 (0.63-1.09)	0.17	0.82 (0.62-1.08)	0.16	0.83 (0.63-1.09)	0.17
	M	191/176	1.00		1.00		1.00

Table 6 Haplotype analysis of two-SNP TNF-α haplotype in IBD status in New Zealand IBD patients and Caucasians

	Haplotype	Case subject frequency (%)	Control subject frequency (%)	Hap-score	Haplotype-specific scores P	Global score statistics
CD	Two SNPs TNF-α region
	[-308 (rs1800629), -238 (rs361525)]
	GG	75.3	76.7	-0.76	0.45	χ2 = 0.85, df = 3, P = 0.82
	GA	6.2	6.0	0.32	0.75
	AG	18.2	17.3	0.59	0.56
UC	Two SNPs TNF-a region
	[-308 (rs1800629), -238 (rs361525)]
	GG	73.9	76.4	-1.31	0.19	χ2 = 3.37, df = 2, P = 0.18
	GA	5.3	6.0	-0.63	0.53
	AG	20.8	17.5	1.79	0.07