Genetic association and epistatic interaction of the interleukin-10 signaling pathway in pediatric inflammatory bowel disease

Table 1 Study samples

Sample	n	Sex, n	Race, n	Age at diagnosis
IBD	159	Male, 85; female, 74	White, 153; black, 6	13.1
CD	136	Male, 77; female, 59	White, 130; black, 6	13.1
CCFA	118	Male, 65; female, 53	White, 112; black, 6	13.0
Hershey	18	Male, 12; female, 6	White, 18	13.4
UC	23	Male, 9; female, 14	White, 23	13.3
CCFA	10	Male, 5; female, 5	White, 10	12.9
Hershey	13	Male, 4; female, 9	White, 13	13.6
Control	129	Male, 65; female, 64	White, 121; black, 7; unknown, 1	17.5

IBD: Inflammatory bowel disease; CD: Crohn’s disease; CCFA: Crohn’s and Colitis Foundation of America; UC: Ulcerative colitis.

Table 2 Study single nucleotide polymorphisms for IL-10, IL10RA, IL10RB, STAT3 and HO1 genes

Gene	SNP ID	Chromosomal position	Variation	Gene location	Disease implication	Ref.
IL-10	rs1800872	206946407	C-592>A	5'-upstream	associated with IBD	[37]
	rs3024498	206941529	c.T>C	3'-untralated region	associated with colorectal cancer	[41]
	rs3024496	206941864	A>G	3'-untralated region	associated with IBD and colorectal cancer, with decreased IL-10, with increased IgE levels	[37-39,41]
IL10RA	rs3135932	117864063	c.A247>G, p.Ser159Gly	coding region	mutations (other than the studied SNP) associated with pediatric IBD	[7,10,12]
IL10RB	rs2834167	34640788	c.A>G, p.Lys(A)47Glu(G)	coding region	mutations (other than the studied SNP) associated with pediatric IBD	[7-11]
STAT3	rs744166	404514201	A>G	Intron 1 (closer to exon2)	associated with IBD	[20]
HO1	rs2071746	3577672	A413>T	5'-upstream	no association with IBD, associated with asthma and allergy, anti-inflammation, anti-oxidant	[30,31]

IBD: Inflammatory bowel disease; SNPs: Single nucleotide polymorphisms.

Table 3 PCR-RFLP method for genotyping IL-10, IL10RA, IL10RB, STAT3 and HO1 genes

Gene	SNP ID	Variant	PCR amplification		RFLP
			Primers1	Product, bp	Restriction enzyme	Recognition site
IL-10	rs1800872	G>T	IL-2f: 5'-AACTTAGGCAGTCACCTTAGG-3'	149	ScaI	T yes; G No
			IL-2r: 5'-CATCCTGTGACCCCTCCAGT-3'
	rs3024498	T>C	IL-5f: 5'-GCTCCtTGGTTTCTCTTCCTA AG-3'	137	HpyCH4V	C yes; T No
			IL-5r: 5'-AGAAGCTTCCATTCCAAGCC TGA-3'
	rs3024496	A>G	IL-4f: 5'-GTATCAGAGGTAATAAATATTCcAT-3'	178	NlaIII	G yes; A No
			IL-4r: 5'-TAGAAGCATACATGACAATGAAG-3'
IL10RA	rs3135932	A>G	RA3f: 5'-CCCGCAAATGACACATATGgA-3'	172	MnI	G yes; A No
			RA3r: 5'-AGTTCCCAATGGCACACAAGG-3'
IL10RB	rs2834167	A>G	RB3f: 5'-GCCATAGAGGAGAACCAAGTG-3'	206	CarI	G yes; A No
			RB3r: 5'-GCTGTGAAAGTCAGGTTCTCTT-3'
STAT3	rs744166	A>G	ST2f: 5'-CAGGAGTGCCAACATTGAGAG-3'	106	AluI	A yes; G No
			ST2r: 5'-G TAATGTCTTGAGGGAATCGAG-3'
HO1	rs2071746	A>T	HO4f: 5'-TCAGCAGAGGATTCCAGCAGG-3'	110	BfaI	A yes; T No
			TG-3' HO4r: 5'-AGGCAGCGCTGCTCAGAGCAC-3'

¹Lowercase letter indicates a mismatched nucleotide.

Table 4 Genetic association of IL-10, IL10RA, IL10RB, STAT3 and HO1 genes with pediatric inflammatory bowel disease

Gene	SNP ID	Genotype	Disease, n	Control, n	OR	95%CI	P value
IL-10	rs1800872	CC	89	78	0.863	0.564-1.313	0.71
		CA	68	50
		AA	2	1
		C allele	246	206
		A allele	72	52
	rs3024498	CC	86	77	0.830	0.552-1.244	0.616
		CT	65	47
		TT	8	5
		C allele	237	201
		T allele	81	57
	rs3024496	AA	57	27	1.487	1.055-2.099	0.022
		AG	69	69
		GG	33	33
		A allele	183	123
		G allele	185	135
IL10RA	rs3135932	AA	108	85	0.925	0.595-1.433	0.160
		AG	39	40
		GG	12	4
		A allele	255	210
		G allele	63	48
IL10RB	rs2834167	AA	91	64	1.318	0.884-1.968	0.203
		AG	66	60
		GG	2	5
		A allele	248	188
		G allele	70	70
STAT3	rs744166	GG	64	49	0.943	0.662-1.340	0.352
		AG	66	63
		AA	29	17
		A allele	194	161
		G allele	124	97
HO1	rs2071746	AA	32	30	0.957	0.680-1.348	0.634
		AT	96	71
		TT	31	28
		A allele	158	131
		T allele	160	127

SNP: Single nucleotide polymorphism.

Table 5 Epistatic interaction between two single nucleotide polymorphisms in three IL-10 single nucleotide polymorphisms studied

	Epistatic model	rs3024496, P = 0.022, Bp = 0.0226	rs3024498, P = 0.616; Bp = 1
rs1800872, P = 0.71; Bp = 1	aa	P = 0.00015; Bp = 0.003	P = 0.638; Bp = 1
	ad	P = 0.057; Bp = 1	P = 0.605; Bp = 1
	da	P = 0.010; Bp = 0.216	P = 0.977; Bp = 1
	dd	P = 0.239; Bp = 1	P = 0.049; Bp = 1
rs3024496, P = 0.022, Bp = 0.0226	aa		P = 0.371; Bp = 1
	ad		P = 0.222; Bp = 1
	da		P = 0.167; Bp = 1
	dd		P = 0.584; Bp = 1

The P and Bp values for each SNP are shown next to each SNP; the P and Bp values for each of two SNP interactions are shown in the 3^rd and 4^th column. In the 2^nd column (epistatic model), a: Additive; d: Dominant. For a two SNP interaction, four different types of interactions may occur: additive-additive (aa), additive-dominant (ad), dominant-additive (da), and dominant-dominant (dd). Bp: Bonferroni P value; SNP: Single nucleotide polymorphism.

Table 6 Gene-gene interaction between IL-10 with IL10RA, IL10RB, STAT3, or HO1 in pediatric inflammatory bowel disease

Gene	SNP	Epistatic model	IL10RA rs3135932, P = 0.160; Bp = 1	IL10RB rs2834167, P = 0.203; Bp = 1	STAT3 rs744166, P = 0.352; Bp = 1	HO1 rs2071746, P = 0.634; Bp = 1
IL-10	rs1800872	aa	P = 0.046; Bp = 1	P = 0.029; Bp = 1	P = 0.248; Bp = 1	P = 0.910; Bp = 1
		ad	P = 0.739; Bp = 1	P = 0.107; Bp = 1	P = 0.954; Bp = 1	P = 0.617; Bp = 1
		da	P = 0.056; Bp = 1	P = 0.376; Bp = 1	P = 0.117; Bp = 1	P = 0.569; Bp = 1
		dd	P = 0.126; Bp = 1	P = 0.168; Bp = 1	P = 0.036; Bp = 1	P = 0.671; Bp = 1
	rs3024498	aa	P = 0.330; Bp = 1	P = 0.062; Bp = 1	P = 0.143; Bp = 1	P = 0.898; Bp = 1
		ad	P = 0.068; Bp = 1	P = 0.629; Bp = 1	P = 0.032; Bp = 1	P = 0.840; Bp = 1
		da	P = 0.884; Bp = 1	P = 0.307; Bp = 1	P = 0.316; Bp = 1	P = 0.607; Bp = 1
		dd	P = 0.265; Bp = 1	P = 0.644; Bp = 1	P = 0.029; Bp = 1	P = 0.790; Bp = 1
	rs3024496	aa	P = 0.021; Bp = 0.433	P = 0.425; Bp = 1	P = 0.538; Bp = 1	P = 0.346; Bp = 1
		ad	P = 0.020; Bp = 0.426	P = 0.495; Bp = 1	P = 0.306; Bp = 1	P = 0.741; Bp = 1
		da	P = 0.081; Bp = 1	P = 0.189; Bp = 1	P = 0.234; Bp = 1	P = 0.297; Bp = 1
		dd	P = 0.967; Bp = 1	P = 0.570; Bp = 1	P = 0.402; Bp = 1	P = 0.457; Bp = 1
IL10RB	rs2834167	aa	P = 0.403; Bp = 1		P = 0.251; Bp = 1	P = 0.128; Bp = 1
		ad	P = 0.384; Bp = 1		P = 0.956; Bp = 1	P = 0.369; Bp = 1
		da	P = 0.518; Bp = 1		P = 0.776; Bp = 1	P = 0.0018; Bp = 0.039
		dd	P = 0.176; Bp = 1		P = 0.072; Bp = 1	P = 0.289; Bp = 1

Bp: Bonferroni P value; SNP: Single nucleotide polymorphism.

Table 7 Epistatic interaction among the two IL-10 single nucleotide polymorphisms, rs1800872 and rs3024496, and IL10RA, IL10RB, STAT3, or HO1

	Epistatic model	IL10RA rs3135932, P = 0.16; Bp = 1	IL10RB rs2834167, P = 0.203; Bp = 1	STAT3 rs744166, P = 0.352; Bp = 1	HO1 rs2071746, P = 0.634; Bp = 1
Two IL-10 SNPs: rs1800872 and rs3024496 aa: P = 0.0002; Bp = 0.003	aaa	P = 0.003; Bp = 0.099	P = 0.080; Bp = 1	P = 0.175; Bp = 1	P = 0.216; Bp = 1
	aad	P = 0.130; Bp = 1	P = 0.340; Bp = 1	P = 0.920; Bp = 1	P = 0.140; Bp = 1

The additive-additive (aa) interaction model for the two IL-10 SNPs (rs1800872 and rs3024496) from Figure 1 was chosen for further analysis, in order to study the interaction of these two IL-10 SNPs with SNPs of four other genes. The two interaction models, aaa and aad, were for the two IL-10 SNPs and SNPs from each of the four genes. A significant P value (P = 0.003) was observed only with the IL10RA SNP (rs3135932). Bp: Bonferroni P value; SNPs: Single nucleotide polymorphisms.

Table 8 Epistatic interaction of IL10RB and HO1 with IL-10 (three single nucleotide polymorphisms), IL10RA, or STAT3

	Epistatic model	IL-10 rs1800872, P = 0.71; Bp = 1	IL-10 rs3024498, P = 0.616; Bp = 21	IL-10 rs3024496, P = 0.022; Bp = 0.0226	IL10RA rs3135932, P = 0.16; Bp = 1	STAT3 rs744166, P = 0.352; Bp = 1
aa IL10RB rs2834167 and HO1 rs2071746	aaa	P = 0.029;	P = 0.545;	P = 0.255;	P = 0.023;	P = 0.049;
		Bp = 1	Bp = 1	Bp = 1	Bp = 0.820	Bp = 1
	aad	P = 0.127;	P = 0.240;	P = 0.928;	P = 0.193;	P = 0.554;
		Bp = 1	Bp = 1	Bp = 1	Bp = 1	Bp = 1
ad IL10RB rs2834167 and HO1 rs2071746	ada	P = 0.394;	P = 0.824;	P = 0.242;	P = 0.444;	P = 0.436;
		Bp = 1	Bp = 1	Bp = 1	Bp = 1	Bp = 1
	add	P = 0.976;	P = 0.406;	P = 0.327;	P = 0.644;	P = 0.649;
		Bp = 1	Bp = 1	Bp = 1	Bp = 1	Bp = 1
da IL10RB rs2834167 and HO1 rs2071746	daa	P = 0.00015;	P = 0.489;	P = 0.047;	P = 0.008;	P = 0.014;
		Bp = 0.005	Bp = 1	Bp = 1	Bp = 0.287	Bp = 0.491
	dad	P = 0.072;	P = 0.140;	P = 0.388;	P = 0.205;	P = 0.977;
		Bp = 1	Bp = 1	Bp = 1	Bp = 1	Bp = 1
dd IL10RB rs2834167 and HO1 rs2071746	dda	P = 0.199;	P = 0.808;	P = 0.568;	P = 0.766;	P = 0.4731;
		Bp = 1	Bp = 1	Bp = 1	Bp = 1	Bp = 1
	ddd	P = 0.694;	P = 0.958;	P = 0.007;	P = 0.181;	P = 0.057;
		Bp = 1	Bp = 1	Bp = 0.242	Bp = 1	Bp = 1

All four interaction models (aa, ad, da and dd) of IL10RB and HO1 were used for this analysis, as well as both of the interaction models (a and d) of each gene studied. A total of eight interaction models for three SNPs are listed. The dad model of IL10RB and HO1 with the IL-10 rs1800872 is significant (Bp = 0.005). The daa models for IL10RB and HO1 with either IL10RA or STAT3 is weak (Bp = 0.287 and Bp = 0.491 respectively). Bp: Bonferroni P value.