Glucocorticoid receptor gene haplotype structure and steroid therapy outcome in IBD patients

Table 1 Oligonucleotides used as polymerase chain reaction primers to amplify the NR3C1 exons

Primer name	Primer sequence	Nested PCR	Primer name	Primer sequence
GR 2_F	CACTTAGGTTGTCTACCTTTCCTAC	Y	GR 2_Fa	TTCAAAAGGCCACTTAAACTTATTC
GR 2_R	GATAGAAACTACTCTTCTGGTAAC	Y	GR 2_Ra	CCTTGGAGATCAGACCTGTTG
		Y	GR 2_Fb	CTGTGCCCAGTTTCTCTTGC
		Y	GR 2_Rb	CAGCCAGATCTGTCCAAAGC
		Y	GR 2_Fc	TTGGAAACTCCTTCTCTGTGG
		Y	GR 2_Rc	AATGTGGCATGCTGAATGG
GR 3_F	CATTAGAGGACCTAGGAGCCAC	N
GR 3_R	GAAGTGAACCAGAACCACACC	N
GR 4_F	TGAATTCAGTGTGTGTAAGAAGAAC	N
GR 4_R	TTGCACTGTTTTCAGTTTGTTG	N
GR 5_F	CACCTGTATTCACCTGACTCTCC	N
GR 5_R	TTTTTTCTCCTTTTCCATGTCAC	N
GR 6_F	GCCCCAAGCACTCATAACTC	N
GR 6_R	TCAGATGACAGAAGAAAACTGTGTC	N
GR 7_F	AATCTTGGTGTCACTTACTGTGC	N
GR 7_R	CCAAGATGCAGGAAGTTTAAGG	N
GR 8_F	CACCAACATCCACAAACTGG	Y	GR 8_Fa	TTGGTCAGTGGGAACATC
GR 8_R	CCACCAGTTCTTCTTACACACAC	Y	GR 8_Ra	ATGGTGGCTTGTGCCTAC
GR 9a_F	TGATGACGACTCAACTGCTTC	N
GR 9a_R	ATCTGGGGAATTCCAGTGAG	N
GR 9b_F	TCCTAAAAGGGCACAGCTTC	N
GR 9b_R	CAATCATTTGCTTTTTGAATGC	N

PCR: Polymerase chain reaction; GR: Glucocorticoid receptor.

Table 2 Predicted haplotypes found to be in best reconstruction for 181 inflammatory bowel diseases patients

Haplotype number1	Haplotype composition2	Absolute haplotype frequency	Relative haplotype frequency	Number of haplotypes not included3
	Reference: GGGATGGCCATGT	(n = 362)
GR_1	1111111111111	165	0.456
GR_24	1111112111211	90	0.249
GR_34	1111211111111	72	0.199	2
GR_44	1112111111111	8	0.022
GR_5	2211112111221	6	0.017
GR_6	1111112111212	4	0.011
GR_7	1111112111111	3	0.008
GR_8	1111111111211	2	0.006	1
GR_9	1111112121212	2	0.006
GR_10	1111221111111	2	0.006	2
GR_11	2211112111211	2	0.006	1
GR_12	1111111111212	1	0.003	1
GR_13	1111112211212	1	0.003
GR_14	1111211112111	1	0.003
GR_15	1111212111111	1	0.003
GR_16	1121111111111	1	0.003
GR_17	2211111111111	1	0.003	1

¹Haplotypes are arranged in the order of decreasing frequency;

²1 indicates the reference allele at a certain position, 2 indicates the variant allele;

³Number of haplotypes not included in the subsequent association analysis (steroid therapy outcome, tables 4, 7 and 8) due to likelihood values ≤ 0.95;

⁴Haplotype GR_2 corresponds to haplotype b, GR_3 corresponds to haplotype c and GR_4 corresponds to haplotype d in Figure 2.

Table 3 Frequencies of single nucleotide polymorphisms detected in the glucocorticoid receptor gene (NR3C1)

SNP number	Alternativename1	Variant number2	DNA position3	DNA region	cDNA position4	Nucleotide reference	Nucleotide variant	Amino acid exchange	Allele frequency(n = 362)	Reported allele frequencies2
1	2.1	rs6189	3943266	Exon 2	558	G	A	E22E	0.025	0.002-0.034
2	2.2	rs6190	3943264	Exon 2	560	G	A	R23K	0.025	0.002-0.034
3	2.3	rs72542742	3942647	Exon 2	1177	G	A	A229T	0.003	0.002
4	2.4	rs56149945	3942244	Exon 2	1580	A	G	N363S	0.025	0.000-0.046
5	3.1	rs4986593	3856773	Intron 3		T	C		0.213	0.008-0.228
6	4.1	rs61753484	3852751	Intron 4		G	C		0.006	0.000-0.009
7	5.1	rs6188	3843271	Intron 5		G	T		0.290	0.000-0.500
8	6.1	rs6194	3841288	Exon 6	2256	C	T	H588H	0.006	0.000-0.091
9	8.1	rs258751	3825207	Exon 8	2526	C	T	D678D	0.006	0.000-0.149
10	8.2	novel SNP	3824968	Intron 8		A	G		0.003	NA
11	8.3	rs258750	3824816	Intron 8		T	C		0.307	0.091-0.362
12	8.4	rs10482704	3824690	Intron 8		G	T		0.017	0.000-0.027
13	9.1	rs6196	3824417	Exon 9	2790	T	C	N766N	0.022	0.058-0.325

¹Defines both the exon/intron localization and the single nucleotide polymorphism (SNP) number;

²According to the National Center for Biotechnology Information (NCBI) SNP database;

³According to the NCBI genomic reference sequence NT_029289.11;

⁴According to the NCBI cDNA reference cDNA Sequence NM_000176.2. NA: Not applicable.

Table 4 Demographic data of 173 inflammatory bowel diseases patients included in the association analysis

Characteristics	Crohn’s disease	Ulcerative colitis	All
Patients	84 (49%)	89 (51%)	173 (100%)
Age (documented for 171 individuals)	37.5 (± 15.3)	41.7 (± 14.2)	39.7 (± 14.9)
mean ± SD	35	42	39
Median	16	18	16
Minimum	72	82	82
Maximum
Known GC treatment outcome in the past	50	50	100
No. of patients currently treated with GCs	83	60	143
Male/female	52 (58.4%)/ 37 (41.6%)	40 (47.6%)/ 44 (52.4%)	92 (53.2%)/ 81 (46.8%)
Wild-type carriers	15 (16.9%)	20 (23.8%)	35 (20.2%)
Carriers of one variant haplotype	48 (53.9%)	39 (46.4%)	87 (50.3%)
Carriers of two variant haplotypes	26 (29.2%)	25 (29.8%)	51 (29.5%)

GCs: Glucocorticoids.

Table 5 Past and current additional medication of 173 inflammatory bowel diseases patients included in the association analysis

Additional medication	n
5-Aminosalicylic acid	142
6-Mercaptopurine	33
Adalimumab	3
Antibiotics	64
Azathioprine	122
Bisphosphonates	8
Certulizumab	1
Cholestyramine	8
Cyclosporine	8
Infliximab	45
Methotrexate	27
Sulfasalazine	12
Ursodeoxycholic acid	3

Table 6 Extraintestinal manifestations

Extraintestinal manifestations	n (%)1
Peripheral arthritis	46 (27.2)
Uveitis/iritis	6 (3.6)
Pyoderma gangrenosum	4 (2.4)
Erythema nodosum	9 (5.3)
Aphthous oral ulcers	10 (5.9)
Ankylosing spondylitis	7 (4.1)
Primary sclerosing cholangitis	6 (3.6)

¹Documented for 169 patients.

Table 7 Predicted frequencies of haplotype combinations in 173 inflammatory bowel diseases patients

Haplotype combination	n	Frequency
GR_1 + GR_2 or GR_2 hom	51	0.295
GR_1 + GR_3 or GR_3 hom	41	0.237
GR_1 hom (wt)	36	0.208
GR_2 + GR_3	23	0.133
GR_1 + GR_4 or GR_4 hom	6	0.035
GR_1 + GR_5	3	0.017
GR_3 + GR_6	2	0.012
GR_1 + GR_7 or GR_7 hom	1	0.006
GR_1 + GR_16 or GR_16 hom	1	0.006
GR_2 + GR_8	1	0.006
GR_2 + GR_6	1	0.006
GR_2 + GR_9	1	0.006
GR_6 + GR_13	1	0.006
GR_7 + GR_15	1	0.006
GR_2 + GR_4	1	0.006
GR_2 + GR_11	1	0.006
GR_5 + GR_9	1	0.006
GR_9 + GR_17	1	0.006

Table 8 Association between glucocorticoids therapy outcome and the haplotype GR_4

Haplotype	Cohortcomposition	Therapy success rate in wt carriers (success/no success)	Therapy success rate in het/hom variant carriers (success/ no success)	P-value	OR (CI)
GR_4 merged	All	0.682 (15/7)	0.4 (2/3)	0.326	3.214 (0.434-23.787)
	Male	0.733 (11/4)	NA (0/0)	NA	NA
	Female	0.571 (4/3)	0.4 (2/3)	1.000	2.000 (0.194-20.614)

NA: Not applicable (at least one cell box was counted as 0, OR and P not calculatable).