Aquaporin-8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis

Table 1 Patients’ characteristics

Disease	Controls	CD	UC
n	4	4	4
Sex (M/F)	1/3	1/3	2/2
Age, range (yr)	52-60	23-43	20-48
Age, mean (yr)	56	32	35

Table 2 Sequences of primers used for PCR amplification of AQP8 promoter and coding region

AQP8 fragment	Primer designation	5’-3’ sequence
Promotor region	Pro-forward	CCGTGTTAGCCAGGATGG
	Pro-reverse	GAAACCTGCACCTGCTGTG
Exon 1	Ex1-forward	CCCTGCCCTGTTGAGATTTA
	Ex1-reverse	AGGGAAAAAGGGACAGAGGA
Exon 2	Ex2-forward	AAGAGTCCGATGTTTGTGCC
	Ex2-reverse	CTTTGCTTTCCACACCCAGT
Exon 3	Ex3-forward	ACACTGTCTCAAGTGCCAGC
	Ex3-reverse	CACACCCACATATGCACCTC
Exon 4	Ex4-forward	GCAGGGTCGCACAGTAAAAT
	Ex4-reverse	CTGGCCCCTAATAGCAACTG
Exon 5	Ex5-forward	AGCCCCTCTGCCTTCTTTAG
	Ex5-reverse	TTCCAAACCCAAGTGAGAGC
Exon 6	Ex6-forward	AGCCTGGAGACATGACGAAG
	Ex6-reverse	AGAGCCTCCTCAGCAGTCAG

Table 3 Sequences of primer and probes used for TaqMan allelic discrimination of AQP8 SNPs

AQP8 SNP	Primer designation	5’-3’ Sequence
AQP8-A212T	Forward primer	CTTGGTGCCTGGGTGTTTG
	Reverse primer	AGCCAGTAGATCCAGTGGAAGTTC
	FAM-Probe	CATGAATCCCACCCGT
	VIC-Probe	TCCCGCCCGTGCT
AQP8-A260P	Forward primer	GAGATGGGAAGACCCGCC
	Reverse primer	GGAATCCCACGAGCTCTGC
	FAM-Probe	TCCTGAAGGCTCGGTGA
	VIC-Probe	TCCTGAAGCCTCGGTGA
AQP8-R261Q	Forward primer	GGCAGGAAATGCAGGAACTC
	Reverse primer	GGGAAGACCCGCCTCATC
	FAM-Probe	AGCTCTGCTTCACTGAG
	VIC-Probe	AGTTCTGCTTCACCGAG

Exchanged nucleotides are given in bold.

Table 4 Selection of differentially expressed genes located in IBD candidate loci

IBDcandidatelocus/Gene	Controlexpressionileum	FCCD	FCUC	Controlexpressioncolon	FCCD	FCUC
Chr. 16q11-12
MMP2	267	1.5	-1.1	220	1.5	3.7
TM4SF11	194	1.3	1.0	151	-2.1	-1.7
SIAH1	208	1.6	1.2	156	3.5	1.3
MT1G	3053	-1.1	1.2	2356	-1.7	-2.0
NETO2	49	-1.6	-1.7	61	2.5	1.4
Chr. 12q13
DGKA	427	-1.5	1.0	342	-2.3	-2.6
MYO1A	1008	1.1	1.1	811	-3.5	-4.3
Chr. 5q32
SPARC	76	2.6	1.5	104	2.0	2.6
TGFBI	236	2.0	1.0	356	1.5	2.5
CAMLG	93	2.5	1.4	91	2.1	1.4
TCF7	357	-2.0	-1.2	279	-1.2	-1.6
SLC35A4	535	-1.5	-1.5	638	-2.3	-1.5
SEC24A	221	2.3	1.1	211	1.7	1.1
SLC26A2	302	2.3	-2.5	388	6.5	-7.5
EGR1	256	2.3	1.1	180	17.1	3.0
PACAP	150	-2.8	-1.7	138	-1.6	2.0
Chr. 19p13
AES	222	-2.1	-1,1	257	-2.5	-2.3
MAP2K2	420	-2.0	-1,1	627	-2.1	-5.7
Chr. 1p36
CDC42	1428	1.1	-1.2	1429	2.0	-1.9
MFN2	304	-1,1	1.0	367	-3.0	-2.5
C1QA	336	1.1	1.1	373	-1.6	-2.5
Chr. 16p12
MIR16	201	-1.7	-1.1	277	-3.0	-1.2
AQP8	575	-8.6	-9.8	438	4.3	3.0
MT1G	3053	-1.1	1.2	2356	-1.7	-2.0
LITAF	500	-1.2	-1.2	670	1.9	2.1
PRKCB1	157	-2.0	1.4	95	1.1	-1.2
USP7	184	1.6	-1.3	192	2.0	-1.1
Chr. 7q11
WBSCR22	172	-1.7	-1.1	207	-1.9	-2.3
POR	213	-2.1	1.0	172	-2.3	-4.9
SEMA3C	79	1.1	1.0	70	2.3	1.9
CLDN3	386	-1.4	-1.2	618	-2.6	-6.5
WBSCR20A	323	-1.6	1.0	281	-2.0	-1.7
TMPIT	928	1.1	1.1	893	-2.3	-2.3
CLDN4	150	1.1	1.0	232	-1.4	-2.3
CD36	153	2.8	1.3	127	2.3	1.1
HSPB1	376	-3.2	-2.3	493	-1.9	-1.5
Chr. 4q25
ELOVL6	112	1.2	-1.6	332	2.1	1.6
ENPEP	578	1.7	1.6	533	1.1	-11.3
T2BP	322	1.2	1.1	224	2.8	1.4
SYNPO2	242	2.3	1.3	160	2.5	1.1
SEC24B	105	2.0	1.6	161	1.6	1.1
Chr. 3p21
ACY1	744	1.1	1.2	485	-2.1	-1.5
Chr. 3q27
AP2M1	417	-1.6	1.0	503	-2.3	-3.5
TNIK	143	2.1	1.1	135	1.4	1.0
PRKCI	68	2.1	1.2	104	2.0	1.1
SI	1313	1.3	1.1	963	1.4	-2.0
EIF4G1	292	-2.5	-1.6	319	-3.7	-6.1
KLHL6	323	-3.2	1.0	110	1.1	1.7
FXR1	137	1.9	1.2	105	2.1	2.0
Chr. 10q22
MYST4	73	2.0	1.4	75	2.0	1.5
RAI17	363	1.6	1.0	250	1.7	2.1

Selection of known genes in IBD candidate loci, which show a two fold change at least in one comparison. CD or UC samples are referred to control specimens. The control expression values are given for the terminal ileum and the colon. At least two fold changes are indicated by bold numbers.

Table 5 Aquaporin gene expression and regulation in IBD

Gene	Controlexpression ileum	FC CD	FC UC	Controlexpression colon	FC CD	FC UC
AQP1	181	1.7	1.7	165	-2.0	-1.6
AQP3	473	1.9	1.5	352	-1.4	-3.2
AQP8	575	-8.6	-9.8	438	4.3	3.0
AQP11	510	1.2	1.0	409	-1.9	-2.0

List of all AQPs whose transcripts were increased or decreased (shown as minus) in Crohns Disease (CD) or Ulcerative Colitis (UC) samples compared with control specimens. The control expression values are given for the terminal ileum and the colon. Genes with an average difference intensity of 50 are considered as significantly expressed in the given cell type. The fold change (FC) of gene expression between controls and patient groups is indicated. No transcripts for AQP0, AQP2, AQP4, AQP5, AQP6, AQP7 and AQP9 could be detected. AQP10 and AQP12 are not represented on the used arrays.

Table 6 Allelic contribution of AQP8 SNPs

SNP		n VIC	% VIC	n FAM	% FAM	n both	% both	P-Value	χ2
AQP8-A212T		G	G	A	A	G/A	G/A
GCC -> ACC	UC	210	95.5	0	0.0	9	4.1	0.57	0.33
	CD	166	91.7	0	0.0	9	5.0	0.98	0.00
	Co	232	92.8	0	0.0	14	5.6
AQP8-A260P		G	G	C	C	C/G	C/G
GCT -> CCT	UC	94	42.7	33	15.0	78	35.5	0.26	2.66
	CD	56	30.9	26	14.4	83	45.9	0.46	1.55
	Co	93	37.2	43	17.2	107	42.8
APQ8-R261Q		G	G	A	A	G/A	G/A
CGG -> CAG	UC	205	93.2	0	0.0	13	5.9	0.62	0.97
	CD	159	87.8	1	0.6	9	5.0	0.97	0.07
	Co	214	85.6	1	0.4	13	5.2