Increased susceptibility for intrahepatic cholestasis of pregnancy and contraceptive-induced cholestasis in carriers of the 1331T>C polymorphism in the bile salt export pump

Table 1 Primers and probes of real-time PCR for allelic discrimination of ABCC2 SNPs in Caucasians

cDNA position1	SNP	Exon	Amino acid change	Eense-/antisense primer	Probes2
1249	G>A	10	V417I	5’-CCAACTTGGCCAGGAAGGA-3’/	VIC 5’-CTGTTTCTCCAACGGTGTA-3’
				5’-GGCATCCACAGACATCAGGTT-3’	FAM 5’-ACTGTTTCTCCAATGGTGTA-3’
3563	T>A	25	V1188E	5’-GCACCAGCAGCGATTTCTG-3’/	VIC 5’-ACACAATGAGGTGAGGAT-3’
				5’-AGGTGATCCAGGAAAAGACACATTT-3’	FAM 5’-ACAATGAGGAGAGGAT-3’
4544	G>A	32	C1515Y	5’-GTAATGGTCCTAGACAACGGGAAG-3’/	VIC 5’-AGAGTGCGGCAGCC-3’
				5’-CCAGGGATTTGTAGCAGTTCTTCAG-3’	FAM 5’-ATTATAGAGTACGGCAGCC-3’

¹cDNA sequence from GenBank accession numbers NM_000392 starting at the ATG;

²For each SNP two probes were designed and labeled with the fluorescent reporter dyes VIC (allele 1) and FAM (allele 2). SNP: Single nucleotide polymorphism.

Table 2 New group of patients with ICP (ICP_new)

Patient ID	Age (yr)	Liver parameters					Comments		Genotypes of SNPs
		ALT (ULN)	AP (ULN)	γ-GT (ULN)	tBili (ULN)	tBA (ULN)	No of preg/No ICP	Others	ABCB111331T>C (V444A)	ABCC23600T>A (V1188E)	ABCC24581G>A (C1515Y)
1	36	0.9	2.3	3.3	0.8	10.7	2/1		CC	TA	GA
2	31	1.6	2.5.	0.3	0.8	17.3	3/2		TC	TA	GA
3	35	8.9	2.3	1	0.5	10	1/1		TC	TT	GG
4	29	1.2	3	0.6	0.5	7	2/1		CC	TT	GG
5	42	11.6	1.4	0.4	0.8	6	1/1		TC	TT	GG
6	28	4.8	1.8	0.2	0.8	4.7	2/1	Twins	CC	TA	GA
7	32	11.9	3	1.2	0.9	3.6	nd		CC	TT	GG
8	16	5.2	2	0.4	nd	3.3	nd		TC	TT	GG
9	38	6.2	1.2	1.1	0.4	3	2/1		TC	TT	GG
10	23	1.6	1.3	1.3	0.8	2.4	1/1	Twins	TC	TT	GG
11	30	0.4	0.9	0.2	0.5	2.4	3/1		TC	TT	GG
12	22	0.3	1.8	0.1	0.5	2.1	nd		TC	TA	GA
13	32	0.2	0.9	0.7	0.2	2	2/1		TT	TT	GG
14	30	0.8	1.2	0.3	0.6	1.7	1/1		CC	TT	GG
15	28	7.5	2.3	0.9	0.6	nd	nd		TC	TT	GG
16	31	0.9	2.1	1.2	0.5	8.1	1/1		CC	TT	GG
17	20	3.4	1.1	0.4	0.5	2.6	1/1		TT	T	GG
18	24	7.2	1.3	0.4	0.8	nd	1/1		CC	nd	nd
19	31	8.9	1.3	1.2	1.5	nd	2/2		CC	nd	nd
20	32	11	2.2	0.4	3.1	nd	3/3	Pruritus with contraceptives	CC	n	nd
21	41	10.9	2.9	5.7	1.2	nd	1/1	Twins	CC	nd	nd
Summary median (Q1; Q3)	31 (28; 32)	4.8 (0.9; 8.9)	1.8 (1.2; 2.3)	0.6 (0.4; 1.2)	0.6 (0.5; 0.8)	3.5 (2.4; 7.3)

n/a: Not available; ALT: Alanine aminotransferase; tBili: Total bilirubin; tBA: Total bile acids.

Table 3 Characteristics of patients with oral CIC

Patient ID	Oral contraceptive	Age (yr)	Exposure time	Liver parameters					Comments		Genotypes of SNPs
				ALT	AP	γGT	tBili	tBA	Clinical features	Histology	ABCB111331T>C (V444A)	ABCC23600T>A (V1188E)	ABCC24581G>A (C1515Y)
				(ULN)	(ULN)	(ULN)	(ULN)	(ULN)
11	30 &mgr;g ethinylestradiol/75 &mgr;g gestodene	32	nd	4.9	1.7	1	10.9	22.3	Jaundice	Intrahepatic cholestasis	CC	TT	GG
2	30 &mgr;g ethinylestradiol/150 &mgr;g levonorgestrel	15	21 d	1	3	1	4.2	nd	Jaundice, nausea, pruritus	Extensive intrahepatic cholestasis	CC	TT	GG
3	35 &mgr;g ethinylestradiol/50 &mgr;g levonorgestrel	40	2 yr	3.9	2.8	3.6	0.5	1.6	Pruritus	Bland	CC	TT	GG
4	35 &mgr;g ethinylestradio/2 mg cyproteron	34	nd	1	1.3	nd	2.8	1.6	Jaundice	Extensive canalicular cholestasis, mild portal inflamation	CC	TA	GA

¹Patient exhibited previous episodes of ICP.

Table 4 Genotype distribution of non-synonymous ABCB11 variant site 1331T>C in patients and controls

Genotype SNP	ICPold		ICPnew		ICPtotal		Pregnant controls		Caucasian controls
	n(%)	95 % CI	n(%)	95 % CI	n(%)	95 % CI	n(%)	95 % CI	n(%)	95 % CI
ABCB11 1331T>C (V444A)	21 (100)		21 (100)		42 (100)		40 (100)		205 (100)
TT (VV)	-	-	2 (9.5)	0.0-22.1	2 (4.8)	0.0-13.9	7 (17.5)	5.7-29.3	38(18.5)	13.2-23.9
CC (AA)	14 (67.7)	46.5 -86.8	10 (47.6)	26.3-69.0	24 (57.1)	36.0-78.3	8 (20)	7.6-32.4	66 (32.2)	25.8-38.6
TC (VA)	7 (33.3)	13.2-53.5	9 (42.9)	21.7-64.0	16 (38.1)	17.3-58.9	25 (62.5)	47.5-77.5	101 (49.3)	42.4-56.1
Frequency C allele	35 (83.3)	67.4-99.3	29 (69.0)	49.3-88.8	64 (76.2)	58.0-94.4	41 (51.3)	35.8-66.7	233 (56.8)	50.1-63.6
Frequency T allele	7 (16.7)	0.7-32.6	13 (31.0)	11.2-50.7	20 (23.8)	5.6-42.0	39 (48.8)	33.3-64.2	177 (43.2)	36.4-50.0
ABCC2 3563T>A (V1188E)	16 (100)		17 (100)		33 (100)		42 (100)		110 (100)
TT (VV)	15 (93.8)	71.3-98.6	13 (76.5)	52.3-90.4	28 (84.8)	68.9-93.3	37 (88.1)	74.3-96.1	95 (86.4)	68.9-93.3
AA (EE)	-	-	-	-	-	-	-	-	1 (0.9)	0.0-5.0
TA (VE)	1 (3.1)	0.0-15.8	4 (23.5)	9.6-47.7	5 (15.2)	6.7-31.1	5 (11.9)	3.9-25.7	14 (12.7)	7.1-20.5
ABCC2 4544G>A (C1515Y)	16 (100)		17 (100)		33 (100)		42 (100)		110 (100)
GG (CC)	15 (93.8)	71.3-98.6	13 (76.5)	52.3-90.4	28 (84.8)	68.9-93.3	36 (85.7)	71.4-94.6	95 (86.4)	68.9-93.3
AA (YY)	-	-	-	-	-	-	-	-	1 (0.9)	0.0-5.0
GA (CY)	1 (3.1)	0.0-15.8	4 (23.5)	9.6-47.7	5 (15.2)	6.7-31.1	6 (14.3)	5.4-28.6	14 (12.7)	7.1-20.5

Results are given with 95 percent confidence interval (95% CI).

Table 5 1331T>C (V444A): Fisher’s exact test and ORs for the presence of homozygous CC variant and the C allele in the different groups

	CC vs TT			C vs T
	Fisher	Odds ratio	95% CI	Fisher	Odds ratio	95% CI
ICPoldvs Pregnant controls	0.0041	nd1	-	0.0004	4.8	2.2-15.0
ICPoldvs Caucasian controls	0.0029	nd1	-	0.0005	3.8	1.9-11.1
ICPnewvs Pregnant controls	0.1082	4.4	0.7-27.2	0.0441	2.1	1.0-4.7
ICnewvs Caucasian controls	0.1461	2.9	0.6-13.8	0.0850	1.7	0.9-3.4
ICPtotalvs Pregnant controls	0.0065	10.5	1.9-63.7	0.0007	3.0	1.7-6.4
ICPtotalvs Caucasian controls	0.0025	6.9	1.6-32.1	0.0006	2.4	1.5-4.5

¹Could not be determined, as TT = 0.