Polymorphisms at cholesterol 7α-hydroxylase, apolipoproteins B and E and low density lipoprotein receptor genes in patients with gallbladder stone disease

Table 1 PCR condition and restriction enzymes

Genes	Primers	Annealing temp	RE
CYP7A[16,17]	5’TGGTAGGTAAATTATTAATAGATGT3’
	5’AAATTAAATGGATGAATCAAAGAGC3’	61°C	Bsa I
apo B[10]	5’GGAGACTATTCAGAAGCTAA3’
	5’GAAGAGCCTGAAGACTGACT3’	60°C	Xba I
apo E[24]	5’ACAGAATTCGCCCCGGCCTGGTACAC3’
	5’TAAGCTTGGCACGGCTGTCCAAGGA3’	60°C	Hha I
LDL receptor[25]	5’CACTTTGTATATTGGTTGAAACTGT3’
	5’CACTGAACAAATACAGCAACCAGGG3’	62°C

Table 2 Demographic characteristics and biochemistry for patients and controls

	Patients	Controls	P value	P value1
No. (male/female)	105 (78/27)	274 (184/90)
Age (yr)	47.53 ± 10.98	47.94 ± 12.21
BMI (kg/m2)	24.47 ± 3.09	23.50 ± 2.16	< 0.01
Triglycerine (mmol/L)	1.32 ± 1.15	1.19 ± 0.82
Cholesterol (mmol/L)	4.66 ± 0.92	4.91 ± 0.96	< 0.05	< 0.01
HDL (mmol/L)	1.33 ± 0.36	1.40 ± 0.34
LDL (mmol/L)	2.56 ± 0.67	2.69 ± 0.73		< 0.05
apo A I (g/L)	1.34 ± 0.21	1.39 ± 0.18	< 0.05
apo B (g/L)	1.00 ± 0.22	1.03 ± 0.24		< 0.05

¹After adjustment for sex, age and BMI.

Table 3 Distribution of genotypes for patients and controls

Genes	Genotypes	Patients (%)	Controls (%)
CYP7A	AA	44 (41.90)	79 (28.83)
	AC	44 (41.90)	140 (51.10)
	CC	17 (16.20)	55 (20.07)
Apo B	X+/+	1 (0.95)	0 (0)
	X+/-	16 (15.24)	22 (8.03)
	X-/-	88 (83.81)	252 (91.97)
Apo E	ε2/2	0 (0)	1 (0.36)
	ε2/3	15 (14.29)	45 (16.42)
	ε2/4	5 (4.76)	6 (2.19)
	ε3/3	73 (69.52)	183 (66.80)
	ε3/4	11 (10.48)	37 (13.50)
	ε4/4	1 (0.95)	2 (0.73)
LDL receptor	AA	41 (39.05)	90 (32.85)
	AB	2 (1.90)	13 (4.74)
	AC	32 (30.48)	104 (37.96)
	BB	3 (2.85)	4 (1.46)
	BC	8 (7.62)	22 (8.03)
	BC	19 (18.60)	41 (14.96)

Table 4 Frequency of alleles for patients and controls (%)

Genes	Alleles	Patients	Control
CYP7A	A	62.86a	54.38
	C	37.14	45.62
Apo B	X+	8.57b	4.01
	X-	91.43	95.99
Apo E	ε2	9.52	9.67
	ε3	81.90	81.75
	ε4	8.57	8.57
LDL receptor	A	55.24	54.20
	B	7.62	7.84
	C	37.14	37.96

^aχ² = 4.44, P < 0.05;

^bχ² = 6.31, P < 0.01.

Table 5 Association of CYP7A gene polymorphism with plasma lipids

	Patients		Controls		All
	AA/AC	CC	AA/AC	CC	AA/AC	CC
No.	88	17	219	55	307	72
Triglyceride (mmol/L)	1.25 ± 0.99	1.70 ± 7.77	1.15 ± 0.82	1.36 ± 0.81	1.18 ± 0.87	1.44 ± 1.11
Cholesterol (mmol/L)	4.64 ± 0.95	4.78 ± 0.79	4.84 ± 0.94c	5.16 ± 0.99c	4.78 ± 0.95g	5.07 ± 0.95g
HDL (mmol/L)	1.32 ± 0.37a	1.35 ± 0.32	1.42 ± 0.34a	1.33 ± 0.72	1.39 ± 0.35	1.33 ± 0.32
LDL (mmol/L)	2.56 ± 0.70	2.59 ± 0.45e	2.63 ± 0.72c	2.91 ± 0.72e,c	2.61 ± 0.71g	2.83 ± 0.67g
Apo AI (g/L)	1.34 ± 0.22a	1.36 ± 0.15	1.39 ± 0.18a	1.36 ± 0.20	1.38 ± 0.19	1.36 ± 0.18
Apo B (g/L)	1.00 ± 0.23	1.02 ± 0.15	1.01 ± 0.24c	1.10 ± 0.23c	1.01 ± 0.23g	1.08 ± 0.22g

^aP < 0.05, patients vs controls with AA/AC genotype,

^cP < 0.05, AA/AC vs CC within control group,

^eP < 0.05, patients vs controls with CC genotype,

^gP < 0.05, AA/AC vs CC in all the subjects.

Table 6 Stepwise logistic regression of multiple variables

Variables	OR	95%CI
A allelea	1.48	1.05-2.09
X+ allelea	2.28	1.14-4.59
Cholesterolb	0.69	0.64-0.74
BMIa	1.13	1.05-1.22

^aP < 0.05;

^bP < 0.01.