Genetic polymorphism in CD14 gene, a co-receptor of TLR4 associated with non-alcoholic fatty liver disease

Table 1 Primer sequences, polymerase chain reaction conditions, and product sizes of different genes

Polymorphism site	Primer sequence (5'-3')	PCR conditions	Product size (bp)
TLR4 (Asp299gly)		(1) 95 °C for 10 min	249
		(2) 35 cycles of :
	F: GATTAGCATACTTAGACTACTACCTCCATG	94 °C for 1 min
	R: GATCAACTTCTGAAAAAGCATTCCCAC	61.5 °C for 1 min
		72 °C for 1 min
		(3) 72 °C for 7 min
TLR4 (Thr399Ile)		(1) 95 °C for 10 min	406
		(2) 35 cycles of :
	F: GGTTGCTGTTCTCAAAGTGATTTTGGGAGAA	94 °C for 1 min
	R: CCTGAAGACTGGAGAGTGAGTTAAATGCT	64.8 °C for 1 min
		72 °C for 1 min
		(3) 72 °C for 10 min
TLR2 (Arg753Gln)		(1) 95 °C for 10 min	289
		(2) 35 cycles of :
	F: 5’-CCTTCAAGTTGTGTCTTCATAAG-3’	94 °C for 1 min
	R: 5’-GGCCACTCCAGGTAGGTCTT-3’	58.6 °C for 1 min
		72 °C for 1 min
		(3) 72 °C for 10 min
CD14 (-550C/T)		(1) 95 °C for 10 min	368
		(2) 32 cycles of :
	F: GGAAGGGGGAATTTTTCTTTAGGC	94 °C for 1 min
	R: -GGCAGTGTCCTGATGACTCA	59.8 °C for 1 min
		72 °C for 1 min
		(3) 72 °C for 7 min
CD14 (-159C/T)		(1) 95 °C for 10 min	296
		(2) 35 cycles of :
	F: ATCATCCTTTTCCCACACC	94 °C for 40 s
	R: AACTCTTCGGCTGCCTCT	61 °C for 40 s
		72 °C for 40 s
		(3) 72 °C for 10 min

Table 2 Restriction fragment length polymorphism conditions

Polymorphic site	Restriction enzyme	Incubation temperature and duration	Genotype and restriction fragment pattern (bp)
	(units used)
TLR4 (Asp299gly)	NcoI	37 °C for 16 h	AA: 249
			AG: 249, 223, 26
			GG: 223, 26
TLR4 (Thr399Ile)	HinfI	37 °C for 8 h	CC: 406
			CT: 406, 377, 29
			TT: 377, 29
TLR2 (Arg753Gln)	AciI	37 °C for 16 h	GG: 252 and 37
			GA: 252, 37, 289
			AA:289
CD14 (-550C/T)	HaeIII	37 °C for 12 h	CC: 23, 236, 109
			CT: 23, 236, 109, 259
			TT: 109, 259
CD14 (-159C/T)	HaeIII	37 °C for 16 h	CC: 141, 154
			CT: 141, 154, 296
			TT: 296

Table 3 Anthropometric and biochemical characteristics of patients with non-alcoholic fatty liver disease and healthy volunteers n (%)

Parameters	NAFLD (n = 200)	HVs (n = 50)	P value
Mean age (yr)	38.27 ± 10.3	36.56 ± 4.2	0.218
Gender	122 M/78 F	38 M/12 F
Mean BMI (kg/m2)	27.16 ± 4.7	22.1 ± 1.2	0.0002
Lean	35 (17.5)	46 (92)	0.0001
Overweight	39 (19.5)	3 (6)	0.02
Class I obesity	87 (43.5)	1 (2)	0.0001
Class II obesity	39 (19.5)	0 (0)	0.003
Waist (cm)	91.20 ± 9.5	78.12 ± 4.6	0.0001
Hip (cm)	91.32 (89.90-93.94)	89.18 (86.18-93.98)	0.3
Waist/hip ratio	0.99 (0.98-1.01)	0.93 (0.89-0.97)	0.016
Central obesity	135 (67.5)	0 (0)	0.0001
Mean AST (IU/L)	60.27 (46.0-78.7)	22.9 (18.75-29.50)	0.0001
Mean ALT (IU/L)	88.04 (68.9-118.9)	24 (17-29.5)	0.0001
Mean fasting sugar (mg/dL)	94.95 (87-105.8)	83.50 (75-89.25)	0.0001
Diabetes mellitus	29 (14.5)	0 (0)	0.005
Mean HDL (mg/dL)	43 (38-48.9)	52 (47.5-56)	0.0001
Mean Triglyceride (mg/dL)	156 (126-200.2)	102 (93-127.5)	0.0001
Hypertension	48 (24)	0 (0)	0.0001

NAFLD: Non-alcoholic fatty liver disease; HVs: Healthy volunteers; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; HDL: High-density lipoprotein.

Table 4 Distribution of CD14 -550C/T genotype, allele frequency and genetic models for CD14-550C/T n (%)

CD14 C (-550) T	CC	CT	TT
genotypes
Cases (n = 200)	120 (60)	65 (32.5)	15 (7.5)
Control (n = 50)	34 (68)	14 (28)	2 (4)
OR	1	1.3 (0.6-2.6)	2.1 (0.4-9.7)
		P = 0.43	P = 0.322
Genetic models
Multiplicative model	Allele C	Allele T
Cases	305 (76.25)	95 (23.75)	P = 0.21
Control	82 (82)	18 (18)	OR = 1.41
			CI: 0.81-2.48
Dominant model	CC	CT + TT
Cases	120 (60)	80 (40)	P = 0.29
Control	34 (68)	16 (32)	OR = 0.7
			CI: 0.3-1.3
Recessive model	TT	CC + CT
			P = 0.37
			OR = 1.9
			CI: 0.4-8.8

Table 5 Distribution of CD14 -159C/T genotype, allele frequency and genetic models for CD14-159C/T n (%)

CD14 C (-159) T	CC	CT	TT
Genotypes
Cases (n = 200)	36 (18)	70 (35)	94 (47)
Control (n = 50)	10 (20)	30 (60)	10 (20)
OR	1	0.6 (0.2-1.4)	2.6 (1-6.7)
		P = 0.29	P = 0.043
Genetic models
Multiplicative model	Allele C	Allele T
Cases	142 (35.50)	258 (64.50)	P = 0.007
Control	50 (50)	50 (50)	OR = 0.8
			CI: 1.1-2.8
Dominant model	CC	CT + TT
Cases	36 (18)	164 (82)	P = 0.74
Control	10 (20)	16 (32)	OR = 0.7
			CI: 0.4-1.9
Recessive model	TT	CC + CT
Cases	94 (47)	106 (53)	P = 0.0005
Control	10 (20)	40 (80)	OR = 3.5
			CI: 1.6-7.4 -8.8

Table 6 Haplotypes for CD14 C (-159) T and C (-550) T polymorphism

Haplotypes	NAFLD (frequency)	HVs (frequency)	P value	OR (95%CI)
			(Fisher’s exact test)
CC	23.4%	50%	1.58E-007	0.3 (0.19-0.48)
TC	52.3%	32%	0.0002	2.3 (1.4-3.7)
TT	12.2%	18%	0.12	0.6 (0.3-1.1)
CT	12.1%	0%	0.0002	Undefined

HVs: Healthy volunteers; NAFLD: Non-alcoholic fatty liver disease.

Table 7 Comparison of non-alcoholic fatty liver disease patients with and without CD14 C (-159) T polymorphism

CD14 C (-159) T polymorphism (NAFLD = 200)
Parameters	Without polymorphism (n = 106)	With polymorphism (n = 94)	P value
TNF-α (pg/mL) (n = 200)	56 (34-80)	62 (40-112)	0.04
Adiponectin (pg/mL)	745 (649-893)	745 (634-928)	0.93
(n = 200)
IL-1β (pg/mL)	43 (32-47)	43 (25-47)	0.53
(n = 200)
HOMA-IR (n = 200)	1.9 (1.3-2.7)	1.8 (1.4-3.8)	0.34
MS (n = 200)
≥ 3 components	43 (40.5%)	35 (37.2%)	0.63
ALT (IU/L)	83 (68-108)	95 (-130)	0.016
NAS score (n = 60)	8	6	0.94
No NASH	11	10
Borderline NASH	13	12
NASH
Severity of steatosis (n = 60)
1	9	6
2	14	15	0.733
3	9	7
Severity of fibrosis (n = 60)
0	13	11	0.90
1	16	13
2	1	2
3	2	2

TNF-α: Tumor necrosis factor-α; NASH: Non-alcoholic steatohepatitis; ALT: Alanine aminotransferases; NAFLD: Non-alcoholic fatty liver disease.