Common genetic variations in CLOCK transcription factor are associated with nonalcoholic fatty liver disease

Table 1 Clinical and biochemical characteristics of the study individuals

Variables	Control group	NAFLD patients	EmpiricP value
Number of subjects	64	136
Age, yr	46.14 ± 1.64	55.58 ± 1.06	0.001
Smoking habit, cigarettes/d	1.58 ± 0.52	1.30 ± 0.75	NS
Physical activity, h/wk	1.4 ± 0.30	1.4 ± 0.32	NS
Drinking habits, g alcohol/d	1.72 ± 0.64	0.75 ± 0.42	NS
BMI, kg/m2	25.61 ± 0.64	36.02 ± 3.11	0.001
Waist circumference, cm	84.13 ± 2.14	103.07 ± 1.42	0.001
Waist-hip ratio	0.84 ± 0.01	0.91 ± 0.007	0.001
SABP, mmHg	121.64 ± 1.87	124.05 ± 2.11	NS
DABP, mmHg	75.86 ± 1.32	78.38 ± 1.49	NS
Fasting plasma glucose, mmol/L	4.73 ± 0.07	5.74 ± 0.22	0.050
Fasting plasma insulin, pmol/L	46.1 ± 3.5	94.5 ± 6.9	0.001
HOMA Index	1.42 ± 0.12	3.44 ± 0.30	0.001
Total cholesterol, mmol/L	5.59 ± 0.17	5.44 ± 0.15	NS
HDL cholesterol, mmol/L	1.27 ± 0.09	1.20 ± 0.05	NS
LDL-cholesterol, mmol/L	3.22 ± 0.29	3.11 ± 0.14	NS
Uric acid, μmol/L	220 ± 51	244 ± 27	NS
Triglycerides, mmol/L	1.74 ± 0.13	2.02 ± 013	NS
ALT, UI/L	24.37 ± 5.2	49.25 ± 5.32	0.080
AST, UI/L	26.4 ± 2.0	36.72 ± 2.27	0.080
γGT, UI/L	36.50 ± 6.8	53.69 ± 5.32	NS
AF, UI/L	221.87 ± 13.0	228.90 ± 11.2	NS

NAFLD: Non-alcoholic fatty liver disease; BMI: Body mass index; SABP and DABP: Systolic and diastolic arterial blood pressure, respectively; HOMA: Homeostatic model assessment; ALT and AST: Serum alanine and aspartate aminotransferase, respectively; γGT: Gamma-glutamyl-transferase; AF: Alkaline phosphatase. Results are expressed as mean ± SE. Nominal P value stands for statistical significance using Student’s t test after log variable transformation. Empiric P value stands for statistical significance adjusted for multiple testing by Bonferoni correction. NS: Non significant.

Table 2 Tag single nucleotide polymorphisms of the CLOCK gene genotyped in the study

NCBI SNPreference1	Location in theCLOCK gene	Hetero-zygosity	dsSNPallele	Minorallele	MAF
rs1554483	Intron 12	0.494	C/G	G	0.491
rs11932595	Intron 10	0.478	A/G	G	0.288
rs4580704	Intron 8	0.395	C/G	G	0.311
rs6843722	Intron 7	0.483	A/C	C	0.476
rs6850524	Intron 1	0.490	C/G	C	0.3557
rs4864548	Upstream	0.490	G/A	A	0.478

MAF: Minor allele frequency (within the study); CLOCK: Circadian locomoter output cycles protein kaput.

¹Single nucleotide polymorphisms on NCBI Reference Assembly.

Table 3 Association of single-tagSNP and multi-marker tag SNP tests (haplotypes) in CLOCK with NAFLD

Single tagSNP	Associated	Frequency	OR (95% CI)	Empiric
	allele			P value
rs1554483	G	0.491	1.51 (0.93-2.45)	0.0599
rs11932595	A	0.712	1.65 (1.10-2.47)	0.0449
rs4580704	C	0.688	1.03 (0.68-1.55)	0.99
rs6843722	C	0.476	1.70 (1.05-2.76)	0.0239
rs6850524	G	0.643	1.40 (0.85-2.29)	0.1458
rs4864548	A	0.478	1.54 (0.95-2.50)	0.0779
Multi-marker tagSNP test	Associated haplotype	Frequency	OR (95% CI)	Empiric P value
rs11932595, rs6850524	AC	0.315	0.88 (1.55-1.41)	0.91
	GC	0.039	0.32 (0.10-0.94)	0.099
rs4580704, rs4864548	CA	0.477	1.55 (0.99-2.44)	0.1066
	GG	0.307	0.90 (0.56-1.46)	0.8764
	CG	0.212	0.62 (0.37-1.07)	0.119
rs4580704, rs6843722	CA	0.213	0.58 (0.34-0.98)	0.0792
rs4580704, rs6850524	GC	0.300	0.90 (0.56-1.47)	0.939
rs6850524, rs4864548	GG	0.171	0.81 (0.46-1.45)	0.806

Empiric P values were obtained by adjusting nominal P values for multiple testing by permutation test.

Table 4 Estimated haplotype frequency distributions of CLOCK gene SNPs in the control group and in NAFLD patients

Haplotype	Control group	NAFLD patients	OR (95% CI)	Empiric P
	(n = 128)	(n = 272)		level
GACCGA	0.275	0.433	1.82 (1.22-2.70)	0.00562
CAGACG	0.363	0.309	0.89 (0.54-1.49)	0.316
CGCAGG	0.160	0.147	0.84 (0.46-1.53)	0.737
GGCCGA	0.118	0.088	0.73 (0.35-1.54)	0.371
CGCACG	0.084	0.024	0.27 (0.09-0.83)	0.00902

NAFLD: Non-alcoholic fatty liver disease. Haplotypes are composed by variants of rs1554483, rs11932595, rs4580704, rs6843722, rs6850524 and 4864548 in that order. n: Number of chromosomes examined. P value for overall comparison by χ² test.