Genome-Wide Association Study of Liver Enzyme Elevation in Rheumatoid Arthritis Patients Starting Methotrexate

Figures & data

Table 1. Characteristics of patients initiating methotrexate treatment included in the discovery and replication cohorts.

Characteristics	Discovery cohort (n = 198)	Replication cohort (n = 160)	p-value
Females, n (%)	132 (67)	122 (77)	0.04
Age at RA diagnosis, mean ± SD, range (years)	55.1 ± 13.8 (18–81)	55.9 ± 14.0 (20–80)	0.60
History of ALT elevation prior to MTX treatment, n (%)	31 (16)^†	36 (29)^‡	0.07
Rheumatoid factor positivity, n (%)	139 (71)^§	97 (62)^¶	0.08
ACPA positivity, n (%)	136 (71)^#	98 (62)^††	0.07
Age at MTX treatment start, mean ± SD, (range), years	55.9 ± 13.8 (18–81)	57.0 ± 14.2 (20–80)	0.46
MTX maximum weekly dose, mean ± SD, min-max (mg)	17.1 ± 4.4 (7.5–25)	19.6 ± 4.0 (7.5–25)	<0.001
Comorbidities
Diabetes, n (%)	14 (7)	14 (9)	0.56
Hepatic disease^‡‡, n (%)	4 (2)	2 (1)	0.57
Other characteristics
BMI, mean ± SD, range (kg/m^2)	26.4 ± 4.5 (17–40.9)^§	25.9 ± 4.1 (18–36.7)	0.22
Ever smoker, n (%)	91 (46)	83 (53)^†	0.18
Alcohol standard glasses/week, mean ± SD, (range)	2.7 ± 2.6 (0–14)	1.9 ± 2.6 (0–17)^†	0.02

† Missing data for four patients.

‡ Missing data for eight patients.

§ Missing data for one patient.

¶ Missing data for three patients.

# Missing data for seven patients.

†† Missing data for two patients.

‡‡ Four patients with NAFLD and two with chronic hepatitis B infection.

ACPA: Anti-citrullinated protein antibody; ALT: Alanine aminotransferase; MTX: Methotrexate; n: Number; NAFLD: Nonalcohol fatty liver disease; RA: Rheumatoid arthritis; SD: Standard deviation; ULN: Upper limit of normal.

Table 2. Top 6 genome-wide results for alanine aminotransferase above 1.5-times the upper limit of normal within 6 months.

CHR	SNP	BP	n	OR	Lower 95% CI	Upper 95% CI	p-value	Major> minor allele	MAF cases	MAF controls	Gene
10	rs72781580	12244217	198	16.68	4.766	58.34	1.063 × 10^-5	C>T	0.222	0.031	CDC123
1	rs3920617	65189791	198	13.00	4.055	41.65	1.589 × 10^-5	A>C	0.250	0.047	near RAVER2
1	rs55889764	65188460	198	13.00	4.055	41.65	1.589 × 10^-5	T>G	0.250	0.047	near RAVER2
1	rs72675408	65214012	198	13.00	4.055	41.65	1.589 × 10^-5	T>A	0.250	0.047	RAVER2
17	rs3110633	36053069	198	7.027	2.887	17.10	1.740 × 10^-5	C>G	0.639	0.242	HNF1B
9	rs144977051	114332975	198	30.12	6.277	144.5	2.084 × 10^-5	C>T	0.167	0.022	ZNF483–PTGR1

Results are adjusted by sex, age and genetic principal components 1 to 4.

BP: Base pair in the genome reference consortium human assembly build GRCh37; CHR: Chromosome; MAF: Minor allele frequency; OR: Odds ratio.

Figure 1. Manhattan plot for maximum alanine aminotransferase within 6 months.

Alanine aminotransferase (ALT) is log₂ transformed and adjusted by log (baseline ALT), age, sex and genetic principal components 1-4. The line denotes the significance level p < 5 × 10^-8.

Table 3. Top 6 genome-wide results for maximum alanine aminotransferase within 6 months.

CHR	SNP	BP	n	Beta	Lower 95% CI	Upper 95% CI	p-value	Major> minor allele	MAF	Gene
1	rs3920617	65189791	194	0.8321	0.5592	1.105	1.160 × 10^-8	A>C	0.064	near RAVER2
1	rs55889764	65188460	194	0.8321	0.5592	1.105	1.160 × 10^-8	T>G	0.064	near RAVER2
1	rs72675408	65214012	194	0.8321	0.5592	1.105	1.160 × 10^-8	T>A	0.064	RAVER2
1	rs17384589	65186954	194	0.7252	0.4800	0.9705	2.887 × 10^-8	C>T	0.080	near RAVER2
1	rs72675414	65227558	194	0.8979	0.5878	1.208	5.293 × 10^-8	C>T	0.054	RAVER2
1	rs72675415	65230942	194	0.7731	0.5023	1.044	7.833 × 10^-8	G>A	0.067	RAVER2

ALT is log2 transformed and adjusted by log (baseline ALT), age, sex and genetic principal components 1-4.

Beta: Beta estimate; BP: Base pair in the genome reference consortium human assembly build GRCh37; CHR: Chromosome; MAF: Minor allele frequency.

Figure 2. Linear regression of rs72675408 in the discovery and replication cohorts.

Meta-analysis was performed using random and fixed effects models in the metafor R-package. SNP effects were modeled as additive. Estimated odds ratios with 95% CI, p-value and numbers (N) included are shown. The cut-off for a statistically significant association was p < 0.05.

Figure 3. The genomic landscape for the SNPs rs72675408, rs72675451 and rs12402976.

The enhancer harboring rs72675451 interacts with JAK1 and RAVER2 promoters in human liver tissue (pink lines) [27]. The insert shows transcription factors binding in ChIP-seq experiments from the Encyclopedia of DNA Elements project [24]. The color intensity is proportional to signal strength (abbreviations at: http://tinyurl.com/watv2v7).

Table 4. Candidate SNP results for maximum alanine aminotransferase within 6 months.

CHR	SNP	BP	n	Beta	Lower 95% CI	Upper 95% CI	p-value	Major > minor allele	Gene
19	rs4808199	19545099	194	0.2058	0.01456	0.3970	0.03627	G>A	GATAD2A
2	rs780094	27741237	194	-0.1203	-0.2680	0.02747	0.11230	C>T	GCKR
2	rs1260326	27730940	194	-0.1171	-0.2656	0.0313	0.12360	C>T	GCKR
16	rs6499186	68660565	194	0.1211	-0.06435	0.3066	0.20220	T>C	intergenic
16	rs698718	68560185	194	0.1193	-0.06386	0.3026	0.20330	G>A	intergenic
1	rs2298191	112048264	194	-0.1025	-0.2649	0.05998	0.2179	T>C	ADORA3
22	rs738409	44324727	194	-0.08791	-0.2650	0.08913	0.33170	C>G	PNPLA3
7	rs343064	35554788	194	0.05902	-0.08308	0.2011	0.41670	C>T	near HERPUD2
1	rs3393	112042149	194	0.04363	-0.1053	0.1926	0.5665	C>T	ADORA3
22	rs738491	44354111	194	0.03134	-0.1189	0.1815	0.68310	C>T	SAMM50
7	rs10272006	21520132	194	-0.03292	-0.1936	0.1278	0.68850	A>G	SP4
22	rs2896019	44333694	194	-0.02776	-0.2160	0.1604	0.77280	T>G	PNPLA3
22	rs2143571	44391686	194	-0.02357	-0.1998	0.1527	0.79360	G>A	SAMM50

ALT is log2 transformed and adjusted by log (baseline ALT), age, sex and genetic principal components 1-4.

Beta: Beta estimate; BP: Base pair in the genome reference consortium human assembly build GRCh37; CHR: Chromosome.

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