VKORC1 variants as significant predictors of warfarin dose in Emiratis

Figures & data

Table 1 Primers and PCR conditions used to amplify and sequence the vast majority of genomic sequence including the reported SNPs on VKORC1

Forward primer	Reverse primer	Annealing temp (°C)	Annealing time (min)	Product size (bp)
F1-GCCAGCAGGAGAGGGAAATA	R1-AGTTTGGACTACAGGTGCCT	58	60	290
F2-GCAGGCACCTGTAGTCCAA	R2-CGCTTACCCTATGCCAAGTC	58	60	842
F3-TGATCCGCTGGTCTCTAGGT	R3-GAGAGCACTAAGCCCGTCAG	58	60	786
F5-GTCTGGCTGTGCGTGATG	R5-GCCATAGCGCCCGATTAATT	58	45	313
F6-AGGCGTTAGCATAATGACGG	R6-GGGTGGAACCAGGTTAGGAC	60	60	725
F7-GTCCTAACCTGGTTCCACCC	R7-CCCTCCAAGGGACTGGTCT	60	60	995
F9-GAATACGTGCGTAAGCCACC	R9-GGTTCAGACTTGGCTGATTG	60	45	659

Table 2 Descriptive statistics for 90 Emirati individuals treated with warfarin recruited for VKORC1 genotyping

Variable	Value
Gender, n (%) Female Male	45 (50) 45 (50)
Age in years, range Mean age (SD)	21–98 60.54 (17.8)
Indication for warfarin therapy, n Atrial fibrillation Prosthetic heart valve Deep vein thrombosis Cardiomyopathy Hypercoagulable state Stroke Pulmonary embolism Left ventricular thrombosis Valve disease	38 26 16 3 2 2 1 1 1

Figure 1 Linkage disequilibrium analysis: a diagram of VKORC1 with the location of selected SNPs (Transcript: VKORC1-205 ENST00000394975.2). Black and blue boxes represent exons (E) and untranslated regions (UTR), respectively. Dark red blocks represent stronger LD than the lighter ones. Values represent pairwise r².

Table 3 Frequencies of minor alleles at the studied SNPs in comparison to allele frequencies in different populations from the 1000 Genomes Project^a

Variant	Minor allele	MAF in current study	MAF in ALL	P-value^b	AFR	AMR	EAS	EUR	SAS
rs9923231	A	0.52	0.356	<0.001	0.054	0.411	0.885	0.388	0.145
rs61742245	T	0.033	˂0.01	<0.001	0.001	–	–	0.001
rs188009042	G	0.01	0.003	^a	–		0.014	–	–
rs17708472	T	0.083	0.094	0.79	0.033	0.140	0.003	0.234	0.092
rs9934438	T	0.52	0.356	<0.001	0.054	0.412	0.885	0.388	0.145
rs8050894	C	0.52	0.416	0.008	0.256	0.442	0.885	0.400	0.148
rs2359612	T	0.53	0.610	0.0003	0.821	0.575	0.115	0.612	0.855
rs7294	A	0.328	0.420	0.013	0.454	0.402	0.112	0.366	0.759

Notes: ^aFrequencies in different populations are adapted from www.ensembl.org; ^bP-values calculated between the current allele frequencies and allele frequency of global populations (1000 Genomes Project) using Fisher’s exact test.

Abbreviations: MAF, minor allele frequency; ALL, all individuals from phase 3 of 1000 Genomes Project; AFR, African; AMR, American; EAS, East Asian; EUR, European; SAS, South Asian.

Table 4 Frequencies of genotypes at the studied SNPs and results of ANOVA

VKORC1 SNP	Genotypes frequencies, n (%)	Average dose±SD	ANOVA
			F (2,87)	P-value
rs9923231			32.955	0.00*
GG	22 (24.4%)	7.05±2.89
GA	43 (47.8%)	4.73±1.63
AA	25 (27.8%)	2.56±1.00
rs61742245
GG	86 (95.6%)	4.53±2.22	5.3.5	0.007*
GT	2 (2.2%)	7.50±2.12
TT	2 (2.2%)	9.25±8.13
rs188009042
AA	89 (98.9%)	4.66±2.47	1.80	0.18
AG	1 (1.0%)	8.00
GG	0	–
rs17708472
CC	76 (84.4%)	4.56±2.44	1.37	0.258
CT	13 (14.4%)	5.27±2.60
TT	1 (1.1%)	8.00
rs9934438			32.955	0.00*
CC	22 (24.4%)	7.05±2.89
CT	43 (47.8%)	4.73±1.63
TT	25 (27.8%)	2.56±1.00
rs8050894			29.541	0.00*
GG	21 (26.7%)	7.03±2.94
GC	45 (50.0%)	4.73±1.71
CC	24 (23.3%)	2.58±1.01
rs2359612			28.37	0.00*
CC	19 (21.1%)	7.09±3.04
CT	47 (52.2%)	4.81±1.76
TT	24 (26.7%)	2.58±1.01
rs7294			9.23	0.00*
GG	39 (43.3%)	3.60±2.48
GA	43 (47.8%)	5.32±1.99
AA	8 (8.9%)	6.71±2.63

Note: *Statistically significant.

Table 5 Analysis of haplotypes and their association with dose

Haplotype	rs9923231	rs61742245	rs9934438	rs8050894	rs2359612	rs7294	F (%)	Difference in mean (CI)*	P
H1	A	G	T	C	T	G	50	0.00	–
H2	G	G	C	G	C	A	32	2.29 (1.6–2.95)	<0.0001**
H3	G	G	C	G	C	G	11	1.87 (0.93–2.81)	0.00015**
H4	G	T	C	G	C	G	2	3.18 (1.9–4.46)	<0.0001**
H5	G	G	C	G	T	G	1.2	1.02 (−1.61–3.65)	0.45
Rare	*	*	*	*	*	*	2.8	2.36 (0.62–4.1)	0.0089**

Notes: *The most frequent haplotype is considered the reference and the difference in means measures the difference between the mean of dose among each haplotype and the reference. **Significant association.

Table 6 Model summary and predictors of logistic regression

Model	R	R square	Adjusted R square	Std error of the estimate
1	0.656^a	0.431	0.424	1.88
2	0.702^b	0.493	0.482	1.79
3	0.724^c	0.524	0.507	1.74

Notes: ^aPredictors (constant), rs9923231. ^bPredictors (constant), rs9923231, Age. ^cPredictors (constant), rs9923231. Age, rs61742245.

Abbreviation: Std, standard.

Table S1 Results of cross-tabulation

	Low dose	Intermediate dose	High dose
GG	2	4	16
GA	6	30	7
AA	20	5	0
Total	28	39	23
GG	2	4	15
GC	7	30	8
CC	19	5	0
Total	28	39	23
CC	2	4	16
CT	6	30	7
TT	20	5	0
Total	28	39	23
CC	2	3	14
CT	7	31	9
TT	19	5	0
Total	28	39	23
GG	22	11	6
GA	4	28	11
AA	2	0	6
Total	28	39	23
AA	28	39	22
AG	0	0	1
GG	0	0	0
Total	28	39	23
GG	28	38	20
GT	0	0	2
TT	0	1	1
Total	28	39	23
CC	26	33	17
CT	2	6	5
TT	0	0	1
Total	28	39	23