Missense Variant rs28362680 in BTNL2 Reduces Risk of Coronary Heart Disease

Figures & data

Table 1 Characteristics of Patients with CHD and Healthy Individuals

Characteristics		Cases	Control	p
		n = 710	n = 709
Age (years)	Mean ± SD	62.58 ± 10.49	62.23 ± 7.44
	≤ 62	362 (51.0%)	366 (51.6%)	0.940^a
	> 62	348 (49.0%)	343 (48.4%)
Gender	Male	481 (67.7%)	479 (67.6%)	0.940^a
	Female	229 (32.3%)	230 (32.4%)
Smoking status	Yes	349 (49.2%)	352 (49.6%)	0.853^a
	No	361 (50.8%)	357 (50.4%)
Drinking status	Yes	344 (48.5%)	341 (48.1%)	0.894^a
	No	366 (51.5%)	368 (51.9%)
Complicated with hypertension	Yes	446 (62.8%)	–
	No	264 (37.2%)	–
Complicated with diabetes	Yes	198 (27.9%)	–
	No	512 (72.1%)	–
Fasting blood glucose		5.97 ± 2.6	6.08 ± 1.63	< 0.0001^b
Triglyceride		1.63 ± 0.99	1.77 ± 1.43	0.106^b
Total cholesterol		4.06 ± 1.07	4.76 ± 0.97	0.011^b
High density lipoprotein		1.11 ± 0.26	1.14 ± 0.23	0.024^b
Low density lipoprotein		2.35 ± 0.87	2.61 ± 0.75	0.001^b

Notes: ^arepresents the p value calculated by the chi-square test. ^b represents the p value calculated by the t-test.

Abbreviation: CHD, coronary heart disease.

Table 2 The Basic Information and HWE About the Selected SNPs of BTNL2

SNP ID	Function	Amino Acid Change	Chr: Position	Alleles (A/B)	MAF		HWE (P value)	Haploreg 4.1
					Cases	Controls
rs35624343	Missense Variant	P (Pro) > L (Leu)	6: 32393985	A/G	0.138	0.164	0.102	Promoter histone marks; Enhancer histone marks; DNAse; Proteins bound; Motifs changed; Selected eQTL hits
rs117896888	Missense Variant	V (Val) > L (Leu)	6: 32394022	G/C	0.061	0.082	0.801	Promoter histone marks; Enhancer histone marks; DNAse; Proteins bound; Motifs changed
rs41441651	Missense Variant	D (Asp) > H (His)	6: 32396111	T/C	0.061	0.083	0.807	SiPhy cons; Motifs changed
rs41417449	Missense Variant	M (Met) > V (Val)	6: 32396234	C/T	0.061	0.082	0.804	–
rs28362680	Missense Variant	A (Ala) > V (Val)	6: 32403039	A/G	0.208	0.279	0.091	Enhancer histone marks; DNAse; Proteins bound; Motifs changed
rs2076523	Missense Variant	K (Lys) > E (Glu)	6: 32403058	C/T	0.377	0.408	0.088	SiPhy cons; Enhancer histone marks; DNAse; Proteins bound; Motifs changed

Notes: P > 0.05 indicates that the genotypes were in Hardy–Weinberg Equilibrium; “-” indicates data missing.

Abbreviations: Pro, proline; Leu, leucine; Val, valine; Asp, aspartic acid; His, histidine; Met, methionine; Ala, alanine; Lys, lysine; Glu, glutamic acid; A, minor allele; B, wild-type allele; HWE, Hardy–Weinberg equilibrium; SNP, single nucleotide polymorphisms; MAF, minor allele frequency.

Table 3 The Association Analysis Between Susceptibility of CHD and Single Nucleotide Polymorphism of BTNL2

SNP ID	Model	Genotype	Control	Case	OR (95% CI)	p-value
rs35624343	Allele	G	1185 (83.57%)	1224 (86.2%)	1
		A	233 (16.43%)	196 (13.8%)	0.81 (0.66–1.00)	0.051
	Genotype	G/G	489 (69%)	525 (73.9%)	1
		A/A	207 (29.2%)	174 (24.5%)	0.78 (0.35–1.76)	0.548
		G/A	13 (1.8%)	11 (1.6%)	0.78 (0.62–0.99)	0.040
	Dominant	G/G	489 (69%)	525 (73.9%)	1	0.036
		G/A-A/A	220 (31%)	185 (26.1%)	0.78 (0.62–0.98)
	Recessive	G/G-G/A	696 (98.2%)	699 (98.5%)	1	0.670
		A/A	13 (1.8%)	11 (1.6%)	0.84 (0.37–1.89)
	Overdominant	G/G-A/A	502 (70.8%)	536 (75.5%)	1	0.045
		G/A	207 (29.2%)	174 (24.5%)	0.79 (0.62–0.99)
	Log-additive	---	---	---	0.80 (0.65–0.99)	0.043
rs117896888	Allele	C	1295 (91.84%)	1333 (93.87%)	1
		G	115 (8.16%)	87 (6.13%)	0.74 (0.55–0.98)	0.036
	Genotype	C/C	595 (84.4%)	625 (88%)	1
		G/G	105 (14.9%)	83 (11.7%)	0.38 (0.07–1.97)	0.250
		C/G	5 (0.7%)	2 (0.3%)	0.75 (0.55–1.02)	0.069
	Dominant	C/C	595 (84.4%)	625 (88%)	1	0.046
		C/G-G/G	110 (15.6%)	85 (12%)	0.73 (0.54–1.00)
	Recessive	C/C-C/G	700 (99.3%)	708 (99.7%)	1	0.250
		G/G	5 (0.7%)	2 (0.3%)	0.40 (0.08–2.05)
	Overdominant	C/C-G/G	600 (85.1%)	627 (88.3%)	1	0.074
		C/G	105 (14.9%)	83 (11.7%)	0.75 (0.55–1.03)
	Log-additive	---	---	---	0.73 (0.55–0.98)	0.035
rs41441651	Allele	C	1301 (91.75%)	1333 (93.87%)	1
		T	117 (8.25%)	87 (6.13%)	0.73 (0.54–0.97)	0.028
	Genotype	C/C	597 (84.2%)	625 (88%)	1
		T/T	107 (15.1%)	83 (11.7%)	0.38 (0.07–1.98)	0.252
		C/T	5 (0.7%)	2 (0.3%)	0.74 (0.54–1.01)	0.054
	Dominant	C/C	597 (84.2%)	625 (88%)	1	0.036
		C/T-T/T	112 (15.8%)	85 (12%)	0.72 (0.53–0.98)
	Recessive	C/C-C/T	704 (99.3%)	708 (99.7%)	1	0.250
		T/T	5 (0.7%)	2 (0.3%)	0.40 (0.08–2.06)
	Overdominant	C/C-T/T	602 (84.9%)	627 (88.3%)	1	0.058
		C/T	107 (15.1%)	83 (11.7%)	0.74 (0.55–1.01)
	Log-additive	---	---	---	0.72 (0.54–0.97)	0.027
rs41417449	Allele	T	1296 (91.78%)	1333 (93.87%)	1
		C	116 (8.22%)	87 (6.13%)	0.73 (0.55–0.97)	0.031
	Genotype	T/T	595 (84.3%)	625 (88%)	1
		C/C	106 (15%)	83 (11.7%)	0.38 (0.07–1.97)	0.250
		C/T	5 (0.7%)	2 (0.3%)	0.74 (0.55–1.01)	0.060
	Dominant	T/T	595 (84.3%)	625 (88%)	1	0.040
		C/T-C/C	111 (15.7%)	85 (12%)	0.73 (0.54–0.99)
	Recessive	T/T-C/T	701 (99.3%)	708 (99.7%)	1	0.250
		C/C	5 (0.7%)	2 (0.3%)	0.40 (0.08–2.05)
	Overdominant	T/T-C/C	600 (85%)	627 (88.3%)	1	0.064
		C/T	106 (15%)	83 (11.7%)	0.75 (0.55–1.02)
	Log-additive	---	---	---	0.73 (0.54–0.97)	0.030
rs28362680	Allele	G	1011 (72.11%)	1125 (79.23%)	1
		A	391 (27.89%)	295 (20.77%)	0.68 (0.57–0.81)	<0.0001
	Genotype	G/G	355 (50.6%)	437 (61.5%)	1
		A/A	301 (42.9%)	251 (35.4%)	0.40 (0.23–0.67)	0.001
		G/A	45 (6.4%)	22 (3.1%)	0.68 (0.54–0.84)	<0.0001
	Dominant	G/G	355 (50.6%)	437 (61.5%)	1	<0.0001
		G/A-A/A	346 (49.4%)	273 (38.5%)	0.64 (0.52–0.79)
	Recessive	G/G-G/A	656 (93.6%)	688 (96.9%)	1	0.003
		A/A	45 (6.4%)	22 (3.1%)	0.47 (0.28–0.78)
	Overdominant	G/G-A/A	400 (57.1%)	459 (64.7%)	1	0.004
		G/A	301 (42.9%)	251 (35.4%)	0.73 (0.59–0.90)
	Log-additive	---	---	---	0.66 (0.55–0.79)	<0.0001
rs2076523	Allele	T	839 (59.17%)	881 (62.31%)	1
		C	579 (40.83%)	233 (37.69%)	0.88 (0.75–1.02)	0.087
	Genotype	T/T	237 (33.4%)	269 (38%)	1
		C/C	365 (51.5%)	343 (48.5%)	0.78 (0.56–1.08)	0.135
		C/T	107 (15.1%)	95 (13.4%)	0.83 (0.66–1.05)	0.113
	Dominant	T/T	237 (33.4%)	269 (38%)	1	0.072
		C/T-C/C	472 (66.6%)	438 (62%)	0.82 (0.66–1.02)
	Recessive	T/T-C/T	602 (84.9%)	612 (86.6%)	1	0.370
		C/C	107 (15.1%)	95 (13.4%)	0.87 (0.65–1.18)
	Overdominant	T/T-C/C	344 (48.5%)	364 (51.5%)	1	0.270
		C/T	365 (51.5%)	343 (48.5%)	0.89 (0.72–1.10)
	Log-additive	---	---	---	0.87 (0.75–1.02)	0.081

Notes: “p-value < 0.05” and bold text represent statistical significance. “-” indicates Log-additive model.

Abbreviations: CHD, coronary heart disease; SNP, single nucleotide polymorphisms; OR, odds ratio; CI, confidence interval.

Table 4 Haplotype Analysis of Candidate BTNL2 Genetic Polymorphisms with CHD Risk

SNP	Haplotype	Freq (Case)	Freq (Control)	Crude Analysis		Adjusted by Gender, Age, Smoking and Drinking
				OR (95% CI)	p	OR (95% CI)	p
rs117896888|rs41441651|rs41417449|rs28362680	CCTG	0.7177	0.7914	1	—	1	—
rs117896888|rs41441651|rs41417449|rs28362680	CCTA	0.1998	0.1473	0.65 (0.53–0.80)	<0.0001	0.65 (0.53–0.80)	<0.0001
rs117896888|rs41441651|rs41417449|rs28362680	GTCA	0.0790	0.0604	0.68 (0.51–0.92)	0.014	0.68 (0.51–0.92)	0.013
rs117896888|rs41441651|rs41417449|rs28362680	****	0.0036	0.0008	0.23 (0.03–2.13)	0.200	0.23 (0.03–2.12)	0.200

Notes: “p-value < 0.05” and bold text represent statistical significance.

Abbreviations: SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval.

Figure 1 Haplotype block map for the BTNL2 SNPs (rs117896888, rs41441651, rs41417449, rs28362680). (A) The numbers inside the diamonds indicate The D’ for pairwise analyses. (B) The numbers inside the diamonds indicate the R² for pairwise analyses. The colors represent the degree of linkage disequilibrium: the redder the color, the stronger the linkage disequilibrium.

Table 5 BTNL2 SNP–SNP Interaction Models Analyzed by the MDR Method

Model	Training Bal. Acc	Testing Bal. Acc	OR (95% CI)	p value	CVC
rs28362680	0.558	0.558	1.60 (1.30–1.98)	< 0.0001	10/10
rs117896888, rs28362680	0.561	0.555	1.64 (1.33–2.02)	< 0.0001	9/10
rs35624343, rs117896888, rs28362680	0.566	0.557	1.72 (1.39–2.13)	< 0.0001	10/10
rs35624343, rs117896888, rs28362680, rs2076523	0.571	0.548	1.81 (1.46–2.24)	< 0.0001	10/10
rs35624343, rs117896888, rs41441651, rs28362680, rs2076523	0.571	0.547	1.81 (1.46–2.24)	< 0.0001	8/10
rs35624343, rs117896888, rs41441651, rs41417449, rs28362680, rs2076523	0.571	0.542	1.81 (1.46–2.24)	< 0.0001	10/10

Notes: p values were calculated using χ² tests; “p-value < 0.05” and bold text represent statistical significance.

Abbreviations: MDR, multifactor dimensionality reduction; Bal. Acc., balanced accuracy; CVC, cross-validation consistency; OR, odds ratio; 95% CI, 95% confidence interval.

Figure 2 Multifactor dimensionality reduction (MDR) analysis of interaction between the candidate SNPs of BTNL2 ((rs35624343, rs117896888, rs41441651, rs41417449, rs28362680 and rs2076523). (A) SNP-SNP Interaction Dendrogram: the color represents the degree of redundancy or synergy between SNP-SNP; the closer the color is to red, the more synergy, and the closer to blue, the more redundancy. (B) Fruchterman-Reingold: values in nodes represent the IGs of individual attribute (main effects). Values between nodes are IGs of each pair of attributes (interaction effects).