The association between PD-1 gene polymorphisms and susceptibility to multiple sclerosis

Figures & data

Figure 1. Amplified fragments of PD1.3, which were digested by PstI. The first lane is 100 bp DNA marker. The PCR product size was 180 bp. If the product was digested, the allele was identified as A; otherwise, it was identified as G.

Table 1. Oligonucleotide primers sequences used in PCR-RFLP.

SNP	Position	Restriction enzyme	Primers
PD-1.1 (rs3608432)	Promotor	MspI	F: 5′-TTCTAGCCTCGCTTCGGTTA-3′
			R: 5′-CTCAACCCCACTCCCATTCT-3′
PD-1.3 (rs11568821)	Intron 4	PstI	F: 5′-CCCCAGGCAGCAACCTCAAT-3′
			R: 5′-GACCGCAGGCAGGCACATAT-3′
PD-1.5 (rs2227981)	Exon5	Alu I	F: 5′-GGACAGCTCAGGTAAGCAG-3′
			R: 5′-AAGAGCAGTGTCCATCCTCAG-3′

Table 2. Thermal cycling conditions for PCR amplification of PD-1.1, PD-1.3 and PD-1.5 polymorphisms.

Polymorphism	Initiation	Denaturation	Annealing	Extension	Final extension
PD-1.1	95 °C (4 min)	95 °C (40 s)	60 °C (50 s)	95 °C (40 s)	72 °C (10 min)
		32 cycles
PD-1.3	95 °C (15 s)	95 °C (40 s)	60 °C (50 s)	72 °C (40 s)	72 °C (10 min)
		32 cycles
PD-1.5	94 °C (10 min)	95 °C (45 s)	64 °C (40 s)	72 °C (40 s)	72 °C (10 min)
		32 cycle

Table 3. Comparison of genotypes and alleles from PD-1.1 polymorphism in patient and control groups.

	Model	Control group	Patient group	OR	95%CI	p-Value
		N = 246	N = 229
Genotype (total population)	AA	0 (0%)	0 (0%)	1.15	0.87–1.45	.21
	AG	13 (5. 3%)	8 (3.5%)	1.17	0.86–1.43	.27
	GG	233 (94.7%)	221 (96.5%)	1.18	0.89–1.44	.21
Allele	A	13 (2.6%)	8 (1.7%)	1.17	0.88–1.49	.35
	G	479 (97.4%)	450 (98.3%)	–	–	–
Allele carriers	AA	0 (0%)	0 (0%)	1.15	0.86–1.46	.07
	AG + GG	246 (100%)	229 (100%)		–
	GG	233 (94.7%)	22 (96.5%)	1.15	0.87–1.16	.09
	AA + AG	13 (5.3%)	8 (3.5%)	–	–	–
		N = 186	N = 175
Genotype (Female)	AA	0 (0%)	0 (0%)	1.15	0.98–1.47	.301
	AG	13 (6.9%)	7 (4%)	1.15	0.87–1.41	.32
	GG	173 (92.1%)	168 (96%)	1.17	0.91–1.49	.77
Allele	A	13 (3.5%)	(2%) 7	1.15	0.88–1.07	.21
	G	259 (96.5%)	343 (98%)	–	–	–
Allele carriers	AA	0 (0%)	0 (0%)	1.16	0.87–1.42	.45
	AG + GG	189 (100%)	175 (100%)	–	–	–
	GG	173 (93.1%)	168 (96%)	0.98	0.98–1.39	.07
	AA + AG	13 (6.9%)	7 (4%)	–	–	–
		N = 60	N = 54
Genotype (Male)	AA	0 (0%)	0 (0%)	1.01	0.98–1.47	.71
	AG	0 (0%)	1 (1.9%)	1.00	0.98–1.06	.052
	GG	60 (100%)	53 (98.1%)	0.98	0.96–1.37	.063
Allele	A	0 (0%)	1 (1%)	0.99	0.95–1.37	.11
	G	36 (30%)	107 (99%)	–	–	–
Allele carriers	AA	0 (0%)	(0%) 0	0.98	0.95–1.09	.27
	AG + GG	60 (100%)	54 (100%)	–	–	–
	GG	60 (100%)	53 (98.1%)	1.02	0.95–1.42	.93
	AA + AG	0 (0%)	1 (1.9%)	–	–	–

OR: odds ratio; CI: confidence interval.

Table 4. Comparison of genotypes and alleles from PD-1.3 polymorphism in patient and control groups.

	Model	Control group	Patient group	OR	95%CI	p-Value
		N = 246	N = 229
Genotype (total population)	AA	35 (14.2%)	2 (0.9%)	0.71	0.38–0.86	.0001
	AG	14 (5.7%)	30 (13.1%)	0.79	0.36–0.83	.0001
	GG	197 (80.1%)	197 (86%)	0.57	0.44–0.75	.0001
Allele	A	84 (17.1%)	34 (7.4%)	0.65	0.50–0.85	.0001
	G	408 (82.9%)	424 (92.6%)	–	–	–
Allele carriers	AA	35 (14.2%)	2 (0.9%)	0.58	0.38–0.89	.0001
	AG + GG	211 (85.8%)	227 (99.1%)		–	–
	GG	197 (18.11%)	197 (86%)	0.57	0.39–1.05	.08
	AA + AG	49 (19.9%)	32 (14%)	–	–	–
		N = 186	N = 175
Genotype (Female)	AA	34 (18.30%)	2 (1.1%)	0.76	0.42–0.90	.0001
	AG	6 (3.2%)	21 (12%)	0.60	0.45–0.94	.0001
	GG	146 (78.5%)	152 (86.9%)	0.73	0.46–0.80	.0001
Allele	A	74 (19.9%)	25 (7.1%)	0.90	0.59–0.98	.0001
	G	298 (80.1%)	325 (92.9%)	–	–
Allele carriers	AA	34 (18.30%)	2 (1.1%)	0.52	0.43–0.76	.001
	AG + GG	152 (81.7%)	173 (98.9%)	–	–
	GG	146 (78.5%)	152 (86.9%)	0.66	0.33–0.72	.03
	AA + AG	40 (21.5%)	23 (13.1%)	–	–
		N = 60	N = 54
Genotype (Male)	AA	11 (1.7%)	0 (0%)	0.78	0.65–1.87	.94
	AG	8 (13.3%)	9 (16.6%)	0.57	0.54–1.79	.84
	GG	51 (85 %)	45 (83.4%)	1.47	0.95–2.77	.81
Allele	A	10 (8.3%)	9 (8.3%)	0.26	0.10–1.03	.80
	G	115 (91.7%)	99 (91.7%)	–	–
Allele carriers	AA	1 (1.7%)	0 (0%)	0.64	0.43–1.76	.82
	AG + GG	59 (98.3%)	54 (100%)	–	–
	GG	51 (85%)	45 (83.3%)	0.18	0.11–1.48	.84
	AA + AG	9 (15%)	9 (16.7%)	–	–	–

OR: odds ratio; CI: confidence interval.

Table 5. Comparison of genotypes and alleles from PD-1.5 polymorphism in patient and control groups.

	Model	Control group	Patient group	OR	95%CI	p-Value
		N = 246	N = 229
Genotype (total population)	CC	123 (50%)	96 (41.9%)	0.80	0.43–1.04	.07
	CT	102 (41.4%)	107 (46.7%)	0.95	0.63–1.39	.28
	TT	21 (8.6%)	26 (11.4%)	0.84	0.66–1.94	.30
Allele	C	348 (70.7%)	299 (65.5%)	0.92	0.43–1.07	.08
	T	144 (29.3%)	159 (34.7%)	–	–
Allele carriers	CC	123 (50%)	96 (41.9%)	0.92	0.53–1.23	.07
	TT + CT	123 (50%)	133 (58.1%)		–
	TT	21 (18.5%)	26 (11.4%)	0.82	0.38–1.74	.30
	CC + CT	225 (91.5%)	203 (88.6%)	–	–
		N = 186	N = 175
Genotype (Female)	CC	93 (50%)	74 (42.3%)	1.0	0.63–1.47	.14
	CT	78 (42%)	83 (47.4%)	0.98	0.67–1.50	.34
	TT	15 (0.8%)	18 (10.3%)	1.01	0.83–1.95	.46
Allele	C	264 (71%)	231 (66%)	1.00	0.60–1.02	.17
	T	108 (29%)	119 (34%)	–	–
Allele carriers	CC	93 (50%)	74 (42.3%)	0.98	0.80–1.13	.14
	TT + CT	93 (50%)	101 (57.7%)		–
	TT	15 (8.1%)	18 (10.3%)	0.99	0.22–1.38	.46
	CC + CT	171 (91.9%)	157 (89.7%)	–	–
		N = 60	N = 54
Genotype (Male)	CC	30 (50%)	22 (40%)	1.01	0.76–1.43	.32
	CT	24 (40%)	24 (44.4%)	1.01	0.80–1.48	.77
	TT	6 (10%)	8 (14.8%)	1.05	0.63–1.91	.43
Allele	C	84 (70%)	68 (63%)	1.09	0.81–2.09	.32
	T	36 (30%)	40 (37%)	–	–
Allele carriers	CC	30 (50%)	22 (10.3%)	1.12	0.52–1.78	.32
	TT + CT	30 (50%)	32 (57.7%)	–	–
	TT	6 (10%)	8 (10.3%)	1.12	0.39–1.44	.43
	CC + CT	54 (90%)	46 (89.7%)	–	–

OR: odds ratio; CI: confidence interval.

Table 6. Frequency of genotypes, alleles and allele carriers of PD-1.5 polymorphism in subjects with different courses of MS disease.

		RRMS	SPMS	PPMS	p 1*	p 2*
Genotype (total population)	CC	56 (41.5%)	20 (42.4%)	2 (25%)	.67	.37
	CT	64 (41.4%)	23 (46.8%)	4 (50%)	.38	.16
	TT	15 (8.6%)	5 (10.6%)	2 (11.4%)	.81	.52
Allele	C	176 (70.7%)	63 (65.6%)	8 (50%)	.45	.21
	T	94 (29.3%)	33 (34.4%)	8 (50%)
Allele carriers	CC	56 (50%)	20 (41.7%)	2 (25%)	.64	.35
	TT + CT	79 (58.5%)	28 (58.3%)	6 (75%)
	TT	15 (11.1%)	5 (10.4%)	2 (25%)	.47	.22
	CC + CT	120 (88.9%)	43 (89.6%)	6 (75%)

*P1: obtained by comparison of RRMS, SPMS, and PPMS; *P2: obtained by comparison of RRMS + SPMS, and PPMS.