World J Oncol

World Journal of Oncology, ISSN 1920-4531 print, 1920-454X online, Open Access

Article copyright, the authors; Journal compilation copyright, World J Oncol and Elmer Press Inc

Journal website http://www.wjon.org

Original Article

Volume 8, Number 5, October 2017, pages 162-170

CTLA-4 Genetic Variants (rs11571317 and rs3087243): Role in Susceptibility and Progression of Breast Cancer

Figure 2. The in-silico analysis showed CTLA-4 gene rs11571317 SNP and possible associated transcription factors.

Figure 3. Comparative allele frequency of CTLA-4 SNP rs11571317 in various study populations. Source: https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=11571317

Figure 4. Comparative allele frequency of CTLA-4 SNP rs3087243 in various study populations. Source: https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=3087243

**Table 1.** Clinical Characteristics of BC Patients
Clinical parameters	Values
Tumor stage
T_1-2	160 (56)
T_3-4	125 (44)
Lymph node status
LN-ve	48 (17)
LN+ve	237 (83)
Metastatic
M0	237 (82)
M1	48 (18)
Duration of disease
< 6 months	150 (52)
7 - 12 months	83 (29)
> 12 months	52 (19)
DOD
< 6 months
T_1-2	100 (66)
T_3-4	50 (44)
LN-ve	34 (22)
LN+ve	116 (78)
M0	130 (87)
M1	20 (13)

Table 1. Clinical Characteristics of BC Patients

Clinical parameters

Values

Tumor stage

T_1-2

160 (56)

T_3-4

125 (44)

Lymph node status

LN-ve

48 (17)

LN+ve

237 (83)

Metastatic

237 (82)

48 (18)

Duration of disease

< 6 months

150 (52)

7 - 12 months

83 (29)

> 12 months

52 (19)

DOD

< 6 months

T_1-2

100 (66)

T_3-4

50 (44)

LN-ve

34 (22)

LN+ve

116 (78)

130 (87)

20 (13)

**Table 2.** Demographical and Clinical Information of Breast Cancer Patients and Healthy Controls
Category	Controls (N, %)	Breast cancer patients (N, %)	t-, z-test (P value)
*t-test: mean age, age at onset of breast cancer (years), and body mass index (kg/m²). z-test: age at marriage(years), age at first pregnancy, parity, consanguinity, familial incidence, diet, lactation, area of living, and occupation.
Mean age*	285 48.45 ± 13.49	285 51.29 ± 10.75	0.005
Range (years)	18 - 90	25 - 86
Age at onset of breast cancer (years)*		48.5 ± 11
≤ 48	-	142 (50) 40.50 ± 5.68	-
> 48	-	143 (50) 57.64 ± 7.09	-
Age at marriage (years)
≤ 18	179 (62)	185 (64)	0.69
19 - 30	105 (37)	93 (33)	0.55
> 30	1 (0.3)	7 (3)	-
Unmarried	0 (0)	0 (0)	-
Age at first pregnancy
≤ 18	138 (48)	107 (37)	0.08
19 - 30	144 (50)	157 (54)	0.48
> 30	3 (2)	9 (3)	0.92
0	0 (0)	12 (4)	-
Parity
Parous	285 (100)	273 (95)	< 0.01
Nulliparous	0 (0)	12 (5)	-
Consanguinity
Yes	0 (0)	29 (10)	-
No	285 (100)	256 (98)	< 0.01
Familial incidence
No		206 (72)	-
BC		15 (5)	-
Other cancer		29 (10)	-
Life style-related diseases		35 (12)	-
Variables
Diet
Vegetarians	35 (12)	40 (14)	0.79
Non-vegetarians	250 (88)	245 (86)	0.50
Body mass index (kg/m²)*	24.5 ± 3.8	24.9 ± 5	0.23
< 25	176 (61) 22.2 ± 1.9	140 (49) 21.3 ± 2	< 0.01
≥ 25	108 (39) 28.27 ± 3.1	145 (51) 27.8 ± 3.2	0.19
Breast feeding
Yes	200 (100)	279 (98)	< 0.01
No	0 (0)	6 (2)	-
Area of living
Rural	95 (33)	66 (23)	0.16
Urban	190 (66)	219 (77)	< 0.01
Occupation
Housewives	107 (37)	165 (58)	< 0.01
Agriculture workers	177 (62)	120 (42)	< 0.01

Table 2. Demographical and Clinical Information of Breast Cancer Patients and Healthy Controls

Category

Controls (N, %)

Breast cancer patients (N, %)

t-, z-test (P value)

*t-test: mean age, age at onset of breast cancer (years), and body mass index (kg/m²). z-test: age at marriage(years), age at first pregnancy, parity, consanguinity, familial incidence, diet, lactation, area of living, and occupation.

Mean age*

285
48.45 ± 13.49

285
51.29 ± 10.75

0.005

Range (years)

18 - 90

25 - 86

Age at onset of breast cancer (years)*

48.5 ± 11

≤ 48

142 (50)
40.50 ± 5.68

> 48

143 (50)
57.64 ± 7.09

Age at marriage (years)

≤ 18

179 (62)

185 (64)

0.69

19 - 30

105 (37)

93 (33)

0.55

> 30

1 (0.3)

7 (3)

Unmarried

0 (0)

Age at first pregnancy

≤ 18

138 (48)

107 (37)

0.08

19 - 30

144 (50)

157 (54)

0.48

> 30

3 (2)

9 (3)

0.92

0 (0)

12 (4)

Parity

Parous

285 (100)

273 (95)

< 0.01

Nulliparous

0 (0)

12 (5)

Consanguinity

Yes

0 (0)

29 (10)

285 (100)

256 (98)

< 0.01

Familial incidence

206 (72)

15 (5)

Other cancer

29 (10)

Life style-related diseases

35 (12)

Variables

Diet

Vegetarians

35 (12)

40 (14)

0.79

Non-vegetarians

250 (88)

245 (86)

0.50

Body mass index (kg/m²)*

24.5 ± 3.8

24.9 ± 5

0.23

< 25

176 (61)
22.2 ± 1.9

140 (49)
21.3 ± 2

< 0.01

≥ 25

108 (39)
28.27 ± 3.1

145 (51)
27.8 ± 3.2

0.19

Breast feeding

Yes

200 (100)

279 (98)

< 0.01

0 (0)

6 (2)

Area of living

Rural

95 (33)

66 (23)

0.16

Urban

190 (66)

219 (77)

< 0.01

Occupation

Housewives

107 (37)

165 (58)

< 0.01

Agriculture workers

177 (62)

120 (42)

< 0.01

**Table 3.** Genotype and Allele Frequencies of CTLA-4 (rs11571317) Gene Polymorphism in South Indian Breast Cancer Patients, Healthy Controls and Multiple Logistic Regression Analysis for BC Clinical Pathological Features
rs11571317	CC, N (%)	CT, N (%)	TT, N (%)	χ² value (P-value)	Minor allele frequency T	Group comparison	OR (95% CI)	P-value
Controls (285) Patients (285)	39 (13.5) 89 (31.1)	246 (86.5) 196 (68.9)	0 (0) 0 (0)	24.19 (< 0.01)	0.43 0.34	CC vs. others CT vs. others TT vs. others	2.86 (1.80 - 4.36) 0.34 (0.22 - 0.53) -	< 0.01 < 0.01 -
HWE: controls: χ² = 164.2 (P < 0.05); patients: χ² = 78.2 (P < 0.05)
Variables	CC vs. CT: OR (CI)	P-value	CT vs. CC: OR (CI)	P-value
AAO	1.05 (0.58 - 1.91)	0.85	1.06 (0.58 - 1.92)	0.83
Tumor status	0.98 (0.50 - 1.91)	0.96	0.98 (0.50 - 1.92)	0.97
LN status	1.86 (0.59 - 5.80)	0.28	1.26 (0.57 - 2.79)	0.55
Metastasis	2.64 (0.92 - 7.60)	0.07	2.64 (0.92 - 7.58)	0.07
DOD < 6 months vs. > 6 months	1.12 (0.61 - 2.07)	0.69	1.15 (0.62 - 2.11)	0.64

Table 3. Genotype and Allele Frequencies of CTLA-4 (rs11571317) Gene Polymorphism in South Indian Breast Cancer Patients, Healthy Controls and Multiple Logistic Regression Analysis for BC Clinical Pathological Features

rs11571317

CC, N (%)

CT, N (%)

TT, N (%)

χ² value (P-value)

Minor allele frequency T

Group comparison

OR (95% CI)

P-value

Controls (285)
Patients (285)

39 (13.5)
89 (31.1)

246 (86.5)
196 (68.9)

0 (0)
0 (0)

24.19 (< 0.01)

0.43
0.34

CC vs. others
CT vs. others
TT vs. others

2.86 (1.80 - 4.36)
0.34 (0.22 - 0.53)
-

< 0.01
< 0.01
-

HWE: controls: χ² = 164.2 (P < 0.05); patients: χ² = 78.2 (P < 0.05)

Variables

CC vs. CT: OR (CI)

P-value

CT vs. CC: OR (CI)

P-value

AAO

1.05 (0.58 - 1.91)

0.85

1.06 (0.58 - 1.92)

0.83

Tumor status

0.98 (0.50 - 1.91)

0.96

0.98 (0.50 - 1.92)

0.97

LN status

1.86 (0.59 - 5.80)

0.28

1.26 (0.57 - 2.79)

0.55

Metastasis

2.64 (0.92 - 7.60)

0.07

2.64 (0.92 - 7.58)

0.07

DOD < 6 months vs. > 6 months

1.12 (0.61 - 2.07)

0.69

1.15 (0.62 - 2.11)

0.64

**Table 4.** Genotype and Allele Frequencies of CTLA-4 rs3087243 Gene Polymorphism in South Indian Breast Cancer Patients, Healthy Controls and Multiple Logistic Regression Analysis for BC Clinical Pathological Features
rs3087243	GG, N (%)	GA, N (%)	AA, N (%)	χ² value (P-value)	Minor allele frequency A	Group comparison	OR (95% CI)	P-value
Controls Patients	74 (26) 71 (25)	202 (71) 197 (70)	9 (3) 17 (5)	1.95 (= 0.376)	0.39 0.41	GG vs. others GA vs. others AA vs. others	0.94 (0.64 - 1.37) 0.91 (0.64 - 1.31) 1.94 (0.85 - 4.4)	0.84 0.71 0.15
HWE: controls: χ² = 69.9 (P < 0.05); patients: χ² = 53.6 (P < 0.05)
Variables	GG vs. GA + AA: OR (CI)	P-value	GA vs. GG + AA: OR (CI)	P-value	AA vs. GG + GA: OR (CI)	P-value
AAO	1.44 (0.70 - 2.94)	0.31	0.57 (0.30 - 1.08)	0.08	1.86 (0.67 - 5.16)	0.23
Tumor status	0.01 (0.0043 - 0.09)	< 0.01*	6.28 (3.01 - 13.07)	< 0.01*	3.46 (1.02 - 11.67)	0.04*
LN status	1.38 (0.61 - 3.11)	0.43	0.68 (0.31 - 1.52)	0.35	0.91 (0.17 - 4.90)	0.91
Metastasis	0.97 (0.17 - 5.53)	0.97	3.60 (0.94 - 13.78)	0.06	-	0.99
DOD < 6 months vs. > 6 months	(0.91 - 4.02)	0.08	0.62 (0.32 - 1.19)	0.15	0.84 (0.29 - 2.46)	0.76

Table 4. Genotype and Allele Frequencies of CTLA-4 rs3087243 Gene Polymorphism in South Indian Breast Cancer Patients, Healthy Controls and Multiple Logistic Regression Analysis for BC Clinical Pathological Features

rs3087243

GG, N (%)

GA, N (%)

AA, N (%)

χ² value (P-value)

Minor allele frequency A

Group comparison

OR (95% CI)

P-value

Controls
Patients

74 (26)
71 (25)

202 (71)
197 (70)

9 (3)
17 (5)

1.95 (= 0.376)

0.39
0.41

GG vs. others
GA vs. others
AA vs. others

0.94 (0.64 - 1.37)
0.91 (0.64 - 1.31)
1.94 (0.85 - 4.4)

0.84
0.71
0.15

HWE: controls: χ² = 69.9 (P < 0.05); patients: χ² = 53.6 (P < 0.05)

Variables

GG vs. GA + AA: OR (CI)

P-value

GA vs. GG + AA: OR (CI)

P-value

AA vs. GG + GA: OR (CI)

P-value

AAO

1.44 (0.70 - 2.94)

0.31

0.57 (0.30 - 1.08)

0.08

1.86 (0.67 - 5.16)

0.23

Tumor status

0.01 (0.0043 - 0.09)

< 0.01*

6.28 (3.01 - 13.07)

< 0.01*

3.46 (1.02 - 11.67)

0.04*

LN status

1.38 (0.61 - 3.11)

0.43

0.68 (0.31 - 1.52)

0.35

0.91 (0.17 - 4.90)

0.91

Metastasis

0.97 (0.17 - 5.53)

0.97

3.60 (0.94 - 13.78)

0.06

0.99

DOD < 6 months vs. > 6 months

(0.91 - 4.02)

0.08

0.62 (0.32 - 1.19)

0.15

0.84 (0.29 - 2.46)

0.76

**Table 5.** The Reference Motif cgtgggtt (78.23) Threshold Value Is Changed to Mutant catgggtt (51.7)
Splice site type	Motif	New splice site	Wild type	Mutant	If cryptic site uses exon length variation	Variation (%)
DONOR	Aacgtgggt	AACatgggt	78.53	51.7	NA	SITE BROKEN -34.17

Table 5. The Reference Motif cgtgggtt (78.23) Threshold Value Is Changed to Mutant catgggtt (51.7)

Splice site type

Motif

New splice site

Wild type

Mutant

If cryptic site uses exon length variation

Variation (%)

DONOR

Aacgtgggt

AACatgggt

78.53

51.7

SITE BROKEN -34.17

**Table 6.** Haplotype Analysis of CTLA-4 (rs11571317 and rs3087243) Gene Polymorphisms in Breast Cancer Patients and Controls and Haplotypes Association With Epidemiologic, Clinical Parameters in South Indian Breast Cancer Patients
Haplotype	Frequency	Odds ratio (95% CI)	P-value
C-A	0.3956	1.00	-
T-G	0.3877	0.40 (0.25 - 0.62)	< 0.01
C-G	0.2167	1.31 (0.91 - 1.89)	0.15
Global haplotype association P-value < 0.0001
S. no.	Haplotype	AAO < 48 years vs. > 49 years patients	Tumor size	LN status	Metastasis	DOD < 6 months vs. > 6 months
C1	C-G	1.00	1.00	1.00	1.00	1.00
C2	C-A	2.00 (0.75 - 5.28) P > 0.05	3.61 (1.10 - 11.86) P < 0.05	2.61 (0.84 - 8.15) P > 0.05	0.35 (0.10 - 1.22) P > 0.05	0.24 (0.07 - 0.77) P < 0.05
C3	T-G	1.68 (0.68 - 4.14) P > 0.05	0.24 (0.03 - 1.67) P > 0.05	1.95 (0.60 - 6.34) P > 0.05	0.60 (0.13 - 2.87) P > 0.05	0.59 (0.19 - 1.79) P > 0.05
C4	T-A	0.65 (0.17 - 2.50) P > 0.05	15.81 (4.96 - 50.40) P < 0.05	2.13 (0.74 - 6.09) P > 0.05	3.98 (1.25 - 12.63) P < 0.05	0.67 (0.18 - 2.53) P > 0.05

Table 6. Haplotype Analysis of CTLA-4 (rs11571317 and rs3087243) Gene Polymorphisms in Breast Cancer Patients and Controls and Haplotypes Association With Epidemiologic, Clinical Parameters in South Indian Breast Cancer Patients

Haplotype

Frequency

Odds ratio (95% CI)

P-value

C-A

0.3956

1.00

T-G

0.3877

0.40 (0.25 - 0.62)

< 0.01

C-G

0.2167

1.31 (0.91 - 1.89)

0.15

Global haplotype association P-value < 0.0001

S. no.

Haplotype

AAO < 48 years vs. > 49 years patients

Tumor size

LN status

Metastasis

DOD < 6 months vs. > 6 months

C-G

1.00

C-A

2.00 (0.75 - 5.28)
P > 0.05

3.61 (1.10 - 11.86)
P < 0.05

2.61 (0.84 - 8.15)
P > 0.05

0.35 (0.10 - 1.22)
P > 0.05

0.24 (0.07 - 0.77)
P < 0.05

T-G

1.68 (0.68 - 4.14)
P > 0.05

0.24 (0.03 - 1.67)
P > 0.05

1.95 (0.60 - 6.34)
P > 0.05

0.60 (0.13 - 2.87)
P > 0.05

0.59 (0.19 - 1.79)
P > 0.05

T-A

0.65 (0.17 - 2.50)
P > 0.05

15.81 (4.96 - 50.40)
P < 0.05

2.13 (0.74 - 6.09)
P > 0.05

3.98 (1.25 - 12.63)
P < 0.05

0.67 (0.18 - 2.53)
P > 0.05