Results in Table 2 showed that there was no statistically significant difference between two groups' age (P=0.065). There was no statistically significant difference in gender frequency between the case and control groups (P=0.635). Smoking ratio in case group was 34.49% higher than control group 27.29%. Alcohol ratio was 21.49% in case lower than 23.30% in control group, alcohol consumption was not statistically significant difference between two groups (P=0.443).
Table 2 Distribution of selected demographic variables and risk factors in cases and controls
Variable
|
Overall Cases (n=577)
|
Overall Controls (n=678)
|
P
|
|
n (%)
|
n (%)
|
|
Age (years)
|
61.34±11.097
|
62.31±7.549
|
0.065
|
Age (years)
|
|
|
|
< 62
|
268 (46.45)
|
324 (47.79)
|
|
≥62
|
309(53.55)
|
354(52.21)
|
0.635
|
Sex
|
|
|
|
Male
|
394 (68.28)
|
456(67.26)
|
|
Female
|
183(31.72)
|
222(32.74)
|
0.698
|
Smoking status
|
|
|
|
Never
|
378 (65.51)
|
493(72.71)
|
|
Ever
|
199(34.49)
|
185 (27.29)
|
0.006
|
Alcohol use
|
|
|
|
Never
|
453 (78.51)
|
520(76.70)
|
|
Ever
|
124 (21.49)
|
158(23.30)
|
0.443
|
Primary information for gene CCND1 rs9344 Polymorphisms
Table 3 shows that rs9344 is located in 11 chromosome, category is protein coding, minor allele frequency(MAF) for Chinese in genecard database is 0.442 and in our controls is 0.451. Hardy-Weinberg equilibrium test in our controls is 0.295(P>0.05), which means that our sample population is representative. We use Snapshot method as genotyping and the percentage of successful tests is 99.20%.
Table 3 Primary information for gene CCND1 rs9344 Polymorphisms
GenotyPed SNPs
|
Gene
|
Chr Pos (NCBI Build 38)
|
Category
|
MAFa for Chinese in database
|
MAF in our controls (n = 678)
|
P value for HWEb test in our controls
|
Genotyping method
|
Genotyping value (%)
|
rs9344
|
CCND1
|
11:69648142
|
Protein Coding
|
0.442
|
0.451
|
0.295
|
Snapshot
|
99.20
|
a MAF: minor allele frequency;
b HWE: Hardy–Weinberg equilibrium;
GeneCards Datebase: https://www.genecards.org/
Table 4 Hardy-Weinberg equilibrium test : There was no statistically significant difference between the case and control groups (P>0.05). Genotype frequency conforms to the law of genetic balance, the selected samples are representative.
Table 4 Hardy–Weinberg equilibrium analysis for rs9344
Group rs9344
|
|
|
AA
|
AG
|
GG
|
2
|
P
|
Case
|
Observed
|
168
|
302
|
107
|
|
|
|
Expected
|
176.4
|
285.3
|
115.4
|
1.983
|
0.159
|
Control
|
Observed
|
182
|
343
|
143
|
|
|
|
Expected
|
187.1
|
332.9
|
148.1
|
0.620
|
0.431
|
P value<0.05 means statistically significant difference;αtakes two sides check;df=1.
Table 5 shows: The A allele frequency in case group is higher than control group (47.43%>45.07%); The G allele distribution in case group is lower than control group (52.57%<54.93%), yet this difference is no statistically significant between the two groups (P=0.237).
Table 5 Analysis of rs9344 A allele and G allele between cases and controls
Variable
|
Case
|
Control
|
P
|
OR(95% CI)
|
A allele
|
638(47.43)
|
516(45.07)
|
|
|
G allele
|
707(52.57)
|
629(54.93)
|
0.237
|
0.91(0.78-1.07)
|
The results of Table 6 showed that occurrence of AG was slightly higher in case than in control (52.34% >51.35%) with no statistical significance (P=0.722). There was still no statistically significant difference(P=0.787) after being logistics regression test.
Occurrence of GG in case group was lower than in control group (18.54%<21.40%), but the difference was not statistically significant (P=0.208). GG were still not statistically different (P=0.189) after being logistics regression test.
In dominant model, mutation(AG+GG)frequency was lower in case group than control group (70.88% <72.75%), but difference was not statistically significant (P=0.464). There was still no statistical difference between the two groups (P=0.496) after logistic regression analysis. For recessive model dominated, GG/(AA+AG) with no statistical difference (P=0.209).
Table 6 Logistic regression analysis of CCND1 gene rs9344 Polymorphism between cases and controls
Genotype
|
GC Cases
(n=577)
|
|
Controls
(n=678)
|
Crude OR
(95%CI)
|
P
|
Adjusted OR a
(95%CI)
|
P
|
n
|
%
|
|
n
|
%
|
|
|
|
|
rs9344
|
|
|
|
|
|
|
|
|
|
AA
|
168
|
29.12
|
|
182
|
27.25
|
1.00
|
|
|
|
AG
|
302
|
52.34
|
|
343
|
51.35
|
0.95(0.74-1.24)
|
0.722
|
0.96(0.74-1.25)
|
0.787
|
GG
|
107
|
18.54
|
|
143
|
21.40
|
0.81(0.59-1.12)
|
0.208
|
0.90(0.76-1.06)
|
0.189
|
AG+GG
|
409
|
70.88
|
|
486
|
72.75
|
0.91(0.71-1.17)
|
0.464
|
0.99(0.85-1.09)
|
0.496
|
GG
|
107
|
18.54
|
|
143
|
21.40
|
0.84(0.63-1.11)
|
0.209
|
0.91(0.79-1.04)
|
0.173
|
AA+AG
|
470
|
81.46
|
|
525
|
78.60
|
1.00
|
|
|
|
Table 7 shows that stratified analysis, we analyzed stratification according to gender, age, smoking and drinking factors. The results showed that differences were not statistically significant in heterozygous mutants, homozygous mutants, dominant models or recessive models.
Table 7 Stratified analyses between rs9344 Polymorphism and risk by sex, age, BMI, smoking status and alcohol consumption
Variable
|
(case/control)
|
Adjusted OR (95% CI); P
|
AA
|
AG
|
GG
|
AA
|
AG
|
GG
|
(AG+GG)VSAA
|
GGVS(AA+AG)
|
gender
|
|
|
|
|
|
|
|
|
Male
|
119/119
|
200/233
|
75/96
|
1.00
|
0.86(0.63-1.18);
P:0.344
|
0.78(0.53-1.16);
P:0.220
|
0.84(0.62-1.13);
P:0.242
|
0.84(0.60-1.18);
P:0.304
|
Female
|
49/63
|
102/110
|
32/47
|
1.00
|
1.19(0.75-1.89);
P:0.454
|
0.88(0.49-1.57);
P:0.655
|
1.10(0.71-1.70);
P:0.678
|
0.78(0.43-1.29);
P:0.329
|
Age
|
|
|
|
|
|
|
|
|
<62
|
68/91
|
151/160
|
49/66
|
1.00
|
1.26(0.86-1.86);
P:0.234
|
0.99(0.61-1.61);
P:0.979
|
1.18(0.82-1.71);
P:0.367
|
0.85(0.56-1.28);
P:0.442
|
≥62
|
100/91
|
151/183
|
58/77
|
1.00
|
0.75(0.53-1.07);
P:0.115
|
0.69(0.44-1.07);
P:0.095
|
0.73(0.52-1.03);
P:0.069
|
0.82(0.56-1.20);
P:0.314
|
Smoking
|
|
|
|
|
|
|
|
|
Never
|
106/136
|
205/246
|
67/102
|
1.00
|
1.07(0.78-1.46);
P:0.077
|
0.08(0.57-1.26);
P:0.401
|
1.00(0.74-1.35);
P:0.985
|
0.81(1.14-0.57);
P:0.219
|
Ever
|
62/46
|
97/97
|
40/41
|
1.00
|
0.74(0.46-1.19);
P:0.217
|
0.72(0.41-1.29);
P:0.273
|
0.74(0.47-1.15);
P:0.181
|
0.88(0.54-1.43);
P:0.601
|
Alcohol
|
|
|
|
|
|
|
|
|
Never
|
128/145
|
241/257
|
84/109
|
1.00
|
1.06(0.79-1.43);
P:0.689
|
0.87(0.60-1.27);
P:0.473
|
1.01(0.76-1.33);
P:0.967
|
0.84(0.61-1.15);
P:0.280
|
Ever
|
40/37
|
61/86
|
23/34
|
1.00
|
0.66(0.38-1.14);
P:0.135
|
0.63(0.31-1.25);
P:0.184
|
0.65(0.38-1.10);
P:0.105
|
0.82(0.46-1.49);
P:0.520
|
For CCND1 gene rs9344 locus, genotype was detected successfully 99.20% in 1255 samples.