Abstract
Objective: The present study aimed at assessing the relationship between Leukocyte-specific protein 1 gene (LSP1) polymorphisms (rs569550 and rs592373) and the pathogenesis of breast cancer (BC). Methods: 70 BC patients and 72 healthy subjects were enrolled in the study. Rs569550 and rs592373 polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Odds ratio (OR) with 95% confidence interval (CI) were calculated by the chi-squared test to assess the relationship between LSP1 polymorphisms and BC risk. Linkage disequilibrium (LD) and haplotypes were also analyzed by HaploView software. Results: Genotype distribution of the control was in accordance with Hardy-Weinberg equilibrium (HWE). The homozygous genotype TT and T allele of rs569550 could significantly increase the risk of BC (TT vs. GG: OR=3.17, 95% CI=1.23-8.91; T vs. G: OR=1.63, 95% CI=1.01-2.64). For rs592373, mutation homozygous genotype CC and C allele were significantly associated with BC susceptibility (CC vs. TT: OR=4.45, 95% CI=1.38-14.8; C vs. T: OR=1.70, 95% CI=1.03-2.81). LD and haplotypes analysis of rs569550 and rs592373 polymorphisms showed that T-C haplotype was a risk factor for BC (T-C vs. G-T: OR=1.74, 95% CI=1.04-2.92). Conclusion: LSP1 rs569550 and rs592373 polymorphisms are both risk factors for BC.
Keywords: LSP1, breast cancer, polymorphisms
Introduction
Breast cancer (BC) is the most frequency diagnosed cancer and the leading cause of cancer-related deaths among the women [1]. The occurrence of BC is still clearly increasing all over the world [2]. The previous studies have shown that the occurrence and development of BC were a complex interaction between genes and environment [3,4]. Only a small number of individuals who expose to the same environment develop BC, so genes play vital roles in the occurrence of BC. But the tumorigenesis mechanism of BC is still unclear [5]. In recent years, a number of studies have reported that some gene polymorphisms could influence the susceptibility to BC, such as CCND1 rs9344, Hif-1α rs11549465, LEP G-2548A, LEPR Q223R and so on [6-9]. Leukocyte-specific protein 1 (LSP1) is an F-actin binding protein specially expressed in endothelial cells and hematopoietic lineage. LSP1 gene is located on 11p15.5 and contains 20 exons [10,11]. It has been demonstrated that LSP1 is related with many diseases, such as HIV, inflammatory diseases, hepatocellular carcinoma and so on [11-13]. So far, the relationship between LSP1 polymorphisms and the development of BC was hardly reported.
So a case-control study was carried out to evaluate the relevance of LSP1 rs569550 (T>G) and rs592373 (T>C) polymorphisms with the risk of BC.
Material and methods
Objects of study
Total 70 female cases with BC in the study were outpatients and inpatients from The General Hospital of Beijing Military Command. They were unrelated with each other and belonged to Chinese Han population. The cases were confirmed by pathological diagnosis. The BC-free group was consisted of 72 healthy women from the examination center at the same period as controls. The controls were frequency-matched with cases by age and race and they all had no family history of breast diseases. The study was approved by the Research Ethics Committee of the hospital.
DNA extraction
Genome DNA of peripheral venous blood was extracted from all the objects using traditional phenol chloroform method and stored in -20°C refrigerator.
Genotyping of LSP1 polymorphisms
Polymerase chain reaction-restriction fragment length of polymorphism (PCR-RFLP) was used to detect LSP1 polymorphisms (rs569550 and rs592373). PCR primers were showed in Table 1. The primers were synthesized by Shanghai SANGON Biological Engineering Technology Co., Ltd. PCR reaction system was a volume of 50 µL solution, including 2 µL DNA template, 2 µL primers (1 µL upstream and 1 µL downstream), 25 µL Master Mix, and 21 µL of ddH2O. The reaction program was as follows: initial degeneration at 95°C for 3 minutes, followed by 36 cycles with degeneration at 94°C for 30 seconds, annealing (at 57-60°C ) for 30 seconds, and extension at 72°C for 40 seconds, the final extension at 72°C for 5 minutes. Then PCR products were digested by NcoI restriction enzyme. All digested products were detected by 2% agarose gel electrophoresis.
Table 1.
Primers sequences and amplified lengths of rs569550 and rs592373
| Primer | Primer sequence |
|---|---|
| rs569550 | Former: 5’-CTGTCACCTGCTCACACCTC-3’ |
| Reverse: 5’-GGAGAGGTGTGAGCAGGTGA-3’ | |
| rs592373 | Former: 5’-GTGCTCCAAAGACGGGCGGT-3’ |
| Reverse: 5’-CTCCTACCGCCCGTCTTTGG-3’ |
Statistical methods
SPSS18.0 software was used for statistical analysis. Genotype distribution of the control group was tested by Hardy-Weinberg equilibrium (HWE). The odds ratio (ORs) and 95% confidence intervals (CIs) were calculated by chi-squared test to evaluate the association of LSP1 rs569550 and rs592373 polymorphisms with BC susceptibility. Linkage disequilibrium (LD) was analyzed to assess the combined effects of the two polymorphisms on the occurrence of BC in the HaploView software. P<0.05 represents the difference with statistical significance.
Results
Essential features of study objects
There were no significant differences between cases and controls in such factors as age, smoking and abortion history (P>0.05 for all). While, the differences were statistically significant between two groups in family history and menopause history (P<0.05 for both) (Table 2).
Table 2.
Essential features of cases and controls
| Essential feature | Cases n (%) | Controls n (%) | P value |
|---|---|---|---|
| Age (mean age) | 53.8±10.8 | 53.4±12.2 | 0.86 |
| ≤50 | 22 (31.4) | 21 (29.2) | |
| >50 | 48 (68.6) | 51 (70.8) | |
| Smoking | 0.87 | ||
| yes | 31 (44.3) | 30 (41.7) | |
| no | 39 (55.7) | 42 (58.3) | |
| Family history | 0.00 | ||
| yes | 45 (64.3) | 16 (22.2) | |
| no | 25 (35.7) | 56 (77.8) | |
| Abortion history | 0.43 | ||
| yes | 19 (27.1) | 15 (20.8) | |
| no | 51 (72.9) | 57 (79.2) | |
| Menopause | 0.02 | ||
| yes | 43 (61.4) | 30 (42.9) | |
| no | 27 (38.6) | 42 (57.1) |
Association between LSP1 rs569550 and rs592373 polymorphisms and the risk of BC
The feasible association of LSP1 polymorphisms with BC susceptibility was showed in Table 3. The homozygous genotype TT and allele T of LSP1 rs569550 significantly increased the risk of BC (TT vs. GG: OR=3.17, 95% CI=1.23-8.91; T vs. G: OR=1.63, 95% CI=1.01-2.64). For rs592373 polymorphism, CC genotype and C allele were both genetic-susceptibility factors for BC (CC vs. TT: OR=4.45, 95% CI=1.38-14.8; C vs. T: OR=1.70, 95% CI=1.03-2.81).
Table 3.
Genotypes and alleles distribution of LSP1 rs569550 and rs592373 polymorphisms
| Genotype | Cases n (% ) | Controls n (% ) | P | OR (95% CI) |
|---|---|---|---|---|
| rs569550 | ||||
| GG | 23 (32.9) | 30 (41.7) | - | 1.00 (Ref.) |
| GT | 30 (42.9) | 35 (48.6) | 0.77 | 1.12 (0.54-2.32) |
| TT | 17 (24.2) | 7 (9.7) | 0.03 | 3.17 (1.23-8.91) |
| G | 76 (54.3) | 95 (66.0) | - | 1.00 (Ref.) |
| T | 64 (45.7) | 49 (34.0) | 0.04 | 1.63 (1.01-2.64) |
| rs592373 | ||||
| TT | 32 (45.7) | 38 (52.8) | - | 1.00 (Ref.) |
| TC | 23 (31.9) | 30 (41.7) | 0.80 | 0.91 (0.44-1.87) |
| CC | 15 (22.4) | 4 (5.5) | 0.01 | 4.45 (1.34-14.8) |
| T | 87 (55) | 106 (73.6) | - | 1.00 (Ref.) |
| C | 53 (45) | 38 (26.4) | 0.04 | 1.70 (1.03-2.81) |
LD test and haplotype analysis
The analysis of pairwise LD between rs569550 and rs592373 suggested that there was notable linkage disequilibrium (D’=0.86, r2=0.67) and two loci formed three haplotypes of T-T, T-C and G-T. The heterozygous haplotype T-C significantly increased the risk of BC (OR=1.74, 95% CI=1.04-2.92) (Table 4). So the T-C haplotype might be a risk factor for BC.
Table 4.
Relationship between haplotypes of LSP1 rs569550 and rs592373 polymorphisms and BC susceptibility
| Haplotype | Cases 2n=140 (%) | Controls 2n=144(%) | P | OR (95% CI) |
|---|---|---|---|---|
| G-T | 76 (15.0) | 95 (6.9) | - | 1.00 (Ref.) |
| T-C | 53 (45.0) | 38 (18.1) | 0.03 | 1.74 (1.04-2.92) |
| T-T | 11 (40.0) | 11 (75.0) | 0.62 | 1.25 (0.51-3.04) |
| Total | 140 (100.0) | 144 (100.0) | - | - |
Discussion
BC is the most common cancer with highest morbidity and mortality, cases in China accounting for 12.2% and 9.6% of all new diagnosed BC and deaths from BC in the world, respectively [14]. In Asia, the morbidity shows a significant rising trend over the years [15]. Moreover, its incidence rate varies due to the difference of races and geographical conditions [16]. Studies also found that long-term exposure to insecticides or pesticides increased the risk of BC [17,18]. Izano M et al. reported that current smoking was related with high risk of BC [19]. While, several studies suggested that isoflavone could reduce the occurrence of BC [20]. Until now, the pathogenesis of BC is still unclear.
In recent years, many studies found important roles of genetic factors in the occurrence of BC. The study by Sun X et al. showed that miR-21 and miR-200c were related with the onset of BC [21]. In addition, MTHFR rs1801133 polymorphism also could increase the risk for BC [22]. Moreover, the study of Wang J et al. showed that Pin1 might serve as a positive diagnosis biomarker for breast cancer [23]. Meanwhile, the function of LSP1 gene on the occurrence of BC has attracted a lot of attention.
LSP1 is mainly expressed in lymphocytes, neutrophils, macrophages and endothelium. It also regulates neutrophil movements, the adhesion of fibrinogen matrix proteins and the traverse of endothelial cell layers. In other words, LSP1 plays a regulatory role in the activation and chemotaxis of neutrophils. It has been reported that LSP1 rs3817198 polymorphism could affect the susceptibility to breast cancer [24,25]. But rs592373 and rs569550 polymorphisms are hardly reported.
The present study investigated the association of LSP1 polymorphisms with BC risk and the results showed that there were significant differences in the distribution of TT genotype and T allele in rs569550 polymorphism as well as CC genotype and C allele in rs592373 between cases and controls, which were significantly associated with BC risk. The haplotype analysis showed that T-C haplotype could increase the risk for BC.
In conclusion, LSP1 rs569550 and rs592373 polymorphisms may be closely correlated with the occurrence of BC. Further study with well-design and enough large sample size is needed to confirm the correlation and some environmental factors should be considered at the same time.
Disclosure of conflict of interest
None.
References
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