Colorectal cancer is the second (in women) or third (in men) most common neoplasm worldwide. In México, colorectal cancer ranks fourth in both sexes 1. Environmental, genetic and epigenetic factors have been related to colorectal cancer development 2-4. Although obesity increases the risk of colorectal cancer by 1.5- to 2-fold 5 and 17.7% of the cases are ascribed to excess body mass 6, the cause of this association is not yet clear.
The relationship between colorectal cancer and obesity may reflect the impaired structure or function of adipocytokines, which are proteins that regulate cell proliferation, angiogenesis, and apoptosis 7. For instance, an increase in leptin appears to promote the development and progression of colorectal cancer 8.
The LEPR (leptin receptor) protein is encoded by a gene in the 1p31.3 locus 9. The LEPR polymorphisms c.326A>G (rs1137100) and c.668A>G (rs1137101) are located in exons 4 and 6, respectively. The former results in a conservative substitution of arginine for lysine in codon 109 (p.K109R) while the latter produces a non-conservative change of glutamine to arginine in codon 223 (p.Q223R) 10. This p.Q223R substitution occurs inside the region coding for the extracellular domain and could affect the interaction with leptin 11. Even if the impact of the LEPR pathway in C colorectal cancer is not yet clear, increased expression in tumor tissue has been related to proliferation and angiogenesis 12.
The objective of this study was to determine the association between c.326A>G and c.668A>G LEPR polymorphisms and colorectal cancer in Mexican patients.
Material and methods
Patients and controls
The analysis included 147 patients diagnosed by histopathological and clinical criteria with sporadic colorectal cancer at the Hospital Civil de Guadalajara “Dr. Juan I. Menchaca”. The group included 87 males and 60 females with an average age of 57 years (range: 20 to 69 years). The control group was integrated by 134 blood donors from the same hospital without a colorectal cancer diagnosis.
All subjects signed the informed consent. This study was approved by the ethics committee of Centro Universitario de Los Altos, Universidad de Guadalajara (CUA/CINV/PCIL-011/2009).
DNA extraction
We used the Miller method 13 combined with the DTAB-CTAB protocol 14 on each 5-ml sample of peripheral blood with EDTA added as an anticoagulant. DNA purity and concentration were determined by spectrophotometry.
PCR-RFLP
We searched for LEPR variants with a PCR-RFLP protocol according to predetermined conditions 15,16. For the c.326A>G variant, we used 5’-TTTCCACTGTTGCTTTCGGA-3’ forward and 5’- AAACTAAAGAATTTACTGTTGAAACAAATGGC-3’ reverse primers under the following conditions: DNA initial denaturation for 5 min at 94°C; 40 cycles of 94°C for 45 s, annealing at 61.8°C for 45 s, extension at 72°C for 90s, and a final extension at 72°C for 10 min. We looked for the c.668A>G LEPR polymorphism using 5’-AAACTCAACGACACTCTCCTT-3’ forward and 5’-TGAACTGACATTAGAGGTGA-3’ reverse primers and amplification conditions similar to those described above except for an annealing temperature of 55°C.
For the single nucleotide polymorphism analysis, we used the restriction enzymes HaeIII for c.326A>G and HpaII for c.668A>G (both of which recognize the polymorphic G allele) for 16 hours. The assays were done in duplicate.
Electrophoresis
DNA fragments were identified in 6% polyacrylamide gels stained with silver nitrate. Their sizes were 101, 70 and 31 bp for c.326A>G and 80, 58 and 22 bp for c.668A>G.
Statistical analysis
Allelic and genotypic frequencies were determined by direct counting. The distribution of genotypes was tested for Hardy-Weinberg equilibrium using the c2 test. Haplotype frequencies and linkage disequilibrium values were calculated by the Arlequin, version 3.5, software 17. Linkage disequilibrium with r2>0.3 was considered to be significant 18. Intergroup differences were established by c2 or Fisher’s exact test and the association was determined by the odds ratio (OR) in SPSS™, version 10.0.
Results
LEPR polymorphisms
Demographic and clinical data of CRC patients are listed in table 1. In the control group, both LEPR polymorphisms were in Hardy-Weinberg equilibrium (p>0.05). For methodological reasons, not all individuals were included in the analysis. The comparison between controls and colorectal cancer patients associated the AG genotype of c.326A>G to an increased risk for colorectal cancer (OR=1.81, 95% CI=1.04-3.16, p=0.04) while no association was found for the c.668A>G polymorphism (table 2).
Variable | n (%) |
---|---|
Gender | |
Female | 60 (41) |
Male | 87 (59) |
Diabetes | |
Yes | 28 (19) |
No | 102 (69) |
Missing data | 17 (12) |
Smoking | |
Yes | 75 (51) |
No | 59 (40) |
Missing data | 13 (9) |
Alcohol consumption | |
Yes | 70 (47.6) |
No | 60 (40.8) |
Missing data | 17 (11.6) |
LEPR haplotypes
The haplotype AA was the most frequent in controls (93/194; 48%) and colorectal cancer patients (132/254; 52%). LD was only evident among the latter (r2=0.36). The AG haplotype showed a significant association with colorectal cancer (OR=0.58, CI=0.35-0.96, p<0.03) (table 3).
Discussion
Our finding of an increased risk of colorectal cancer in heterozygous AG individuals for the LEPR c.326A>G polymorphism compares with a similar association documented in Korean patients with gastric cancer 19. Regarding the same single nucleotide polymorphism (SNP), an increased risk (OR=1.64; 95%CI=1.10-2.45; p=0.016) for luminal A breast cancer was observed in GG patients 20 whereas the G allele was associated with prostate cancer mortality (hazard ratio=0.82: 95%CI=0.67-1.00; p=0.027) in another study 21. Moreover, this missense c.326A>G (p.K109R) variant was associated with increased birth weight in the Human Gene Mutation Database (HGMD-public: CM032948) 22. However, no association was found in other cancer studies 16,23,24.
Inasmuch as lysine (K) and arginine (R) are basic large-size amino acids 25, the Ensembl project― as predicted by SIFT and Polyphen― classified this variant as tolerable and benign 26. Yet, the higher propensity of lysine to post- translation modifications (methylation, ubiquitination, acetylation, phosphorylation, and sumoylation) indicates that its substitution could lead to protein structure alterations and increase colorectal cancer susceptibility 27.
The lack of association of the LEPR c.668A>G polymorphism with colorectal cancer in this and previous studies 11,28-31, and also documented in other neoplasms 16,23,32-37, probably reflects that the change of glutamine (Q), a polar amino acid of medium size, to arginine (R), a large-sized and basic amino acid, is harmless.
However, opposite results have been reported. In Caucasian women with breast cancer, the G allele was related to differentiation grade (OR=2.45, 95%CI 1.40-4.31) 38. Likewise, an increased risk for breast 39, oral 40, and lung 41 cancer development ascribed to the same variant has been proposed. Additionally, two meta-analyses revealed no association with cancer 42,43. These controversial results could be explained by the genetic structure of populations or the presence of additional variants modifying the LEPR gene.
The apparent protective effect of the AG haplotype disclosed in the present analysis (c.326A>G/c.668A>G) is consistent with the view that the accumulation of LEPR variants and gene interactions modulates colorectal cancer risk and development.
In conclusion, our results indicate that in the Mexican population, the AG genotype of the c.326A>G LEPR variant increases the risk of CRC, whereas the AG haplotype (c.326A/c.668G) protects against such tumors.