Functional polymorphisms of FAS and FASL gene and risk of breast cancer - pilot study of 134 cases

Abstract

Fas/Fas ligand (FasL) system is one of the key apoptotic signaling entities in the extrinsic apoptotic pathway. De-regulation of this pathway, i.e. by mutations may prevent the immune system from the removal of newly-formed tumor cells, and thus lead to tumor formation. The present study investigated the association between -1377 G/A (rs2234767) and -670 A/G (rs1800682) polymorphisms in Fas as well as single nucleotide polymorphisms INV2nt -124 A/G (rs5030772) and -844 C/T (rs763110) in FasL in a sample of Iranian patients with breast cancer. This case-control study was done on 134 breast cancer patients and 152 normal women. Genomic DNA was extracted from whole blood samples. The polymorphisms were determined by using tetra-ARMS-PCR method. There was no significant difference in the genotype distribution of FAS rs2234767 polymorphism between cases and controls. FAS rs1800682, FASL rs5030772, and FASL rs763110 genotypes showed significant associations with an increasing risk of breast cancer (odds ratio OR = 3.18, P = 0.019; OR = 5.08, P = 0.012; OR = 2.40, P = 0.024, respectively). In conclusion, FAS rs2234767 was not associated with breast cancer risk. Though, FAS rs1800682, FASL rs5030772, and FASL rs763110 polymorphisms were associated with the risk of breast cancer in the examined population.

Figure 1. Maps of the human Fas and FasL genes with polymorphism positions indicated.

(A) Map of the human Fas gene. Exons 1–9 are numbered and represented by black boxes. (B) position of the single nucleotid variations within the core promoter of the Fas gene, a G>A polymorphism at position −1377 and a A>G polymorphism at position 670. (C) Map of the human Fas Ligand gene. Exons 1–4 are numbered and represented by black boxes. (D) Position of single nucleotide polymorphisms (SNPs) within the Fas Ligand promoter, a C>T substitution at position −844 and a A

Figure 2. Schematic representation of Tetra-Primer Amplification Refractory Mutation System.

T-ARMS-PCR was used for the detection of SNPs of Fas rs1800682 (A), Fas rs2234727 (B), FasL rs763110 (C) and FasL rs5030772 (D). Two forward and two reverse specific primers are used to produce three potential products. Product sizes were 158 bp for A allele, 207 bp for G allele, and 309 bp for two outer primers (control band) for Fas rs1800682 (A). Product sizes were 216 bp for G allele, 340 bp for A allele, and 507 bp for control band for Fas rs2234727 (B). Product sizes were 145 bp for T allele, 192 bp for C allele, and 284 bp for control band for FasL rs763110 (C). Product sizes were 197 bp for G allele, 300 bp for A allele, and 438 bp for control band for FasL rs5030772 (D).

Figure 3. Electrophoresis pattern of tetra-ARMS-PCR for detection of polymorphisms.

Agarose gel electrophoresis was used to detect band-pattern of tetra-ARMS-PCR for Fas rs1800682 (A), Fas rs2234727 (B), FasL rs763110 (C), and FasL rs5030772 (D). M = DNA marker.

Figure 4. Examples of DNA sequencing results of Fas and FasL prlymorphisms.

DNA sequencing results of Fas rs1800682 (A), Fas rs2234727 (B), FasL rs763110 (C) and FasL rs5030772 (D) for tetra-ARMS-PCR results depicted in figure 3 are shown.