Identification Of Novel Variants Of Metadherin In Breast Cancer

Object:we investigated the genetic polymorphisms in MTDH by direct sequencing in a cohort of breast cancer cases and controls, with the intention of discovering of novel variants and comparing the distribution of SNP genotype frequencies of these 2 affected vs. non-affected cohorts to determine whether a particular SNP may influence susceptibility to breast cancer development.Method:DNA from each sample of whole blood was extracted with the TIANamp Genomic DNA Kit.Then the MTDH gene was amplified with polymerase chain reaction (PCR) prior to sequencing. And we tested the object gene by the software Maglign7.0 and Chromas2.33 to trace all variants.The genotypes and allele frequencies of the known and novel SNPs were tested for equilibrium using a validated, public statistical web-tool based on the Hardy-Weinberg model with a p value of>0.5cm suggesting equilibrium. The distribution of known and novel SNPs between the case and control groups were analyzed using chi-squared and the Fisher's exact test was used when one cell count was less than 5. All p values were exacted two-sided and p values of<0.05 were considered statistically significant. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression analysis to evaluate the association between genotype frequencies and risk of breast cancer development.Results:Thirteen variants were detected in the control group compared with eleven variants in the affected breast cancer cohort; of these,9 unnamed, novel variants were discovered in sum. The distribution of all variants in both control and case groups is displayed in Figure 1. The novel and known variants appear to gather at both ends of MTDH gene, with 3 novel variants displayed in the central part of the gene in case group, but not in the control group. The 3 variants are located at exon₆ (untitled₇), exon₇ (untitled₈) and intron₇ (untitled₉) individually.Among all the variants detected,3 SNPs (rs2438211, rs2449512 and rs1311) were found with complete linkage in both groups; the sequencing chromatogram results representing the linkage are shown in figure 2. In addition to the known SNPs, several new SNPs were discovered:one novel variant in the flanking sequence; one in the 5'UTR,; three in the 3'UTR; and one in intron 11 for the control group. Of note, the high frequency variant in the intron 11 was also detected in the case group. Strikingly, two low frequency novel variants were detected in case group located in exon₆ and exon₇, respectively. The variant in exon₇ (1390 G>A, untitled₈) was a synonymous SNP while the variant in exon₆ (1333 C>G, untitled₇) converted a Asp to Glu in the MTDH protein. Only one patient with breast cancer carried the SNP (G>C, untitled₉) just one nucleotide prior to exon₈, which was against the "GT-AG rule" in mRNA splicing.Conclusion:In addition to finding some known variants of MTDH, several novel SNPs of MTDH were discovered in our study that appear to be correlated to breast cancer susceptibility Since MTDH has been demonstrated as a key regulator in the complex network of oncogenic pathways, additional investigations further clarifying the functional role of this gene and its association to breast cancer development and recurrence are needed. While additional study on the MTDH gene is necessary, variants of MTDH may prove to be potential markers for breast cancer development, prognostic indicators for disease progression, and possible foci for future targeted therapy.