Genetic variants of ATP-binding cassette, sub-family B, member 5 (ABCB5) in hepatocellular carcinoma

Abstract

Genetic variations including single nucleotide polymorphisms (SNPs), small insertions and deletions (INDELs) have been reported to affect the expression levels and functions of protein. A number of ATP-binding cassette(ABC) transporters have sequence variations and the alterations were associated with differential expression levels, clinico-pathological characteristics and cancer risk in various cancer studies. Our research group has previously shown that ABCB5 conferred resistance to chemotherapeutic agents in hepatocellular carcinoma (HCC) cell lines and ABCB5 expression levels were significantly associated with recurrence-free survival of HCC patients. Notably, whether ABCB5 demonstrated unique genetic variations in HCC patients was not clear. The aims of this study are to identify the genetic variants of ABCB5 gene in HCC patients with reference to healthy individuals, and to evaluate the significances of genetic variants with HCC clinico-pathological features.

In Phase 1 study, exons and exon/intron boundaries of ABCB5 gene were examined by Sanger sequencing in twenty HCC tumor DNA samples. A total of 47SNPs and 4 INDELs were identified in the first small HCC cohort. Five SNPs (-132C>T, IVS6+17G>T, IVS14+50T>C, 2166T>G and 4609A>G) were novel sequence variations.

In Phase 2 study, unique SNPs and INDELs from Phase 1 study were examined in 300 HCC blood DNA, 90 paralleled HCC tumor DNA, and compared with 300 healthy blood DNA samples. The sequence variations were investigated by Sequenom MassArray genotyping or pyrosequencing as appropriate in the second large cohort. The associations of genetic variants with clinico-pathological characteristics were evaluated including gender, age, HCC family history, hepatitis B virus (HBV) status, tumor size, number of tumor nodule, tumor stage, Edmondson-Steiner grade, venous infiltration, AFP level and recurrence-free survival rates.

Comparison of HCC blood and healthy blood samples demonstrated that specific ABCB5 genotypes (IVS2+135C>T, IVS22-123C>T and 2802G>A) were associated with higher HCC risks (P<0.05). In addition, eight SNPs (-214C>G, -132C>T, IVS3+1G>C, 392C>T, 784C>A, IVS14+50T>C, IVS22-123C>T and 2802G>A) had significant association with tumor stage (P<0.05). Four SNPs (-90C>T, IVS2+135C>T, IVS5+46T>G and IVS7+192A>G) were significantly associated with tumor size. Three SNPs (IVS3+1G>C, 392C>T and 784C>A) were associated with the number of tumor nodules (P<0.05). Twelve SNPs (-238A>G, -132C>T, IVS2+108C>T, IVS2+135C>T, IVS5+46T>G, IVS9+25G>A, IVS9+139T>C, 1005C>T, 1337T>C, IVS14+50T>C, 2166T>G and 4609A>G) were significantly associated with recurrence-free survival (log-rank test, P<0.05). Furthermore, one SNP (392C>T) was significantly associated with mRNA expression level (P=0.025). Notably, comparison of the paralleled blood and tumor DNA sequences from HCC patients indicated that ABCB5 mutation in tumors has not been observed.

In summary, unique ABCB5 genetic variants were observed in HCC patients. Specific genotypes were associated with increased HCC risk, aggressive HCC features including large tumor size, presence of venous infiltration, late tumor stage and poor recurrence-free survival. Further investigations on the functional significance of ABCB5 variants are warranted.