[Establishment of a multiplex ligation-dependent SNP genotyping method and its application in the detection of genes related to chemotherapeutic drugs in breast cancer].
OBJECTIVE
To establish a method for SNP genotyping of multi-genes by allele-specific oligonucleotide probe ligation mediated by a thermostable ligase, and to explore the genetic polymorphisms of drug-metabolizing enzymes in breast cancer patients and their association with chemotherapeutic responses.


METHODS
10 SNP loci of enzyme genes related to chemotherapeutic drugs such as taxanes, anthracyclines and cyclophosphamide were selected, and were genotyped for blood samples from 126 breast cancer patients by the established method. Their correlations with therapeutic responses were retrospectively evaluated.


RESULTS
The lower detection limit of genomic DNA by this developed method was 6.25 ng. The fluorescent peak locations of ligation products on ABI PRISM 377 DNA sequencer were accurate and consistent with prospective sizes in bases (Bias range 0.08 - 0.69 bp, x(-) = 0.31 bp, s = 0.18 bp). Same genotyping results were obtained for repeat tests of 8 random samples, which were further confirmed by sequencing analysis. The 10 SNP loci were polymorphic of different frequency in the breast cancer patients. The combinations with GSTP1 genotypes and GSTM1 genotypes were related to anthracycline-based chemotherapy efficacy (P = 0.037), and the low GSTs activity group (GSTP1 variant allele + GSTM1 null) showed the best effects (85.7%). GSTM1 genotypes and their combinations with GSTP1 and/or CYP3A5*3 genotypes were related to taxane-based therapy efficacy (P < 0.05 for all), and both the low GSTs activity group and the drug slow-metabolising group (low GSTs activity group + CYP3A5*3 wild allele) showed better effects (100%).


CONCLUSIONS
The established method is reliable and applicable in multiplex SNPs genotyping of multi-genes. SNPs combination may have a better clinical application value for prediction of chemotherapeutic responses.
Tang
Jin-hai
JH
Department of General Surgery, Jiangsu Tumor Hospital, Nanjing 210009, China.
Zhao
Jian-hua
JH
Wu
Jian-zhong
JZ
Lu
Jian-wei
JW
Pan
Li-qun
LQ
Xu
Zhi-yin
ZY
D000328:Adult; D000368:Aged; D018943:Anthracyclines / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics*; D003520:Cyclophosphamide / Q000627:therapeutic use; D051544:Cytochrome P-450 CYP3A / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D051549:Glutathione S-Transferase pi / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D012189:Retrospective Studies; D043823:Taxoids / Q000627:therapeutic use; D016896:Treatment Outcome
D018943:Anthracyclines; D000970:Antineoplastic Agents; D003520:Cyclophosphamide; D051544:Cytochrome P-450 CYP3A; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D043823:Taxoids; C510164:CYP3A5 protein, human; C496556:GSTP1 protein, human