Dataset reporting detection of breast cancer-related HER2I655V polymorphism using allele-specific polymerase chain reaction

The dataset presented in this article is related to the research article entitled “Detection of HER2 Gene Polymorphism in Breast Cancer: PCR Optimization Study” (B.R. Budiarto, Desriani, 2016) [1] with some modification in primers used and in PCR optimization strategy to eliminate false-positive result that may occur in HER2I655V polymorphism detection. Combining a new set of primers with PCR gradient, The allele-specific PCR well performs to detect all type of breast cancer-originated HER2I655V genotypes. The validation of this method was done using Sanger DNA sequencing, offering an alternative tool for HER2I655V polymorphism detection in another type of cancer.


a b s t r a c t
The dataset presented in this article is related to the research article entitled "Detection of HER2 Gene Polymorphism in Breast Cancer: PCR Optimization Study" (B.R. Budiarto, Desriani, 2016) [1] with some modification in primers used and in PCR optimization strategy to eliminate false-positive result that may occur in HER2 I655V polymorphism detection. Combining a new set of primers with PCR gradient, The allele-specific PCR well performs to detect all type of breast cancer-originated HER2 I655V genotypes. The validation of this method was done using Sanger DNA sequencing, offering an alternative tool for HER2 I655V polymorphism detection in another type of cancer.
& 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Biology More specific subject area

Molecular Biology, Cancer research
Type of data The data provide a straightforward strategy for clarifying the possibility of a false-positive amplification generated by allele-specific PCR.
The data provide the technique to determine the proper dosage of DNA template used in allelespecific PCR for the success of HER2 I655V genotyping.
Data comparison of HER2 I655V polymorphism between our improved allele-specific PCR with Sanger DNA sequencing is provided in a way to validate the method easily.
Allele frequency data for breast cancer -related HER2 I655V polymorphism can be used as a reference for researchers who conduct similar experiments for HER2 I655V polymorphism studies with breast cancer risk related to specific demography or other races.

Data
The detection of breast cancer-related HER2 I655V polymorphism using allele-specific PCR using mismatch-specific primers strategy as shown in Fig. 1. The optimum annealing temperature for breast cancer-related HER2 I655V genotyping in allele-specific PCR was done using gradient PCR strategy with SNP-known DNA template (Fig. 2). To test sensitivity of the method, we used two types of genomic DNA which contain AA genotype or AG genotype in HER2 gene as allele representative with diluted ranging from 0.019 up to 10 ng of the template at optimized PCR condition (Fig. 3). Then, the reliability of allele-specific PCR was compared with data of Sanger DNA sequencing by looking the consistency of HER2 I655V genotypes data between two methods ( Fig. 4 and Table 1). The allele frequency of breast cancer-originated HER2 I655V obtained from allele-specific PCR was similar with an allele frequency data of breast cancer-originated HER2 I655V from Asian population ( Table 2).

Sample collection and genomic DNA extraction
Sixty-one breast cancer tissues from archived biopsies as the frozen section were collected from M. Djamil Hospital Padang, West Sumatera Province. Genomic DNA was then extracted followed manual tissue DNA extraction protocol (Pure Link Genomic DNA Mini Kit; Invitrogen). We have also prepared two types of genomic DNA with SNPs have previously been determined using a Sanger DNA sequencing method. No.6 tissue-retrieved genomic DNA with HER2 gene carries AG genotype while DEV (abbreviaton of patient's name) tissue-retrieved genomic DNA with HER2 gene carries AA genotype. All subjects enrolled in this experiment were approved by the local ethics committee, issued by the Ministry of Health, Republic of Indonesia.

Allele-specific PCR optimization
The optimum annealing temperature for allele-specific PCR was determined using genomic DNA with known SNPs. Each of 12.5 mL reaction mixture was contained 11.25 mL of PCR Super Mix (Invitrogen), 0.1 mM of allele-specific forward primer (5 0 -CCAGCCCTCTGACGTCCAGCA-3 0 ), 0.06 mM of long allele-specific forward primer (5 0 -GCGGGCAGGGCGGCGGGGGCGGGGCCCCAGCCCTCTGACGTC-CACCG-3 0 ), 0.15 mM of reverse primer (5 0 -TCCGTTTCCTGCAGCAGTCTCC-3 0 ), and 0.25 ml ( 3-6 ng) of DNA template. The Primers ratio used in this experiment followed the optimized PCR method as suggested by Germer and Higuchi, [2] and Gaundet et al. [3] with minor modification. The PCR amplification profile was as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 20 s, gradient temperature annealing from 54.3°C, 54.9°C, 57.3°C, 59.7°C, 60.8°C to 62.3°C for 20 s, and extension at 72°C for 30 s (Kyratec Super Cycler Thermal Cycler, Australia). All PCR tubes, distilled water, pipette tips, and pipettes were pre-treated by exposing them on UV-light for 7 15-20 minute prior to use. All PCR reagent mixing was done under laminar air flow.

Sensitivity test of allele-specific PCR
Two genomic DNA with known SNPs (No.6 genomic DNA for AG genotype and DEV genomic DNA for AA genotype) were fixed at 50 ng/mL to make serial dilution ranging from 10 ng declining to   0.019 ng. These diluted DNA then were applied as a template for allele-specific PCR reaction at optimum annealing temperature with PCR reagents and PCR amplification profile followed method mentioned above.

Validating and genotyping test using allele-specific PCR
Sixty-one samples of breast cancer were tested for HER2 I655V polymorphism using optimized allele-specific PCR. The PCR condition and temperature profile were the same as mentioned above except for annealing temperature was fixed at 54.3°C. The method was validated by direct Sanger DNA sequencing of 61 sample of breast cancer patients by firstly amplified DNA fragment using primer pairs HER2_F (5 0 -CCAGCCCTCTGACGTCCAT-3 0 ) and HER2_R (5 0 -TCCGTTTCCTGCAGCAGTCTCC-3 0 ) generating 142 bp PCR product. The PCR amplification profile was as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, an nealing temperature at 60°C for 30 s, and extension at 72°C for 30 s. This PCR product was sequenced using HER2_R primer only done by First-Base Asia Ltd. Fig. 3. Sensitivity test of allele-specific PCR in genotyping HER2 I655V polymorphism. PCR was performed using optimized PCR condition where annealing temperature was at 54.3°C. NTC was Non-Template Control. The PCR product was run on 3% agarose under UV illumination.

Comparing allele frequency of HER 2I655V obtained from our data to published data
Data were tested for statistical significance by using Statistical Package for the Social Sciences software system SPSS-17 statistical software (SPSS, Chicago,IL). Frequency of each HER2 I655V allele was calculated using formulation frequency of Ile ¼f(Ile/Ile) þ0.5f(IleVal), whereas frequency of Val¼ f(Val/ Val) þ0.5f (IleVal). Chi-square test was used to analyze differences in genotype frequencies between our data with published dataset to evaluate any possible deviation from Hardy-Weinberg equilibrium of our genotyping data. Genotype frequencies are interpreted as statistically significant different if only the P value is less than 0.05 [4].

Acknowledgments
We thank Dr.dr. Wirsma Arif Harahap, SpB(K)Onk. for providing us samples of breast cancer patients for this study. Percentage means the number of samples produces correct genotype in total samples tested Table 2 Genotype frequency of HER2 I655V polymorphism showed by our data versus published data.

Author
Year of publication Note: Each of P value represented the different of genotype frequency of HER2 I655V obtained from Chi-Square test. a no siginificant event was observed between genotype frequency of HER2 I655V in our experiment with Asian population. b no siginificant event was observed between genotype frequency of HER2 I655V in our experiment with European population. c a siginificant event was observed between genotype frequency of HER2 I655V in our experiment with African population.

Transparency document. Supporting information
Transparency data associated with this article can be found in the online version at http://dx.doi. org/10.1016/j.dib.2016.09.033.