Balkan Journal of Medical Genetics

APLASIA RAS HOMOLOGOUS MEMBER I GENE AND DEVELOPMENT OF GLIAL TUMORS
Yakut S1, Tuncer MR2,* Berker M3, Goksu E2, Gurer I4, Ozes ON1, Luleci G1, Karauzum SB1
*Corresponding Author: Sibel Berker Karauzum, Department of Medical Biology and Genetics, Faculty of Medicine, Akdeniz University Antalya, Turkey; Tel.: +90 242 2496971; Fax: +90 242 2274482; E-mail: sibelkarauzum@akdeniz.edu.tr
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MATERIALS AND METHODS

Materials. This study involved 21 primary tumors consisting of 13 glioblastoma multiforme (GBM), WHO grade IV; five oligodendrogliomas (O), WHO grade III and two anaplastic astrocytomas (AA), WHO grade III and one anaplastic oligoastrocytoma (AOA), WHO grade III. The age range of the patients (13 male, eight female) were 21-72 years old. Primary tumor tissues were obtained from patients who underwent surgery, subtotal (seven patients) and gross total (14 patients) with their informed consent. For LOH analysis, peripheral blood samples from 21 normal healthy volunteers was also involved. We used RNA samples from normal brain tissues of the seven autopsies with their relatives’ informed consent as reference tissue for mRNA expression analyses. DNA and RNA Extraction. DNA was isolated from tumor tissue using the salting-out method [15]. Control DNA samples were prepared from peripheral blood lymphocytes of the same cases for LOH analyses. RNA was extracted from tumors and normal brain tissue samples using the SV Total RNA Isolation Kit (Promega, Madison, WI, USA) and the manufacturer’s protocol. Real-Time Quantitative Reverse Transcriptase- Polymerase Chain Reaction. Reverse transcription of total RNA was performed using TaqMan Reverse Transcription Reagents (Cat. N8080234; Applied Biosystems, Foster City, CA, USA). cDNA was synthesized from 2 μg of RNA using random hexamer and Super- Script II reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions. The amplification comprised one cycle at 25°C for 10 min. and one cycle at 48°C for 60 min. Real-time quantitative PCR was performed using two gene-specific primers (ARHI, 5’-TCT CTC CGA GCA GCG CA-3’ and ARHI, 5’-TGG CAG CAG GAG ACC CC-3’), a labeled probe (5’-TGT CTT CTA GGC TGC TTG GTT CGT GCC-3’) (5’-FAM; 3’-TAMRA) (9), 2 μL of reverse transcriptase reaction mixture and 12.5 μL of Master Mix (Applied Biosystems) on an ABI PRIMS™ 7700 Sequence Detection System (Applied Biosystems), according to the manufacturer’s protocol. Ribosomal RNA was amplified in the same reaction. Both the rRNA primers and probes were obtained from PE Applied Biosystems (Cat. 4308329). Amplification used a touch-down PCR protocol beginning with 94°C for 5 min., followed by seven cycles at 95°C for 45 seconds, annealing temperatures starting at 62°C for 45 seconds (decreasing by 1°C/cycle) and 72°C for 90 seconds for extension. This step was followed by 40 cycles at 95°C for 45 seconds, 55°C for 45 seconds, 72°C for 90 seconds and a final extension at 72°C for 10 min. The expression level of the tumors was calculated by normalizing that of ARHI to that of the rRNA. Tumors that showed a lower ARHI level than normal brain tissue were interpreted as decreased. Expression results were analyzed by the comparative threshold cycle (ΔΔCt) method according to the User Bulletin No. 2 (Applied Biosystems). All experiments were performed in duplicate. BisulfiteTreatment and Combined Bisulfite Restriction Analysis. Twenty-one glial brain tumor samples were evaluated for loss of functional allele resulting from allele-specific methylation of CpG is land I, II and III. Bisulfite treatment of DNA was performed according to a modified standard protocol. The COBRA was performed as described in [16]. DNA from lymphocytes of a healthy volunteer was treated with Sss1 methyltransferase (New England Biolabs, Beverly, MA, USA), then subjected to bisulfite treatment as a positive control. After cleaning the modified DNAs using the GeneClean III Kit (Bio 101 system; Q-Biogen, Vista, CA, USA), 1 μL (100 ng/ml) DNA was amplified in 50 μL reaction mixtures containing 5 μL of 10 × PCR buffer, 3 μL of 25 mM MgCl2, 1.5 μL of 40 mM dNTPs, 2.5 μL of each primer (10 pmol/ μL) and 0.2 μL (2 U/μL) of Platinum AmpliTaq Gold DNA polymerase (Invitrogen). Primers were designed based on the nucleotide sequence of the ARHI gene submitted to GenBank (AF202543). Primers used for COBRA were: CpGI-F/R, 5’-GTA AGG GAG AAA GAA GTT AGA-3’/5’-TAC TAT CCT AAC AAA ACC CTC-3’; CpGII-F/R, 5’-GTT GGG TTA GTT TTT TAT AGT TGG TT-3’/5’-AAC CAA ACA ACC TAA AAA ACA AAT AC-3’; CpGIII-F/R, 5’-GTT TTT AAG TTT TAT AGG AAG ATT-3’/5’-ATA ATA TAC AAA AAA AAC ACA CC-3’. Amplification used 94°C for 5 min.; 35 cycles at 94°C for 50 seconds, 57°C for 1 min. (for CpG II); 60°C for 1 min. (for CpG I and III) and 72°C for 50 seconds and with a final extension at 72°C for 7 min. After amplification, the PCR products were digested with restriction enzyme TagI (New England Biolabs) for CpG I and BstUI (New England Biolabs) for CpG II and III, (both enzymes recognize and cut methylated DNA). DNA samples were fractionated using 8.0% polyacrylamide gel. The gels were stained with ethidium bromide and the intensity of methylated alleles was calculated using DigiDoc 1000 software (Alpha Innotech, San Leandro, CA, USA). Results above 75.0% and 35.0- 74.0% were interpreted as hypermethylation and partial methylation, respectively. Loss of Heterozygosity Analysis. To study the allelic deletion at chromosome 1p31, the five highly polymorphic microsatellite markers D1S226, D1S488, D1S430, D1S207 and D1S2638 (http://www.gdb.org) were used. Primers for detection of LOH were as follows: D1S226-F/R, 5’-6-FAM-GCT AGT CAG GCA TGA GCG-3’/5’-6-FAM-GGT CAC TTG ACA TTC GTG G-3’; D1S488-F/R,5’-6-FAM-GCA AAA CAG AGA CTT CAC CT-3’/5’-6-FAM-CTT CCA GGG ACT AGA ATG G-3’; D1S430-F/R, 5’-6-FAM-TCC AGA TTT AGT GTC ATT TCC C-3’/5’-6-FAM-CAC TTA CAG TAA CAA GCC CCA G-3’; D1S207-F/R, 5’-6-FAM-CAC TTC TCC TTG AAT CGC TT-3- ’/5’-6-FAM-GCA AGT CCT GTT CCA AGT CT-3’; D1S2638-F/R, 5’-6-FAM-CTT GGA TTG GTG GGT ACT A-3’/5’-6-FAM-AGG TTT CAG GGT GGC T-3’. The PCR used 100 ng/mL of DNA in a 25 μL reaction mixture that contained 2.5 μL of 10 × PCR buffer, 0.2 μL of 5 U/μL of Taq DNA polymerase (Invitrogen), 3 μL of 25 mM MgCl2, 1.5 μL of 20 mM dNTPs and 1.5 μL of 10 pmol/μL of the primers mentioned above. Amplification used: initial denaturation at 94°C for 5 min.; 32 cycles at 94°C for 45 seconds, 50°C for 45 seconds and 72°C for 1 min. and with a final extension at 72°C for 7 min. for markers D1S226, D1S488, D1S207, D1S2638. For the D1S430 marker: initial denaturation at 94°C for 5 min.; 32 cycles at 94°C for 45 seconds, 55°C for 45 seconds and 72°C for 1 min. with a final extension at 72°C for 7 min. The PCR products were diluted 30-fold, then 1.5 μL was added to 23.5 μL formamide with 0.5 μL Genescan™ 500 LIZ TM size standards (Applied Biosystems). DNA fragments were separated by capillary electrophoresis and the signals were detected with DNA sequencer 310 (Applied Biosystems, Perkin-Elmer Corporation) The results were analyzed by Genescan collection and analysis software (Applied Biosystems, Perkin-Elmer Corporation). Scoring of LOH was performed by Genescan output. If the ratio of the normal and tumor DNA peak heights were ≤0.5 and ≥1.5, it was intrepeted as LOH of the long allele and of the small allele, respectively. The presence of LOH was calculated with a formula mentioned below.

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