Balkan Journal of Medical Genetics

RAPID DETECTION OF FETAL ANEUPLOIDIES BY QUANTITATIVE FLUORESCENT-POLYMERASE CHAIN REACTION FOR PRENATAL DIAGNOSIS IN THE TURKISH POPULATION
Guzel AI, Yilmaz MB, Demirhan O, Pazarbasi A, Kocaturk-Sel S, Erkoc MA, Inandiklioglu N, Ozgunen FT, Sariturk C
*Corresponding Author: Associate Professor Ali Irfan Guzel, Department of Medical Biology and Genetics, Faculty of Medicine, Rize University, 53100, Rize, Turkey; Tel.: +90-464-212-30-09; Fax: +90- 464-212-30-15; E-mail: aliirfanguzel@hotmail.com
page: 11

MATERIALS AND METHODS

The majority of prenatal samples (98.99%) were AF (the investigated pregnancies were from the South Anatolia region of Turkey) (n = 1874), collected between 14 and 26 weeks of gestation for our routine diagnostic tests at the Department of Medical Biology and Genetics, Faculty of Medicine, Çukurova University, Adana, Turkey. A total of four FB samples (0.2%) and 15 fetal tissues (FT) from aborted fetuses (0.8%) were also investigated. The most frequent indications for QF-PCR diagnosis of common chromosome aneuploidies were advanced maternal age, abnormal serum a-fetoprotein (AFP) levels and abnormal ultrasound fi ndings. All the women who participated were informed about the advantages and limitations of the assay. Some AF samples that were dark in color instead of bright transparent yellow or contaminated with blood, were treated with distilled water in order to wash away possible maternal cell contamination (MCC). As a result of MCC, if additional peaks were observed in the electrophoretograms, these suspected samples were tested simultaneously with corresponding maternal blood DNA samples to detect fetal and maternal STR patterns by comparing them to each other. All trisomic samples were validated with conventional karyotype analysis following QF-PCR testing. DNA extraction for QF-PCR was performed on AF, FB and FT samples by incubating cell pellets with InstaGeneTM Matrix (Bio-Rad Laboratories, Hercules, CA, USA). The QF-PCR amplifi cations were performed using AneufastTM (Molgentix SL, Barcelona, Spain) trisomy detection kit that included dNTPs, Hot Start Taq DNA polymerase and fl uorescently- labeled primers for 27 STR markers (Table 1) located on chromosomes 13, 18, 21, X, and Y, and primer sets for amelogenin (AMXY) gene and sexdetermining region Y (SRY) regions, which were used for the detection of fetal sex in six different multiplex (employing two or more sets of primers in one reaction condition) PCR mixtures containing specifi c primers for the chromosome of interest STR regions in an optimized reaction mixture. Reactions were prepared by combining 10 μL of the STR/PCR mixtures (referred as S1 and S2, which contain primer sets for the STR regions of chromosomes 13, 18, 21, X and Y), 5-10 ng of amniotic cell DNA and PCR-grade water up to a total volume of 15 μL and thermal cycled (15 min. at 95°C followed by 25 cycles of 40 seconds at 95°C, 90 seconds at 60°C and 40 seconds at 72°C, with a fi nal extention at 60°C for 30 min.) according to the manufacturer’s protocol. The QF-PCR products [1.5 mL from each of the two main mixes (S1 and S2)] were added into 40 μL Hi-Di™ Formamide (Applied Biosystems, Foster City, CA, USA), containing 0.3 μL of GeneScan™-500LIZ™ (Applied Biosystems) used as a size standard. After denaturation at 95°C for 3 min., the mixtures were allowed to cool down to 4°C and then capillary electrophoresis was carried out on an ABI PRISM™ 3130 Genetic Analyzer (Applied Biosystems) using a POP7 polymer. Analysis of the results and calculations of the peak areas (peak area is the area under a specifi c STR peak) were performed using GeneMapper 4.0 software (Applied Biosystems). The criteria and guidelines for the determination of the QF-PCR results of normal and pathological cases were as follows: in normal individuals who were heterozygous for a given STR, the same amount of fl uorescence was generated for both alleles. Therefore, the ratio between the fl uorescent peak areas was 1:1 (ratios are calculated by dividing the area of the shorter allele in base pairs by the area of the longer one). In normal individuals who are homozygous (have the same repeat number) for a specifi c STR allele, the quantifi cation is not possible so the marker is regarded as uninformative, and the ratio is therefore considered as 1. In trisomic cases, the three copies of a specifi c chromosome can be detected as a 1:1:1 ratio (trisomic triallelic) or as a 2:1 ratio (trisomic diallelic) patterns when their peak areas are compared. Assessment of a normal or trisomic copy number is concluded when at least two informative markers were detected for the chromosome of interest (Aneufast User Manual; Molgentix SL).

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