Balkan Journal of Medical Genetics

ANXA5 AND VEGFA GENE VARIANTS IN WOMEN WITH EARLY PREGNANCY LOSSES FROM NORTH MACEDONIA
Terzikj M, Bozhinovski Gj, Branoski A, Dimkovska M, Kubelka-Sabit K, Plaseska-Karanfilska D
*Corresponding Author: Corresponding author, Prof. Dijana Plaseska-Karanfilska, MD, PhD. Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Science and Arts, Skopje, North Macedonia, e-mail: dijana@manu.edu.mk
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MATERIALS AND METHODS

Materials For this study, a total of 380 DNA samples were observed using a multiplex single-base extension reac- tion assay. The samples were taken from 190 women experiencing EPLs, 104 from Macedonian and 86 from Albanian ethnic backgrounds and the same number of samples were obtained from women without a history of pregnancy loss as well as at least one healthy live birth as control group. Following a methodology outlined by Noveski et al., the examined women with EPLs were additionally selected because no fetal chromosomal abnormalities were detected in their POCs [32]. Of the total number of women with pregnancy losses, 81 had single pregnancy loss (sporadic), and 109 had two or more pregnancy losses (recurrent). Also, from the total number of women with early pregnancy losses, 151 did not have a previous live birth, and 39 had. All participants gave informed consent for participa- tion in the study. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Macedonian Academy of Sciences and Arts (09-1047/6 from 04.05.2016). This work was financially supported by the project “Molecular basis of spontaneous abortion” (project number: 08-707) funded by the Macedonian Academy of Sciences and Arts. Methods Blood samples were collected from each participant and utilized for DNA isolation from leukocytes using the conventional method involving phenol/chloroform extrac- tion and ethanol precipitation, as described by Efremov et al. (1999). All individuals were analyzed for the presence of 4 different variants in the ANXA5 gene: rs28717001 (c.-210A>C), rs28651243 (c.-184T>C), rs112782763 (c.- 229G>A), rs113588187 (c.-135G>A) and 2 variants in the VEGFA gene: c.-1154G>A (rs1570360) and c.*237C>T (rs3025039). Taking inspiration from the methodology previously employed in our laboratory [33, 34, 35], we de- signed a multiplex single-base extension method for simul- taneous detection of the 6 variants utilizing the Multiplex SNaPshot kit (Multiplex SNaPshot; Applied Biosystems, Warrington, WA) for the reaction, followed by capillary electrophoresis on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The PCR primers were designed to function in a mul- tiplex mix and to produce PCR fragments of 427–550 bp. Briefly, the PCR amplification reaction contained 2 µL of 100 ng genomic DNA, 1.5 µL of a multiplex mix where each specific forward and reverse oligonucleotide primers are with a 10 pmol concentration (Table 1), 2 µL 2.5mM of each deoxyribonucleotide triphosphate (dNTP), 1.5 µL 25mM magnesium chloride, 5 µL GC rich enhancer and 0.2 µL of 1U Taq polymerase (Hot Fire polymerase; Solis Biodyne, Tartu, Estonia) in a total volume of 25 µL. The PCR conditions were as follows: 10 min. initial denaturation at 95°C, followed by 33 cycles of 1 min at 95°C,1 min at 61°C for the primer annealing and 1 min at 72°C. The final elongation was set at 72°C for 10 min. Afterwards, purification was carried out using 1µL of ExoSAP-IT® (USB, Cleveland, OH) per 2.5 µL of PCR product. The process included an incubation step at 37°C for 20 minutes and subsequent inactivation of the enzyme at 86°C for 20 minutes. The refined PCR products served as the basis for identifying the six mutations. In the subsequent single- base extension reaction, the detection primers (SNaPshot primers) were aligned adjacent to the single-nucleotide polymorphism position. Various lengths of poly (dC) tails were appended to the single-base extension primers (re- fer to Table 2). The SNaPshot reaction included a 1.5µL primer mix comprising all 6 single-base extension prim- ers at 3.5µL of purified PCR products, and 1.5µL of the SNaPshot Multiplex kit (Applied Biosystems), adding up to a final volume of 6.5 µL. The cycling profile com- prised of 25 cycles of 95°C for 10 s, 55°C for 10 s, and 60°C for 30 s. After the reaction, the 5’-phosphophoryl groups of unincorporated dideoxynucleotide triphosphates were eliminated by adding 1.0U of shrimp alkaline phos- phatase (SAP; USB), followed by an incubation step at 37°C for 40 min and at 86°C for 20 min to deactivate the enzyme. Capillary electrophoresis was conducted using an ABI PRISM 3130 Genetic Analyzer, and the results were analyzed usiBiosystems, Foster City, CA). Specifics of the multiplex PCR and SNaPshot reaction products can be found in Table 2. Due to the impact of the dye, size, and nucleotide composition on the mobility shift of DNA fragments, the reported sizes may deviate by a few bases from the actual sizes, especially with shorter fragments where the relative contribution of the dye is more pronounced. Figure 1 dis- plays a representative electrophoretogram from a patient heterozygous for all six variants. The Chi-square test as well as Fisher’s exact test, were employed to assess the significance of the results in both the examined and control groups was determined based on the p-value. A p-value <0.05 was considered statistically significant.ng Gene Mapper, Version 4.0 (Applied

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