Balkan Journal of Medical Genetics

DELINEATION OF PARTIAL CHROMOSOMAL ABNORMALITIES IN EARLY PREGNANCY LOSSES
Bozhinovski Gj1, Terzikj M1, Kubelka-Sabit K2,3, Plaseska-Karanfilska D1,*
*Corresponding Author: *Corresponding Author: Prof. Dijana Plaseska-Karanfilska, M.D., Ph.D. Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov,” Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000, Skopje, Republic of North Macedonia. Tel: +389-2-3235-410 E-mail: dijana@manu.edu.mk
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MATERIALS AND METHODS

Study group and selection of samples In a prior investigation, partial chromosomal abnor- malities were identified in 16 out of 900 analyzed EPL samples [4]. More recently, an additional four samples exhibiting partial chromosomal abnormalities were de- tected and incorporated into the current analysis, bringing the total number of EPL samples included in this study to 20. These samples consisted of single chromosomal ab- normalities in half, while the other half exhibited double chromosomal abnormalities. All EPL samples referred to the Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, at the Macedonian Academy of Sciences and Arts, Skopje for analysis of chromosomal aneuploidies were selected by a collaborative team of gynecologists and pathologists. Standard histopathological analysis was performed on all EPLs, as described previously [14, 15]. To ensure ethical compliance, written informed consent was obtained from all study participants, and the research protocol received approval from the institutional ethics committee (Macedonian Academy of Sciences and Arts, 09-1047/6 from 04.05.2016). All EPL samples were processed using standard phe- nol-chloroform or automated extraction methods to obtain genomic DNA for further analysis. To initially exclude maternal contamination and to screen for common ane- uploidies, we employed quantitative fluorescent polymer- ase chain reaction (QF-PCR) with a panel of short tandem repeat (STR) markers targeting chromosomes 13, 18, 21, and the sex chromosomes [16]. This method allowed for rapid detection of trisomies and monosomies involving chromosomes 13, 18, 21 and X, as well as triploidies. To complement QF-PCR, multiplex ligation-dependent probe amplification (MLPA), using P036-Subtelomere Mix 1 and P070-Subtelomere Mix 2B probemixes, was implemented to identify chromosomal imbalances on all chromosomes. Array comparative genomic hybridization (aCGH) To further elucidate the genomic architecture of the observed partial chromosomal abnormalities, array com- parative genomic hybridization (aCGH) analysis was con- ducted following Agilent Technologies’ SureTag Complete DNA Labeling Kit (Agilent Technologies, Santa Clara, CA, USA). Genomic DNA extracted from the analyzed samples, along with sex-matched control DNA, was enzy- matically fragmented using the restriction enzymes AluI and RsaI to ensure uniform DNA fragment sizes. Subse- quently, the fragmented DNA samples were fluorescently labeled: the experimental DNA was labeled with Cyanine 5 (Cy5), while the control DNA was labeled with Cyanine 3 (Cy3). After purification, the labeled experimental and control samples were combined and co-hybridized onto Agilent Technologies’ CGH Constitutional G3 4x180k microarrays. Following hybridization, data acquisition was performed using Agilent Technologies’ DNA microarray scanner with surescan technology, ensuring accurate cap- ture of fluorescence signals. The resulting data were subse- quently processed and analyzed using Agilent’s Genomic Workbench software platform. This analysis provided a comprehensive visualization of CNVs, with detailed genomic annotations based on the UCSC hg19 genome assembly, facilitating precise mapping and interpretation of the chromosomal imbalances observed in the study. Identifying Recurrent Chromosomal Regions and Gene Ontology (GO) enrichment analysis To gain insights into the genetic factors underlying early pregnancy loss, we conducted a comprehensive anal- ysis of a total of 32 chromosomal abnormalities detected by aCGH in 20 EPLs with partial chromosomal abnormalities. We have established a criterion for identifying recurrent chromosomal regions: a chromosomal region, included in deletion or duplication, had to be observed in more than two EPLs. We determined the smallest overlapping region (SOR) for each recurrent chromosomal region. This approach enabled us to pinpoint the specific genes and pathways that are likely involved in the pathogenesis of early pregnancy loss. To gain a deeper understanding of the biological func- tions and processes associated with the identified genes, we conducted a Gene Ontology (GO) enrichment analysis utilizing the Panther software [12]. This analysis compares the frequency of specific GO terms within a group of genes involved in SORs. Significantly enriched GO terms were visualized using a bar plot, thus providing a clear and intui- tive way to interpret the results of the enrichment analysis and identify the biological functions and processes and that are most likely to be involved in EPL.

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