A HOMOZYGOUS DELETION OF THE DPY19L2 GENE IS A CAUSE OF GLOBOZOOSPERMIA IN MEN FROM THE REPUBLIC OF MACEDONIA
Noveski P1, Madjunkova S1, Maleva I1, Sotiroska V2, Petanovski Z2, Plaseska-Karanfilska D1,*
*Corresponding Author: Professor Dijana Plaseska-Karanfilska, Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology, Av. Krste Misirkov 2, POB 428, 1000 Skopje, Republic of Macedonia; Tel.: +389-2-3235-410; Fax: +389-2-3115-434; E-mail: dijana@manu.edu.mk
page: 73

MATERIALS AND METHODS

Among more than 200 infertile men, two patients with 100.0% of globozoocephalic spermatozoa with no acrosome in their semen were recruited for genetic study at the Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology (RCGEB) “Georgi D. Efremov”, Skopje, Republic of Macedonia laboratories. Patient 1 was a 30-year-old man with two unsuccessful intra cytoplasmic sperm injection (ICSI) attempts, while patient 2 was a 35-year-old man who has previously experienced three unsuccessful ICSI attempts. Both patients were of Macedonian ethnic origin, living in Tetovo, a town in the western part of the Republic of Macedonia. Although they originated from the same town, they were not related and no consanguinity was reported in their families. The search for mutations in the SPATA16 and PICK1 genes was performed on genomic DNA by sequencing of all exons and exon/intron boundaries using BigDye™ Terminator Cycle Sequencing Ready Reaction Kit on an ABI PRISM™ 3100 Genetic Analyzer (Life Technologies Corporation, Carlsbad, CA, USA). The oligonucleotide primers used for polymerase chain reaction (PCR) and sequencing were designed by Primer 3 software [14] and synthesized by Integrated DNA Technologies, Coralville, IA, USA. The search for copy number variants (CNVs) was performed by oligonucleotide array comparative genomic hybridization (aCGH) using the 180K Agilent Human Genome CGH Microarrays and Genomic Workbench software (Agilent Technologies, Santa Clara, CA, USA). The deletion at 12q14.2, including the entire DPY19L2 gene, was confirmed with classical PCR using seven loci, of which three were intragenic, located on exons 1, 11, and 22 of the DPY19L2 gene, while the other four were located in the low copy repeat (LCR) regions. The primers in the centromeric LCR1 region, named LCRa and LCRb, were localized approximately 25 and 9 kb 3’ of DPY19L2, respectively, and the primers in the telomeric LCR2 region, named LCRc and LCRd, were localized approximately 62 and 77 kb 5’ of DPY19L2, respectively [11]. For further confirmation of the deletion we performed a gap-PCR, using primers flanking the DPY19L2 deletion [10]. This PCR generates a fragment of 1700 bp only in the patients carrying the DPY19L2 deletion.



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