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
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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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