Balkan Journal of Medical Genetics

STUDY OF THREE SINGLE NUCLEOTIDE POLYMORPHISMS IN THE SLC6A14 GENE IN ASSOCIATION WITH MALE INFERTILITY
Noveski P1, Mircevska M1, Plaseski T2, Peterlin B3, Plaseska-Karanfilska D1,*
*Corresponding Author: 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 Macedonia. Tel. +389-2-3235-410. Fax: +389-2-3115-434. E-mail: dijana@manu.edu.mk
page: 61

MATERIALS AND METHODS

Study Samples. A total of 370 infertile males (247 from Slovenia and 123 from Macedonia) and 237 fertile controls (114 from Slovenia and 127 from Macedonia) were studied. The infertile group consisted of 137 patients with idiopathic azoospermia (87 from Slovenia and 50 from Macedonia) and 243 with oligozoospermia (160 from Slovenia and 73 from Macedonia). Patients with sex chromosome aneuploidies and Y chromosome AZF microdeletions were excluded from the study. Fertile controls have fathered at least one child and their paternity was proven by DNA analysis. Informed consent was obtained from all men and the study was approved by the Ethics Committee of the Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia. DNA Isolation. DNA was isolated from peripheral blood using the standard phenol/chloroform protocol. Multiplex Polymerase Chain Reaction. The PCR primers were designed to produce different PCR fragment sizes, so as to make their separation by agarose gel electrophoresis possible. Primer sequences for PCR amplification of the three SLC6A14 SNPs are as shown in Table 1. Polymerase chain reaction multiplexes were performed in a final volume of 20 μL, containing: 10 × KLA buffer [50 mM Tris base, 16 mM (NH4)2SO4, 0.1% Tween 20, pH 9.2], 25 mM MgCl2, 0.25 mM dNTPs, 10 pmol of each primer, Tth DNA polymerase and approximately 100 ng of genomic DNA. The cycling conditions were: 2 min. 95 °C initial denaturation, followed by 28 cycles of 40 seconds at 95 °C, 40 seconds at 59 °C, 45 seconds at 72 °C, and final elongation at 72 °C for 10 min. A rapid thermal ramp at 4 °C followed and the PCR products obtained were analyzed with electrophoresis on a 1.5% agarose gel. Multiplex SNaPshot Analysis. Before performing the SNaPshot reaction, 1 μL of PCR product was treated with 0.6 μL of ExoSAP-IT (Exonuclease I and Shrimp Alkaline Phosphatase; USB Corporation, Clevelend, OH, USA) at 37 °C for 2 hours or overnight. The reaction mixture was incubated at 86 °C for 20 min. to inactivate the ExoSAP-IT. The purified PCR products were used as templates to detect the three polymorphic positions in the SLC6A14 gene. The detection primers were mixed with final concentration of 1 pmol/μL each. Multiplex single base extension reactions were performed in a 4.6 μL final volume, combining 1 μL of SNaPshot Multiplex Ready Reaction Mix (Life Technologies, Carlsbad, CA, USA), 1 μL of deionized H2O, 1.6 μL of purified PCR product and 1 μL (1 pM) SNaPshot primer cocktail. The minisequencing primers were 5’-tailed with a polyT sequence to produce extension products 25, 30 and 35 nucleotides long to allow separation by capillary electrophoresis (Table 2). Cycling conditions were: 25 cycles of 10 seconds at 96 °C, 10 seconds at 50 °C and 30 seconds at 60 °C, followed by rapid thermal ramp to 4 °C. To remove unincorporated fluorescently-labeled ddNTPs, the final products were incubated with 1 U of shrimp alkaline phosphatase (USB Corporation) for 1 hour at 37 °C (or overnight) and then at 86 °C for 20 min. to inactivate, the enzyme. Capillary Electrophoresis. The SNaPshot products were separated by capillary electrophoresis on an ABI PRISM™ 3130 Genetic Analyzer (Life Technologies). Analysis of electropherograms was performed using the GeneMapper software (Life Technologies) and the sizes of the fragments was determined relative to the GeneScan120 LIZ size standard (Life Technologies). Representative electropherograms that show the three SNPs in the SLC6A14 gene in a patient with ACG (a) and a patient with TTC haplotypes (b), are shown in Figure 1. Statistical Analysis. Allelic and haplotype frequencies were compared with statistical non parametric tests for categorical variables Pearson c2 using the Statistical Package for Social Sciences Version 19 (SPSS, Chicago, IL, USA). Haplotype frequencies were calculated with Haploview 4.2 (Broad Institute, Cambridge, MA, USA) [17]. A p value of less than 0.05 was considered to be statistically significant. Analysis of RNA Secondary Structure. Effect of different SNP alleles on RNA secondary structure was analyzed with RNAsnp web tool [18] that is a freely available web tool (http://rth.dk/resources/ rnasnp/submit). We used two of the proposed modes of operation, Mode 1 and Mode 2, with default settings for folding window (200 bp) and default associated parameters for each mode. The two modes use different methods of calculation; Mode 1 uses a global folding method RNAfold, while Mode 2 uses a local folding method RNAplfold. Structural changes with a p value of less than 0.2 were considered to be statistically signifi cant.

Number 26 VOL. 26(1), 2023	Number 25 VOL. 25(2), 2022
Number 25 VOL. 25 (1), 2022	Number 24 VOL. 24(2), 2021
Number 24 VOL. 24(1), 2021	Number 23 VOL. 23(2), 2020
Number 22 VOL. 22(2), 2019	Number 22 VOL. 22(1), 2019
Number 22 VOL. 22, 2019 Supplement	Number 21 VOL. 21(2), 2018
Number 21 VOL. 21 (1), 2018	Number 21 VOL. 21, 2018 Supplement
Number 20 VOL. 20 (2), 2017	Number 20 VOL. 20 (1), 2017
Number 19 VOL. 19 (2), 2016	Number 19 VOL. 19 (1), 2016
Number 18 VOL. 18 (2), 2015	Number 18 VOL. 18 (1), 2015
Number 17 VOL. 17 (2), 2014	Number 17 VOL. 17 (1), 2014
Number 16 VOL. 16 (2), 2013	Number 16 VOL. 16 (1), 2013
Number 15 VOL. 15 (2), 2012	Number 15 VOL. 15, 2012 Supplement
Number 15 Vol. 15 (1), 2012	Number 14 14 - Vol. 14 (2), 2011
Number 14 The 9th Balkan Congress of Medical Genetics	Number 14 14 - Vol. 14 (1), 2011
Number 13 Vol. 13 (2), 2010	Number 13 Vol.13 (1), 2010
Number 12 Vol.12 (2), 2009	Number 12 Vol.12 (1), 2009
Number 11 Vol.11 (2),2008	Number 11 Vol.11 (1),2008
Number 10 Vol.10 (2), 2007	Number 10 10 (1),2007
Number 9 1&2, 2006	Number 9 3&4, 2006
Number 8 1&2, 2005	Number 8 3&4, 2004
Number 7 1&2, 2004	Number 6 3&4, 2003
Number 6 1&2, 2003	Number 5 3&4, 2002
Number 5 1&2, 2002	Number 4 Vol.3 (4), 2000
Number 4 Vol.2 (4), 1999	Number 4 Vol.1 (4), 1998
Number 4 3&4, 2001	Number 4 1&2, 2001
Number 3 Vol.3 (3), 2000	Number 3 Vol.2 (3), 1999
Number 3 Vol.1 (3), 1998	Number 2 Vol.3(2), 2000
Number 2 Vol.1 (2), 1998	Number 2 Vol.2 (2), 1999
Number 1 Vol.3 (1), 2000	Number 1 Vol.2 (1), 1999
Number 1 Vol.1 (1), 1998

About the journal ::: Editorial ::: Subscription ::: Information for authors ::: Contact