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.



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