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Infertility, defined as inability of couples to achieve clinically or biochemically recognizable pregnancy after at least one year of sexual intercourse, occurs in about 15% of the world's population. Male infertility is responsible for 40-50% of the cases. In men, the genetic causes account for about 30% affecting fecundity by endocrinologic, anatomic and idiopathic mechanisms. Cystic fibrosis (CF) mutations are involved in many forms of obstructive azoospermia: congenital bilateral absence of vas deferens (CBAVD), congenital unilateral absence of vas deferens (CUAVD) (Morrela A. et al. 2005). CFTR express in epithelial cells of exocrine tissues such as the lungs, sweat glands, pancreast, vas deferens and in postmeiotic round spermatids for converting haploid spermatids into spermatozoa, lots of events occur such as nucleus condensation and decrease in cytoplasm volume by reduction of intracellular water content (Blau et al., 2002, Radpour et al. 2008a). Maximal CFTR expression needs for promoting this developmental stage (Rosenberg et al. 2004, Radpour et al. 2006). A genital form of cystic fibrosis (CF) is congenital bilateral absence of the vas deferens (CBAVD). This abnormality is responsible for 2%-6% of male infertility. The incidence of CF and thus CBAVD differ in various populations. 97%-98% of men with cystic fibrosis are CBAVD. In these patients transfer of spermatozoa from testis or epididymal structures to external genital tract is blocked. The normal CFTR gene encodes a 1480 amino acid glycoprotein known as CFTR (Daudin et al. 2000, Claustres et al. 2005, Radpour et al. 2005, Xu et al. 2007, Radpour et al. 2008b).
Certain biological processes including translocation of various substrates across membranes and non-transport-related process such as translation of RNA and DNA repair are performed by ABC transporters, this system contains transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis (Pohl et al. 2005, Rees et al. 2009). They transport many different substrates across extra - and intracellular membranes, such as metabolic products, sterols, lipids and drugs.
ABC transporters are involved in tumour resistance, cystic fibrosis, bacterial multidrug resistance, and a range of other inherited human diseases (Hollenstein et al. 2007, Hvorup et al. 2007, Kadaba et al. 2008). The protein has two 6-membered membrane spanning domains linked by a polypeptide chain called the R-domain which contains numerous phosphorylation sites (Naama et al.1995, Jarvi et al. 1998, Anzai et al.2003, Grangeia et al. 2004, Wong et al. 2004). There are also two nucleotide binding folds (NBF). In this study we investigated frequency of Î”F508 and Î”I507 mutations in 100 infertile men.
Materials & methods:
Patients: One hundred twenty patients were precisely selected from different infertile men treated for infertility in the Isfahan Infertility Center (IIC), between 2008 and 2009. By surveying profile of different patients, obstructive and non-obstructive azoospermia men were selected. The mean age of the patients was 31.5 years, ranging from 23 to 48 years. Eighty five non-obstructive azoospermias, 15 patients with congenital bilateral absence of vase deferens (CBAV) and 60 fertile healthy men (as controls) are studied here.
Primer design: Two Î”F508 and Î”I507 mutations, from the exon 10 of CFTR gene, were selected here. The sequence of the gene CFTR was achieved from NCBI nucleotide website. The specificity of the primers was analyzed using Oligo®6 software. More comparison between designed primers and the exon 10 sequence was performed by CLC software. Sequences of primers are shown in table1.
DNA extraction: Two ml blood was collected from each patient and/or normal men as controls. Tubes contain EDTA was used here, for preventing blood clotting. The collected blood samples were promptly shacked gently and kept on ice until extraction. Genomic DNA was extracted from blood by using salting-out procedure. All DNA samples were diluted to the same concentration (50 ng/µl).
Multiplex-ARMS-PCR: Newton et al. (1989) described the amplification refractory mutation system (ARMS) as a general technique for the analysis of any point mutation or small deletion. The basis of the system is the observation that an oligonucleotide containing mismatch at its 3' OH residue will not function as primers in the PCR under optimised conditions. A typical ARMS test consists of two complementary reactions. The first reaction contains an ARMS primer specific for the normal DNA sequence and cannot amplify mutant DNA at a given locus. Similarly, the second reaction contains a mutant-specific primer and does not amplify normal DNA (table 2). The genotype of an individual can be determined by analysis of the amplification products. A normal individual generates PCR product only in the normal reaction; a heterozygote gives products in both reactions, and a homozygous mutant individual does so only in the mutant reaction. In this study Multiplex-ARMS-PCR was used for detection of Î”F508 and Î”I507 mutations.
Four primers were used here for amplification of three fragments. Amplification of normal sequence by certain ARMS primers gives two fragments a 173 bp and a 123 bp. On the other hand, amplification of mutant sequence by specific ARMS primers also gives two fragments a 170 bp and a 120 bp. Therefore, FC and RC primer pair produces a 240 bp fragment. DFN and FC primers produce 173 bp fragment. A 123 bp fragment is produced by using IFN and RC primers. In similar, DFM and FC primers produce 170 bp fragment, and IFM and RC primers produce 120 bp fragment. DNA amplification was carried out in duplicate for all the samples. The PCR machine Ependorff Mastercycler AG22331 was used here. Each 25 reaction mixture contained 3 µl template DNA (50-100 ng), 1.6µl MgCl2 (2.0 Mm), 2.5 µl 10X PCR buffer, 1.5 µl FC (20 pmol mlË‰¹), 1.5µl RC (20 pmol mlË‰¹), 0.75 µl DFN (20pmol mlË‰¹), 0.75 µl IFN (20 pmol mlË‰¹), 1 µl dNTP mix (10 mM), 0.4 µl Taq polymerase (5U), 12 µl ddH2O. The reaction mixtures were prepared and kept on ice until the heating block of the thermal cycler reached the denaturation temperature (94°C). The PCR amplification was carried out at 94°C for 10 minutes and then subjected to 32 amplification cycles; each cycle was 40" minutes at 94°C, 1 minute at 58.8°C, 1 minute at 72°C. This was followed by a final extension at 72°C for 10 minutes. Amplification products were separated by electrophoresis using a 2.5% metaphor agarose gel, stained with Ethidium Bromide DNA gel stain, and visualized by ultraviolet illumination. The gel images were captured using a CCD camera linked to an image processing system.
DeltaF508 and Î”I507 mutations were studied by Multiplex-ARMS technique. The CTT/CTT, CTT/-, -/- genotypes of Î”F508 mutation were observed in 100%, 0%, 0% of the control group and in 97%, 2%, 1% of patients (table3). Compared with CTT/CTT wild genotype, a significant correlation wasn't found between CTT/- , - /- genotype and infertility(OR= 0.970, confidence interval CI= 0.937-1.004, P= 0.179). None of the samples had Î”I507 mutation. CAT/CAT wild genotype of Î”I507 mutation was observed in 100% of the control and patient groups.
One hundred infertile men (Iranian) have been studied here. Normal fertile fathers were considered and studied here as the control group. Two different sets of multiplex reaction were used in order to screen two different mutations (Î”F508 and Î”I507) in these patients. Four kinds of primers (FC, RC, IFN, and DFN) were included in the first reaction mixture. The second one had also four kinds of primers (FC, RC, IFM, and DFM). The later reaction could amplify the mutant CFTR. The location of two deletion mutations Î”F508 and Î”I507 are shown in Fig 1. The sequences of two mutations are aligned with the normal CFTR sequence in this Fig. The locations of the all primers are illustrated on the normal CFTR sequence in the Fig 2. The PCR amplified fragments are shown in Fig 3. If there is Î”F508 mutation, DFM and FC primers amplify CFTR and produce a 170 bp fragment. These are schematically drawn in the Figure 1. In this figure the control primers are shown in black horizontal oblongs. So the sizes of different amplified fragments are also indicated in this Figure. IFM and RC primers amplify either the sequence with Î”I507 or Î”F508 mutation, but they don't amplify normal sequence (Fig 2 and 3). IFN and RC primers also amplify normal sequence, so they couldn't amplify any of Î”F508 and Î”I507 mutations. DFN and FC primers amplify either normal sequence or Î”I507 mutation. DFM and FC primers amplify Î”F508 mutation (170), so they don't amplify neither normal nor Î”I507 mutation (these are summarized in table 1). Two 170 bp and 120 pb fragments were seen in two different infertile men, indicating the existence of Î”F508 in their CFTR gene. Amplification of three fragments in their PCR with the normal primers set, determined that they also have the normal CFTR gene too. It was concluded that these two patients are heterozygote for the Î”F508 (Fig. 3). One patient was also detected as a homozygote of Î”F508, so the mutant primer set could amplify three fragments for this person (Fig. 3). While there was no amplification with the normal primers set. Obviously, the control fragment was amplified with the mentioned primers set. The accuracy of the performance of PCR was confirmed by the observation of this amplified fragment. The Frequencies of CTT genotypes in control & patients are summarized in Table 3. No individuals (of the patients under investigation in this research) were identified to carry the Î”I507 mutation.
Multiplex-ARMS was performed by Ferrie et al. for detection of Î”F508 and Î”I507 (1992) in cystic fibrosis patients of England. They have found that 80.7% of these patients had Î”F508 and 0.5% of them had Î”I507. Radpour et al. (2007) investigated Î”F508 frequency in Iranian infertile men by SSCP -PCR. They have found that 17 men out of 112 CBAVD patients were heterozygote for Î”F508. Schijver et al. (2005) investigated Î”I507 Hispanics that had below 1% frequency. Schijver et al. (2005) investigated Î”I507 mutation in the ethnic diversity of the U.S. population by Microarray method. Richard M. Ferrie et al. (1992) were carried out Multiplex-ARMS for detection Î”F508 and Î”I507 mutations. They used four Multiplex-ARMS reactions for each sample. Although they have included four primers in each of their PCR sets, but two amplified fragments were produced from those sets. So, they had to use AAT1/2 primers (in order to amplify a 220 bp fragment of exon V of Î±1-antitrypsin gene) or AAT3/4 primers (in order to amplify a 360bp fragment of exon III of this gene) (Newton et al., 1989) as their control primers. Detection of the frequencies of these two mutations was carried out her in Iran for the first time, by the Multiplex-ARMS-PCR. Here we used two different primer sets, each capable of amplifying three different sizes fragments. Only four primers were included in each set, in which two of them considered as control. Here we reported that 2% of the patients under investigation were detected to carry Î”F508 mutation. On the other hand, it's been observed that about 1% of this population is homozygote for Î”F508 mutation (Table 3).