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Technical Briefs |
Kámorya1 Genodia Molecular Diagnostics Ltd., Bajaki Ferenc 1-3, 1211 Budapest, Hungary
aauthor for correspondence: fax 36-1-4270350, e-mail kamory.eniko{at}genodia.hu
Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasian populations and is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The CFTR gene encodes a transmembrane protein that forms a cAMP-regulated chloride channel. Wide variations in disease manifestations are observed among affected CF patients, and a multitude of disease-causing mutations have been found in the CFTR gene (1). In 97% of men with CF, bilateral congenital absence of the vas deferens (CBAVD) blocks the transport of spermatozoa from testicular structures to the distal genital tract, causing azoospermia (2). Infertility attributable to CBAVD does not necessarily coincide with other manifestations of CF. CBAVD accounts for 
1–2% of all male infertility and at least 6% of the cases of obstructive azoospermia.
Isolated CBAVD patients carry either a CF mutation (
F508 in 16–83% of cases and R117H in 6–29% of cases) and/or a 5T variant in intron 8 (12–47% of patients), supporting the hypothesis that CBAVD represents a mild, primary genital form of CF (2)(3)(4)(5)(6)(7). Three length variations of a polythymidine (polyT) tract within the splice acceptor site in intron 8 of the CFTR gene (GenBank accession no. M55106) have been associated with variable efficiency of exon 9 splicing (8). On the basis of the increased frequency (compared with the general population) of the five-thymidine (5T) variant [vs seven or nine thymidines (7T or 9T)] in CBAVD patients, the 5T variant was classified as a CBAVD mutation (7).
Currently available but time-consuming methods for 5T/7T/9T genotyping include PCR amplification followed by acrylamide gel electrophoresis (9)(10), detection of mRNA length (2), restriction endonuclease digestion (7), single-strand conformation polymorphism analysis and direct sequencing (11), capillary zone electrophoresis (12), and dot-blot hybridization (12).
We have developed a much faster, sensitive, single-step method to detect the IVS8 5T variant in CBAVD patients. Our method is based on the LightCycler technology (Roche) using rapid PCR followed by analysis of the melting behavior of fluorescently labeled hybridization probes (13). The sensor probe covers the 5T/7T/9T polymorphic site and was designed against the 5T allele. In probe design, we use the fact that a long (TG)9–12 repetitive sequence is located adjacent to the 5T/7T/9T polythymidine tract (Fig. 1A
). By design, in the cases of the 7T and 9T alleles, the sensor probe slips on the TG tract and anneals to the template DNA with one and two mismatches, respectively (Fig. 1A
). Hence, the sensor probe targeting the polymorphic mutation site melts off the 7T and 9T alleles at lower temperatures than the 5T allele. The anchor probe, which is in a head-to-tail arrangement with the sensor probe and plays role in the fluorescence resonance energy transfer, was designed to have a significantly higher melting temperature than the completely matching sensor probe. Because the sensor probe does not completely cover the entire length of the TG tract, the length of the variable TG repeat should not affect the melting curve. Thus, the TG repeat length cannot be detected by this technique, although the TG repeat may influence alternative splicing when it is activated by the 5T allele (14)(15)(16).
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Genomic DNA was extracted from the peripheral blood of patients with oligozoo-azoospermia by use of genomic DNA purification reagents (High Pure PCR Template Preparation Kit; Roche). Polymorphism analysis was obtained from PCR amplification and melting curve analysis using a LightCycler instrument. In the reaction, the LightCycler DNA Hybridization Probes buffer (Roche) was used with a final MgCl2 concentration of 3 mM. PCR was performed in a reaction volume of 10 µL containing 0.5 µM each primer (forward, 5'-tgacaaactcatcttttatttttgatg-3'; reverse, 5'-caaccgccaacaactg-3'), 0.2 µM each of the anchor (5'-tgttattgttttgttttgctttctcaaataattcccca-Bodipy 630/650-3') and the sensor (5'-6-FAM-tccctgttaaaaacacacacacacaca-phosphorylated-3') probes. The anchor probe was labeled at the 3' end with Bodipy 630/650, whereas the sensor probe was labeled at the 5' end with 6-carboxyfluorescein (6-FAM) and modified at the 3' end by phosphorylation to block extension. The primers and probes were designed and manufactured by Genodia Molecular Diagnostics Ltd. The PCR program included an initial denaturation at 95 °C for 30 s (temperature transition rate, 20 °C/s), followed by 40 cycles of denaturation at 95 °C for 0 s (temperature transition rate, 20 °C/s), annealing at 58 °C for 10 s (temperature transition rate, 20 °C/s), and extension at 72 °C for 10 s (temperature transition rate, 20 °C/s). Melting curve analysis was performed after amplification, starting with heating (95 °C for 0 s) and cooling (50 °C for 30 s) steps for renaturation of the hybridization probes. Subsequently, the temperature was increased to 75 °C with a slow transition rate (0.1 °C/s). The fluorescent signal (F) was continuously monitored at 640 nm during the temperature ramp and plotted against temperature (T) to obtain melting curves for the samples. The melting curves were converted to derivative melting curves (–dF/dT). The derivative melting curves of the 5T/9T and the 7T/9T genotypes and a negative control are shown in Fig. 1B
. The melting peaks were at 56 °C for the 9T allele, 61.5 °C for the 7T allele, and 65.5 °C for the 5T allele.
To confirm the LightCycler genotyping results, we tested three samples with different genotypes from the CF Center of the University of North Carolina and three other samples from the Ludwig Boltzmann Institute of Vienna. To validate the LightCycler method, all samples with known genotypes, as well as 24 samples from randomly selected patients, were analyzed by SNaPshot minisequencing on an ABI 310 genetic analyzer. Although the length of the (TG)9–12 repetitive sequence preceding the polymorphic region could not be determined by this technique, the genotyping results were identical to our real-time PCR data in all of the cases.
To validate the applicability of the new method and to investigate whether the frequency of the 5T allele differs between CBAVD patients and controls, we analyzed a larger patient population. One hundred and fifty-seven patients with oligozoospermia and azoospermia (sperm count <5 x 106/mL) were selected from in vitro fertilization centers and hospital departments of andrology. Samples from these patients were analyzed for a small CFTR mutation panel by real-time PCR using hybridization probes followed by melting curve analysis (data not shown). This mutational panel tested consisted of the 5T variant and frequent CF mutations in CBAVD: F508 and R117H.
The genotypes of the 157 samples tested for the panel of CFTR mutations are shown in Table 1
: 11 samples (7%) carried the heterozygous 5T variant without other CF mutations, whereas 3 samples were heterozygous for both F508 and 5T (2%). Regarding this configuration, Groman et al. (16) and Noone et al. (14) found that the 5T allele is always in trans position with F508, producing male infertility, nonclassic CF, or a normal phenotype. Four of our samples (2.5%) carried only one mutation (F508) without the 5T variant, but no homozygous 5T was detected. There were no homozygous carriers for F508 or R117H, or carriers of both F508 and R117H alleles. All of the patients carrying the 5T allele had been diagnosed with azoospermia; one of them had ductus deferens agenesis. For the rest of the cases. no such clinical data were available. The frequency of the 5T genotype (9%) in our patient population was higher than the frequency published (7) for the general European Caucasian population (5%).
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In summary, the proposed real-time PCR using hybridization probes coupled with melting curve analysis is a rapid and sensitive alternative to other known methods for detection of the CFTR 5T/7T/9T polymorphism.
Acknowledgments
The control samples with different genotypes were generously provided by Kenneth Friedman and Peadar Noone of the CF Center from the University of North Carolina (Chapel Hill, NC) and Klaus Huber at the Ludwig Boltzmann Institute for Molecular Genetic Laboratory Diagnostics (Vienna, Austria). The technical help of Judit Bali is acknowledged.
References
The following articles in journals at HighWire Press have cited this article:
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  J. K. Bickmann, W. Kamin, M. Wiebel, F. Hauser, J. J. Wenzel, C. Neukirch, M. Stuhrmann, K. J. Lackner, and H. Rossmann A Novel Approach to CFTR Mutation Testing by Pyrosequencing-Based Assay Panels Adapted to Ethnicities Clin. Chem., June 1, 2009; 55(6): 1083 - 1091. [Abstract] [Full Text] [PDF]
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