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Technical Briefs |
1 Medical Genetics Unit and 2 Biomedical Centre Applied Research CRBA, S.Orsola-Malpighi University Hospital, Bologna, Italy; 3 Department of Experimental Evolutionary Biology, Bologna University, Bologna, Italy;
aaddress correspondence to this author at: U.O. Genetica Medica, Policlinico S.Orsola-Malpighi, Via Massarenti, 9, 40138 Bologna, Italy; fax 39-051-6363851, e-mail mantovan{at}med.unibo.it)
Abstract
Background: The 5T allele of the polyT tract located within intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is a variant that in trans with a severe CFTR mutation can result in normal phenotype, congenital bilateral absence of vas deferens (CBAVD), or mild cystic fibrosis. The 5T allele has been associated with the skipping of exon 9, a process that seems to be influenced by an adjacent 9–13TG tandem repeat. The 12- or 13TG repeats are often associated with an abnormal phenotype. We present here a single-step method for direct haplotyping of the TG repeats in 5T carriers.
Method: The method is based on a single-step PCR, using a fluorescently labeled forward primer and a reverse allele-specific primer matching the 5T allele. We validated the test in 30 control samples of known 5T-poly(TG) haplotype and then used this method to evaluate 57 clinical samples.
Results: The expected TG genotypes were obtained for all 5T control samples, and no nonspecific amplification of either the 7T or 9T alleles was detected. In our 5T-positive collection 9 of 9 (100%) CBAVD patients, 6 of 12 (50.0%) chronic pancreatitis patients, and 12 of 36 (33.3%) individuals undergoing assisted reproduction showed 5T-12TG haplotype.
Conclusions: Our method is an accurate, specific, and simple tool to characterize the 5T poly(TG) haplotype. Our results confirm the high frequency of 5T-12TG in CBAVD patients and do not preclude a potential effect also in pancreatitis. This assay can be useful in assessment of the disease risk in 5T carriers.
Incompletely penetrant mutations that cause disease phenotypes in some but not all individuals can make the interpretation of genetic testing particularly difficult. One such problematic mutation is the abbreviated 5T variant of the polyT tract in intron 8 (IVS8 5T) of the cystic fibrosis transmembrane conductance regulator (CFTR [MIM 602421]) gene.
The CFTR gene is positioned on chromosome 7q31 and spans 250 kb and 27 exons. Mutations in this gene can cause cystic fibrosis (CF [MIM 219700]), the most common recessive disease in the Caucasian population, with an incidence of 1 of 2500 births and a carrier frequency of 1 of 25. The CFTR gene has more than 1500 different mutations (see http://www.genet.sickkids.on.ca/cftr/), which have verifiable pathogenic effects (1)(2).
If the 5T variant occurs in trans with a severe CFTR mutation, it can result in variable phenotypes, ranging from normal phenotype to congenital bilateral absence of the vas deferens (CBAVD) or mild CF (3)(4). Compared with the 2 other known alleles (IVS8 7T and 9T), the 5T allele is associated with decreased efficiency of the intron 8 splice acceptor site, resulting in frequent skipping of exon 9. Experimental evidence indicates that a repeat of 9–13TG dinucleotides, placed immediately upstream of the polyT tract, can further modulate the exon 9 skipping: exon skipping increases proportionally with the number of TG repeats (5)(6). Individuals carrying 5T adjacent to either 12- or 13TG repeats are more likely to exhibit an abnormal phenotype than those with 5T adjacent to <12TG (7)(8)(9). The high prevalence of 5T carriers makes the assessment of the TG repeat number of great interest as a reliable predictor of the 5T allele penetrance.
We describe here a rapid and simple allele-specific fluorescent PCR method for direct haplotyping of the TG repeats in 5T individuals. This method is based on a single-step PCR, using a reverse primer matching the 5T allele plus 1 additional nucleotide at the 3' end. The forward primer was fluorescently D4-labeled (Invitrogen, Corp).
To develop the test we used 30 control samples derived from known carriers of the 5T allele, in which the T-TG tract had previously been genotyped. The specificity of this test was checked against 8 5T-negative control samples. In a further step, we tested our method in a collection of 5T-positive samples. This study complied with the Declaration of Helsinki, and written informed consent was obtained from each participant.
Genomic DNA was isolated from EDTA whole-blood with a QIAamp DNA Blood Kit (Qiagen Inc.). The mutation screening of the CFTR gene was performed with a reverse dot blot-based commercial reagent set, validated for the simultaneous detection of 29 mutations and polyT genotyping (INNO-LiPA CFTR 12 and 17+Tn, Innogenetics Group).
The TG repeat number was genotyped in the control samples by direct sequencing of the T-TG tract amplified with forward 5'-ACC CCG CTT ATA GGA GAA GA-3' and reverse 5'-CGC CAA CAA CTG TCC TCT TT-3' primers by automated capillary electrophoresis (CEQ8000, Beckman Coulter Inc.) and analyzed with Sequencer software (Gene Codes Corp.). In heterozygous ambiguous samples, PCR-amplified products were cloned into a plasmid vector (TOPO TA Cloning, Invitrogen, Corp.) and resequenced.
The allele-specific fluorescent PCR was performed on an GeneAmp PCR System 9700 (Applied Biosystems) with fluorescently-labeled forward primer 5'-D4-GGC CAT GTG CTT TTC AAA CT-3' and the 5T allele-specific reverse primer 5'-CCC CAA ATC CCT GTT AAA AAC-3'; 25 ng of DNA was amplified with 4 pmol of each primer and 35 cycles (95 °C for 30 s, 63 °C for 30 s, and 72 °C for 40 s) were used to amplify products of variable length in a range of 120 bp (5T-10TG) to 126 bp (5T-13TG). The 10-µL PCR mixture contained 10 mmol/L Tris-HCl, 50 mmol/L KCl, 2.0 mmol/L MgCl2, 200 µmol/L each dNTP, and 0.4 units of Taq Gold DNA polymerase (PE Applied Biosystems). PCR product (0.5 µL), 0.5 µL size standard (CEQ DNA Size Standard 400), and 39 µL sample loading solution (CEQ SLS) were run and sized by CEQ 8000 automated capillary electrophoresis.
Peaks of 122–124 bp (respectively 5T-11TG and 5T-12TG) were obtained for all 5T control samples, giving the expected TG genotypes. The specificity of our method was successfully tested in 5T heterozygous samples, and no nonspecific amplification of either the 7T or 9T alleles was detected. The assay gave completely specific amplification in the presence of at least 5T allele. When the PCR was performed in 5T-negative samples, nonspecific PCR products were obtained, yielding peaks that were no more than one tenth the height of the specific ones (Fig. 1
). Good reproducibility of the assay was obtained both within runs [mean (SD) retention times and estimated sizes were 28.99 (0.24) min and 122.74 (0.11) nucleotides for 5T-11TG; 29.41 (0.60) min and 124.71 (0.31) nucleotides for 5T-12TG] and between runs [mean (SD) retention times and estimated sizes were: 29.13 (0.58) min and 122.51 (0.41) nucleotides for 5T-11TG; 29.30 (0.61) min and 124.65 (0.43) nucleotides for 5T-12TG].
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To confirm the effectiveness of the method we next screened a wider collection of 5T-positive cases. CBAVD and pancreatitis patients were included in the study (10). In addition, non-CBAVD individuals undergoing assisted reproduction techniques (ART) were selected. Samples and results are shown in Table 1
.
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Among 14 CBAVD patients, 9 (64.3%) exhibited the 5T allele combined with a severe CFTR mutation. The 12TG allele was detected in all 9 5T-positive cases. These data confirm the known association of the 5T allele with CBAVD (4)(11) and strongly support the reported role of the 12TG repeats in the penetrance of the 5T variant in this condition.
We found that 12 (14.3%) of 84 chronic or acute pancreatitis patients were 5T carriers; among these 50% (6 of 12) exhibited the 5T-12TG haplotype. The prevalence of 5T carriers in 677 persons undergoing ART was 5.3% (36 of 677), and the 5T-12TG haplotype was detected in 33.3% (12 of 36) of these cases.
The 5T-carrier frequency was slightly higher than the general population (5%–10%) (12)(13) among pancreatitis patients but was in the normal range among non-CBAVD ART individuals. The prevalence of the 12TG repeats is more difficult to assess, because few population data are currently available (8). The 12TG prevalence was increased, though not significantly, in our pancreatitis patients compared to 12TG-positive ART individuals. Further studies comparing the prevalence of the 5T-poly(TG) haplotypes to the general population are needed to define their role in pancreatic disease.
The TG repeats affect the phenotype of 5T allele carriers only in cis; thus for a correct evaluation the 5T-poly(TG) haplotype must be identified. Direct sequencing of CFTR intron 8 (14) and melting-curve analysis of hybridization probes (15) have been proposed for the genotyping of poly(TG) tracts in conjunction with the polyT tract. These methods have the advantage of alleviating the need to do poly(T) screening first, and they can also detect very rare variants such as 3T or 6T (16). The sequence analysis is time-consuming and expensive, however, and interpreting and phasing complex electropherograms in heterozygous samples is often difficult. The melting-curve assay could be ambiguous in the differentiation of some haplotypes (17).
Many laboratories perform a first routine CFTR screening with mutation panels that include IVS8 5T, 7T, and 9T genotyping, although the diagnostic value of the detection of just the 5T allele is uncertain. The method presented here is a simple and inexpensive assay to improve the analysis of CFTR gene status.
Growing evidence supports the idea that more TG repeats increase the penetrance of the 5T allele as disease-causing mutation. The disease risk for males with 5T-12TG or 13TG in trans with a severe CF mutation is reported to be 0.78 and 1.0, respectively, whereas for females it seems to be lower (8)(9). Thus, the knowledge of the TG repeat number allows for more accurate prediction of benign vs pathogenic 5T alleles and may be helpful in the interpretation of genetic testing. Although further investigation is needed of TG repeat number reliability as a predictor of penetrance for 5T, it should be routinely tested for a better disease risk assessment.
Our data confirm the high frequency of 5T-12TG in CBAVD patients and do not preclude an effect in pancreatitis as well. We suggest that current practice for CFTR mutation testing would benefit from including the method outlined in the current study.
Acknowledgments
We are grateful to Paul Massa for scientific editing. This study was partially supported by Fondazione Cassa di Risparmio in Bologna.
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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 P.-H. Kuo, L. G. Doudeva, Y.-T. Wang, C.-K. J. Shen, and H. S. Yuan Structural insights into TDP-43 in nucleic-acid binding and domain interactions Nucleic Acids Res., April 1, 2009; 37(6): 1799 - 1808. [Abstract] [Full Text] [PDF]
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C. Costa, J.-M. Costa, J. Martin, B. Boissier, M. Goossens, and E. Girodon Multiplex Allele-Specific Fluorescent PCR for Haplotyping the IVS8 (TG)m(T)n Locus in the CFTR Gene Clin. Chem., September 1, 2008; 54(9): 1564 - 1567. [Abstract] [Full Text] [PDF]
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