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Letters to the Editor |
AP-HP, CHU Henri Mondor, Laboratoire de, Génétique Moléculaire, Unité INSERM U654, Créteil 94010, France
aAddress correspondence to this author at: Laboratoire de Génétique Moléculaire, CHU Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France. Fax 33-149-812-842; e-mail catherine.costa{at}im3.inserm.fr.
To the Editor:
Millson et al. (1) recently reported the use of melting curve analysis of hybridization probes to direct molecular haplotyping of both the IVS8 poly(TG) and poly(T) repeat tracts of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Precise genotyping at this locus may be clinically relevant in CFTR pathology because longer (TG)m associated with shorter (T)n repeats are less favorable for the efficiency of exon 9 splicing. In particular, the (TG)m locus influences penetrance of the T5 allele, which may be associated with male infertility by congenital bilateral absence of the vas deferens or by atypical cystic fibrosis, with discrimination between (TG)11(T)5, (TG)12(T)5,and (TG)13(T)5 being clinically relevant (2). Growing interest in this area has led to the development of several assessment methods, most of them multistep and time-consuming. The method described by Millson et al. (1) is attractive because it aims to determine genotypes at these 2 loci simultaneously through a single-step assay, thus avoiding family linkage study.
Because melting curve analysis with hybridization probe technology is routinely used in our laboratory (3), we conducted a study to implement IVS8 genotyping and confirm its reliability. With the protocol described, we tested patients presenting with a wide combination of already known haplotypes. Preliminary results obtained during the initial phase revealed repetitive poor fluorescence values, even when a perfect match (TG)12(T)5 DNA sample was tested, leading to uninterpretable results. When we used a new set of primers, the same probes allowed correct analysis of melting curves to be achieved. We thus used our own primers (forward, 5'-GTAATGGATCATGGGCCATGT3'; reverse, 5'-CACCCATACATTCTCCTAATG-3') during the second part of the study, carried out on 75 DNA samples. The melting curves as well as melting temperatures (Tm) were similar to those obtained by Millson et al. (1). Genotypes with Tm shifts >2 °C could be easily differentiated, whereas those with Tm shifts <1 °C were more difficult to differentiate. Intra- and interrun studies showed ranges in Tm variations for all tested haplotypes of 0.10–0.33 and 0.5–1.3 °C, respectively (Table 1
). These results might be attributable to interDNA variations, salt concentration variations, and the fact that many DNA samples tested came from laboratories that used different extraction methods. It has been suggested that use of the 
Tm calculation (difference between wild-type and mutant peaks in heterozygous samples) (4) might overcome this problem because both alleles are affected equally and thus Tm is less affected by sample-to-sample variation. However, the accurate assignment of some genotypes did not appear realistic; indeed, some haplotypes, in particular the (TG)12(T)5 and (TG)13(T)5 alleles, could not be clearly distinguished, which lessens the clinical usefulness of the test. Although discrimination of (TG)11(T)5 from (TG)12(T)5 and (TG)13(T)5 may be sufficient, (TG)12(T)5 and (TG)13(T)5 are considered true, mild, disease-causing alleles; therefore, differentiation between (TG)12(T)5 and (TG)13(T)5 might be important in some clinical situations. In combination with a cystic fibrosis–causing allele in trans, the disease risks for males bearing (TG)12(T)5 and (TG)13(T)5 have been assessed as 78% and 100%, respectively (2). More studies on the outcome of (T)5 patients are required to determine whether the (TG)13(T)5 allele is associated with predisposition to multisymptomatic disease with regard to (TG)12(T)5.
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The challenge in the assay described is the use of a single probe for simultaneous detection of adjacent TG and T repeats and accurate genotyping. The reporter probe was designed to match perfectly with the (TG)12(T)5 allele, and depending on the DNA haplotype under study, single or multiple mismatches as well as loops may occur within the probe, whose melting behavior could not be easily predicted. Combinations of highly destabilizing mismatches at n-n pairs containing guanosine (TG tract) with less destabilizing mismatches at n-n pairs containing thymidine (T tract) are particularly difficult to interpret.
In conclusion, we did not find the assay described by Millson et al. (1) robust enough to be implemented in a diagnostics laboratory. Improvements such as use of high-resolution melting curve analysis or additional or modified probes allowing better discrimination between genotypes would be necessary before such a test can be introduced in a clinical setting.
References
The following articles in journals at HighWire Press have cited this article:
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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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 C. Bareil, C. Guittard, J.-P. Altieri, C. Templin, M. Claustres, and M. des Georges Comprehensive and Rapid Genotyping of Mutations and Haplotypes in Congenital Bilateral Absence of the Vas Deferens and Other Cystic Fibrosis Transmembrane Conductance Regulator-Related Disorders J. Mol. Diagn., November 1, 2007; 9(5): 582 - 588. [Abstract] [Full Text] [PDF]
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V. Mantovani, P. Garagnani, P. Selva, C. Rossi, S. Ferrari, M. Cenci, N. Calza, V. Cerreta, D. Luiselli, and G. Romeo Simple Method for Haplotyping the Poly(TG) Repeat in Individuals Carrying the IVS8 5T Allele in the CFTR Gene Clin. Chem., March 1, 2007; 53(3): 531 - 533. [Abstract] [Full Text] [PDF]
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