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Simultaneous molecular haplotyping of both IVS8 [(TG).sub.m] and [(T).sub.n] tracts in the CFTR gene: still a challenge.

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).sub.m] associated with shorter [(T).sub.n] repeats are less favorable for the efficiency of exon 9 splicing. In particular, the [(TG).sub.m] locus influences penetrance of the TS 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).sub.11][(T).sub.5], [(TG).sub.12][(T).sub.5], and [(TG).sub.13][(T).sub.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).sub.12][(T).sub.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' -GTAATGGATCAT000CCATGT3'; reverse, 5'-CA000ATACATTCTCCTAATG-3') during the second part of the study, carried out on 75 DNA samples. The melting curves as well as melting temperatures ([T.sub.m]) were similar to those obtained by Millson et al. (1). Genotypes with [T.sub.m] shifts >2[degrees]C could be easily differentiated, whereas those with [T.sub.m] shifts <1[degrees]C were more difficult to differentiate. Intra- and interrun studies showed ranges in [T.sub.m] variations for all tested haplotypes of 0.10-0.33 and 0.5-1.3[degrees]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 [DELTA][T.sub.m] calculation (difference between wildtype and mutant peaks in heterozygous samples) (4) might overcome this problem because both alleles are affected equally and thus [DELTA][T.sub.m] 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).sub.12][(T).sub.5] and [(TG).sub.13][(T).sub.5] alleles, could not be clearly distinguished, which lessens the clinical usefulness of the test. Although discrimination of (TG)11[(T).sub.5] from [(TG).sub.12][(T).sub.5] and [(TG).sub.13][(T).sub.5] may be sufficient, [(TG).sub.12][(T).sub.5] and [(TG).sub.13][(T).sub.5] are considered true, mild, disease-causing alleles; therefore, differentiation between [(TG).sub.12][(T).sub.5] and [(TG).sub.13][(T).sub.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).sub.12][(T).sub.5] and [(TG).sub.13][(T).sub.5] have been assessed as 78% and 100%, respectively (2). More studies on the outcome of [(T).sub.5] patients are required to determine whether the [(TG).sub.13][(T).sub.5] allele is associated with predisposition to multisymptomatic disease with regard to (TG)1z[(T).sub.5].

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).sub.12][(T).sub.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.

DOI: 10.1373/clinchem.2005.065383


(1.) Millson A, Pont-Kingdon G, Page S, Lyon E. Direct molecular haplotyping of the IVS-8 poly(TG) and polyT repeat tracts in the cystic fibrosis gene by melting curve analysis of hybridization probes. Clin Chem 2005;51:161923.

(2.) Groman JD, Hefferon TW, Casals T, Bassas L, Estivill X, Des Georges M, et al. Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign. Am J Hum Genet 2004;74: 1322-5.

(3.) Costa C, Pissard S, Girodon E, Huot D, Goossens M. A one-step real-time PCR assay for rapid prenatal diagnosis of sickle cell disease and detection of maternal contamination. Mol Diagn 2003;7:45-8.

(4.) Lyon E. Discovering rare variants by use of melting temperature shifts seen in melting curve analysis. Clin Chem 2005;51:1331-2.

Catherine Costa * Michel Goossens Emmanuelle Girodon

AP-HP, CHU Henri Mondor Laboratoire de Genetique Moleculaire Unite INSERM U654 Creteil 94010, France

* Address correspondence to this author at: Laboratoire de Genetique Moleculaire, CHU Henri Mondor, 51 avenue du Marechal de Lattre de Tassigny, 94010 Creteil, France. Fax 33-149-812-842; e-mail

Response to the letter "simultaneous molecular haplotyping of both IVSS (TG). and (T), Tracts in the CFTR gene: still a challenge" by costa et al

To the Editor:

In their letter, Costa et al. addressed issues related to the clinical implementation of haplotype analysis of the poly(TG) and poly(T) repeat tracts in the cystic fibrosis transmembrane regulator (CFTR) gene by melting curve analysis. In esponse to their comments, we will describe the technical and logistic approaches we used to successfully implement this assay.

We and Costa et al. have observed that some haplotype melting temperatures ([T.sub.m]s) are very close together, as exemplified by the TG10-7T and TG11-9T alleles. Differentiation of clinical samples is facilitated by comparing both the [T.sub.m] and the curve shape with those for identical controls in the same run, based on intrarun reproducibility. These controls have fully characterized [(TG).sub.m]/[(T).sub.n] haplotypes and are preferably extracted by the same method as was used for the clinical samples. On receipt of patient whole-blood samples, we perform DNA extractions with a standard in-house method, a procedure that alleviates problems with [T.sub.m] shifts caused by different extraction methods or potentially degraded stored samples.

After the publication of our report (1), we obtained data for 2 haplotype combinations that were especially challenging to discriminate. The TG11-9T/TG11-7T and TG11-7T/ TG10-7T combinations each produce a single, broad peak as opposed to 2 distinct peaks. The TG11-9T/ TG11-7T combination has a [T.sub.m] of 53.2[degrees]C, whereas TG11-7T/TG10-7T produces a peak with a [T.sub.m] of 51.5[degrees]C and a shoulder at 54.8[degrees]C. The distinct shapes are reproducible between different samples and on different runs. When we performed runs that included controls for each combination, we were able to confidently identify both haplotype combinations.

We use this haplotyping assay in our clinical workflow in conjunction with other CFTR assays that genotype the poly(T) repeat. In one scenario, patients with suspected cystic fibrosis (CF) symptoms are tested first by a mutation panel, including the poly(T) tract. Using molecular haplotyping, we can then determine the poly(TG) repeat number as well as the [(TG).sub.m]/[(T).sub.n] haplotype. Because we have already determined the [(T).sub.n], we can detect a miscall between the TG10-7T or TG11-9T haplotypes. Thus we have clearly identified these and other haplotypes, with the exception of TG12-5T and TG13-5T, which have indistinguishable [T.sub.m]s, as confirmed by Costa et al. If warranted by the clinical situation, the haplotype can be confirmed with bidirectional sequencing when a sample has a [T.sub.m] coinciding with the [T.sub.m] of the TG12-5T control. In a more frequent scenario, results of the IVS-8 region generated from full CFTR gene sequence analysis may require clarification, because it is often difficult to read a heterozygous sequence through this highly repetitive and variable region. In this case, melting curve analysis is the confirmatory test.

Since its development in the fall of 2001, this assay has been very reliable, although we believe it is best used to complement other detection methods for CF sequence variations rather than in isolation. When combined with either a mutation panel or full gene analysis, these haplotypes can be used in conjunction with clinical symptoms to identify and possibly predict compound effects with other alleles.

DOI: 10.1373/clinchem.2006.072348


(1.) Millson A, Pont-Kingdon G, Page S, Lyon E. Direct molecular haplotyping of the IVS-8 poly(TG) and polyT repeat tracts in the cystic fibrosis gene by melting curve analysis of hybridization probes. Clin Chem 2005;51:161923.

Alison Millson [1] Genevieve Pont-Kingdon [1] Elaine Lyon [1, 2] *

[1] ARUP Institute for Clinical and Experimental Pathology Salt Lake City, UT

[2] Pathology Department University of Utah School of Medicine Salt Lake City, UT

* Author for correspondence.
Table 1. Inter--and intrarun variations in [T.sub.m] for the
different haplotypes.

 Mean Range [T.sub.m]
 variation, [degrees]C,

Haplotypesa Samples, n Interrun Intrarun

[(TG).sub.9][(T).sub.9] 2 43.25-44.25 43.62-43.68

[(TG).sub.10] [(T).sub.9] 10 47.65-50.25 48.30-48.86

[(TG).sub.10][(T).sub.7] 11 51.55-52.55 51.86-52.38

[(TG).sub.11][(T).sub.9] 6 51.7-52.7 52.13-52.35

[(TG).sub.11][(T).sub.7] 14 53.63-55.85 54.54-55.2

[(TG).sub.12][(T).sub.7] 7 58.8-60.4 59.55-59.65

[(TG).sub.11][(T).sub.5] 5 59.6-61.2 60.41-60.65

[(TG).sub.12][(T).sub.5] 13 63.2-64.2 63.25-63.45

[(TG).sub.13][(T).sub.5] 7 62.9-63.9 63.14-63.36

(a) Indistinctly identifiable haplotypes are indicated in bold.
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Title Annotation:Letters
Author:Costa, Catherine; Goossens, Michel; Girodon, Emmanuelle
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Aug 1, 2006
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