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Letters to the Editor |
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
aAuthor for correspondence.
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 (Tms) are very close together, as exemplified by the TG10–7T and TG11–9T alleles. Differentiation of clinical samples is facilitated by comparing both the Tm and the curve shape with those for identical controls in the same run, based on intrarun reproducibility. These controls have fully characterized (TG)m/(T)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 Tm 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 Tm of 53.2 °C, whereas TG11–7T/TG10–7T produces a peak with a Tm of 51.5 °C and a shoulder at 54.8 °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)m/(T)n haplotype. Because we have already determined the (T)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 Tms, 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 Tm coinciding with the Tm 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.
References
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