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Clinical Chemistry 51: 1619-1623, 2005. First published July 14, 2005; 10.1373/clinchem.2005.052159
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(Clinical Chemistry. 2005;51:1619-1623.)
© 2005 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

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

Alison Millson1, Genevieve Pont-Kingdon1, Sam Page1 and Elaine Lyon1,2,a

1 Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT.
2 Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT.

aAddress correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Molecular haplotyping is a developing technology with great potential for use in clinical diagnostics. We describe a haplotyping method that uses PCR combined with hybridization probes.

Methods: We designed a LightCycler assay that uses fluorescence resonance energy transfer hybridization probes to haplotype the poly(TG) and polyT (TG-T) tract in the IVS-8 region of the CFTR gene. The reporter probe was designed as a perfect match to the TG12-5T allele.

Results: Analysis of 132 samples revealed 9 unique derivative melting temperatures (Tms); the lowest was 42.4 °C and the highest was 63.6 °C. The lowest Tms were in the TGn-9T group, the intermediate Tms in the TGn-7T group, and the highest Tms in the TGn-5T group. Haplotype frequencies were highest (39%) for TG11-7T and lowest (0.4%) for TG13-5T.

Conclusions: Different combinations of polymorphisms under the reporter hybridization probe had unique and characteristic Tms. This property enables genotyping as well as determination of the phase of multiple variants under the probe, a principle we demonstrated by haplotyping the TG-T repeat tract in the IVS-8 region of the CFTR gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cystic fibrosis (CF)1 gene on chromosome 7q31 spans ~250 kb of DNA, and 27 exons encode the cystic fibrosis transmembrane conductance regulator (CFTR) protein (1). Mutations in this protein give rise to CF, which is one of the most common autosomal recessive disorders in Caucasians, with an incidence of ~1 in 2500 Caucasian births and a carrier frequency of ~1 in 25. More than 1000 mutations have been described in the CFTR gene (2). Although some of these mutations, such as {Delta}F508, are clearly classified as severe, the phenotype/genotype correlation of other mutations is not well established. Studies have demonstrated polymorphisms outside the CFTR gene (3)(4) as well as polymorphisms within the gene that modify the phenotype of some CF mutations. The polyT tract located in the CFTR IVS-8 intronic region, immediately 5' of exon 9, is one example. The number of thymidines (T) present, 5, 7, or 9, affects the splicing efficiency of exon 9. If the 5T allele is present, a proportion of CFTR transcripts will lack exon 9, producing a nonfunctional protein and variable CF symptoms (5). When the 5T allele is found in conjunction with the mutation R117H, it has the potential to compound the already affected CFTR transcript. If the R117H mutation and the 5T allele are found on the same chromosome (in cis), the result is a phenotypically more severe CF chromosome (6). The poly(TG) repeat, 5' of the polyT repeat, also influences splicing of exon 9 (7), and when present on the same allele as a 5T repeat, the longer the poly(TG) repeat, the higher the proportion of CFTR transcripts that will lack exon 9. 5T repeats adjacent to either TG12 or TG13 repeats are more likely to exhibit an abnormal phenotype than are 5T repeats adjacent to TG11 (8). The TG repeat number also exerts an effect on a 7T background. Compared with TG10, TG11 repeats decrease almost 3-fold and TG12 repeats decrease 6-fold the number of CFTR transcripts lacking exon 9 (7). We present a molecular method that allows direct detection of the phase (cis vs trans or haplotype) of the TG and the T tracts of IVS-8.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
samples
We analyzed a collection of 132 samples, including normal (n = 58), CF-suspected (n = 60), and Coriell Cell Repository CF-panel samples (n = 14). The clinical case study sample was received in ARUP’s clinical molecular laboratory for CF screening. Human genomic DNA was extracted either by phenol–chloroform extraction followed by ethanol precipitation (9) or by MagNA Pure extraction [Roche Molecular Diagnostics; (10)]. All samples were deidentified in accordance with an Internal Review Board protocol.

primers and probes
Primers were designed to amplify a 345-bp product of the region containing the IVS-8 polyT tract, using GenBank accession no. AH006034. The forward primer sequence was 5'-CCTCTAGAAACCGTATGC-3', and the reverse primer sequence was 5'-CAACCGCCAACAACTG-3'. Fluorescence resonance energy transfer probes were synthesized by Idaho Technology. The reporter probe, 5'-CCCTGTTAAAAACACACACACAC-3' with the 3'-end labeled with fluorescein, was designed to be a perfect match of the 5T variant (underlined). The anchor probe, located directly 5' from the reporter probe, was 5'-ACACACACACACATCAAAAATAAAAGATGAGTTTG-3' with the 5'-end labeled with LCRed 640 (Roche Molecular Diagnostics) and the 3'-end blocked with a phosphate group.

LIGHTCYCLER pcr amplification
PCR was performed with LightCycler-DNA Master Hybridization Probes (Roche Molecular Diagnostics), and 1 µL of the 10x hybridization master mixture in a 10-µL reaction containing Taq, deoxynucleoside triphosphates, and 10 mM MgCl2. An additional 0.8 µL of 25 mM MgCl2/10-µL reaction was added, giving a final MgCl2 concentration of 3 mM. Primer concentrations were 0.5 µM for both the forward and reverse primers, with probe concentrations at 0.2 µM. DNA concentrations were 50–100 ng. The PCR conditions were 95 °C for 2 s, 58 °C for 10 s, and 72 °C for 15 s for 40 cycles. The programmed transition rates were 20 °C/s from denaturation to annealing, 1 °C/s from annealing to extension, and 20 °C/s from extension to denaturation. Fluorescence was detected once per cycle at the end of the annealing stage. After amplification, samples were denatured at 95 °C for 0 s, cooled to 45 °C at a transition rate of 20 °C/s, and held for 2 min. The samples were then heated to 75 °C at a rate of 0.1 °C/s with continuous fluorescence monitoring. The data were plotted as negative-derivative fluorescence curves with respect to temperature (–dF/dT).

The haplotypes of 25 samples were confirmed with bidirectional sequencing using BigDye terminator chemistry (Applied Biosystems) as described in Lucarelli et al. (11). The sample set contained at least one example of all of the different haplotypes found in this study.

Five samples containing representative chromosomes of the 6 most frequent haplotypes were assayed to determine the within- and between-run melting temperature (Tm) variations. Each sample was run in replicate to determine within-run precision. The between-run precision was determined by running each sample on 5 separate runs.

We used allele-specific amplification targeting the R117H locus combined with long-range PCR (12) to amplify two 17.7-kb amplicons from an R117H/{Delta}F508 compound heterozygote. As a control, a common forward primer was used to amplify both chromosomes in a third 17.7-kb amplicon. We mixed 8 µL of each of the 3 amplicons with 1 µL of each haplotyping probe (0.2 µM). The samples were melted with the following protocol: denatured at 95 °C for 5 min, cooled to 30 °C at a transition rate of 20 °C/s, held for 2 min, and then heated to 75 °C at a rate of 0.1 °C/s with continuous fluorescence monitoring. The data were plotted as negative-derivative fluorescence curves with respect to temperature (–dF/dT).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The study sample set comprised 264 chromosomes. The characteristic Tm was identified for each haplotype (Table 1 ). The derivative melting curve of an individual heterozygous at either (or both) the TG and T locus generated 2 distinct peaks, each peak representing 1 chromosome (Fig. 1 ). When multiple samples were analyzed, each chromosome was still identifiable by its characteristic Tm (Fig. 2 ).


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  		Table 1. Frequency and characteristic Tm for each haplotype found (n = 264 chromosomes).



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  		Figure 1. Derivative melting curve of 2 haplotypes, TG10-9T and TG11-5T, obtained from an individual heterozygous at both the TG and T loci.



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  		Figure 2. Derivative melting curves displaying combinations of TG and T repeats.

All results were confirmed by sequencing.

Because the reporter probe was designed to be a perfect match to the 5T repeat tract, the Tms increased as the T repeat number decreased to 5 (the perfect match). Within each T repeat category, the Tm increased with increasing TG repeat number, with the exception of TG13-5T, for which the Tm was slightly lower than the Tms of the TG12-5T samples. Only 1 sample had the TG13-5T haplotype; therefore, the Tm reproducibility of that haplotype in multiple samples could not be determined. Without multiple samples to confirm this Tm, we cannot exclude the possibility that different salt concentrations from different extraction procedures affected the Tm. The most common haplotypes in our study were TG11-7T with a frequency of 39%, TG10-9T with a frequency of 29%, and TG10-7T with a frequency of 19% (Table 1Up ). The percentage of 9T haplotypes was somewhat higher than previously described in the general population (6). This difference is most likely because our CF sample set had an increased percentage of {Delta}F508 chromosomes, which are known to be commonly in cis with 9T. One rare haplotype was discovered with a 9TG-10T haplotype. The Tm of this sample was 6 °C lower than the next Tm. Eight haplotypes were found in all, and at least one example of each was confirmed by sequencing (data not shown).

Within- and between-run precision studies assessing the Tm variation of the 6 most common haplotypes were performed (Table 2 ). All SDs were within ± 0.3 °C.


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  		Table 2. Within- and between-run precision for Tm.1

One {Delta}F508/R117H compound heterozygous sample with 9T/5T repeats was haplotyped with long-range allele-specific PCR (12) and the TG-T haplotyping probes. Haplotype analysis showed {Delta}F508-TG10-9T on one chromosome and R117H-TG12-5T on the other (Fig. 3 ). On the basis of the current literature (6)(13), these results would be supportive of a clinical CF presentation; however, the clinical scenario was a healthy 23-year-old male, with no obstructive pulmonary disease, who was referred for CF testing as part of a work-up for congenital bilateral absence of the vas deferens.



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  		Figure 3. R117H/polyT/poly(TG) haplotyping.

Three 17.7-kb amplicons were generated by use of 3 different forward primers and a common reverse primer. Two were allele-specific, for either the wild type or mutant R117H, and the third amplified both alleles. Addition of the TG-T haplotyping probes shows the mutant R117H allele, carrying the TG12-5T, and the wild-type R117H allele, containing the {Delta}F508 mutation, in cis with the TG10-9T.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
With the growing number of clinical correlations to haplotypes, new methods are being developed that are more applicable to clinical testing. Traditionally, family studies have been used to identify parental chromosomes but may pose a challenge in obtaining samples required for accurate diagnoses. Most molecular approaches for haplotyping require either physical separation of the chromosomes into haploid states (14)(15) or analysis of individual DNA molecules (16)(17). Various analysis methods include allele-specific amplification (18), PyrosequencingTM (19), combination of Pyrosequencing with allele-specific amplification (20), and POLONY (21), to name a few. Recently, cycle sequencing has been used to assess the TG-T tract (11). Our assay uses melting curve analysis to haplotype the TG-T tract directly from a single individual.

This assay demonstrates the effect of multiple mismatches under the probe on Tm. Hybridization probes have been used extensively to genotype single-nucleotide polymorphisms. Once it was recognized that a reporter probe covering several polymorphisms would melt as a unit, thereby characterizing the complete sequence residing under the probe, the probes were applied as haplotyping agents (22). Depending on the haplotype, the probes may bind perfectly or have single or multiple mismatches to the template, allowing the "loop out" of template sequences between the polymorphisms. Fig. 4 shows 3 examples illustrating possible probe-binding scenarios for the TG-T region. The reporter probe was designed to be a perfect match to the 5T template. In the first example, a TG11-7T template, there was 1 mismatch with the probe and lowering of the Tm. In the second example, a TG10-9T template, there were 2 mismatches with further destabilization of the reporter probe and further lowering of the Tm. The third template had 5 Ts and was a perfect match in this area of the probe, but because of the TG11 repeats, the probe either had a dangling end or the reporter probe looped out 2 bases of the TG repeat, causing destabilization of the reporter probe and lowering of the Tm, but to a lesser extent than the other template examples.



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  		Figure 4. Possible scenarios of probes binding to 3 different templates.

The probes are a perfect match to the TG12-5T template.

The haplotyping result for the R117H/{Delta}F508 compound heterozygote was surprising in view of the current theory regarding the effect of the TG influence on CFTR transcripts. The chromosome containing {Delta}F508-TG10-9T is considered severe because of the effect of {Delta}F508. Although the genotype/phenotype correlation is not well established, the chromosome containing R117H-TG12-5T can lead to classic CF because of the combined effects of the 3 variants. With both chromosomes carrying mutations, this patient would be predicted to suffer from CF. Instead, the only clinical symptom is infertility. Other CFTR polymorphisms, such as M470V, have been shown to exert influence on the production of the CFTR protein and might help explain the penetrance of the R117H-TG12-5T chromosome (7). The phenotype could also be influenced by the presence of modifier polymorphisms outside the CFTR gene. Further clinical studies are required to better understand the clinical consequences of these haplotypes.

We found haplotyping by derivative curve analysis to be inexpensive, and the procedure could be completed in ~1 h. Interpretation of the derivative melting curves was straightforward. With the increasing evidence of the physiologic influence of the TG-T haplotype, the ability to easily assay the haplotype has become clinically important, and our experience demonstrates the advantages of hybridization probes as a molecular haplotyping tool.


   Footnotes
 
1 Nonstandard abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; and Tm, melting temperature.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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This Article
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