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1 Department of Clinical Chemistry, Georg-August-University, Robert-Koch-Strasse 40, 37075 Goettingen, Germany
aAuthor for correspondence. Fax 49-551-39-8551; e-mail nahsen{at}gwdg.de.
To the Editor:
We have read with interest the recent reports of Marziliano et al. (1) and Pirulli et al. (2), who used melting temperature assays to genotype different types of mutations. Because detection of the underlying mutation is only indirect in these methods, they differ in their molecular detection principle from hybridization probe-based genotyping (3) or allele-specific amplification coupled to SYBR-green I detection (4). Indirect detection methods demand extra caution in the assignment of genotypes solely on the basis of product melting temperature (Tm). From a theoretical standpoint, some genotypes will not be detected.
The UDP-glucuronosyltransferase 1 (UGT1A1) (TA)n insertion/deletion polymorphism is of significance for the manifestation of Gilbert disease (3)(5). TA repeats are intrinsically unstable, and, therefore, it is not surprising that five to eight TA repeats occur in humans and have functional significance (5). The method of Marziliano et al. (1) uses Tm as a measure of amplicon length to discover the UGT1A1 promoter genotype. In the described assay, a 2-bp difference is detected by the resulting Tm shift in a 130-bp amplicon. A similar technique was successfully used in the screening for a 9-bp deletion in a 55-bp PCR amplicon (6). However, for the assay of Marziliano et al. (1), it has to be noted that using only an indirect measure of strand length and composition is insufficient for the assignment of a genotype on the basis of the theoretical reasons described below.
In addition to the points stated by Marziliano et al. (1), the melting curve analysis of a DNA strand is related to (a) the ionic strength of the buffer, (b) the DNA concentration, and (c) the DNA bases’ nearest neighbors (n-n) (3)(7). Within an assay system, buffer conditions can be considered constant. The genotyping of heterozygous samples requires special consideration because a mixture of two homoduplexes and two heteroduplexes results after PCR amplification, denaturation, and reannealing. An insertion/deletion polymorphism causes the formation of base bulges in the heteroduplexes. The bulge size is a major determinant of the destabilization caused by the disturbed base stacking (3). For example, the DNA base bulge size is 4 bp in the case of a 6TA/8TA duplex and 2 bp in the case of a 6TA/7TA or 7TA/8TA heteroduplex. Reported Tms for the 6TA and 7TA genotype differ by only 1.3 °C in the 130-bp amplicon (1). A heterozygous 6TA/7TA sample has an apparent Tm in between the 6TA and 7TA sample because the different Tms of the underlying homo- and heteroduplexes cannot be adequately resolved. On the basis of these respective considerations, we anticipate that in patients heterozygous for the 6TA/8TA genotype, a Tm undistinguishable from that of a homozygous 7TA genotype results. The presence of an 8TA genotype was already reported in an Italian patient with Gilbert syndrome (8). Other allelic combinations are possible where the same problem is present. Care must also be taken to ensure a constant DNA concentration in the assay before melting curves are acquired. Variation in the purity or amount of DNA in the assay can lead to different amounts of DNA after a constant number of PCR cycles. In the described assay (1), this will add to the total error and will cause variation in the Tm with the risk of wrong genotyping results.
This is not acceptable for a genotyping assay, and we recommend the use of more specific methods for allele assignment. Methods that ensure reliable genotyping for this locus have already been published and include polyacrylamide gel electrophoresis resolution of the PCR amplicon size (5), hybridization probes (3), sequencing, denaturing gradient gel electrophoresis, and denaturing HPLC [see references in (3)]. All of these methods also have the potential to detect the exact TA repeat numbers, which is not necessarily the case if only the amplicon Tm is used for screening.
Pirulli et al. (2) claim "sensitivity" and "specificity" of the DNA melting assay for the detection of different types of alanine:glyoxylate aminotransferase (AGXT) mutations. In addition to what was already mentioned above, we want to point out some results in Table 1 of Ref. (2). Both sample 9 and sample 13 share a homozygous G
A mutation. However, the resulting Tm shifts are 1.6 °C and -0.9 °C, respectively. In general, n-n pairs containing guanosine are more stable than n-n pairs containing adenosine. When a mutation in a strand causes the change of a guanosine n-n pair to an adenosine n-n pair, we would expect this to be destabilizing in most of the cases. Sample 2 is, in contrast to sample 11, stabilized by the presence of mismatches. The observed Tm of sample 2 is 0.8 °C higher than for the wild type, although the duplex is destabilized by two mismatches in both cases. This illustrates the deviation from n-n behavior in longer DNA strands and makes the results difficult to interpret. Furthermore, sometimes a product Tm differs by only 0.1 °C, whereas mean SDs are 0.14 °C (samples 15 and 2; samples 6 and 11). This makes it impossible to "easily distinguish different types of AGXT mutations" as claimed by Pirulli et al. (2). This is exemplified in Fig. 1
, which does not show melting curves, but the expected probability distribution defined by 88.0 °C with an SD of 0.14 °C and 88.1 °C with an SD of 0.18 °C. The large overlap in the distributions is obvious and substantiates that it is impossible to assign certain Tms to underlying genotypes.
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Our conclusion is that DNA Tm assays based on SYBR Green I melting curves are powerful tools for screening various types of mutations but lack both sequence and mutation specificity. A lack of sensitivity must be expected if stable mismatches occur among stable neighboring bases (high GC content). Results obtained with these methods at highly polymorphic loci must be confirmed by a mutation-specific detection method. Interpretation of results arising from only the inspection of SYBR Green I melting curves requires great caution. In our opinion, such methods should not be used for routine genotyping applications.
References
The following articles in journals at HighWire Press have cited this article:
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  E. Lyon and C. T. Wittwer LightCycler Technology in Molecular Diagnostics J. Mol. Diagn., March 1, 2009; 11(2): 93 - 101. [Abstract] [Full Text] [PDF]
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 K. E Liyanage, A. J Hooper, J. C Defesche, J. R Burnett, and F. M van Bockxmeer High-resolution melting analysis for detection of familial ligand-defective apolipoprotein B-100 mutations Ann Clin Biochem, March 1, 2008; 45(2): 170 - 176. [Abstract] [Full Text] [PDF]
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 M. G. Herrmann, J. D. Durtschi, L. K. Bromley, C. T. Wittwer, and K. V. Voelkerding Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping: Cross-Platform Comparison of Instruments and Dyes Clin. Chem., March 1, 2006; 52(3): 494 - 503. [Abstract] [Full Text] [PDF]
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N. von Ahsen Two for Typing: Homogeneous Combined Single-Nucleotide Polymorphism Scanning and Genotyping Clin. Chem., October 1, 2005; 51(10): 1761 - 1762. [Full Text] [PDF]
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R. Graham, M. Liew, C. Meadows, E. Lyon, and C. T. Wittwer Distinguishing Different DNA Heterozygotes by High-Resolution Melting Clin. Chem., July 1, 2005; 51(7): 1295 - 1298. [Full Text] [PDF]
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H. El Housni, P. Heimann, J. Parma, and G. Vassart Single-Nucleotide Polymorphism Genotyping by Melting Analysis of Dual-Labeled Probes: Examples Using Factor V Leiden and Prothrombin 20210A Mutations Clin. Chem., October 1, 2003; 49(10): 1669 - 1672. [Full Text] [PDF]
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N. von Ahsen Labeled Primers for Mutation Scanning: Making Diagnostic Use of the Nucleobase Quenching Effect Clin. Chem., March 1, 2003; 49(3): 355 - 356. [Full Text] [PDF]
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C. N. Gundry, J. G. Vandersteen, G. H. Reed, R. J. Pryor, J. Chen, and C. T. Wittwer Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes Clin. Chem., March 1, 2003; 49(3): 396 - 406. [Abstract] [Full Text] [PDF]
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