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High Resolution Melting Analysis (HRMA) for the identification of a rare UGT1A1 promoter polymorphism
Clinical Biochemistry 44 (2011) 1359–1360
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Letter to the Editor High Resolution Melting Analysis (HRMA) for the identification of a rare UGT1A1 promoter polymorphism Keywords: HRMA UGT1A1 promoter polymorphism
To the Editor, In 2009, our paper [1] showed that the three most frequent genotypes of the UGT1A1 gene promoter can be identified in Caucasian population by HRMA. Furthermore, the analysis of Tms gave interesting findings: in a 70 bp long amplicon, containing a TA repeats region, the Tm variation is affected by the number of TA repeats rather than the amplicon length [2]. In fact the wild type genotype (TA)6/6 has a Tm 0.5 °C higher than the (TA)7/7 genotype. We do not know if this behavior is similar to that of other rare genotypes such as (TA)5/5 or (TA)8/8. Recently, Ostanek et al. [3] confirmed not only our results but also the high reproducibility of HRMA for the study of the UGT1A1 promoter. These authors analyzed the less frequent Caucasian UGT1A1 genotypes, but they were not able to discriminate all genotypes by mean of HRMA analysis both on the non-spiked amplicon and on the 1:1 spiked DNAs. Thus, they assumed that “further improvements in HRMA method are necessary in order to achieve complete genotyping of micro satellite promoter repeats”. During a routine HRMA screening run, including ten different samples of patients referred to our laboratory for Gilbert's syndrome diagnosis, we analyzed a sample showing (Fig. 1A) melting profile (MP) and Tm behavior slightly different from the standard UGT1A1 genotypes (TA6/6, 7/6, 7/7). This sample was therefore analyzed by direct sequencing which revealed the presence of (TA)5/6 genotype, a rare UGT1A1 promoter polymorphism. In order to optimize also the identification of this genotype by HRMA, we initially decided to add to each standard and (TA)5/6 sample a known amount of a (TA)6/7 in the ratio 3/1. Unfortunately, this strategy failed since the rare genotype and the wild type presented similar MPs (Fig. 1B). Finally, we spiked all samples with the (TA)7/7 sample in the ratio 3/1. Adding this genotype we identified four MPs (Fig. 1C), corresponding to the three most common genotypes and the rare (TA)5/6. The results obtained with the PCR and HRMA on triplicate samples, using our previously published protocol [1], confirmed the robustness of our method in terms of reproducibility and sensitivity [1].
Therefore, the spiking with (TA)7/7 makes possible the identification of the rare (TA)5/6 genotype. Differently from Ostanek et al, we used as spiking a concentration of (TA)7/7 not exceeding 35%: although our technical conditions were slightly different from those reported in literature [4,5]. We can therefore conclude that a) we can correctly identify (TA)5/6 UGT1A1 genotype by HRMA applying our protocol which works only when the spiking with (TA)7/7 is performed; b) HRMA technique is able, alone, to identify the unknown genotypes, but only after technical adjustments, the precise genotype attribution may be obtained; c) finally, we think that the use of the same methodology described above may allow the identification of other rare UGT1A1 promoter genotypes, probably not exceeding a concentration of 35% between the spiked genotype and the unknown sample. References [1] Minucci A, Concolino P, Giardina B, Zuppi C, Capoluongo E. Rapid UGT1A1 (TA)(n) genotyping by high resolution melting curve analysis for Gilbert's syndrome diagnosis. Clin Chim Acta 2010;411:246–9. [2] Minucci A, Mello E, Tripodi D, Zuppi C, Capoluongo E. Contribution of the TA repeats on melting temperature (T(m)) in a double strand DNA: comparison of two methods and implications in molecular diagnostics. Clin Biochem 2011;44:736–8. [3] Ostanek B, Furlan D, Bratanič BB. Genotyping UGT1A1(TA)(n) polymorphism rare variants by high resolution melting curve analysis. Clin Chim Acta 2011;412:489-9. [4] Seipp MT, Herrmann M, Wittwer CT. Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting. J Biomol Tech 2010;21:163–6. [5] Montgomery JL, Sanford LN, Wittwer CT. High-resolution DNA melting analysis in clinical research and diagnostics. Expert Rev Mol Diagn 2010;10:219–40.
Angelo Minucci⁎ Enrica Mello Domenico Tripodi Paola Concolino Cecilia Zuppi Ettore Capoluongo Laboratory of Clinical Molecular Biology, Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Italy ⁎Corresponding author at: Laboratory of Clinical Molecular Diagnostics, Department of Biochemistry & Clinical Biochemistry, Catholic University, Rome, Italy. Fax: +39 0630156783. E-mail addresses: [email protected], [email protected] (A. Minucci).
0009-9120/$ – see front matter © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.08.1130
15 March 2011
1360
Letter to the Editor
A
6TA/6TA 6TA/7TA 7TA/7TA Unknown (5TA/6TA)
B
6TA/6TA and 5TA/6TA 6TA/7TA 7TA/7TA
C
6TA/6TA 6TA/7TA 7TA/7TA 5TA/6TA
Fig. 1. HRMA of four UGT1A1 promoter polymorphisms. HRMA was performed on the LightCycler® 480 Real-Time PCR System (Roche Diagnostics). The dye used was Dye Reso Light (Roche Diagnostics) belonging to the new generation dyes. In panel A the software classifies (TA)5/6 in a manner not significantly different from the standards in terms of melting profiles and Tm. In panel B the spiking with (TA)6/7 does not improve the separation but makes (TA)5/6 more similar to the wild type genotype. In 1 °C after the (TA)7/7 addition in all samples the instrument associates melting profiles for each genotype.
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Letter to the Editor High Resolution Melting Analysis (HRMA) for the identification of a rare UGT1A1 promoter polymorphism Keywords: HRMA UGT1A1 promoter polymorphism
To the Editor, In 2009, our paper [1] showed that the three most frequent genotypes of the UGT1A1 gene promoter can be identified in Caucasian population by HRMA. Furthermore, the analysis of Tms gave interesting findings: in a 70 bp long amplicon, containing a TA repeats region, the Tm variation is affected by the number of TA repeats rather than the amplicon length [2]. In fact the wild type genotype (TA)6/6 has a Tm 0.5 °C higher than the (TA)7/7 genotype. We do not know if this behavior is similar to that of other rare genotypes such as (TA)5/5 or (TA)8/8. Recently, Ostanek et al. [3] confirmed not only our results but also the high reproducibility of HRMA for the study of the UGT1A1 promoter. These authors analyzed the less frequent Caucasian UGT1A1 genotypes, but they were not able to discriminate all genotypes by mean of HRMA analysis both on the non-spiked amplicon and on the 1:1 spiked DNAs. Thus, they assumed that “further improvements in HRMA method are necessary in order to achieve complete genotyping of micro satellite promoter repeats”. During a routine HRMA screening run, including ten different samples of patients referred to our laboratory for Gilbert's syndrome diagnosis, we analyzed a sample showing (Fig. 1A) melting profile (MP) and Tm behavior slightly different from the standard UGT1A1 genotypes (TA6/6, 7/6, 7/7). This sample was therefore analyzed by direct sequencing which revealed the presence of (TA)5/6 genotype, a rare UGT1A1 promoter polymorphism. In order to optimize also the identification of this genotype by HRMA, we initially decided to add to each standard and (TA)5/6 sample a known amount of a (TA)6/7 in the ratio 3/1. Unfortunately, this strategy failed since the rare genotype and the wild type presented similar MPs (Fig. 1B). Finally, we spiked all samples with the (TA)7/7 sample in the ratio 3/1. Adding this genotype we identified four MPs (Fig. 1C), corresponding to the three most common genotypes and the rare (TA)5/6. The results obtained with the PCR and HRMA on triplicate samples, using our previously published protocol [1], confirmed the robustness of our method in terms of reproducibility and sensitivity [1].
Therefore, the spiking with (TA)7/7 makes possible the identification of the rare (TA)5/6 genotype. Differently from Ostanek et al, we used as spiking a concentration of (TA)7/7 not exceeding 35%: although our technical conditions were slightly different from those reported in literature [4,5]. We can therefore conclude that a) we can correctly identify (TA)5/6 UGT1A1 genotype by HRMA applying our protocol which works only when the spiking with (TA)7/7 is performed; b) HRMA technique is able, alone, to identify the unknown genotypes, but only after technical adjustments, the precise genotype attribution may be obtained; c) finally, we think that the use of the same methodology described above may allow the identification of other rare UGT1A1 promoter genotypes, probably not exceeding a concentration of 35% between the spiked genotype and the unknown sample. References [1] Minucci A, Concolino P, Giardina B, Zuppi C, Capoluongo E. Rapid UGT1A1 (TA)(n) genotyping by high resolution melting curve analysis for Gilbert's syndrome diagnosis. Clin Chim Acta 2010;411:246–9. [2] Minucci A, Mello E, Tripodi D, Zuppi C, Capoluongo E. Contribution of the TA repeats on melting temperature (T(m)) in a double strand DNA: comparison of two methods and implications in molecular diagnostics. Clin Biochem 2011;44:736–8. [3] Ostanek B, Furlan D, Bratanič BB. Genotyping UGT1A1(TA)(n) polymorphism rare variants by high resolution melting curve analysis. Clin Chim Acta 2011;412:489-9. [4] Seipp MT, Herrmann M, Wittwer CT. Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting. J Biomol Tech 2010;21:163–6. [5] Montgomery JL, Sanford LN, Wittwer CT. High-resolution DNA melting analysis in clinical research and diagnostics. Expert Rev Mol Diagn 2010;10:219–40.
Angelo Minucci⁎ Enrica Mello Domenico Tripodi Paola Concolino Cecilia Zuppi Ettore Capoluongo Laboratory of Clinical Molecular Biology, Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Italy ⁎Corresponding author at: Laboratory of Clinical Molecular Diagnostics, Department of Biochemistry & Clinical Biochemistry, Catholic University, Rome, Italy. Fax: +39 0630156783. E-mail addresses: [email protected], [email protected] (A. Minucci).
0009-9120/$ – see front matter © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.08.1130
15 March 2011
1360
Letter to the Editor
A
6TA/6TA 6TA/7TA 7TA/7TA Unknown (5TA/6TA)
B
6TA/6TA and 5TA/6TA 6TA/7TA 7TA/7TA
C
6TA/6TA 6TA/7TA 7TA/7TA 5TA/6TA
Fig. 1. HRMA of four UGT1A1 promoter polymorphisms. HRMA was performed on the LightCycler® 480 Real-Time PCR System (Roche Diagnostics). The dye used was Dye Reso Light (Roche Diagnostics) belonging to the new generation dyes. In panel A the software classifies (TA)5/6 in a manner not significantly different from the standards in terms of melting profiles and Tm. In panel B the spiking with (TA)6/7 does not improve the separation but makes (TA)5/6 more similar to the wild type genotype. In 1 °C after the (TA)7/7 addition in all samples the instrument associates melting profiles for each genotype.