| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1 Institute of Clinical Pharmacology, Charité, Humboldt University of Berlin, 10117 Berlin, Germany
2 TIB Molbiol, Syntheselabor, 10829 Berlin, Germany
aaddress correspondence to this author at: Institute for Clinical Pharmacology, University Hospital Charité, Schumannstrasse 20/21, 10117 Berlin, Germany; fax 49-30-450-525932, e-mail konstanze.diefenbach{at}charite.de
The melanocortin-1 receptor (MC1R), localized on chromosome 16q24.3, is a G-protein-coupled receptor expressed mostly in melanocytes. Melanotropic ligands such as 
-melanocyte-stimulating hormone and adrenocorticotropic hormone act via MC1R and regulate the proportion of the photo-protective melanins eumelanin and pheomelanin, which may contribute to ultraviolet (UV) radiation-induced skin damage (1) by favoring the synthesis of eumelanin. Individuals with red hair have a predominance of pheomelanin in hair and skin and/or a reduced ability to produce eumelanin, which may explain why they fail to tan and suffer from increased cutaneous UV sensitivity and why UV irradiation is more dangerous for them. Fair skin and red hair are also associated with an increased risk of cutaneous malignant melanoma (2)(3). Recently, some polymorphic variants of MC1R have been associated with red hair and found to be overrepresented in individuals with fair skin (4)(5)(6)(7), particularly the Arg151Cys, Arg160Trp, and Asp294His variants (1)(4). These variants correlate with an increased risk of malignant melanoma (7)(8)(9). The association between MC1R variants and malignant cutaneous melanoma suggests that the MC1R gene is a susceptibility gene for this skin malignancy (10).
Heritable factors should be taken into consideration when measuring cutaneous UV sensitivity of substances with a phototoxic potential. Because MC1R may be a genetic determinant of individual skin sensitivity toward UV irradiation, we developed LightCycler assays for the detection of common MC1R polymorphisms. This rapid-cycle PCR combined with real-time fluorescence monitoring and melting point analysis is able to detect polymorphisms in 
1 h.
LightCycler methods were established for all common (>1%) and/or functional polymorphisms (11)(12)(13) (see Table 1
). Genomic DNA from 100 volunteers was extracted from peripheral blood by standard methods. Primers and LightCycler hybridization probes (see Table 1
) were developed according to the MC1R sequence (OMIM 15555; GenBank accession no. NM_002386). For some primers and hybridization probes, mismatches were incorporated either to destabilize folded motifs in the target or to optimize the differentiation in the melting analysis (see Val60Leu, Arg160Trp, and Arg163Gln in Table 1
). The probes were labeled using the respective LightCycler Red640 ester or Red705 amidite according to the manufacturer’s instruction (Roche Diagnostics). Two of the assays were established for simultaneous detection of two mutations each (see Val92Met and Thr95Met, and Arg160Trp and Arg163Gln in Table 1
). Because of low allele frequencies (<1%), two polymorphisms (Thr95Met and Asp294His) were not detected in our 100 volunteers. Melting data of these variants are given according to assays using synthetic oligonucleotides covering the wild type or the mutated variant (see Table 1
).
|
All PCRs were performed in a total volume of 20 µL in LightCycler glass capillaries. The reaction mixture contained 0.2 µM each primer, 0.1 µM each of the anchor and sensor, 0.1 mM deoxynucleotide triphosphates, 30 mg/L bovine serum albumin, 50 mL/L dimethyl sulfoxide, 2 µL of 10x Taq Buffer, 1 µL of the sample (containing 20–30 ng/µL genomic DNA), 1 U of Taq polymerase, and assay-specific amounts of MgCl2 (2.5–6.3 mM; see Table 1
) and was adjusted to the final volume of 20 µL with distilled water. The PCR conditions in the LightCycler (Roche Diagnostics) were as follows: initial denaturation at 95 °C for 40 s, followed by 45 cycles of denaturation at 95 °C for 5 s, 10 s of annealing at assay-specific temperatures (see Table 1
), and extension at 72 °C for 10 s.
After amplification the melting analysis was performed by denaturation at 98 °C for 0 s and annealing at 40 °C for 20 s; the temperature was increased continuously up to 85 °C with a ramp rate of 0.1 °C/s. Fluorescence was recorded during the heating period, and the melting curves (F/T) were converted to melting peaks (-dF/dT). The melting peak analysis showed well-differentiated temperatures for all polymorphisms (see Table 1
). The melting curves for the typing of two variants (Arg160Trp and Arg163Gln) are shown in Fig. 1
as examples.
|
LightCycler results were compared with the results achieved by PCR–restriction fragment length polymorphism typing (11)(14)(15) and DNA sequencing (PrismTM 310 Genetic Analyzer; Applied Biosystems Applera) and correlated perfectly.
In conclusion, the established LightCycler-based assays offer a fast, accurate, and reproducible method for detecting point mutations in MC1R by melting curve analysis. Thus, they can be valuable tools for the screening of volunteers or patients when evaluating cutaneous UV sensitivity.
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Trp) and 163 (Arg