In order to detect possible dysplastic melanocytic naevi (DMN) associated melanoma risk factors and lesion specific differences in the mutation spectrum of dysplastic and congenital melanocytic naevi (CMN), we screened 19 specimens of human sporadic DMN derived from 19 patients for the presence of mutations in five genes, which we had investigated in a former study in 19 CMN 1 and which have been reported to be associated with human cutaneous melanoma (N-ras, 2 p53, 3 CDKN2A, 4 CDK4, 5 and MC1R 7). METHODSDNA was extracted from selected paraffin embedded DMN resection specimens using the QIAamp DNA Mini Kit (Qiagen) according to the recommendations of the supplier. The relative number of atypical melanocytes in the DMN and the histological subtype of the DMN were determined in parallel slides by an experienced dermatologist (Dr Regina Zimmermann) (table 1). The screening strategy for the detection of activating point mutations in the oncogenes N-ras and CDK4 as well as for germline sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis, and the screening strategy for the detection of homozygous deletions and point mutations in the tumour suppressor genes p53 and CDKN2A by combined multiplex-PCR/SSCP analysis, have been described previously.1 In order to find out if the SSCP screening system that is used in our laboratory is suitable to detect point mutations in minor cellular subpopulations of the DMN lesions investigated (for example, N-ras point mutations in the atypical melanocyte fraction), we added gradually decreasing amounts of N-ras mutation harbouring genomic DNA (CAA to AAA mutation at one allele) to genomic N-ras wild type DNA before PCR and SSCP analysis. As a result, we could show that the aberrant mutation associated SSCP band is still visible at an admixture of less than 1% of mutation harbouring DNA (fig 1).In the present study we extended our MC1R screening system in order to allow the detection of two additional sequence variants (R151C and R160W), which like V92M and D294H 6 have been reported to be associated with red hair and light skin. 8 For reamplification of the 899 bp MC1R PCR preamplification product 1 with primer pair MC1R4A (5′ TCGCC GTGGA CCGCT ACATC 3′)/MC1R4B (5′ GCGTG CTGAA GACGA CACTG 3′) (120 bp PCR product, suitable for codon 151 and Key points• Nineteen specimens of human sporadic dysplastic melanocytic naevi (DMN) were screened for the presence of mutations in N-ras, p53, p16INK4a, p14ARF, CDK4, and MC1R.• In contrast to human congenital melanocytic naevi, a very low frequency of N-ras mutations seems to be characteristic of DMN.
Nanotechnology presents countless opportunities to develop new and improved consumer products for the benefit of society. However, as the industrial production and use of nanotechnology products continue to expand at a fast scale, potential human health concerns and ecological safeguards for the environment need to be addressed. Health risk assessment involving different animal species for multi-organ toxicity complimented with molecular investigations in cells is essential for investigating the potential toxic effects of nanomaterials. The purpose of this review is to present the current state of knowledge regarding the potential routes of human exposure to nanomaterials and their biological health effects. Although anthropogenic nanosized particles emitted in the environment are known to produce adverse human health in susceptible populations, much remains to be explored. Exposures can occur from direct exposure or from the use of commercial products made of nanomaterials. Safe manufacturing guidelines for prevention of exposures and recommendations on safe handling and use need to be established on a proactive basis to prevent adverse outcomes.
Eighteen congenital melanocytic naevi (CMN) from 17 patients and 18 dysplastic melanocytic naevi (DMN) from 18 patients were screened for mutations in the BRAF oncogene (present study) and the N-ras oncogene (in the course of two foregoing studies) by single-strand conformational polymorphism (SSCP)/sequencing analysis. BRAF mutations were demonstrated in both types of lesion. As a whole, 17 of 18 CMN (94.4%) and five of 18 DMN (27.7%) harboured either BRAF or N-ras mutations. As the BRAF oncogene is frequently found to be mutated in human cutaneous melanomas, it may constitute a risk factor for melanoma formation within CMN and DMN.
We have analyzed the Ha-ras, Ki-ras and N-ras gene for point mutations at codons 12, 13 and 61 via restriction fragment length polymorphism/polymerase chain reaction analysis and subsequent direct sequencing in non-cultured fresh-frozen tissues of 16 superficial spreading melanomas (SSM), 13 nodular malignant melanomas (NMM), 2 lentigo malignant melanomas (LMM), 1 dysplastic nevus, 1 congenital nevus and 5 normal nevi from 38 patients. Mutations were found in 4 melanoma samples, all belonging to the nodular malignant type. Three of them were mutated in N-ras and one in the Ha-ras gene. Mutation in N-ras was also detected in the congenital nevus. All mutations were exclusively located at the first two base pairs of codon 61. No Ki-ras mutation was detected in any lesion. No mutation could be found in SSM and LMM in addition to dysplastic and normal nevi. The frequency of ras mutation in NMM was 31%, whereas in SSM it was 0%. Our study suggests (a) an association between ras mutations (mainly N-ras) and the NMM as a subgroup of human melanoma; (b) that activation of Ki-ras is not involved in the pathogenesis of melanoma. The role of UV radiation in point mutations of ras genes in human melanoma is discussed.
In this study we analysed snap-frozen surgical resections of 16 superficial spreading melanomas, 13 nodular malignant melanomas, 2 lentigo maligna melanomas, 1 dysplastic nevus, 1 congenital nevus and 5 normal nevi from 38 patients for point mutations in the human p53 gene at exons 5-8 by polymerase chain reaction/single-strand conformation polymorphism as well as for loss of heterozygosity of p53 by restriction-fragment-length polymorphism/polymerase chain reaction in order to determine whether p53 aberrations are associated with melanoma subtypes. In addition, we analysed six melanoma cell lines for point mutations in p53. Our results revealed the absence of point mutations and loss of heterozygosity in all fresh resected lesions. However, a TAC (Tyr) to TGC (Cys) transition at codon 163 in exon 5 was found in one cell line.
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