Detection and Analysis of Genetic Alterations in Normal Skin and Skin Tumours Åsa Sivertsson Med.Lic. Royal Institute of Technology Department of Biotechnology Stockholm 2002  Åsa Sivertsson Department of Biotechnology Royal Institute of Technology Stockholm Center for Physics, Astronomy and Biotechnology SE-106 91 Stockholm Sweden Printed at Universitetsservice US AB Box 700 14 100 44 Stockholm Sweden ISBN 91-7283-379-3 Åsa Sivertsson (2002): Detection and analysis of genetic alterations in normal skin and skin tumours Department of Biotechnology, Royal Institute of Technology Stockholm, Sweden ISBN 91-7283-379-3 ABSTRACT The investigation of genetic alterations in cancer-related genes is useful for research, prognostic and therapeutic purposes. However, the genetic heterogeneity that often occurs during tumour progression can make correct analysis challenging. The objective of this work has been to develop, evaluate and apply techniques that are sufficiently sensitive and specific to detect and analyse genetic alterations in skin tumours as well as in normal skin. Initially, a method based on laser-assisted microdissection in combination with conventional dideoxy sequencing was developed and evaluated for the analysis of the p53 tumour suppressor gene in small tissue samples. This method was shown to facilitate the analysis of single somatic cells from histologic tissue sections. In two subsequent studies the method was used to analyse single cells to investigate the effects of ultraviolet (UV) light on normal skin. Single p53 immunoreactive and non- immunoreactive cells from different layers of sunexposed skin, as well as skin protected from exposure, were analysed for mutations in the p53 gene. The results revealed the structure of a clandestine p53 clone and provided new insight into the possible events involved in normal differentiation by suggesting a role for allele dropout. The mutational effect of physiological doses of ultraviolet light A (UVA) on normal skin was then investigated by analysing the p53 gene status in single immunoreactive cells at different time-points. Strong indications were found that UVA (even at low doses) is indeed a mutagen and that its role should not be disregarded in skin carcinogenesis. After slight modifications, the p53 mutation analysis strategy was then used to complement an x-chromosome inactivation assay for investigation of basal cell cancer (BCC) clonality. The conclusion was that although the majority of BCC’s are of monoclonal origin, an occasional tumour with apparently polyclonal origin exists. Finally, a pyrosequencing-based mutation detection method was developed and evaluated for detection of hot-spot mutations in the N-ras gene of malignant melanoma. More than 80 melanoma metastasis samples were analysed by the standard approach of single strand conformation polymorphism analysis (SSCP)/DNA sequencing and by this pyrosequencing strategy. Pyrosequencing was found to be a good alternative to SSCP/DNA sequencing and showed equivalent reproducibility and sensitivity in addition to being a simple and rapid technique. Keywords: single cell, DNA sequencing, p53, mutation, UV, BCC, pyrosequencing, malignant melanoma, N-ras  Åsa Sivertsson, 2002 There is much pleasure to be gained from useless knowledge BB BB ee ee rr rr tt tt rr rr aa aa nn nn dd dd RR RR uu uu ss ss ss ss ee ee ll ll ll ll ISBN 91-7283-379-3 This thesis is based on the following publications, which in the text will be referred to by their Roman numerals: LIST OF PUBLICATIONS I. Persson Å, Ling G, Williams C, Bäckvall H, Ponten J, Ponten F, Lundeberg J. (2000) Analysis of p53 mutations in single cells obtained from histological tissue sections. Anal Biochem 287(1): 25-31. II. Persson Å/Ling G, Berne B, Uhlén M, Lundeberg J, Ponten F. (2001) Persistent p53 mutations in single cells from normal human skin. Am J Pathol 159:1247-1253. III. Persson Å, Wiegleb Edström D, Bäckvall H, Lundeberg J, Pontén F, Ros A- M, Williams C. (2002) The Mutagenic Effect of Ultraviolet A1 in Human Skin-demonstrated by sequencing the p53 gene in single keratinocytes. Photodermatology, Photoimmunology and Photomedicine. In press. IV. Asplund A, Sivertsson Å, Lundeberg J, Pontén F (2002) Analysis of x- chromosome inactivation patterns in human basal cell carcinoma reveals diverse clonal organization: evidence of multicellular origin. Manuscript. V. Sivertsson Å, Platz A, Hansson J, Lundeberg J. (2002) Pyrosequencing as an alternative to SSCP for detection of N-ras mutations in human melanoma metastases. Clin Chem. In press. INTRODUCTION___________________________________________________ 1 Mutation detection_______________________________________________________ 1 Scanning methods _______________________________________________________ 2 DNA sequencing ______________________________________________________________ 2 Sanger sequencing_____________________________________________________________ 2 SSCP _______________________________________________________________________ 4 Other conformation-based methods________________________________________________ 6 Cleavage –based techniques _____________________________________________________ 8 Specific methods ________________________________________________________11 Hybridisation-based techniques__________________________________________________ 11 Methods based on allele-specific amplification ______________________________________ 12 Oligonucleotide ligation assays __________________________________________________ 12 Techniques based on polymerase extension _________________________________________ 13 Pyrosequencing ______________________________________________________________ 16 CARCINOGENESIS________________________________________________ 19 Cancer of the skin _______________________________________________________21 Ultraviolet radiation __________________________________________________________ 21 Normal skin _________________________________________________________________ 24 The tumour suppressor p53 _____________________________________________________ 26 The p53 patch _______________________________________________________________ 27 Non-melanoma skin cancer _____________________________________________________ 29 Malignant melanoma__________________________________________________________ 30 PRESENT INVESTIGATION________________________________________ 32 Genetic analysis of skin and skin tumour samples. _____________________________32 Sample handling and preparation for analysis_______________________________________ 32 Sample preparation _________________________________________________________ 32 Microdissection____________________________________________________________ 33 Laser microdissection _______________________________________________________ 33 p53 gene analysis ________________________________________________________35 Development of method for single cell genetic analysis (I). _____________________________ 35 Applications ____________________________________________________________38 Effects of UV radiation on the p53 gene in normal human epidermis (II, III) _______________ 38 Sun-exposure and persistent p53 mutations (II) _____________________________________ 38 p53 mutations induced by UVA1 (III) _____________________________________________ 41 The clonality of BCC (IV) ______________________________________________________ 43 Detection of N-ras hot-spot mutations in melanoma using pyrosequencing (V)______________ 45 Abbreviations______________________________________________________ 47 Acknowledgements _________________________________________________ 48 References ________________________________________________________ 50 [...]... sequence composition of the samples The detection sensitivity is greater in GC rich templates which is due to the higher proportion of hydrogen bonds formed between G and C residues resulting in a more intricate, and thus more easily influenced folding of the strands (Highsmith, 1999) 5 Detection and analysis of genetic alterations in normal skin and skin tumours The detection of fragments after electrophoresis... dUTP incorporation and at dGTP sites 9 Detection and analysis of genetic alterations in normal skin and skin tumours using two different excision reactions involving uracil-N-glycosylase/E coli endonuclease IV and dGTP modifications respectively The resulting sets of fragments correspond to the positions of deoxyguanine and deoxythymidine in the sequence and can be analysed using standard sequencing... ionisation - time -of- flight - mass spectrometry) emerged as a method for DNA sequencing in the late 1980’s An innovation in the field of ionisation of macromolecules made it possible to perform 13 Detection and analysis of genetic alterations in normal skin and skin tumours analysis of DNA in a system which was earlier limited to peptide analysis (Karas, 1988) DNA samples are desorbed, ionised and subjected... therefore of great interest for diagnostic and prognostic purposes as well as in understanding gene function Mutation detection Gross chromosomal aberrations can usually be detected by cytogenetical methods or by DNA fragment analysis, while methods that are more sensitive and usually more expensive must be used for detection of subtle alterations A wide range of methods already exists for detection of single... signals, evasion of apoptosis, a limitless replicative potential, sustained angiogenesis and the capacity of tissue invasion and metastasis Self-sufficiency in growth signals can be achieved through alterations in 19 Detection and analysis of genetic alterations in normal skin and skin tumours growth signalling, for example by receptor over-expression, autocrine stimulation or activation of the ras oncogene... portion of the hair follicle (Nishimura, 2002) A Cornified layer Granular layer Epidermis Spinous layer Dermis Basal layer Cornified layer B Granular l Epidermis Spinous l Basal l Dermis Subcutis Hair follicle Sebaceous gland Sweat gland Fat Figure 5 Histological section of normal epidermis (A) and a schematic picture of the different layers of the skin (B) 25 Detection and analysis of genetic alterations. .. Detection and analysis of genetic alterations in normal skin and skin tumours identify previously characterised sequence variations such as polymorphisms and mutation hotspots Some of the more widely used methods as well as some promising upcoming techniques will be described briefly below Scanning methods In order to perform a comprehensive mutation analysis of a stretch of DNA the use of scanning... photoproduct S S P P 5-OH-dC 8-OXO-dG Figure 4 Structure of UV-induced photoproducts (A) and the DNA lesions commonly formed after UVA irradiation (B) 23 Detection and analysis of genetic alterations in normal skin and skin tumours Normal skin The skin including the underlying subcutis amounts to 15% of our bodyweight and is thereby our largest organ and also the most important barrier to protect us from... at defined positions 15 Detection and analysis of genetic alterations in normal skin and skin tumours Pyrosequencing The principle of sequencing-by-synthesis was described in 1985 (Melamede, 1985) and led to the development of two approaches for DNA sequencing; direct detection of labelled nucleotides (Canard, 1994; Metzker, 1994) and indirect detection of released pyrophosphate (Hyman, 1988; Nyren,... 2001; Gustafsson, 2001; Fakhrai-Rad, 2002; Wasson, 2002; Andreasson, 2002) but it has been used in the typing of bacteria (Monstein, 2001) and viruses (Gharizadeh, 2001; O'Meara, 2001), mutation screening (Garcia, 2000) and tag library sequencing (Agaton, 2002; Nordstrom, 2001) 17 Detection and analysis of genetic alterations in normal skin and skin tumours Polymerase 5' TAGAAGGACCGTTTAAGT 3' 5' ATCTT . intricate, and thus more easily influenced folding of the strands (Highsmith, 1999). Detection and analysis of genetic alterations in normal skin and skin tumours 6 The detection of fragments. 91-7283-379-3 Åsa Sivertsson (2002): Detection and analysis of genetic alterations in normal skin and skin tumours Department of Biotechnology, Royal Institute of Technology Stockholm, Sweden ISBN. methods Detection and analysis of genetic alterations in normal skin and skin tumours 2 identify previously characterised sequence variations such as polymorphisms and mutation hotspots. Some of the