Detection and Analysis of Genetic Alterations in Normal Skin and Skin Tumours

81 Pages·2002·0.715 MB·English

by  Sivertsson A.

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

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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