Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Sun‐induced Missense Mutations Are Extensively Accumulated and Tolerated in Phenotypically Intact Stem Cell Compartments of Human Skin
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0002-2207-7370
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Proteomics.
Show others and affiliations
(English)Article in journal (Refereed) Submitted
Abstract [en]

Here we demonstrate that intermittently sun‐exposed human skin contains an extensive number of phenotypically intact stem cell compartments bearing missense mutations in the p53 tumor suppressor gene. Deep sequencing of sun‐exposed and shielded microdissected skin from mid‐life individuals revealed that persistent p53 mutations had accumulated in 14% of all epidermal cells, with no apparent signs of a growth advantage of the affected cell compartments. Furthermore, 6% of the mutated epidermal cells encoded a truncated protein. The abundance of these events, not taking into account intron mutations and mutations in other genes that also may have functional implications, suggests an extensive tolerance of human cells to severe genetic alterations caused by ultraviolet light, with an estimated annual rate of accumulation of approximately 35,000 new persistent protein altering p53 mutations in sun exposed skin of a human individual.

Identifiers
URN: urn:nbn:se:kth:diva-12404OAI: oai:DiVA.org:kth-12404DiVA: diva2:310852
Note
QC 20100416Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2011-11-15Bibliographically approved
In thesis
1. Methods for Analyzing Genomes
Open this publication in new window or tab >>Methods for Analyzing Genomes
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human genome reference sequence has given us a two‐dimensional blueprint of our inherited code of life, but we need to employ modern‐day technology to expand our knowledge into a third dimension. Inter‐individual and intra‐individual variation has been shown to be larger than anticipated, and the mode of genetic regulation more complex. Therefore, the methods that were once used to explain our fundamental constitution are now used to decipher our differences. Over the past four years, throughput from DNA‐sequencing platforms has increased a thousand‐fold, bearing evidence of a rapid development in the field of methods used to study DNA and the genomes it constitutes. The work presented in this thesis has been carried out as an integrated part of this technological evolution, contributing to it, and applying the resulting solutions to answer difficult biological questions.

Papers I and II describe a novel approach for microarray readout based on immobilization of magnetic particles, applicable to diagnostics. As benchmarked on canine mitochondrial DNA, and human genomic DNA from individuals with cystic fibrosis, it allows for visual interpretation of genotyping results without the use of machines or expensive equipment. Paper III outlines an automated and cost‐efficient method for enrichment and titration of clonally amplified DNA‐libraries on beads. The method uses fluorescent labeling and a flow‐cytometer to separate DNA‐beads from empty ones. At the same time the fraction of either bead type is recorded, and a titration curve can be generated. In paper IV we combined the highly discriminating multiplex genotyping of trinucleotide threading with the digital readout made possible by massively parallel sequencing. From this we were able to characterize the allelic distribution of 88 obesity related SNPs in a population of 462 individuals enrolled at a childhood obesity center. Paper V employs the throughput of present day DNA sequencingas it investigates deep into sun‐exposed skin to find clues on the effects of sunlight during the course of a summer holiday. The tumor suppressor p53 gene was targeted, only to find that despite its well‐documented involvement in the disease progression of cancers, an estimated 35,000 novel sun‐induced persistent p53 mutations are added and phenotypically tolerated in the skin of every individual every year. The last paper, VI, describes a novel approach for finding breast cancer biomarkers. In this translational study we used differential protein expression profiles and sequence capture to select and enrich for 52 candidate genes in DNA extracted from ten tumors. Two of the genes turned out to harbor protein‐altering mutations in multiple individuals.

Publisher
53 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:4
Keyword
array, sequence capture, genotyping, trinucleotide threading, sequencing, massively parallel sequencing, single molecule sequencing, Visual DNA, p53, single nucleotide polymorphism, biomarker
National Category
Genetics
Identifiers
urn:nbn:se:kth:diva-12407 (URN)978-91-7415-596-9 (ISBN)
Public defence
2010-05-07, FD5, AlbaNova Universitetscentrum, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2010-04-19 Created: 2010-04-16 Last updated: 2010-08-26
2. Enabling massive genomic and transcriptomic analysis
Open this publication in new window or tab >>Enabling massive genomic and transcriptomic analysis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid advances have enormously expanded sequencing opportunities and applications, but also imposed heavy strains on steps prior to sequencing, as well as the subsequent handling and analysis of the massive amounts of sequence data that are generated, in order to exploit the full capacity of these novel platforms. The work presented in this thesis (based on six appended papers) has contributed to balancing the sequencing process by developing techniques to accelerate the rate-limiting steps prior to sequencing, facilitating sequence data analysis and applying the novel techniques to address biological questions.

 

Papers I and II describe techniques to eliminate expensive and time-consuming preparatory steps through automating library preparation procedures prior to sequencing. The automated procedures were benchmarked against standard manual procedures and were found to substantially increase throughput while maintaining high reproducibility. In Paper III, a novel algorithm for fast classification of sequences in complex datasets is described. The algorithm was first optimized and validated using a synthetic metagenome dataset and then shown to enable faster analysis of an experimental metagenome dataset than conventional long-read aligners, with similar accuracy. Paper IV, presents an investigation of the molecular effects on the p53 gene of exposing human skin to sunlight during the course of a summer holiday. There was evidence of previously accumulated persistent p53 mutations in 14% of all epidermal cells. Most of these mutations are likely to be passenger events, as the affected cell compartments showed no apparent growth advantage. An annual rate of 35,000 novel sun-induced persistent p53 mutations was estimated to occur in sun-exposed skin of a human individual.  Paper V, assesses the effect of using RNA obtained from whole cell extracts (total RNA) or cytoplasmic RNA on quantifying transcripts detected in subsequent analysis. Overall, more differentially detected genes were identified when using the cytoplasmic RNA. The major reason for this is related to the reduced complexity of cytoplasmic RNA, but also apparently due (at least partly) to the nuclear retention of transcripts with long, structured 5’- and 3’-untranslated regions or long protein coding sequences. The last paper, VI, describes whole-genome sequencing of a large, consanguineous family with a history of Leber hereditary optic neuropathy (LHON) on the maternal side. The analysis identified new candidate genes, which could be important in the aetiology of LHON. However, these candidates require further validation before any firm conclusions can be drawn regarding their contribution to the manifestation of LHON.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. 45 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:24
Keyword
DNA, RNA, sequencing, massively parallel sequencing, alignment, assembly, single nucleotide polymorphism, LHON
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-45957 (URN)978-91-7501-164-6 (ISBN)
Public defence
2011-12-02, Petrén‐salen, Nobels väg 12B, Karolinska Institute Campus Solna, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20111115Available from: 2011-11-15 Created: 2011-11-01 Last updated: 2011-11-15Bibliographically approved

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Ståhl, Patrik L.Stranneheim, HanrikBerglund, LisaLundeberg, Joakim
By organisation
Gene TechnologyProteomics

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 60 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf