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
­A strategy for identifying nuclear modifier genes by massively parallel whole-genome sequencing
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
Show others and affiliations
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Leber hereditary optic neuropathy (LHON) results from mutations in mtDNA, butadditional factors are required for disease expression. LHON is thus a model for theconcept of modifiers affecting expression of single gene diseases. No modifier factorhas yet been clearly identified. Here we describe a large, consanguineous familyaffected by LHON with offspring showing variable disease expression. This providesan opportunity to investigate the presence of nuclear modifiers in homozygousgenomic regions. We analyzed genomes from six members, parents and foursiblings. Each genome was sequenced to >23x coverage and approximately 3.8million single nucleotide variants and small indels per individual were called, where17,000‐20,000 were located in the exome. As a first step, we hypothesize that amodifier gene affecting penetrance of the LHON mutation, and another modifiergene predisposing to an aggravated phenotype, are located in the protein‐codingparts of the genome (the exome). As we gain experience in data analysis, this can befollowed by extended analyses of additional genomic regions. Our initial, simplehypothesis generated five lists of candidate modifier genes, conforming to fivedifferent models of inheritance. In total, 86 candidate genes were identified and 11of these genes contained 14 variants that were further validated by Sangersequencing. Additional Sanger validation in another two affected siblings reducedthe number of candidate genes to two potential disease‐causing variants.

Keyword [en]
LHON, whole-genome sequencing, DNA, SNVs
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-48041OAI: oai:DiVA.org:kth-48041DiVA: diva2:456666
Note
QS 2011Available from: 2011-11-15 Created: 2011-11-15 Last updated: 2011-11-15Bibliographically approved
In thesis
1. 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
Stranneheim, HenrikJemt, AndersHolmberg, KristinaLundeberg, Joakim
By organisation
Gene TechnologyScience for Life Laboratory, SciLifeLab
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 297 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