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Nucleotide variations
in the human genome
Suitable markers for tissue identification
and allelic instability testing
Ronald Huijsmans
The work in this thesis was conducted at the laboratory of Molecular Diagnostics at the
Jeroen Bosch Hospital in close cooperation with the department of Medical Microbiology
and Infection Control at the Vrije Universiteit (VU) University Medical Center.
Financial support for the publication of this thesis was kindly provided by:
- The Jeroen Bosch Academy
- Roche Diagnostics Nederland B.V.
- Roxane de Beaumont and Rob van der Meij
- BioMérieux Benelux B.V.
- Life Technologies
ISBN
978-94-6108-485-9
Lay-out & printer
Gildeprint drukkerijen, Enschede, The Netherlands
Cover image
Irma Verhoofstad
VRIJE UNIVERSITEIT
Nucleotide variations
in the human genome
Suitable markers for tissue identification
and allelic instability testing
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad Doctor aan
de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus
prof.dr. F.A. van der Duyn Schouten,
in het openbaar te verdedigen
ten overstaan van de promotiecommissie
van de Faculteit der Geneeskunde
op dinsdag 22 oktober 2013 om 13.45 uur
in de aula van de universiteit,
De Boelelaan 1105
door
Cornelis Johannes Jacobus Huijsmans
geboren te Roosendaal en Nispen
Promotor
prof.dr. P.H.M. Savelkoul
Copromotor
dr. M.H.A. Hermans
Contents
Chapter 1 General introduction 7
1.1 History of DNA research 9
1.2 Impact of molecular diagnostics in pathology 9
1.3 DNA variations 10
1.3.1. Single nucleotide polymorphisms 10
1.3.2. SNP associated traits and disorders 11
1.4 Somatic mutations and cancer 13
1.4.1. Loss of heterozygosity 13
1.4.2. Oncogene amplification 16
1.5 Applications of SNP detection in molecular diagnostics 16
1.5.1. SNP genotyping methods 16
1.5.2. Tissue identification 17
1.5.3. Cancer diagnostics 18
1.5.3.A. LOH of the JAK2 locus in myeloproliferative disease 18
1.5.3.B. HER2/TOP2A locus in breast cancer 19
Chapter 2 Comparative analysis of four methods to extract DNA from paraffin- 33
embedded tissues: effect on downstream molecular applications
Chapter 3 Single nucleotide polymorphism profiling assay to confirm the identity 49
of human tissues
Chapter 4 Single nucleotide polymorphism profiling assay to exclude serum 69
sample mix-up
Chapter 5 Sensitive detection and quantification of the JAK2V617F allele by 81
real-time PCR: blocking wild-type amplification by using a peptide
nucleic acid
Chapter 6 Single nucleotide polymorphism (SNP)-based loss of heterozygosity 97
(LOH) testing by real time PCR in patients suspect of myeloproliferative
disease
Chapter 7 Allelic imbalance at the HER2/TOP2A locus in breast cancer 117
Chapter 8 8.1 Summary and general discussion 139
8.1.1. Technological advances 139
Chapter 2 | Optimizing DNA extraction 140
Chapter 3 and 4 | Confirming the identity of human tissues 141
Chapter 5 | Determining JAK2V617F mutational status 142
Chapter 6 | Loss of heterozygosity of the JAK2 genomic region 143
Chapter 7 | Allelic imbalance at the HER2/TOP2A locus 144
8.2 Venturing beyond the scope of this thesis: bacterial SNP genotyping 146
8.3 Concluding remarks 147
Chapter 9 Nederlandse samenvatting en discussie 157
Dankwoord 169
Curriculum vitae 171
List of Publications 173

1
General introduction
8 | Chapter 1
1.1 History of DNA research
1
During the winter of 1868-1869 Friedrich Miescher, a Swiss physician, performed experiments
at Germany’s University of Tübingen on the composition of leukocytes, which eventually led
to the discovery of DNA. Miescher noticed an unknown substance in the precipitates derived
from the leukocytes. Further characterization showed that the substance was neither lipid
nor protein. Because the nuclei were the source of isolation he named the novel substance
Nuclein [1]. In 1919 the base, sugar and phosphate were identified by Phoebus Levene to be
the building blocks of DNA [2]. It took more than 30 years thereafter before Watson and
Crick revealed the 3D-structure of DNA in 1953 [3].
Since these basic discoveries, tremendous scientific progress has been made in the fields of
genetics and biotechnology. These developments have led to a new era in science and
medicine, accelerated by the development of DNA sequencing [4] and the Polymerase Chain
Reaction (PCR) [5].
Determining the sequence of a specific DNA stretch of interest was made possible by the
introduction of DNA sequencing. Subsequently, the PCR technique made it possible to
amplify small amounts of DNA and the variations therein, further enhancing the potential of
DNA sequencing by combining both techniques. This allowed scientists to study specific DNA
regions of interest. These developments resulted in the identification of variations in
oncogenes and tumor suppressor genes (TSGs).
A very important initiative was the Human Genome Project (HGP), which started in 1990. The
completion of this project revealed the human genome sequence [6-8]. The results of the HGP
have led to an extremely detailed dataset with regard to the similarities in and differences between
human genomes. The human genome was estimated to consist of about 98.5% non-coding
sequences and only 1.5% coding sequences and to contain approximately 30,000 genes.
1.2 Impact of molecular diagnostics in pathology
Molecular diagnostics, referring to the field of detection and analysis of nucleic acids, is
currently the most rapidly expanding discipline within the field of pathology.
Advances in molecular biology led to the discovery of numerous types of DNA variations
amongst individuals and DNA alterations in cancer, some of which confer a selective growth
advantage to the affected (tumor) cell. These include mutations that lead to changes in DNA
copy numbers such as gains and losses of a chromosomal locus [9].
The applications of molecular diagnostics in pathology therefore range from tissue
identification to various purposes in oncology. The applications in oncology include disease
diagnosis and prognosis of disease susceptibility, prognosis of clinical outcome and therapy
selection and prediction of therapy efficacy.
General introduction | 9