Bachelor Degree - Thesis

Genetic cause of congenital thrombocytopenia
in a Dutch family with 2 affected individuals
Natasja Eland
UMCU Division Medical Genetics
Avans University of Applied Science.
Version 1, Feb 2012

UMC Utrecht, Location Wilhelmina Children hospital
Genetic cause of congenital thrombocytopenia in a Dutch family with 2 affected individuals.
2
By: Natasja Eland, Avans University of Applied Science.
Jan, 2012
Internship location
UMC, Location Wilhelmina Children Hospital
Department of Medical Genetics, section genome diagnostics
Utrecht, the Netherlands
Mentor (Intern UMCU WKZ)
M. Albring
088-7555195
m.albring@umcutrecht.nl
Internship coordinator (Intern UMCU WKZ)
Dr. M.E. van Gijn
Clinical Molecular Geneticist
Department of Medical Genetics, UMC Utrecht
Location Wilhelmina Children Hospital
Lundlaan 6
3584 EA Utrecht
088-7554090
m.e.vangijn@umcutrecht.nl
Internship mentor (University)
Dr. W. van Gils
Avans Hogeschool, ATGM
Lovensdijkstraat 61/63
4818 AJ Breda
Tel: 076-5250418
w.vangils@avans.nl
Intern
Natasja Eland
0643232888
neland@student.avans.nl (contact for internship mentor)
natasja_eland@hotmail.com (contact for internship coordinator and mentor)
Education
Major Biological en Medical Laboratory Research
Minor Forensic DNA Research
Avans University
Breda, the Netherlands
Internship period:
5 September 2011 t/m 23 January 2012

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Jan, 2012
Table of contents
Table of contents..................................................................................................................................................... 3
Abstract ................................................................................................................................................................... 4
Introduction.............................................................................................................................................................. 5
Theoretical background ........................................................................................................................................... 6
Genetic disorders................................................................................................................................................. 6
Mutations ............................................................................................................................................................. 6
Pathogen mutations.......................................................................................................................................... 6
Non-pathogen mutations .................................................................................................................................. 6
Unclassified variants......................................................................................................................................... 6
Inheritance ........................................................................................................................................................... 6
Dominant inheritance........................................................................................................................................ 6
Recessive inheritance ...................................................................................................................................... 7
Genes involved in this project .............................................................................................................................. 7
MPL.................................................................................................................................................................. 7
THPO ............................................................................................................................................................... 8
RUNX1 ............................................................................................................................................................. 9
Genotype/phenotype relation............................................................................................................................... 9
Materials and methods .......................................................................................................................................... 10
Methods and used protocols.............................................................................................................................. 10
Mutation analysis............................................................................................................................................ 10
Genomic copy number variation detection ..................................................................................................... 12
Optimization....................................................................................................................................................... 14
PCR for sequencing purposes........................................................................................................................ 14
(You)MAQ-assay............................................................................................................................................ 14
Results casus ........................................................................................................................................................ 16
Sanger sequencing on MPL and THPO gene.................................................................................................... 16
(You)MAQ-assay on the MPL and RUNX1 gene ............................................................................................... 16
Discussion, conclusion and proposition................................................................................................................. 18
Acknowledgements ............................................................................................................................................... 20
Literature ............................................................................................................................................................... 21
Other resources ................................................................................................................................................. 22
Appendix A: PCR................................................................................................................................................... 23
Appendix B: Sanger Sequencing........................................................................................................................... 24
Appendix C: (You)MAQ-assay............................................................................................................................... 26
Appendix D: Experiments outside the project........................................................................................................ 28

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Jan, 2012
Abstract
Thrombocytopenia is a condition in which your blood has a lower than normal number of platelets. This causes
problems with blood clotting which can lead to severe haemorrhage (internal and external). Thrombocytopenia
can be caused by different factors like toxic chemicals, medicines, viruses but can also be inherited. Some
genetic disorders can cause a low number of blood platelets like for example Wiskot-Aldrich syndrome. Multiple
studies have shown that there are several genes, besides WAS, involved in the development of
thrombocytopenia, like the RUNX1 and MPL gene.
In a Dutch family 2 sisters were born with severe thrombocytopenia. To determine the genetic cause of their
congenital thrombocytopenia they were tested for mutations in the RUNX1 and WAS genes using the Sanger
sequencing method. Unfortunately no mutations were found.
In the current project Sanger sequence analysis was set up and implemented in the diagnostic DNA laboratory of
the UMC Utrecht for the MPL and THPO genes. Moreover a YouMAQ-assay was designed, set up and
implemented for MPL and RUNX1 genes to analyse genomic deletions and duplications. Subsequently the two
sisters were analysed for disease causing mutations and/or deletions in the MPL, THPO and RUNX1 genes. We
could not detect disease causing mutations. These findings imply that, mutations in other so far unknown genes
or genetic regions are responsible for the severe congenital thrombocytopenia.
Used abbreviations
Abbreviation Meaning
HSC Hematopoietic stem cell
ET Essential thrombocythemia
CAMT Congenital Amegakaryocytic thrombocytopenia
FPD Familial Platelet Disorder
AML Acute myeloid leukaemia
DNA Deoxyribonucleic acid
cDNA Copy DNA
dsDNA Double-stranded Deoxyribonucleic acid
ssDNA Single-stranded Deoxyribonucleic acid
PCR Polymerase Chain Reaction
dNTP Deoxyribonucleotide triphosphate
ddNTP Dideoxyribonucleotide triphosphate
MAQ Multiplex Amplicon Quantification
MPL Myeloproliferative Leukaemia
THPO Thrombopoietin
RUNX1 Runt-related transcription factor 1
JAK2 Janus Kinase 2
SH2B3 Adaptor Protein 3
OMIM Online Mendelian Inheritance in Man
RefSeq Reference Sequence
HGVS Human Genome Variation Society
SNP Single Nucleotide Polymorphism
UV Unclassified variant
MAQ-assay Multiplex Amplicon Quantification assay
MPLA Multiplex ligation-dependent probe amplification

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Introduction
When not enough blood platelets are produced in the bone marrow we speak of thrombocytopenia. This causes
problems with blood clotting which can lead to severe haemorrhage (internal and external). In a healthy person
the blood platelet count is about 150.000 to 450.000 platelets per microliter blood. A platelet count below 150.000
is seen as below normal. Mild haemorrhage has platelet counts of 50.000 platelets per microliter blood. In severe
haemorrhage the counts are below 20.000 platelets per microliter blood.
Thrombocytopenia is caused by different factors. For example the bone marrow doesn’t make enough blood
platelets, the bone marrow makes sufficient blood platelets but the body uses up too many/destroys them, the
spleen piles up to many blood platelets (under normal conditions the spleen contains to 1/3rd
of the total blood
platelet count). In some cases all of the named problems can add up to cause thrombocytopenia. (NHLBI)
There are different diseases that cause thrombocytopenia. Like bone marrow cancer, vitamin B12- and folate
deficiency, medications like chemotherapy or genetic disorders like the Wiskot-Aldrich syndrome. (R.McMillan,
2007)
Blood platelets are produced in the bone marrow by megakaryocytes which are derived from the hematopoietic
stem cells (HSC). Though different cytokines and factors contribute to the growth and maturation of these
megakaryocytes, TPO is the major regulator of the platelet production. Mice models provide with proof that TPO
is very important for the signalling of megakaryocyte development. When the TPO gene or the receptor is deleted
in these mice it results in severe reduction in megakaryocytes and peripheral thrombocytopenia. Though the mice
do not develop anaemia or neutropenia, when they lack the MPL gene they have approximately 10% of the
normal HSC numbers and the marrow progenitors for all the hematopoietic lineages are reduced. This finding
provides an important insight in the function and signalling of TPO, meaning it is not only important for the
production of platelets but also for the maintenance of HSC. (A.E.Geddis, 2011)
Other studies (Pikman et al., 2006; Ghilardi et al., 1999 and Wiestner et al., 1998) have shown that MPL and the
THPO gene are also involved in the development of thrombocythemia, the mutations causing this phenotype are
dominantly inherited.
CAMT, congenital amegakaryocytic thrombocytopenia, is a genetic disorder caused by mutations in the
thrombopoietin (TPO) receptor; MPL. MPL is expressed on megakaryocytes and platelets. These represent the
majority of the receptors for TPO to bind to. Under normal circumstances when the production of platelets in
megakaryocytes is low the plasma TPO levels will raise. Next to the severely reduced thrombopoiesis the
megakaryocytes and platelets that are present in the blood stream do not express a functional MPL. This causes
the TPO levels in the plasma of children to rise up to a 10 fold of the normal levels. But because MPL is not
functional because of pathogenic mutations, the production of platelets is not increased. (A.E.Geddis, 2011)
In the diagnostic genetic testing of a Dutch family with 2 members effected with congenital thrombocytopenia
indicated that there are no pathogenic mutations present in the WAS and RUNX1 genes. These genes are known
to cause thrombocytopenia (J.Michaud)). In this project I set up DNA diagnostic tests for the THPO, MPL and an
additional YouMAQ assay for the RUNX1 gene in the DNA diagnostic laboratory of UMC Utrecht and screened a
family with 2 sisters that presented severe thrombocytopenia at birth(family tree is shown in figure 1).
At first some theoretical background information is given about genetic disorders, types of mutations, additional
information about the genes and the genotype/phenotype relation between described mutations. Hereafter some
information is given about the performed techniques like the PCR, sanger sequencing and the (You)MAQ-assay
which were used to determine the genetic cause of the 2 sisters’ thrombocytopenia followed up by the results. As
the last part of the paper I will discuss the results, give my conclusions and propose experiments to continue this
research.
Figure 1: The family tree of the 2 sisters
with congenital thrombocytopenia. Both
parents show no signs of thrombocytopenia
and are expected to be healthy.

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Jan, 2012
Theoretical background
Genetic disorders
Complex organisms like humans have two copies of every chromosome in every cell (called diploid). This means
that all genes are normally present in a duplicate form. A genetic disorder is a disease caused by abnormalities in
these genes or chromosomes. These abnormalities are called mutations. When a mutation is present on both
alleles this mutation is called homozygote, and when a mutation is present on one of the alleles this mutation is
called heterozygote. A mutation can be de novo (a newly created mutation) or hereditary. Hereditary mutations
are germline, meaning they are present in all body cells, the opposite of novo mutations which are present in the
cell where it emerged plus all daughter cells (i.e. a tumour).
Mutations
Pathogen mutations
Mutations in the DNA of encoded proteins may lead to changes in the structure, to a decrease or complete loss of
expression. Since this change in the DNA sequence affects all copies of the encoded proteins these mutations
can be damaging to cells and organisms. There are different kinds of pathogenic mutations; missense mutations,
nonsense mutations and frameshift mutations. Al of these mutations encode for a mutant protein which cannot
perform the task it was originally designed to do, causing defects and diseases.
Non-pathogen mutations
Next to pathogenic mutations there are also mutations which are non-pathogenic. These are called SNP’s (Single
Nucleotide Polymorphism) and are mostly very common in the human population. Most SNP’s have been
screened in different populations to calculate the frequencies of each allele and explore the different genotypes.
Unclassified variants
In some cases a nucleotide change is found which is very rare or has never been seen before. When literature or
databases have no information about that change it is unknown if it is a pathogenic mutation or not. These types
of changes are called unclassified variants and extra experiments have to be set up to reveal the nature of the
change.
Inheritance
Dominant inheritance
In a dominant inherited disorder the patient carries a normal and an affected gene. Dominant inheritance can be
caused by mutations that cause a gain of function of the encoded protein. For example a mutation can increase
the activity of a gene product, create more functions for the gene product or even lead to its inappropriate spatial
and temporal expression. Dominant inheritance can also be caused by mutations that lead to loss of function
mutations. In some cases both healthy alleles are needed for a normal protein function so removing a single allele
will lead to abnormal functions. (H.Lodish, 2000) I.e. thrombocythemia is caused by a dominant gain of function
Point mutations
Mutations that involve changes in a single nucleotide are mostly called point mutations.
These changes in a single base pair may produce one of the following three types of
mutations:
Missense mutation
A missense mutation results in a protein in which one amino acid is substituted
for another which may lead to changes in protein folding, function and
expression.
Nonsense mutation
A nonsense mutation causes the implementation of a stop codon in the place for
a regular amino acid which leads to premature termination of translation and
therefore creating a defect protein.
Frame shift mutation
Frame shift mutations cause a change in the reading frame which leads to the
introduction of unrelated amino acids to the protein and are majorly followed by a
stop codon creating a defect protein.
(H.Lodish, 2000)

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mutation leading to an overactive MPL and THPO transcript. (Y.Pikman, 2006) (N.Ghilardi, 1999) (A.Wiestner,
1998)
Recessive inheritance
A recessive inherited disorder is distinguished from dominant inherited disorders because it needs 2 affected
alleles for the expression of a phenotype. This means that a recessive inherited disorder is caused by
homozygote or compound heterozygote and in the majority of cases inactivates the affected gene which leads to
a loss of function. For example the recessive mutation will cause a removal of a part or entire gene from the
chromosome, inhibit expression of the gene or alter the structure of the encoded gene which leads to an
alternation of function (H.Lodish, 2000). For example thrombocytopenia is a recessive disease and can be caused
by loss of function mutations in the MPL gene (CAMT).
Genes involved in this project
MPL
Gene function
The MPL gene encodes for a protein which is homologue for members of the hematopoietic receptor superfamily.
This transmembrane protein, named CD110, is 635 amino acid long and contains 2 extracellular cytokine
receptors and 2 intracellular cytokine receptor box motifs. Presence of anti-sense oligonucleotides of
MPL inhibits colony formation of megakaryocyte which are responsible for the production of thrombocytes (blood
platelets). The MPL gene contains 12 exons and is located on chromosome position 1p34. (NCBI) (OMIM)
Pathogenic mutations
Thrombocytopenia can be caused by homozygote or compound heterozygote mutations in the MPL gene.
Mutations like nonsense, missence and splicing mutations have been found all over the MPL gene but most
common mutations are found in exon 2 and 3. These encode the first cytokine receptor domain (as seen in figure
2) and mutations in this region that create a frame shift or premature stop codon disrupt the entire intracellular
domain of the receptor. Mutations like these are expected to result in a complete loss of receptor function. Figure
3 shows a few MPL proteins examples where a mutation causes a defect protein. (A.E.Geddis, 2011)
Homozygote deletions or nonsense mutations are predicted to result, if translated, into a premature terminated
MPL protein which lacks a transmembrane and intracellular domain. These mutations should result in a complete
loss of MPL function. Homozygote and heterozygote missense mutations result in a protein with a residual
function of MPL which leads to a less severe form of CAMT. (M.Ballmaier, 2001)
Also a point mutation in exon 4 and a nucleotide deletion in exon 10 are responsible for a premature termination
of the MPL protein which after translation lacks 2 intracellular receptors which are essential for the signal transfer.
The point mutation in exon 4 is responsible for an amino acid change. Because of this change one of the two
cytokine receptors no longer functions as a TPO receptor which leads to a defect response to TPO. (K.Ihara,
1999)

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Figure 2 (Right): Schematic presentation of the MPL protein. The boxes and numbers on the
left side represent the exons of the coding sequence of the MPL gene. The boxes on the left side
represent the different regions that are produced by the corresponding exons. In which the signal
peptide (SP), the hydrophilic insert in the first cytokine receptor domain (HR) and the
transmembrane region (TM) are indicated with grey boxes. (M.Ballmaier, 2001)
Figure 3 (Left): MPL protein products derived from different transcripts with mutations. The
mutations in the MPL gene alter the receptor function in different ways. The wild-type receptor
(most right) is glucosylated and expresses on the cell surface where it can interact with TPO.
F104S MPL is also expressed on the surface but unable to bind TPO. R102P MPL is barely
glycosylated and poorly expressed on the cell surface. P635L is unstable but if the degradation is
inhibited it can go to the cell membrane and signal. (A.E.Geddis, 2011)
THPO
Gene function
The THPO gene encodes for TPO, a humoral growth factor which is needed for the megakaryocyte proliferation,
maturation as well as the thrombopoiesis. This protein functions as a ligand for the protein encoded from the MPL
gene. The THPO gene contains 6 exons and is located on chromosome position 3q27. (NCBI)
TPO is produced at a constant rate by the liver and removed from the circulation by receptor-mediated uptake
and destruction.
Gain of function mutations
The 5’-untranslated region contains 2 alternative promoters P1a and P1 with multiple transcription initiation sites.
10% of the total transcripts are synthesized from P1a and 90% from P1. This study (C.Dördelmann, 2008) found 6
SNP’s and a 58-bp deletion variant. Three of these variants are located in the 5’-flanking (C-920T [RS2855306],
A-622G and C-413T [RS885838]) and three in the 5’-untranslated region (C+5A, G+115A and C+135T). The
deletion is located between positions -1450 and -1507(all the locations are based on the sequence of accession
number U17071). Four of these SNP’s were tested for their effect on promoter activity. MolHap 3 (Molecular
Haplotype 3), like seen in figure 4, leads to a significant loss in transcript activity which results in lowered TPO
production and is followed up in lowered blood platelet counts (thrombocytopenia). MolHap 2 leads to significantly
more transcript activity compared to the wild type. (C.Dördelmann, 2008)

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Figure 4: 5 MolHaps of the THPO 5UTR upstream promoter P1. 5 MolHaps of the THPO upstream promoter P1 were
identified. The major allele is presented in uppercase letters and the minor allele is in lowercase letter. (C.Dördelmann, 2008)
There is also proof of a mutation in the splice donor site of intron 3 (5’ region of exon 2) which leads to exon 3
skipping. This mutation leads to improved translation and elevated TPO levels in the serum. (A.Wiestner, 1998)
Loss of function mutations
No studies have described patients with the presence of a loss of function mutation in the THPO gene.
Knowing the function of this gene we hypothesized that a mutation in this gene might cause thrombocytopenia.
Therefore the family in this study is tested on research base for mutations in this gene.
RUNX1
Gene function
The RUNX1 gene encodes for a protein which represents the alpha subunit of CBF (Core Binding Factor) and is
thought to be involved with the development of normal haematopoiesis. The CBF is a heterodimeric transcription
factor which binds to the core element of many enhancers and promoters. The gene RUNX1 contains 8 exons
and is located on chromosome position 21q22,3. (NCBI) (OMIM)
8 Point mutations and a deletion of the entire RUNX1 gene are linked to thrombocytopenia which can eventually
lead to AML. The mutation Y260X is located in the start of a transactivating domain and removed a part of the
negative regulator region for DNA binding. Functional analysis of 7 mutations (2 frameshift, 2 nonsense and 3
missense mutations) were performed to map the mechanics that contribute to FPD/AML.
The ability to bind DNA was reduced or abolished in all affected proteins as expected from the sites of
substitutions in the Runt domain. The missense and nonsense mutant proteins (largely intact Runt domain) kept
the ability to heterodimerize with PEBP2β/CBFβ. The frameshift mutant proteins, which lack a part of the Runt
domain, fail to heterodimerize. (J.Michaud)
Partial and entire gene deletions
Large deletions of the haematopoietic transcription factor RUNX1 have been identified in 13 families. This
heterozygote germline mutation in the RUNX1 gene causes FPD with predisposition to AML. In addition
microdeletions of the RUNX1 gene and several surrounding genes are found in patients with mental impairment
and multiple abnormalities in which platelet disorders (thrombocytopenia and/or abnormal platelet aggregation)
are a characteristic phenotype manifestation. (Chettouh et al., 1995 and Theodoropoulos et al., 1995) (M.Béri-
Dexheimer, 2008) (E.S.Click, 2011)
Genotype/phenotype relation
All the previously described genotypes have different effects on the phenotype. Table 1 shows a summary of
some of the important mutations described in the previous sections.
The majority of the gain of function mutations in the coding region of the MPL and THPO gene are missense
mutations which are dominant and cause thrombocythemia. For example the splice donor site mutation in intron 3
causes exon 3 skipping which leads to an improved translation of the remaining coding region producing higher
amounts of TPO causing the TPO serum levels to rise. (A.Wiestner, 1998)

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Homozygote of compound heterozygote nonsense mutations and microdeletions in the coding regions of the MPL
and RUNX1 gene are recessive mutations and cause thrombocytopenia. For example a homozygote point
mutation in exon 10 causes a premature termination of the MPL protein translation which lacks intracellular
receptors. This causes loss of signal transfer into the nucleus leading to thrombocytopenia. (K.Ihara, 1999)
Table 1: Summary of the important mutations explained in the previous section. Displayed is the genotype with the
nomenclature from the associated literature. Next to the associated phenotype, the expected inheritance and the followed
consequence are described.
Genotype Expected
autosomal
heritance
Consequence Phenotype Literature
MPLW515L Dominant Gain of function Thrombocythemia (Y.Pikman, 2006)
THPO C-413t Dominant Gain of function Thrombocythemia (C.Dördelmann, 2008)
THPO splice donor site
intron 3 mutation
Dominant Gain of function Thrombocythemia (A.Wiestner, 1998)
MPL exon 4 point
mutation
Recessive Loss of function Thrombocytopenia (K.Ihara, 1999)
MPL exon 10 1 bp
deletion
Recessive Loss of function Thrombocytopenia (K.Ihara, 1999)
THPO A-622g/C-413t Recessive Loss of function Thrombocytopenia (C.Dördelmann, 2008)
RUNX1 21q22.11-12
micro deletion
Recessive Loss of function Thrombocytopenia (M.Béri-Dexheimer,
2008)
(E.S.Click, 2011)
Materials and methods
Before a new gene analysis or technique can be implemented in regular diagnostic work the test must be
validated and meet the set quality requirements. Validation terms and conditions are set up by the rules of the
accredited quality system (NEN-EN-ISO 15189:2007). All the tests used in this study were validated according to
this system.
There are different steps to walk through before a test is ready for validation:
1. Validationplan
2. Testing the methods and primers.
3. Optimization.
4. Validation.
Next to meeting the set quality requirements the method must also be reproducible. This means that running the
experiment once, getting great results and meeting all the requirements doesn’t make the test robust and
reproducible. Before all methods can be validated the experiment must have been done at least 2 times to be
sure that the primers are specific for the target region. When the test meets all requirements after 2 experiments it
will be validated.
Methods and used protocols
Mutation analysis
To determine which small mutations, polymorphisms, insertions, deletions or duplications are present in the
template DNA, the DNA was sequenced with the Sanger method. The Sanger sequencing method is an
enzymatic method which uses a controlled termination of the in vitro DNA replication. A blank sample, normal
male and normal female sample were included in each run to serve as an internal control in the PCR and
sequencing process. (Appendix A; PCR and Appendix B; Sanger Sequencing for the protocols)
Quality requirements
Primer design and PCR:
1. Amplicon size preferably between 120 and 380 bp.
2. Make sure that the annealing temperature of the primers is between 60,7 and 62,0 ˚C. This way the
experiments are easier to perform in regular diagnostics because all the primers can be used around
the same temperature and thus the same program.
3. Primer size must be between 17 and 30 bp. This is to ensure the specificity and when made any
larger the chance of degradation is higher.
4. Every primer set must include the flanking donor and acceptor splice site sequences. This means a
5’- and 3’ exclusion buffer of 39 (position -20 must be shown for the donor/acceptor splice site) is put
in. This exclusion buffer is also implemented because the ABI prism 3730 starts reading the
sequence properly after about 53 bp.

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5. Blank control sample must be blank at all times.
Sanger Sequencing:
1. The PCR-amplification product (Amplicon) is specific and shows in relation to the position of the
chosen PCR-primers the right length.
2. The PCR-amplification product (Amplicon) shows the specific sequence of the exon and the flanking
donor and acceptor splice site sequences.
3. The PCR-amplification product (Amplicon) shows after the ABI automatic sequencer process, during
analysis, an unambiguously baseorder. In which the objective is to get a relative fluorescence of
more than 100 RFU (Relative Fluorescent Units) with Seqscape or Sequence Pilot and a lane quality
of at least 20 by analysing with Mutation Surveyor.
4. The normal control must show no pathogenic variations.
PCR for sequencing purposes
The template DNA was multiplied in a PCR by adding 1 µl DNA to 24 µl Master mix. In the master mix11 µl
distilled water, 12.5 µl AmpliTaq Gold 360 Master Mix 1
(Hot start) and 0.25 µl of forward and reverse primer mix
were put together for 1 sample. The primer mix contained 25 nM forward primer and 25 nM reverse primer.
The PCR program (in a TProfessional Basic Thermocycler 2
) consisted of 10 minute incubation at 95˚C where
after a cycle of 1 minute at 95˚C, 1 minute at 60˚C and 1 minute at 72˚C was repeated 33 times. The last step is 4
minute incubation at 72˚C. The product was checked on a 2% agarose gel. By adding 3,5 µl Orange G 3
with
Gelred 4
to 5 µl PCR product and running that for 25 minutes by 140 V (PowerPac 300 and Sub-cell GT 5
) and
checked together with an allelic ladder (O’GeneRuler #SM11736
) under an UV-light (ChemiDoc XRS 5).
Sanger sequencing
10 µl of the PCR product was presented to an automatic purifying system (SciClone Workstation 7
) and
sequencer where after it was presented on a Genetic Analyzer (ABI Prism 3130/3730 1
).
By using a universal M13 primer (a unique sequence which is not present in the entire human genome) consisting
out of the following sequence 5’GTTTTCCCAGTCACGAC 3’, attached to one of the exon primers (forward or
reverse) this universal primer can be used for every sequence reaction, no matter what gene or exon is
sequenced.
Analyses of the results
After the data was processed the sequence could be viewed in different software like Mutation Surveyor 8
,
Sequence Scanner 1
and for the reference sequence with internal/external SNP/mutation databases we used
Alamut 9
. In Mutation Surveyor the sequence of the patient’s DNA and the normal control were put beside the
reference sequence from NCBI. The base location and other information were gathered from Alamut. This
software was used to determine if a variant in the sequence is neutral and common (SNP) or if this variant is
possible pathogenic (mutation/UV).
1 Applied Biosystems, Foster City, U.S.A.
2 Biometra, Goetingen, Germany.
3 Sigma-Aldrich, Zwijdrecht, the Netherlands.
4 GelRed NAC STAIN, VWR, Radnor, U.S.A.
5 Bio-Rad, Hercules, U.S.A.
6 Fermentas, St.Leon-Rot, Germany
7 Caliper, Hopkinton, U.S.A.
8 Soft Genetics, State College, U.S.A.
9 Interactive Biosoftware, San Diego, U.S.A.

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Jan, 2012
Genomic copy number variation detection
Multiplex Amplicon Quantification (MAQ) is a method for quantifying the relative copy number of specific target
sequences. The PCR amplicons can be designed in random region’s (>30kb) so this technique can be used for
whole genome analysis. The design of a flagged MAQ assay starts with the design of primers by either the web
based tool of the manufacturer or using the program Light Scanner Primer Design1
, though with the last the assay
will called a YouMAQ-assay instead of a MAQ-assay since the primers are self-designed. By using the
fluorescent label component from the MAQ kit, which binds to the primers, the amplicons will be fluorescent
labelled during the PCR amplification. This YouMAQ kit also contains 6 primer pairs which form the control peaks.
These control products must be present when the results are analysed and are included in the panel. A blank
sample, samples of normal men(x50) and normal women(x50) pools were included in each run to serve as an
internal control in the PCR and genetic analyzing process. (Appendix C; YouMAQ-assay protocol)
Quality requirements
1. DNA concentrations and purity of the samples meet the protocol requirements.
2. Minimal 3 and at least half of the peaks of the region specific amplification products and all 6 control
amplification products must be unambiguously visible.
3. The signal strength of all amplification products must be above 10 (MPLA analyse lane score
threshold).
4. The results of the positive control must always be pointing out a deletion or duplication of the exons
or entire gene.
5. The test is reproducible and robust, blanks are negative and duplicate samples show similar results.
1 Idaho Technologies, Salt Lake City, U.S.A.
Nomenclature
The HGVS guidelines are used for the nomenclature of the variations found in this project.
The base of the name can be derived from the nucleotide number in the cDNA reference
sequence or the number of the corresponding amino acid (codon). This number will be
combined with the corresponding nucleotide or amino acid change.
The name of a variant with an amino acid change is build up from:
- c. notation: i.e. c.1610 G>A
- Amino acid: i.e. R = Arginine (Arg)
- Number amino acid: i.e. p.537
- New amino acid code: i.e. Gln
These 4 things together create the name for the codon change from Arginine to
Glutamic Acid on amino acid number 537 by the mutation G to A on nucleotide
number 1610.
 c.1610G>A_(p.Arg537Gln)
The name of a variant with no amino acid change is build up from:
- c. Notation: i.e. c.1469-70 C>T
In this case the c. notation is from a non-coding region. In which the first number
(1469) indicates the first nucleotide of the exon and the second number (-70) the
amount of nucleotides the variant is placed before the starting of the exon. (When
the variant lies after the coding region of an exon the first number will be the
number of the last nucleotide in the exon and the second number will be indicated
with * and the number of nucleotides it is placed after the end of the exon.)
The name of a variant with a insertion of 1 or more nucleotides is build up from:
- c. notation of the nucleotides the insertion is between: i.e c.229-20 and
c.229-19.
- The abbreviation “Ins”
- The amount of nucleotides inserted or the specific nucleotides inserted: i.e.
CTTC
These 3 things together create the name of a CTTC insertion between the 19th
and 20th
nucleotide before c.229.
 c.229-20_229-19InsCTTC

13
Jan, 2012
6. The YouMAQ-amplification product (Amplicon) is specific and shows in relation to the position of the
chosen YouMAQ-primers the right length.
7. The YouMAQ-amplification product (Amplicon) shows the specific sequence of the exon and the
flanking donor or acceptor splice site sequences.
YouMAQ-assay
Primer pools were made by making a solution of primers with an end concentration of 100 nM forward primer and
900 nM reverse primer (Following manufacturer guidelines 1
).
The master mix contains 3 µl YouMAQ PCR mix3
, 1 µl of the forward primer pool, 1 µl of the reverse primer pool
and 0.075 µl Taq DNA polymerase2
for 1 sample. 5 µl of this master mix was added to 8 µl water and 2 µl of the
diluted DNA (conc. 10 ng/µl) sample where after this mix was amplified on a preheated PCR machine 3
. The used
program consisted of 10 minutes incubation at 98˚C where after cycle of 45 seconds at 95˚C, 45 seconds at 60˚C
and 2 minutes at 68˚C was repeated 25 times with a ramprate of 2˚C/sec. As a last step the products are
incubated 10 minutes by 72˚C. Before the products could be presented onto the ABI prism 3730 Genetic
analyzer4
they were denaturized by putting 0.3 µl LIZ marker, 10 µl HiDi and 2 µl product together and incubate 3
minutes at 95˚C in a preheated PCR machine. The total volume of 12 µl is presented onto the Genetic analyzer
for fragment analyzing.
Analyses of the results
When the data was gathered the program Genemarker 5
was used to analyze the data. In this program we
created a panel that contained all the 6 control peaks and all the products that the primer pool is expected to
create. An example of a panel is shown in figure 5. From this panel the program will determine if all products are
present and with the peak height it will calculate the relative copy number of alleles. The calculation is based on a
synthetic control sample which contains the peak heights of all run samples. The results are given in peak ratios
and a graph like shown in figure 7(page 17). A peak ratio of 1 means there are 2 allele copies present in the
sample, meaning a normal ratio. A ratio between 0,8 and 1,2 is considered normal while everything below 0,8 is
considered a deletion and everything above 1,2 is duplicated.
Figure 5: Peak pattern created by 8 exon primer sets and 6 control primer sets. The x-axis shows the size of the products
in bp and the y-axis shows the relative fluorescent units (RFU). The peaks smaller than 50 bp are considered to be primer
dimmers.
1 Multiplicon, Niel, Belgium.
3 Biometra, Goetingen, Germany.
5 Soft Genetics, State College, U.S.A.

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Jan, 2012
Optimization
PCR for sequencing purposes
About 99% of the time the primers designed for a sequence analyses work straight away. In some cases the
primer needs something extra to work. For example the generally used annealing temperature is too low, the
target region is very GC rich or the primer amplifies 2 regions of the genome creating 2 amplicons. For example a
primer set which doesn’t function on an annealing temperature of 61˚C it is possible to test that primer set in a
gradient PCR. This will perform a PCR on 5 different annealing temperatures between 60 and 70˚C at the same
time. Afterwards it is possible to see if the primer works at a higher annealing temperature, if so it would mean
that this particular primer set has to be amplified differently from the “main stream”. For continuously diagnostics
this isn’t the best solution and therefore most of the time a new primer set is made.
(You)MAQ-assay
An important part of the design of a YouMAQ assay kit is the optimization of the peak heights. The assay must fall
between set requirements to be validated according to the quality system. This includes that the peak height of all
the control and exon peaks must be over 500 RFU (Relative Fluorescent Units). These control products are
designed to perform about the same and expected is that the RFU of these products is the same. Preferably the
exon peak is also round the same height as the control peaks.
During the optimization of the RUNX1 (You)MAQ-assay some problems were encountered; the primer sets
weren’t stable, primer sets didn’t generate enough products and some primer sets didn’t generate any product.
Shown in figure 5(page 13) is the peak pattern created in the first test with all the RUNX1 primers together in 1
pool. Clearly visible is the difference in RFU between the control peaks and all the exon peaks. Also the peak for
exon 9 is missing, possibly because this primer was designed to create a product over 350 bp long which can
cause problems during amplification. To address these problems, new primer pools were made adding a double
concentration of forward and reverse primer for exon 3, 7, 8 and 9. This was done because the peaks for exon 3,7
and 8 were the lowest ones and the peak for exon 9 was totally absent. After performing a YouMAQ with the new
primer pools the results were analysed and the peaks pattern is shown in figure 6A.
Figure 6A: Peak pattern created by 8 exon primer sets, with double concentration forward and reverse primers, and 6
control primer sets. The x-axis shows the size of the products in bp and the y-axis shows the relative fluorescent units (RFU).
The peaks smaller than 50 bp are considered to be primer dimmers.
Again no product was visible for exon 9 so a new primer was designed with a smaller amplification product.
The adding of more primers also caused a new problem, extremely high primer dimmer peaks. This indicates that
primers interact with each other rather than interact with the template DNA causing low exon and control peaks.
Also no product was detected for exon 9 indicating that this new primer set doesn’t work in the current set up and
possibly that the sequence where the 2 primer sets are designed in a region which is hard to amplify in the PCR
set up from the YouMAQ protocol. Since the adding extra primers didn’t change the height of the peak the next
option in the optimization process was to split up those primers sets. Several different pools were created with
different primer combinations to figure out which of the primers can be put together to create 1 or 2 pools, with as
many primers as possible in 1 pool, to improve easy handling in the regular diagnostic workflow. 5 different pools
were created, each with a different mix up of primers, to ensure the best combination of primers could be
selected.
The results of these experiments (data not shown) indicated that the exon 4 primer set was very unstable, and
therefore is left out for the remaining experiments in this project. In addition the exon 9 primer set did work in

15
Jan, 2012
these spilt up pools tough the created product was smaller than 500 RFU in all different pools and didn’t meet the
set requirements for validation. Therefore the exon 9 primer set was also left out for the remaining experiments in
this project. Eventually 2 primer pools were created. This way the less efficient primer sets get more PCR mix
compounds to amplify their target DNA and create a larger amount of amplicons. The first primer pool contained
primers for exon 1, 3 and 7 whose peak pattern is shown in figure 6B. And the second primer pool contained
primers for exon 2, 5, 6 and 8 whose peak pattern in shown in figure 6C. These 2 primer sets are validated for
diagnostics in the DNA laboratory and were used to test the 2 thrombocytopenia patients plus to perform the
screening on 19 excising patients.
Figure 6B: Peak pattern created by primer sets for exon 1, 3 and 7 plus the 6 control primer sets. The x-axis shows the
size of the products in bp and the y-axis shows the relative fluorescent units (RFU). The peaks smaller than 50 bp are
considered to be primer dimmers.
Figure 6C: Peak pattern created by primer sets for exon 2, 5, 6 and 8 exon plus the 6 control primer sets. The x-axis
shows the size of the products in bp and the y-axis shows the relative fluorescent units (RFU). The peaks smaller than 50 bp are
considered to be primer dimmers.

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Jan, 2012
Results casus
Sanger sequencing on MPL and THPO gene
We tested the hypothesis that the congenital thrombocytopenia in this Dutch family, with 2 affected individuals,
might be caused by loss-of-function mutations in the MPL or THPO gene. To confirm this hypothesis the DNA of
both patients was sequenced and results were checked for mutations and SNP’s. The found changes with
Mutation Surveyor are presented in table 2.
Table 2: Summary of found SNP's in the family’s 2 affected individuals, including database numbers of Alamut for reference.
The notation of the SNP location is based on the notations in Alamut (transcript number NM_005373.2 and NM_000460.2)
where c.1 is the starting codon. Frequencies and heterozygosity numbers are taken from Alamut based on Caucasian
populations. (*UTR = Untranslated Region. 5’UTR is the region before exon 1 and 3’UTR is the region after the exon 6
termination codon).
As seen in table 2 all the found changes in sequence are SNP’s which are common found amongst the
Caucasian population. This means that none of the found changes in the sequences of the 2 patients are
pathogenic and therefore responsible for their congenital thrombocytopenia.
(You)MAQ-assay on the MPL and RUNX1 gene
We tested the hypothesis that the congenital thrombocytopenia in this Dutch family, with 2 affected individuals,
might be caused by a deletion or duplication in the RUNX1 or MPL gene since no mutations were found in the
THPO and MPL gene. To confirm this hypothesis the DNA of both patients was checked for a deletion or
duplication through a MAQ-assay confirming the relative copy number or all exons in both genes.
The results of this assay are shown in table 3 and in figure 7A and 7B for the MPL gene and in figure 8A to 8C for
the RUNX1 gene.
MPL
Exon
Patient
Database
number
Frequency
Average
Heterozygosity1 2
Alleles
C T
7
Homozygote
c.981-41 G>A
Homozygote
c.981-41 G>A
RS 1760670 0.606 0.394 0.36
9
Homozygote
c.1469-70 T>C
Homozygote
c.1469-70 T>C
RS 839995 0.608 0.392 0.5
THPO (TPO)
Exon
Patient
Database
number
Frequency
Average
Heterozygosity1 2
Alleles
C T
5’UTR*
Homozygote
c.-861 T>C
Heterozygote
c.-861 T>C
RS 885838 0.508 0.492 0.5
2
Heterozygote
c.-136 T>C
- RS 956732 0.542 0.458 0.5
5
Heterozygote
c.229-20_229-
19InsCTTC
Heterozygote
c.229-20_229-
19InsCTTC
RS 72396770 - - -
6 (3’UTR*)
Homozygote
c.*35 G>A
Homozygote
c.*35 G>A
RS 6141 0.62 0.38 0.41
6 (3’UTR*) -
Heterozygote
c.*59 G>A
RS 78565404 0.917 0.083 0.5

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Jan, 2012
Table 3: Summary of the YouMAQ results of the MPL and RUNX1 genes in the family’s 2 affected individuals. The different
exons are put together as they were tested in the primer pools. Included is the positive control for this test with a diagnostically
proven deletion (data not shown).
MPL
Exon
Patient
Positive control
1 2
1 to 6 Normal Normal N.A.
7 to 12 Normal Normal N.A.
RUNX1
Exon
Patient
Positive control
1 2
1, 3, 7 Normal Normal Deleted
2, 5, 6, 8 Normal Normal Deleted
Figure 7A: Results of the MPL (You)MAQ on patient 1. Left are the results of pool 1 containing the primer sets for exon 1 to 6.
Right are the results of pool 2 containing the primer sets for exon 7 to 12.The light gray squares represent the exons and the
black squares represent the control peaks.
Figure 7B: Results of the MPL (You)MAQ on patient 2. Left are the results of pool 1 containing the primer sets for exon 1 to 6.
Right are the results of pool 2 containing the primer sets for exon 7 to 12.The light gray squares represent the exons and the
black squares represent the control peaks.
Figure 8A: Results of the RUNX1 (You)MAQ on the positive control. Left are the results of pool 1 containing the primer sets
for exon 1, 3 and 7. Right are the results of pool 2 containing the primer sets for exon 2, 5, 6 and 8. The red squares represent
the deleted exons and the blue squares represent the control peaks.

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Jan, 2012
Figure 8B: Results of the RUNX1 (You)MAQ on patient 1. Left are the results of pool 1 containing the primer sets for exon 1,
3 and 7. Right are the results of pool 2 containing the primer sets for exon 2, 5, 6 and 8. The green squares represent the exons
and the blue squares represent the control peaks.
Figure 8C: Results of the RUNX1 (You)MAQ on patient 2. Left are the results of pool 1 containing the primer sets for exon 1,
3 and 7. Right are the results of pool 2 containing the primer sets for exon 2, 5, 6 and 8. The green squares represent the exons
and the blue squares represent the control peaks.
Screening
Next to the testing of the patients a screening is performed on RUNX1 for 19 patients in the database who have
been tested on mutations in the RUNX1 gene and on MPL for 2 patients who have been tested on mutations in
the MPL and THPO gene. All patients appeared to have normal copy numbers of the RUNX1 and MPL (data not
shown). During the analysis of the screening 2 types of normalisation were used; internal probe normalisation and
population normalisation. The difference between both types is that with the population normalisation all the
results were a bit lowered. Eventually the results were interpreted while using the internal probe normalisation.
Discussion, conclusion and proposition
In this project diagnostic tests were set up according to the quality system where after they were implemented in
the workflow and used to test the family described in the casus.
Results gained from the sanger sequencing method and the YouMAQ-assay indicates that there are no mutations
found in the MPL, THPO and RUNX1 genes of the 2 sisters. We concluded that both coding sequences for the
receptor and cytokine are normal and contain no protein structure and function changes.
The 2 sisters present a different THPO genotype indicating that they did not inhered the same alleles from both
healthy parents, which would argue against a role for THPO in this presumed recessive disorder. This conclusion
is derived from the different homozygote and heterozygote SNP’s found in this gene which also contribute to the
conclusion that THPO gene isn’t fully deleted. Therefore no YouMAQ-assay was set up, though an exon deletion
isn’t fully excluded.
Curiously the two affected individuals showed lowered TPO serum levels (data not shown) which is not consistent
with former studies. These studies (described in the previous chapter) all show that patients with
thrombocytopenia suffer from significantly higher TPO serum levels then healthy persons. These findings imply
that, since the coding sequence of the THPO gene isn’t damaged, there could be a defect within the non-coding
sequence or the mRNA stability/expression. The non-coding sequence contains important promoters and branch
points responsible for the activation of transcription and correct mRNA splicing. Variants in the promoters of the
THPO gene that were described to increased THPO levels were examined but no pathogenic mutations were
found. (C.Dördelmann, 2008) No other regulatory mechanisms were examined.
I’d recommend setting up a non-coding sequence and gene expression analysis of the MPL and THPO gene
since both of these genes are expected to play a part in the thrombopoiesis signalling pathway. By analyzing the

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Jan, 2012
entire 5’ and 3’ untranslated regions (UTR) and the gene expression of these 2 genes it is possible to determine if
the coding regions are properly activated and when activated, if they are correctly expressed.
Next to these additional experiments, for the MPL and THPO gene, another option is to develop new research
diagnostics for the JAK2 and SH2B3 genes. As explained in a previous section about MPL mutations, a described
mutation in JAK2 (JAK2V617F (Y.Pikman, 2006)) causes a mutant protein which is a constitutively active tyrosine
kinase that activates downstream signal transduction pathways and transforms haematopoietic cells to cytokine-
independent cells. This indicates that these cells do not need cytokines to activate the downstream signalling,
causing essential thrombocythemia by overactivation of the JAK-STAT pathway shown in figure 9. The JAK-STAT
pathway not only plays an important role in the thrombopoiesis but is also activated through different processes
indicating that a mutation in the JAK2 gene can cause a more complex phenotype then displayed in the 2
patients.
Figure 9: An illustrative overview of the JAK-STAT signalling pathway. Shown on the left is the signalling pathway with no
mutations in any of the genes involved in this cycle. Shown on the right is 3 different abnormalities in which the receptor
becomes cytokine-independent. The middle situation shows a mutant MPL receptor which is unable to bind cytokines like TPO.
The most right situation shows a mutant JAK2 which is overactive and does not require cytokine induced signals to
phosphorylate tyrosines to activate STAT molecules. (MPN)
The SH2B3 gene mutation E208Q (Glu to Gln) is found in patients with somatic essential thrombocythemia. The
mutation occurs in the PH domain and in vitro expression showed that the mutated protein lost the ability to inhibit
TPO-mediated growth. This indicates that the protein which stops the TPO signalling to produce thrombocytes is
defect and the thrombopoiesis continues causing the blood platelet counts to rise. (OMIM) Though no loss of
function mutations are found and described for these genes, a rare variant might be present in the DNA of the 2
sisters.
Finally the feedback loop of the thrombopoiesis is not fully elucidated and therefore genes involved in this process
are undefined and the variants and mutations in these genes are yet unrelated to the described phenotype. This
could mean that a defect in the signalling or detection could cause a shortage of TPO and/or thrombocytes which
will be unnoticed. A recently new technique; next gen analysis can offer new insights in this situation. This
technique is a method to analyse all the 20.000 genes present in the genomic DNA, also called exoom analysis.
Unfortunately this technique isn’t fully tested and implemented in the diagnostics in this laboratory. Though in the

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Jan, 2012
future when the tests are validated and used for diagnostics it is certainly an advisable possibility if no other
experiments have demonstrated a mutation or deletion to explain the phenotype.
Based upon the results gathered in this study, were extensive analysis of the THPO, MPL and RUNX1 gene were
performed, we conclude that a defect in the MPL, THPO and RUNX1 gene is not associated with the phenotype
in the patients. Further tests will be necessary to determine the cause of the congenital thrombocytopenia in this
family.
Acknowledgements
I would like to thank Dr. M.E. van Gijn for all the help with the theoretical information and the writing of this paper,
and also for answering all my questions during the internship period.
I would also like to thank Mirjan Albring for all the practical information and tips on how to perform certain
protocols needed to perform this study and for answering all questions about them.
Last I’d like to thank Aafke Terlingen and Edith Peters for all the help in performing the (You)MAQ-assay and
understanding this technique and analysis methods/program.
My thanks also go out to all colleagues and the other internship student for guidance and information.

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Jan, 2012
Literature
A.E.Geddis Congenital Amegakaryocytic Thrombocythopenia [Journal] // Pediatric Blood & Cancer. : Wiley-
Blackwell Science. - Vol. 57, No. 2 : Vol. 2011. - pp. 199-203.
A.E.Geddis H.M.Linden, K.Kaushansky. Thrombopoietin: a pan-hematopoietic cytokine [Journal] // Cytokine &
Growth Factor Reviews. : Elsevier Science, 2002. - Vol. 13, No. 1 : Vol. 2002. - pp. 61-73.
A.L.B.M.Biemans A.A.F.Jochems and J.A.P.Spranger DNA een blauwdruk [Book]. - Houten : Bohn Stafleu
Van Loghum, 1999. - p. Ch. 2/4/42.
A.Wiestner R.J.Schlemper, A.P.C.van der Maas et al. An activating splice donor mutation in the thrombopoietin
gene causes hereditary thrombocythaemia [Journal] // Nature Genetics. : Nature Publishing Group, January
1998. - Vol 18. - pp. 49-52.
C.Dördelmann R.Telgmann, E.Brand et al. Functional and structural profiling of the human thrombopoietin gene
promotor [Journal] // The Journal of Biological Chemistry. - Rockville : The American Society for Biochemistry and
Molecular Biology, September 2008. - Vol. 283, No. 36. - pp. 24382-24391.
E.S.Click B.Cox, S.B.Olson et al. Fanconi Anemia-Like Presentation in an infant with constitutional deletion of
21q including the RUNX1 gene. [Journal] // American Journal of Medical Genetics. : Wiley-Blackwell Science,
January 2011. - Vol. 155, No. 7 : Vol. 2011. - pp. 1673-1679.
F.C.Schuit Medische biologie: moleculaire benadering van de geneeskunde [Book]. - Houten : Bohn Stafleu Van
Loghum, 2000.
H.Lodish Mutations: Types and Causes [Book Section] // Molecualr Cell Biology / book auth. H.Lodish A.Berk,
S.L.Zipursky et al. - New York : W.H.Freeman and Company, 2000.
J.Michaud F.Wu, M.Osato et al. In vitro analyses of known and novel RUNX1/AML1 mutations in dominant
familial platelet disorder with predisposition to acute myelogenous leukemia: implications for mechanisms of
pathogenesis. [Journal] // Blood. : American Society of Hematology. - Vol. 99, No. 4 : Vol. 2002.
K.Ihara E.Ishii, M.Eguchi et al. Identification of mutations in the c-mpl gene in congenital amegakaryocytic
thrombocytopenia. [Journal] // Proceedings of the National Acadamy of Sciences. : National Acadamy of
Sciences, March 1999. - Vol. 96, No. 6 : Vol. 1999. - pp. 3132-3136.
M.Ballmaier M.Germeshausen, H.Schulze et al. C-MPL mutations are the cause of congenital amagakaryocytic
thrombocytopenia. [Journal] // Blood. : American Society of Hematology, January 2001. - Vol. 97, No. 1 : Vol.
2001. - pp. 139-146.
M.Béri-Dexheimer V.Latger-Cannard, C.Philippe et al. Clinical phenotype of germline RUNX1
haploinsufficiency: from point mutations to large genomic deletion. [Journal] // European Journal of Human
Genetics. : Nature Publishing Group, May 2008. - Vol. 16 : Vol. 2008. - pp. 1014-1018.
N.Ghilardi A.Wiestner, M.Kikuchi et al. Hereditary thrombocythaemia in a Japanese family is caused by a novel
point mutation in the thrombopoietin gene. [Journal] // British Journal of Haematology. : Wiley-Blackwell Science,
April 1999. - Vol. 107, No. 2 : Vol. 1999. - pp. 310-316.
R.McMillan Hemorrhagic disorders: Abnormalities of platelet and vascular function [Journal] // Cecil Medicine. -
Philadelphia : Sauders Elsevier, 2007. - 23. - p. Ch. 179.
Y.Pikman B.H.Lee, T.Mercher MPLW515L is a novel somatic activating mutation in Myelofibrosis with Myeloid
metaplasia. [Journal] // Public Library of Science, Medicine. : Public Library of Science, July 2006. - Vol. 3, No. 7 :
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[Online] // National Center for Biotechnology Information. - NCBI. - September 2011. -
www.ncbi.nlm.nih.gov/gene?term.
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[Online] // Myeloproliferative neoplasms. - Novartis oncology. - January 2012. -
http://www.exploringmpn.com/role-of-the-jak-pathway.jsp.

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Appendix A: PCR
Theoretical background
In a polymerase chain reaction (PCR) the template DNA is multiplied. In this PCR the template is amplified with
primers designed on specific regions. Mostly these regions contain 1 or more exons from the expected affected
gene. The PCR is a cyclic process of denaturation, annealing and elongation. These three steps are preformed
each on another temperature and by repeating these steps the concentration of the specific DNA amplicon with
increase exponential.
The first step in this cycle is the denaturation of the dsDNA to ssDNA which is done by a temperature of 94˚C.
Then the mix is cooled down to a temperature between 50 and 60˚C. The exact temperature is dependent on the
annealing temperature of the designed primers which anneal to the template DNA in this step.
The last step is the elongation of the chain on a temperature of 72˚C by DNA-polymerase which is active with
temperatures until 100˚C. The dNTP’s are attached to the 3’ site of the primer complimentary to the template
DNA. This cycle is repeated 25-35 times dependent on the starting concentration of the template DNA.
(A.L.B.M.Biemans, 1999)
Protocol
PCR with AmpliTaq Gold 360 Master Mix (HOTSTART)
The PCR with AmpliTaq Gold 360 Master Mix is a HOTSTART PCR en can be preformed at
roomtemperature. Thaw the AmpliTaq Gold 360 Master Mix only once and save it in the refridgerator.
Majorly the PCR is performed without GC-Enhancer, but when an amplicon is GC rich or the PCR is a-
specific the GC-Enchancer can be added (25 µl reaction: with 65-70% HC add 2,5 µl GC-Enhancer;
with >75% HC add 5 µl GC-Enhancer. In general, when extra specificity is needed add 0,5-1 µl GC-
Enchancer to a 25 µl reaction). Make sure to adapt the volume of Aqua dest. Caution: The forward
and reverse primer can be added together in 1 working solution.
 Aqua dest. 7,25 l
 AmpliTaq Gold 360 Master Mix 12,5 l (endconcentration= 1x)
 (Optional) 360 GC Enhancer 0,5-5 l
 Primer F + R (25M) 0,25 l (endconcentration= 0,25M)
DNA (20ng/ul) 5 l
The above mix is based on using a primer working solution where the forward and reverse primer are
added into a 25 uM solution.
The mix below is used when the 25 uM working solutions of the forward and reverse primers are in
seperate cups.
 Aqua dest. 7,00 l
 AmpliTaq Gold 360 Master Mix 12,5 l (endconcentration= 1x)
 (Optioneel) 360 GC Enhancer 0,5-5 l
 Primer F (25uM) 0,25 l (endconcentration= 0,25M)
 Primer R (25uM) 0,25 l (endconcentration= 0,25M)
DNA (20ng/ul) 5 l
Add 100 ng DNA to a reaction of 25 µl.
It doesn’t matter if 5 µl is used out of a [20 ng/µl] solution or 1 µl out of a [100 ng/µl] sultion. But make
sure to adapt the volume of Aqua dest. in the reaction.
Start the following program on the PCR-machine:
1 cycli of 10 min. 95C
1 min. 60C
1 min. 72C
1 cycli of 4 min. 72˚C

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Appendix B: Sanger Sequencing
Sequence analysis
To determine which mutations, polymorphisms, insertions/
deletions or duplications are present in the template DNA the
DNA will be sequenced with the Sanger method. In this
sequence analysis method the base sequence is mapped
and compared to the reference sequence (RefSeq, mostly
taken from the NCBI database). This way all changes can be
found and compared to databases. The Sanger sequencing
method is an enzymatic method which uses a controlled
termination of the in vitro DNA replication. It uses the
mechanism in the PCR where there must be a free 3’-OH
group on the end of the chain to build in the new nucleotide.
To terminate the reaction a 2’3’-dideoxynucleotide (ddNTP)
was designed with a missing 3’-OH group. This way when a
ddNTP is build into the chain reaction instead of a dNTP the
reaction is terminated. By adding a small amount of these
ddNTP’s to the reaction mix next to the normal dNTP’s the
chain is terminated on different lengths of the amplicon with
1 base difference. A schematic reproduction of this process
is displayed in the figure to the right. Because the 4 different
ddNTP’s are fluorescent labelled, each with a specific colour,
they can be recognized during the electrophoresis. Because
the amplicons are sorted in length and terminated with a
coloured fluorescent label the sequence can be formed
which later can be analysed in different programs.
(F.C.Schuit, 2000)
Protocol
1. Thaw the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Mix before using.
2. Vortex the reaction cup several seconds
3. Centrifuge the reaction cup several seconds in an eppendorf centrifuge at 14.000 rpm.
4. Place the reaction cup in a cold Block till use.
5. Add together in a 0.2 ml MicroAmp Reaction Tube:
Big Dye Terminator Ready Reaction Mix 1 μl
5x Sequence buffer 1 μl (Caution!)
Primer (3M) 1 μl
Template (PCR product: ~1 - 20 ng) x μl
Sterile distilled water (Add till a Total of 10 μl) 7 - x μl
Total volume 10 μl
The primer can be a forward or reverse M13 primer or a specific sequencing primer, dependant on
the nature of the test.
6. With multiple templates, make a reaction mix. Make Caution that every reaction has the same
volume of template else the volume of water in the reaction mix is wrong and so will the end volume.
7. Cover the 0.2 ml MicroAmp reaction tubes straight away with MicroAmp caps.
8. Spin the PCR baseholder for several seconds in an eppendorf centrifuge 5810.
9. Place the PCR rack in a PE gene Amp PCR system 9700
10. Start the PE 9700 with the following program:
25 cycli of 10 sec. 98C
5 sec. 55C
2 min. 60C
∞ 4°C
Schematic reproduction of the Sanger sequencing
method. (F.C.Schuit, 2000)

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Jan, 2012
The extension time is dependent on the length of the end product, with products smaller than 800 bp
an extension time of 2 minutes at 60˚C is sufficient. With products of 800 bp or larger an extension
period of 4 minutes at 60˚C is regular.
11. Purify the sequence reaction according to the protocol.
12. Add 7,5 µl of the purified products to 7,5 µl distilled water in a 96-wells plate for the ABI 3730.
13. Save the products in a cold storage after analyzing.

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Jan, 2012
Appendix C: (You)MAQ-assay
(You)MAQ assay
Multiplex Amplicon Quantification (MAQ) is a method for quantifying the relative copy number of specific target
sequences. The PCR amplicons can be designed in random region’s (>30kb) so this technique can be used for
whole genome analysis. This method can also be used for patients where, after sequence analysis, the suspicion
rises that a deletion of a part (one or more exons) or whole gene is involved in the development of a disease.
The design of a flagged MAQ assay starts with the design of primers through a web-based design tool. It is also
possible to design primers by hand and then the assay will be called a flagged YouMAQ. By using the fluorescent
label component from the MAQ kit, which binds to the primers, the amplicons will be fluorescent labelled during
the PCR amplification. With the ABI prism 3730 the amplicons are sorted in length through electrophoresis after
which the peaks are identified by pre-defined panels. The area of the peak is calculated and used by software to
determine the relative copy number of alleles.
Protocol
Preparing the primer pools
The youMAQ primers arrive in a 100M solution and are saved at -80°C.
The primer pools contain:
a. Forward primer pool: contains of every Forward primer 150nM:
(Per Forward primer 1,5 l (100M) in total 1000l H2O)
b. Reverse primer pool: contains of every Reverse primer 900nM.
(Per Reverse primer 4,5 l (100M) in total 500l H2O)
These pools can be put into several cups and stored at -20˚C. Avoid repeating freezing and
thawing a primer pool.
Make sure all the cups and the storage case have accurate and clear content descriptions.
Preparing the DNA-sample(s)
The quality and concentration of the DNA samples is very important to get good results from
the MAQ analysis. 20-50 ng DNA is required and must meet the following demands:
 OD260/280 ratio ≥ 1.7
 Concentration ≥ 10 ng/l in H2O
 No DNA degradation visible on an agarose gel.
Measure the DNA-concentration and dilute the DNA to 100-150 ng/µl in 1x TE.
Mix the DNA dilution for at least 2 hours (possible over night) on a vortex mixer to
homogenize.
DNA-sample
1. Re-measure the DNA concentration of the samples after homogenizing.
2. Dilute the DNA further to 10 ng/µl in ~ 100 µl H2O end volume.
3. Use only water as blank sample.
4. Use a duplicate men- and women- 50x reference pool as control sample. (see SOP-SDD-
A.067) Dilute these also to 10 ng/µl in 50 µl H2O end volume.
YouMAQ procedure
Thaw the needed primer mix on ice.
Thaw the YouMAQ PCR mix (from the kit) on ice, vortex and spin.
Make all the mixes on ice and start the PCR as quick as possible after adding the Taq DNA
polymerase.
Begin with pipetting the DNA in the PCR reaction mix, afterwards make the master mix and
add it straight away to the PCR reaction mix in the plate.
1. Make PCR reaction mix directly in a 96 well’s PCR plate or PCR strips
- 2 l DNA (total input ~20 ng)
- 8 l water
- 5 l master mix*
- 15 l total volume
Mix the DNA well with the master mix by resuspending with the pipette.
Cover the cups with caps.

27
Jan, 2012
Save the plate on ice.
2. Make Master mix* for #=n reactions+1 vb. (n=11+1)
- 3 l flagged youMAQ PCR mix 36 l
- 1 l For primer pool 12 l
- 1 l Rev primer pool 12 l
- 0.075 l Taq DNA Polymerase 0,9l
Mix the Total mix thoroughly and spin down, save on ice.
Amplification
1. Start flagged youMAQ PCR program and wait till the Block is on temperature.
2. Place the plate in the PCR machine
3. Perform the PCR under the following conditions:
Caution: ramprate: 2C/sec.
1. 10 min. 98C
2. 45 sec. 95C
3. 45 sec. 60C (25 cycli, 2 t/m 4)
4. 2 min. 68C
5. 10 min. 72C,
The program is ended with incubation at 4C.
After the Amplification the plate can be kept in the refrigerator for max. 1 week if not
directly sequenced. If it is needed to be kept longer place the plate at -20C.
Making the Genescan samplesheet
Caution: samplesheet must be filled in before the samples are put into the plate.
1. Depending on what part of the samplesheet is filled in you must continue after.
2. Fill in the sample names in the intended koloms starting with your own initials.
Caution: Pipet order for the lanes: 1,3,5,7,9,11,2,4,6,8,10,12. (The analyzer starts with all
the uneven lanes for run 1 and the even lanes for run 2)
Prepare en denaturing for Genescan analysis
1. Make a mix of LIZ and HiDi-formamide. Make sure the LIZ isn’t kept outside the
refrigerator to long.
Per sample; 0,3 µl LIZ + 10 µl HiDi.
2. Pipette 10 µl LIZ/HiDi into a plate.
3. Add 2 µl YouMAQ product
De remaining plate will be kept at 4C.
4. Denature the samples in the PCR machine for 3 min. at 95C.
5. Place the plate directly on ice
6. Pipet the entire mix in the fragment analysis plate according to the samplesheet.
Results
Processing and interpretation of the results is done after the samples are processed by the
analyst working the ABI 3730.
The program Genemarker is used to analyse the ABI-files.

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Jan, 2012
Appendix D: Experiments outside the project
Since the project didn’t contain a lot of lab work extra task were added to learn all the aspects needed to work in a
DNA laboratory (i.e. planning of experiments, pipetting very small volumes of liquid, putting together a series of
experiments with the goal to validate it for diagnostics).
Those extra tasks included:
 Primer design and ordering;
o Exon 7 of the IL33 gene.
o All the exons of TPM3 for a fellow internship student.
 YouMAQ for NPHP1 and MNX1;
o Primer design and ordering.
o Testing the primers
o Optimization
Since the internship period only lasted 4,5 months the major experiments were priority number 1, therefore the
optimization of the NPHP1 and MNX1 YouMAQ’s wasn’t completed yet. Because of this a proposition for a new
experiment to optimize these tests was put together and written on the last page of the journal for these tests.
This way the laboratory can easily pick up the optimization after this internship period.

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