Searching an FHL causing dysfunctional gene Chapter Name

Thesis work report/Exjobbsrapporten

NADA Model:

1. Introductional part

2. Main part

3. Reference part

Aurell Model:

1. Introduction and Background Theory, 1/3

2. Method, 1/3

3. Results and conclusion, 1/3

1. Introductional part

1.1.Front-page and title

1.2.Title and summary (abstract?)

1.3.Table of Contents

1.4.Preword

2. Main part

2.1.Introduction

2.1.1.Thesis

2.2.Background/Theory

2.2.1.About FHL

2.2.1.1. What is known today and how we got here (historical

research?)

2.2.1.1.1.Symptom description:

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how first described in literature, when, by whom; how

described/classified today

2.2.1.1.2.Patient groups:

who are the patients, i.e. family/hereditary background,

ethnicity/origin, age of insjuknande

2.2.1.1.3 Geographical regions statistics ?

2.2.1.1.4Disease causing dysfunction – the mechanism

protein disease Chromoso Cytoban

gene expression status me d exons
9q21.3-
Unknown Unknown FHL1 9 22
10q21-
PRF1 Perforin FHL2 10 22 7
UNC13D Munc 13-4 FHL3 17 17q24 33
STX11 Syntaxin 11 FHL4 6 6q24 4
MYHIIA Myosin ? 22 22q12.3

2.2.1.2. What has already been done (historical research?);

previous works, publications

2.2.1.3. The “Henter-team” (key-people and their position in the

group/their role in the FHL-research)

2.2.2.Diagnostics in FHL

2.2.3.Treatment and cure

2.2.4.Statistics – disease frequency within different patient groups

(and geographical region?), severeness, recovery frequency and
level

2.2.5.Analysis methods utilized by previous teams/in previous

projects

2.3.Objective

The project turned out to be complex, actually consisting of two separate problems.
The main objective of this research is to find a solution to the medical problem.
However, to do this an appropriate analysis method must be developed and
established.

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2.3.1.Medical problem identification/formulation

Move to ---?: The idea to this project sprung from an actual need to diagnose a
group of patients with FHL-symptoms. By the year of 2006 (???) the department of
childhood oncology (?) had 15 cases where the patients were diagnosed as having
FHL, based on their symptoms, although none of them were positive to mutations in
any of the known FHL-involved genes. Obviously there must exist further, undefined
regions in their genomes, possessing defects causing the FHL disease state.

2.3.1.1.Project Objective/aim/goal (“the Missing Gene”)

Presentation of the patient group subject for the research
and the lack of diagnostics/knowledge

The genetic information was available. The main problem was that there exists no
target mutation to screen for; nobody knew what to search/look for. Without a
known disease causing mutation in a defined gene, it is impossible to make a
diagnosis. Without a diagnosis, it is impossible to cure the disease, or to guarantee
an/provide effective treatment. Conclusively, it is necessary to find the disease
causing dysfunctional gene in the genome of each of these patients, in order to
make a diagnosis and offer treatment and cure.

The main objective/the objective of this part of the project is to map the disease loci
of FHL. That is, to identify one or more homozygous regions in the human genome
where undefined genes involved in the regulation of the immune response are
located, potentially contributing to/involved in the disease causing mechanism of
FHL.

2.3.1.2.Far-reaching aim finding the solution; what could be

achieved if the problem is solved? (diagnostics)

Development of diagnostics is a prerequisite to:

• detect FHL-patients in an early stage

• offer screening and eventually in vitro fertilization to parents in the target

group

• compensate for the missing/non-functional gene expression product (i.e. the

subject protein)

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• exchange the damaged DNA region/genetic material through genetic

engineering

2.3.2.Analytical problem identification/formulation

The need to identify the disease causing dysfunctional genes to be able to provide
effective treatment to these patients revealed another problem. Namely, to find the
target regions obviously existing in the genomes of the patients, the genetic
material must be analyzed by a suitable method. Although other diseases with
similar autosomal recessive hereditary patterns are subject to research by other
teams, there seems to be no established method that completely satisfies the
analytical needs.

2.3.2.1.Development of a bioinformatic analysis method

The genetic material to analyze was available. The first problem to attack was how
to analyze this material in an adequate way, in order to find the target regions
specific to each patient. This is performed by bioinformatic methods. Thereby, the
need of bioinformatic competence in the research was recognized.

The prior objective/objective of this part of the project is to develop a bioinformatic

method by which homozygous regions in the genome can be mapped and
effectively registered, overviewed, analyzed (comparison to known genes in gene
banks) and compared to other genomes (comparison to related or unrelated
individuals, affected or unaffected).

2.3.2.2. Far-reaching objective/aim/goal (Future achievements) –

what further researches will be based on this achievement
and what could it lead to? (other autosomal recessive
diseases)

The method developed for mapping FHL-causing genes can be applied as well for
other autosomal recessive diseases.

2.4.Method

To solve the medical main problem, it is necessary to first solve the analytical
problem. That is, in order to find the unidentified mutated genes involved in the
disease mechanism of the affected patients, a bioinformatic method must be
developed, by/through which the genetic material can be analyzed.

2.4.1.Materials - the genetic material to analyze (500K SNP arrays of

15 unrelated patients) (Introduction to the method-part)

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The blood of each patient was analyzed by SNP-arrays and the material was sent to
an external laboratory. (The samples were analysed by Affymetrix GCOS, to
generate data files containing the SNP-maps.) The data received was constituted by
two CEL-files for each patient, one containing Sty-data and one containing Nsp-data.

Small Nucleic Polymorphisms

Small Nucleic Polymorphisms (SNPs) are naturally occurring variations in the

genome------(something about SNPs)--------. More precisely, the available genetic
material from the 15 unrelated patients that is subject for the research is
constituted by Affymetrix 500K SNP-arrays.

Affymetrix 500K SNP-arrays

Each 500K-array consists of two chips, one 250K Sty (Ser-Thr-Tyr-phosphorylating

enzyme???) chip and one 250K Nsp chip respectively. Sty and Nsp are type I
restriction enzymes used during the PCR amplification, which produce (slightly?)
different PCR-products. The restriction enzyme cuts the DNA-molecule at a
restriction site specific for each individual restriction enzyme. Practically, that
means that the Sty-chip and the Nsp-chip do not possess an identical set-up of
SNPs. This is used as a control during the analysis, as each genetic region will be
represented by two SNP-maps that can be compared. Thereby dissimilarities
potentially signaling laboratory errors can be detected.

2.4.2. Method Development – the long roundabout pathway (Main

part of the method-part)

Phone meeting with Affymetrix support -> Turned out that files were incomplete ->
need complete DDT-files, constituted by CEL, CAB, CHP, EXP. DDT is created by
GCOS. We had received only the CEL-file, which is ONLY an image of the chip (???),
no SNP-signal data (chp???), no experimental data (exp), cab=??

Thorigh DataTransfer Tool – transfer CEL (???) to txt, subsequently (ie export cel-file
as txt).

Txt- can be used in AutoSNPa. The Uppsala group first restructured the columns in
the txt-files according to the recommendations in the AutoSNPa manual by I. Carr,
To do this they used MatLab, algorithm written by Affymetrix platform responsible
Hanna Göransson.

However, not necessary. Instead, use DDT to create ddt-files from CAB (???), which
can be imported into GTYPE

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2.4.2.1.Bioinformatical aids available – possible ways/methods to

analyze the genetic information

2.4.2.1.1.Copy Number and Loss of Heterozygosity

2.4.2.1.1.1.Affymetrix – GCOS, GTYPE, CNAT

Use ddt-files.

2.4.2.1.1.2. Others?

2.4.2.1.2.Autozygozity mapping

2.4.2.1.2.1.The Autozygousity center of Leeds University

Hospital

Use txt-files.

2.4.2.1.2.1.1. AutoSNPa

2.4.2.1.2.1.2.IBDfinder – a new tool

2.4.2.1.2.2.The Uppsala Group (MatLab, AutoSNPa)

Used AutoSNPa to find the “best” autozygous regions. i.e. those containing greatest
number of homozygous SNP.

Used Affymetrix softwares to examine (zoom) a specific, arbitraryregion.

Used MatLab to examine as well other homozygous regions, which were not
selected bu AutoSNPa as “best regions”.

2.4.2.1.2.3.Other possibilities? (GRID Allegro)

2.4.3.Result and Conclusion – the outcome of method development

(Conclusion of the method-part)

2.5.Results

2.5.1.Homozygous regions

2.5.2.Candidate regions

2.5.3.Gene list

2.5.4.Candidate genes

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2.6.Analysis and Conclusion

2.6.1.The candidate regions

2.6.2.Narrowing the regions and the gene list

2.6.3.Candidate genes

2.6.4.Next step

2.6.4.1.further narrowing

2.6.4.2.sequencing of candidate genes

2.6.4.3.expanding regions

2.6.4.4.analyzing other interesting regions

2.7.Summary

3. Reference part

3.1.Reference literature

3.2.Study visits

3.2.1.Leeds University Hosptal

3.2.2.Uppsala University Hospital

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Checklist for report structure and content

• Vad är problemet och syftet med exjobbet?

• Vad har andra gjort förut på liknande problem?
• Vad har gjorts inom detta exjobb?
• Vad har man kommit fram till, dvs resultatet?
• Vilket arbetssätt/arbetsmetod har använts?
• Vad betyder resultaten? Hur tolkas de i ett specifikt sammanhang, och vad
kan de leda till i större perspektiv?
• Vilka slutsatser har man dragit av detta arbete?

Check as well:

• Påståenden som inte är uppenbara, bör antingen förklaras och motiveras,

bevisas eller styrkas med referens. Eljest strykas.
• Fakta som hämtats från någon källa, skall det finnas referens till.
• Man skall klart skilja på vad man har kommit fram till, och hur man har
kommit fram till, dvs resultatet och metodiken är två skilda aspekter av
rapporten.
• Om inget anges är målgruppen för exjobbsrapporten 4e årets D-teknologer
före valet till ett fördjupningsblock. Om det finns termer, som inte är
självklara för målgruppen, skall de förklaras, t ex med fotnot. När antalet
fotnoter eller andra typer av förklaringar börjar bli för stort, kan det vara dags
att definiera en målgrupp, t ex personer med grundläggande kunskap i
datasäkerhet. Då kan man utgå från den målgruppen, och endast förklara
termer utanför den nya målgruppens kompetensområde.

Checklist for oppsosition (ur “Anvisningar för examensarnete 20 poäng”)

• Struktur
o Följer rapporten en klar struktur, från problem till resultat eller hoppar
man fram och tillbaka i rapporten?
• Innehåll
o Kapitel 1 bör innehålla en beskrivning som sätter läsaren in i
problemställningen, själva problemet skall framgå, examensarbetets
syfte, så att läsaren kan bedöma om syftet är uppfyllt, och metoden
eller arbetssättet som använts. Finns alla dessa ingredienser i kapitel
1?
o Finns det klart uttryckta avgränsningar? Om ja, är det avgränsningar
av syftet? Om nej, har man i arbetet gjort avgränsningar som borde ha
uttryckts explicit?

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o Finns det i slutet av rapporten ett resonemang, som klargör hur väl
man har uppfyllt syftet? Framgår det att relevant bakgrundslitteratur
har lästs?
o Genomförande: finns arbetet beskrivet så att man förstår omfattningen
och problemen i samband med genomförandet? Följer genomförandet
det man förväntar sig av ett arbete inom den aktuella inriktningen, dvs
tillämpar man de metoder och tekniker som ingår i respektive
inriktningens ram?
• Form och språk
o Är layouten på rapporten tilltalande?
o Har avstavningar gjorts i tillräcklig omfattning?
o Är språkliga formuleringar klara och tydliga?
o Är meningarna lagom långa?
o Är meningarna korrekta enligt det aktuella språkets grammatik?
o Finns det en ordlista? Om ja, behövs den till den aktuella målgruppen?
Om nej, borde en ordlista varit med?
o Följer litteraturförteckningen någon allmänt accepterad standard?
o Finns det fler än 10 slarv- och stavfel?
o Finns det bilagor? Om ja, behövs de? Finns det i rapporten hänvisning
till bilagorna? Om nej, skulle man kunnat lyfta något av rapporten till
bilagor? Vilka delar?

Frågor

Jan-Inge

1. Patientinformation: när/var blev de diagnostiserade?, av vem?, på

vilka grunder?, ålder för insjuknande, familjebakgrund,

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