Thesis Work Report V

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 expression

disease status

Unknown

Unknown

FHL1

9

PRF1

Perforin

FHL2

10

Cytoban d 9q21.322 10q2122

UNC13D

Munc 13-4

FHL3

17

17q24

STX11

Syntaxin 11

FHL4

MYHIIA

Myosin

?

gene

Chromoso me

6 22

6q24

exons

7 33 4

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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•

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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) KTH School of Biotechnology

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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

o

•

Form o o o o o o o o o

Chapter Name

Finns det i slutet av rapporten ett resonemang, som klargör hur väl man har uppfyllt syftet? Framgår det att relevant bakgrundslitteratur har lästs? 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? och språk Är layouten på rapporten tilltalande? Har avstavningar gjorts i tillräcklig omfattning? Är språkliga formuleringar klara och tydliga? Är meningarna lagom långa? Är meningarna korrekta enligt det aktuella språkets grammatik? Finns det en ordlista? Om ja, behövs den till den aktuella målgruppen? Om nej, borde en ordlista varit med? Följer litteraturförteckningen någon allmänt accepterad standard? Finns det fler än 10 slarv- och stavfel? 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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