The European CF twin and sibling study [Elektronische Ressource] : genetic susceptibility to infectious diseases / von Vinod Kumar Magadi Gopalaiah

Vinod Kumar Magadi Gopalaiah

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• Gesundheit

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2007
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

The European CF Twin and Sibling Study;
Genetic Susceptibility to Infectious Diseases




Von der Naturwissenschaftlichen Fakultät der

Gottfried Wilhelm Leibniz

Universität Hannover

zur Erlangung des Grades

DOKTOR DER NATURWISSENSCHAFTEN

- Dr. rer. nat. -



genehmigte Dissertation

von




M.F.Sc, VINOD KUMAR MAGADI GOPALAIAH
geboren am 01.06.1977 in Bangalore, Indien





Hannover 2007















This work is dedicated to
my dearest Parents
and to my wonderful brothers


















Die vorliegende Arbeit wurde in der Klinischen Forschergruppe „Molekulare
Pathologie der Mukoviszidose“, Zentrum Biochemie und Zentrum Kinderheilkunde der
Medizinischen Hochschule Hannover in der Zeit vom 01.10.2003 bis zum 30.09.2006
unter der Leitung von Prof. Dr. Dr. Burkhard Tümmler angefertigt.














Tag der Promotion 24.01.2007

Referent: Prof. Dr. Dr. Burkhard Tümmler
Klinische Forschergruppe OE6711
Zentrum Biochemie und Zentrum Kinderheilkunde
Medizinische Hochschule Hannover


Koreferent Prof. Dr. Gerald-F. Gerlach
Institut für Mikrobiologie
Zentrum Infektionsmedizin
Stiftung Tierärztliche Hochschule Hannover



A token of gratitude

I would like to express my whole hearted thanks and sincere gratitude to my major advisor,
Prof. Dr. Dr. Burkhard Tümmler, Klinische Forschergruppe, Medizinische Hochschule,
Hannover, for giving me the scientific freedom and the wonderful opportunity to
accomplish this thesis. His pragmatic guidance and constant encouragement throughout the
period of my research is gratefully acknowledged.

I would like to thank Prof. Dr. Gerald-F. Gerlach for reviewing my thesis, Prof. Dr. Helmut
Holtmann and Prof. Dr. Karl-Heinz Bellgardt for being part of the review committee.

I owe my profound thanks to my supervisor Dr. Frauke Stanke for giving me an unfailing
guidance, friendly nature, scholarly advice and the confidence she has showered in me and
in my project. Her amazing patience, terrific organizing skills, generous support and deep
insights enabled me to succeed in my profession.

I am very much thankful and grateful to,
Frau Silke Jansen and Ms. Stephanie Tamm for their friendly attitude and technical
assistance
Dr. Andrea van Barneveld for helping me in setting up the western blot experiments
Dr. Lutz Wiehlmann and Dr. Jens Klockgether for their valuable suggestions and
scientific discussions
Dr. Antje Munder for providing me the human cell lines
Ms. Gesa Puls for her help in this thesis preparation
Prof. Christoph Klein for letting me use the FACS and real-time PCR facilities
Dr. Chozhavendan Rathinam and Mr. Giridharan Appaswamy for their help in FACS
and real-time PCR experiments
Dr. Kaan Boztug for his help in FACS experiments
Prof. Thomas F. Wienker and Prof. T. Becker for providing me the statistical analysis
Prof. Dave Ussery for performing functional annaotation of non-coding variants
The Deutsche Forschungsgemeinschaft (DFG)-sponsored European Graduate College
“Pseudomonas: Pathogenicity and Biotechnology” for providing me the
fellowship.


I am thankful to Frau Helga Riehn-kopp, co-ordinator of the graduate college, for her kind
support and timely help in bureaucratic issues.

My heartfelt thanks to Mr. Dieco Wuerdemann, Ms. Tammy Chang and Dr. Andrea van
Barneveld for their friendship and caring, which have supported me in many ways.

I take this opportunity to express my sincere thanks to all the members of “Klinische
Forschergruppe” and members of the EGK for their help and necessary support.

My special thanks to my “GURU’S” Dr. Bob Kennedy, Dr. Devaraja and Dr. Mohana
Kumara for their moral support, affection, and caring.

I pay my whole hearted thanks to my dear friends Rama, Shanthi, Pal, Kummi, Benki, Umi,
Viji, Mona, Giri, Bhanu, Shashi, Anu, Shilpa, Asha, Selvan, Hari, Raghu, Yadhu, Prajeeth
and Shivu for their encouragement and cheerful attitude.

Last but not least, I am indebted to my parents and family members, whose love, blessings
and sacrifice made it possible for me to accomplish this thesis.


















Abstract

Cystic Fibrosis (CF) is the most severe common autosomal recessive congenital disorder in the
Caucasian population. It is caused by molecular lesions in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene located on the long arm of human chromosome 7. While
the CFTR determines the susceptibility of the airways to the opportunistic pathogen
Pseudomonas aeruginosa in CF patients, progression and severity of the CF disease can not be
predicted by the CFTR mutation genotype. Thus, the main objective of this study was to
investigate TLR2, TLR4, TLR5, TLR9, SP-D, CXCR2, PON, TNFR1, CD14, and CD95 as
modulators of CF disease severity and susceptibility to P. aeruginosa infection. All ten
candidate genes were targeted initially by one informative SNP (Single nucleotide
polymorphism) to investigate them as CF disease severity modulators in European CF Twin and
Sibling Study cohort containing 37 families with F508del-CFTR homozygous, exhibiting
extreme clinical phenotypes. SNPs on TLR2, TLR5 and TLR9 failed to show the association.
Polymorphisms on surfactant protein-D, CXCR2 and PON locus showed only a minor
association with CF disease severity. The significant association was found between
polymorphisms within TNFR1, TLR4, CD14, CD95 and CF disease severity. Hence, these four
genes were investigated further by typing more SNPs and haplotype analysis. The genomic
fragment containing the causative variant was identified by direct comparison of two-marker-
haplotype-distributions between sib pair sets displaying a phenotypic contrast. Individuals
carrying contrasting haplotypes were subjected to confirmatory sequencing at the outlined
genomic fragment and the coding region. Sequencing analysis did not reveal any coding
variants. The functional role of non-coding causative variants was explored by in-silico analysis
and suitable phenotypic assays. Furthermore, these causative variants were analysed for their
role in CF infectious disease in two independent CF cohorts stratified for P. aeruginosa-related
endophenotypes such as onset of initial and chronic colonisation.
The haplotype analysis showed that the causative haplotype in TLR4 was located
upstream of TLR4 exon 1 and was associated with CF disease severity but not with P.
aeruginosa early or late colonisation.
TNFR1 first intron harboured a disease modifying haplotype. In-silico analysis
predicted that the alterations in DNAse hypersensitive sites, conserved non-coding sequences
and inverted local repeats due to intron 1 variants may cause differential transcription.
Consistently, western blot analysis showed that the levels of TNFR1 in CF patients’ serum
correlated with TNFR1 causative haplotype. The haplotype analysis on CD95 gene found a
causative variant within intron 2. The in-silico analysis predicted a transcription regulatory
region within intron 2 and the causative SNP was located within this regulatory region. Thus, it
altered the binding site for c-Rel transcription factor. CF patients heterozygous for this intron 2
SNP had significantly lower levels of CD95 mRNA, isolated from rectal suction biopsies of
F508del-CFTR homozygous patients. This effect was specific to either epithelial cells or to CF
context as mRNA quantification by real-time PCR and surface expression by FACS on
peripheral blood mononuclear cells from healthy individuals did not reveal any association.
CD95 polymorphisms were not associated with P. aeruginosa early or late chronic colonisation.
CD14 promoter polymorphism and 3’ UTR polymorphism were associated with CF
disease severity and age at onset of P. aeruginosa chronic colonisation. The diplotype analysis
on CD14 locus revealed a significant association with age-dependent risk to acquire P.
aeruginosa colonisation, the level of sCD14 in serum of CF patients and also associated with P. O-antigen phenotype. The 3’ UTR polymorphism was predicted to alter the binding
site for microRNA on CD14 RNA and also binding site for mRNA processing proteins.

In summary, we identified TLR4, TNFR1, and CD95 as potential genetic modulators of CF
disease severity and CD14 as both the CF disease modulator as well as the modulator of age
dependent risk to acquire P. aeruginosa colonisation among CF patients. Furthermore, the
importance of non-coding variants in CF disease modulation was illustrated.

Key Words: Cystic Fibrosis, Genetic modulators, Innate immunity
Kurzfassung

Cystische Fibrose