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Genetic mapping studies - Asthma and allergy

Genetic mapping studies - Asthma and allergy. Clinical expertise Diagnostic classification. Genetic analysis Disease modelling. Nature of disease gene projects. Hopes and aims: what does one want to find?. Development of therapies New bioactive factors or immediate drug targets

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Genetic mapping studies - Asthma and allergy

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  1. Genetic mapping studies - Asthma and allergy

  2. Clinical expertise Diagnostic classification Genetic analysis Disease modelling Nature of disease gene projects

  3. Hopes and aims: what does one want to find? • Development of therapies • New bioactive factors or immediate drug targets • New pathways or disease mechanisms • New associations for known pathways • Development of diagnostics • Specific assays for disease screening • Specific diagnostic assays for clinical use • Informative and useful new assays

  4. How to think of gene effects in multifactorial diseases? • Pedigrees and penetrance • The threshold model of susceptibility • Quantitative gene effects • Diversity of disease-associated variants

  5. Map location Expression pattern Polymorphism Tissue How to find the asthma gene?

  6. Autosomal dominant, 100% penetrance

  7. …67% penetrance…

  8. …33% penetrance

  9. Threshold model of susceptibility healthydisease gene — gene + Number of people Quantitative measure

  10. Promoter variants • altered transcription • Missense variants • altered protein function • Splice site variants • altered transcript • UTR variants • transcript instability • Nonsense variants • truncated transcript • Intron variants • regulatory elements Diversity of mutations

  11. Population simulation and disease modelling Multilocus association analysis Etc. Linkage analysis Etc. Etc. A gene mapper’s lunchbasket for an excursion to multifactorial diseases

  12. Population movement in the 1500’s Little later immigration Small permanent settlement of south and west coasts >2000 y A brief population history Rapid late population growth (10 x / 250 y)

  13. Why study a multifactorial Why study a multifactorial disease in a founder isolate? disease in a founder isolate?

  14. Kainuu Kainuu Asthma Study Asthma Study 15-20 generations 15-20 generations Department of Medical Genetics and Department of Medical Genetics and Department of Pulmonary Diseases, Department of Pulmonary Diseases, University of Helsinki and HUCH University of Helsinki and HUCH Department of Clinical Genetics, the Finnish Department of Clinical Genetics, the Finnish Family Federation (Väestöliitto) Family Federation (Väestöliitto) Kainuu Central Hospital, Kajaani Kainuu Central Hospital, Kajaani

  15. Disease gene mapping project Design of study Obtaining permissions Recruitment of families Verification of diagnoses Collection of samples Genotyping Analysis of data Identification of gene Functional analysis Utilization

  16. Genome scan • A set of 312 microsatellite markers were chosen in order to find out genomic regions co-segregating with the disease status • All markers genotyped in all individuals of the families recruited • Linkage analysis was carried out

  17. Linkage results of the genome scan for asthma with 304 autosomal and 8 X-chromosomal markers in 86 Finnish pedigrees. Laitinen et al., Nature Genetics 28:87, 2001

  18. A susceptibility gene for asthma in chromosome 7p • Genome scan in Finnish families gave significant evidence for linkage to chromosome 7 (NPL=3.9 for high IgE phenotype; NPL=3.0 for asthma) • Result replicated in French-Canadian pedigrees from Saguenay-Lac-St-Jean (NPL=2.7 for asthma) • Second replication in North Karelian pedigrees (NPL=1.9 for high IgE) Laitinen et al., Nature Genetics 28:87, 2001

  19. Linkage disequilibrium mapping

  20. Fine mapping • Exact location of the gene was mapped by subsequent analysis of linked regions • Laitinen et al. 2004: Science Vol 304, Issue 5668, pages 300-304.Characterization of a Common Susceptibility Locus for Asthma-Related Traits.

  21. Fine mapping after linkage finding • Fig. 1. (A) Hierarchical gene mapping strategy. The linkage region of 20 cM implicated by the genome scan was refined by genotyping 76 microsatellite markers in families from Kainuu. We used the HPM algorithm for finding haplotypes associated with high serum IgE. Haplotype patterns spanning 12 microsatellite markers within 3.5 cM were found associated by a permutation test implemented in HPM. At the next round of fine mapping, 10 additional microsatellites implicated a 301-kb haplotype pattern (5 markers yielded the highest associations). A further five microsatellites and 13 SNPs were genotyped next, implicating a 47-kb haplotype pattern (10 markers) between NM51 and SNP563704. All together, a 133-kb region was sequenced around this segment from a homozygous patient with asthma. Eighty polymorphisms were identified by comparison to the public genomic sequence. (D) Phylogenetic analysis of haplotypes H1 to H7 within a 77-kb segment in Kainuu, North Karelia, and Quebec. The same seven haplotypes occur in all three populations at frequencies >2%. H4 and H5 are the most common risk-associated haplotypes in Kainuu, H7 in North Karelia, and H2 among French Canadians. H1, H3, and H6 are nonrisk haplotypes in all three populations.

  22. Gene structure in the 133-kb region • Fig. 2. Gene content around the conserved 133-kb haplotype segment (gray box). (A) The 133-kb segment spans from intron 2 to intron 5 of GPRA. GPRA undergoes alternative splicing with multiple variants; the three longest variants are shown (thin lines joining exons marked E1 to E9b). Exon 2 donor site may join to alternative exon 3 acceptor sites, separated by 33 bp in the same reading frame, and there are two alternative 3' exons, 9a and 9b. Further splice variants may skip exon 3 or 4 or both, suggesting an involvement of the associated polymorphisms in regulation of splicing and protein isoform production. (B) In the opposite DNA strand, there is a previously unknown gene, AAA1, with at least 18 exons (numbered 1 to 18) with complex alternative splicing. AAA1 spans a total of 500 kb of genomic sequence. Eight exons of GPRA (E1 to E8) are shown for orientation. (C) Northern blot hybridization with a 1285-bp full-length GPRA-A cDNA probe (left) and a mixed splice variant probe for AAA1 (right). A 2.4-kb transcript is visible in all nine lanes (upper arrow) and a 1.8-kb transcript (lower arrow) in four tissues for GPRA. Several alternative transcripts are seen for AAA1 (arrows).

  23. GPRA expression patterns in tissues Fig. 4. (A) Expression of GPRA isoform B in bronchial biopsies from a healthy control (left) and an asthma patient (right). E, epithelium; BM, basement membrane; LP, lamina propria; SM, smooth muscle. (Top) The airway epithelium in the control sample shows only faint staining. Results are typical of 8 asthmatic and 10 control biopsies studied. (B) Relative expression levels of Gpra mRNA in lungs from sensitized (n = 7) and control (n = 8) mice after inhaled ovalbumin challenge. Gpra was significantly up-regulated in sensitized compared with control mice. (C) Variable alternative splicing for AAA1 depending on genotype.

  24. GPRA • The properties of GPRA make it a strong candidate for involvement in the pathogenesis of asthma and other IgE-mediated diseases, as well as a possible drug target. • GPRA might act as a receptor for an unidentified ligand • The putative ligand, isoforms of GPRA, and their putative downstream signaling molecules may define a new pathway critically altered in asthma. • GPRA encodes isoforms that are produced in distinct patterns by bronchial epithelial cells and smooth muscle cells in asthmatic and healthy individuals. • GPRA is also expressed by gut epithelia and keratinocytes of the skin, suggesting a potential role in a wider spectrum of allergic diseases.

  25. Key group members Asthma: Tarja Laitinen, Siru Mäkelä, Anne Polvi, Johanna Vendelin • Computational methods: Päivi Onkamo, Petteri Sevon, Vesa Ollikainen Collaborators Asthma mapping: Lauri A. Laitinen, Mark Daly, Tom Hudson, Eric Lander Computational methods: Heikki Mannila, Hannu T.T. Toivonen Gene expression: Riitta Lahesmaa Acknowledgements

  26. One day I’llmutate…

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