Identification of disease-related genes. Heymut Omran Department of Pediatrics and Adolescent Medicine; Freiburg; Germany

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1 Identification of disease-related genes Heymut Omran Department of Pediatrics and Adolescent Medicine; Freiburg; Germany

2 Gene identification strategies A. Position-independent strategies for identifying genes B. Positional cloning of disease genes C. Combination of both strategies Functional approach: Hemophila A patients lacked blood clotting factor VIII. Protein was purified, partially sequenced

3 Gene identification strategies A. Position-independent strategies for identifying genes B. Positional cloning of disease genes C. Combination of both strategies Positional approach: Gene localization on the human genome. Functional data are not necessary.

4 Position-independent strategies for identifying genes A. Detailed knowledge of the pathophysiology is mandatory

5 Position-independent strategies for identifying genes A. Enzyme deficiency Aminoacylases divide N-acetylated amino acids. Currently two distinct aminoacylases are known.

6 Gas chromatography mass-spectrometry (GC-MS) analysis (Urin)

7 NMR spectroscopy (urine) Metabolite OS-104 II-1 OS-17II- 1 OS-14 II- 1 OS-10 II- 1 sample 1 [mmol/ mol creatinine] sample [mmol/ mol creatinine] sample 3 [mmol/ mol creatinine] [mmol/ mol creatinine] [mmol/ mol creatinine] [mmol/ mol creatinine] N-acetylalanine N-acetylasparagine N-acetylglutamic acid N-acetylglutamine N-acetylisoleucine 18 3 < N-acetylglycine N-acetylvaline <10 <10 <10 30 nd nd N-acetylserine < N-acetylthreonine < N-acetylmethionine

8 Demonstration of ACY1-mutations

9 Position-independent strategies for identifying genes A. Detailed knowledge of the pathophysiology is mandatory B. Adequate disease models (mouse, C. elegans, D. melanogaster, S. cerevesiae, D. rerio, )

10 Position-independent strategies for identifying genes Zellweger Syndrome: Peroxysomal biogenesis disorder (increased very long chain fatty acids)

11 Position-independent strategies for Zellweger Syndrome: identifying genes A. In somatic cell hybridization analyses (fibroblasts) ten distinct complementation groups could be identified B. In Saccharomyces cerevisiae several complementation groups were found C. Cloning of the human S. cerevesiae orthologues allowed PEX gene identifications Reuber et al. Nat Genet 1997; Portsteffen et al. Nat Genet 1997

12 Positional cloning of disease genes A. In positional cloning, disease genes are identified using only knowledge of their chromosomal location

13 Positional cloning of disease genes A. In positional cloning, disease genes are identified using only knowledge of their chromosomal location B. Chromosomal aberrations

14 Positional cloning of disease genes Chromosomal aberrations Alagille Syndrome Autosomal dominant Cholestatic jaundice: Biliary tract paucity peripheral pulmonic stenosis others

15 Positional cloning of disease genes Chromosomal aberrations (Alagille syndrome) Deletions JAG1 gene Chromosomal breakpoint mapping (balanced translocations)

16 Positional cloning of disease genes A. In positional cloning, disease genes are identified using only knowledge of their chromosomal location B. Chromosomal aberrations C. Linkage studies

17 Positional cloning of disease genes Linkage studies Affected Affected Affected Affected Affected Affected

18 Positional cloning of disease genes Linkage studies (microsatellite markers) 1 3 Alleles:

19 Positional cloning of disease genes Linkage studies (SNP analyses) gcgtgattgttagtgc[a/g]gatctgtggtactgct SNP = single nucleotide polymorphism Most frequent genetic variation (appr bp) Frequency of the variation is >1% in gen. population < 1% of SNPs affect protein coding sequence Humane genome contains >3 x 10 6 SNP

20 SNP distribution along the human genome: 10K SNP Array Red: min. 1 SNP per 100 kb Black: Gaps (Median der Distanz zw. Markern: 108 kb)

21 SNP distribution along the human genome: 100K ( x 50K) SNP Array Red: min. 1 SNP per 100 kb Black: Gaps (Median der Distanz zw. Markern: 8 kb)

22 Positional cloning of disease genes Problems of linkage studies Genetics Environment Phenotype

23 Positional cloning of disease genes Problems of linkage studies Gene 1 Disorder Incomplete Penetrance

24 Positional cloning of disease genes Problems of linkage studies Gene 1 Environment Disorder Phenocopy

25 Positional cloning of disease genes Problems of linkage studies Gene 1 Gene Gene 3 + Disorder Polygenic Disorder

26 Positional cloning of disease genes Problems of linkage studies Gene 1 Gene Gene 3 Disorder Genetic Heterogenity

27 Positional cloning of disease genes Genetic heterogeneity Nephronophthisis loci NPHP1: q1-q13 NPHP: 9q-q31 NPHP3: 3q1-q NPHP4: 1p36 NPHP5: 3q1 NPHP6: 1q1

28 Positional cloning of disease genes Homozygosity mapping strategies autosomal rezessive

29 Positional cloning of disease genes Linkage studies (microsatellite markers) 1 3 Alleles:

30 (P) 1 14 Positional cloning of disease genes Homozygosity mapping strategies

31 HOMOZYGOSITY MAPPING STRATEGY Marker Chromosome 3 D3S178 D3S167 D3S370 D3S3573 D3S3607 D3S3606 D3S1587 D3S3548 D3S1541 D3S19 D3S1596 D3S173 D3S3 D3S190 D3S3713* D3S3657 D3S1485 D3S138 D3S3684 D3S3637 D3S1549 D3S3617 D3S1576 DS3554 LOD- Score: ZMAX = 5,9

32 (P) 1 14 Positional cloning of disease genes Physical mapping and search for deletions

33 NPH3 PATHOLOGY pcy Cysts at the corticomedullary junction Tubular atrophy and cystic dilatation Interstitial infiltration and fibrosis Tubular basement changes

34 (P) 1 14 Positional cloning of disease genes Orthology analyses

35 (P) 1 14 Positional cloning of disease genes Mutational analyses

36 (P) 1 14 Positional cloning of disease genes Mutational analyses

37 Gene identification strategies A. Position-independent strategies for identifying genes B. Positional cloning of disease genes C. Combination of both strategies D. Computational analyses