identification of disease-related genes · nmr spectroscopy (urine) n-acetylmethionine 41 45 46 80...
TRANSCRIPT
Identification of disease-related genes
Heymut Omran
Department of Pediatrics and Adolescent Medicine; Freiburg; Germany
Gene identification strategies
A. Position-independent strategies foridentifying genes
B. Positional cloning of disease genes
C. Combination of both strategies
Functional approach:
Hemophila A patients lacked bloodclotting factor VIII. Protein was purified, partially sequenced
Gene identification strategies
A. Position-independent strategies foridentifying genes
B. Positional cloning of disease genes
C. Combination of both strategies
Positional approach:
Gene localization on the human genome. Functional data are notnecessary.
Position-independent strategies foridentifying genes
A. Detailed knowledge of thepathophysiology is mandatory
Position-independent strategies foridentifying genes
A. Enzyme deficiency
Aminoacylases divide N-acetylated amino acids. Currently two distinct aminoacylases are known.
NMR spectroscopy (urine)
507580464541N-acetylmethionine
2841<10172318N-acetylthreonine208393<1099111104N-acetylserine
ndnd30<10<10<10N-acetylvaline
51542917592125108N-acetylglycine
304058<102318N-acetylisoleucine
1435261488698111N-acetylglutamine
284741281169186193N-acetylglutamicacid
133356155667989N-acetylasparagine
156312699111593N-acetylalanine
[mmol/ mol creatinine]
[mmol/ mol creatinine]
[mmol/ mol creatinine]
sample 3[mmol/ mol creatinine]
sample 2[mmol/ mol creatinine]
sample 1 [mmol/ mol creatinine]
OS-120 II-1
OS-124 II-1
OS-127II-1
OS-104 II-1Metabolite
Position-independent strategies foridentifying genes
A. Detailed knowledge of thepathophysiology is mandatory
B. Adequate disease models (mouse, C. elegans, D. melanogaster, S. cerevesiae, D. rerio, …)
Position-independent strategies foridentifying genes
Zellweger Syndrome:
Peroxysomal biogenesis disorder (increased verylong chain fatty acids)
Position-independent strategies foridentifying genes
Zellweger Syndrome:
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. cerevesiaeorthologues allowed PEX gene identifications
Reuber et al. Nat Genet 1997; Portsteffen et al. Nat Genet 1997
Positional cloning of disease genesA. In positional cloning, disease genes
are identified using only knowledgeof their chromosomal location
Positional cloning of disease genesA. In positional cloning, disease genes
are identified using only knowledgeof their chromosomal location
B. Chromosomal aberrations
Positional cloning of disease genesChromosomal aberrations
Alagille Syndrome
Autosomal dominant
Cholestatic jaundice: Biliary tractpaucity
peripheral pulmonic stenosis
others
Positional cloning of disease genesChromosomal aberrations (Alagille syndrome)
• Deletions
• Chromosomal breakpoint mapping (balancedtranslocations)
JAG1 gene
Positional cloning of disease genesA. In positional cloning, disease genes
are identified using only knowledgeof their chromosomal location
B. Chromosomal aberrations
C. Linkage studies
Positional cloning of disease genesLinkage studies
Affected AffectedAffectedAffected AffectedAffected
Positional cloning of disease genesLinkage studies (microsatellite markers)
Alleles:
1
2
23
12
22
22
22
13
12
12
22
3
SNP = single nucleotide polymorphism
Most frequent genetic variation (appr. 100 - 300 bp)
Frequency of the variation is >1% in gen. population
< 1% of SNPs affect protein coding sequence
Humane genome contains >3 x 106 SNP
gcgtgattgttagtgc[A/G]gatctgtggtactgct
Positional cloning of disease genesLinkage studies (SNP analyses)
SNP distribution along the human genome: 10K SNP Array
Red: min. 1 SNP per 100 kbBlack: Gaps(Median der Distanz zw. Markern: 108 kb)
SNP distribution along the human genome: 100K (2 x 50K) SNP Array
Red: min. 1 SNP per 100 kbBlack: Gaps(Median der Distanz zw. Markern: 8 kb)
Positional cloning of disease genesProblems of linkage studies
Incomplete Penetrance
Disorder
Gene 1
Positional cloning of disease genesProblems of linkage studies
Phenocopy
Disorder
Gene 1 Environment
Positional cloning of disease genesProblems of linkage studies
Polygenic Disorder
Gene 1
Disorder
Gene 3Gene 2
+
Positional cloning of disease genesProblems of linkage studies
Genetic Heterogenity
Gene 1
Disorder
Gene 3Gene 2
NPHP3: 3q21-q22
Nephronophthisis lociNPHP1: 2q12-q13NPHP2: 9q22-q31
NPHP4: 1p36NPHP5: 3q21NPHP6: 12q21
Positional cloning of disease genes
Genetic heterogeneity
Positional cloning of disease genesLinkage studies (microsatellite markers)
Alleles:
1
2
23
12
22
22
22
13
12
12
22
3
HOMOZYGOSITY MAPPING STRATEGY
LOD-Score:
ZMAX = 5,9
Marker
D3S1278D3S1267D3S3720D3S3573D3S3607D3S3606D3S1587D3S3548D3S1541D3S1292D3S1596D3S1273D3S2322D3S1290D3S3713*D3S3657D3S1485D3S1238D3S3684D3S3637D3S1549D3S3617D3S1576DS3554Chromosome 3
NPH3 pcyPATHOLOGY
• Cysts at thecortico-medullaryjunction
• Tubularatrophy and cystic dilatation
• Interstitialinfiltration and fibrosis
• Tubularbasementchanges