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.

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

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

Demonstration of ACY1-mutations

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)

Genetics Environment

Phenotype

Positional cloning of disease genesProblems of linkage studies

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

autosomal rezessive

Positional cloning of disease genesHomozygosity mapping strategies

Positional cloning of disease genesLinkage studies (microsatellite markers)

Alleles:

1

2

23

12

22

22

22

13

12

12

22

3

Positional cloning of disease genesHomozygosity mapping strategies

39

47

12 14

(P2)99 100

38

85

31

HOMOZYGOSITY MAPPING STRATEGY

LOD-Score:

ZMAX = 5,9

Marker

D3S1278D3S1267D3S3720D3S3573D3S3607D3S3606D3S1587D3S3548D3S1541D3S1292D3S1596D3S1273D3S2322D3S1290D3S3713*D3S3657D3S1485D3S1238D3S3684D3S3637D3S1549D3S3617D3S1576DS3554Chromosome 3

Positional cloning of disease genesPhysical mapping and search for deletions

39

47

12 14

(P2)99 100

NPH3 pcyPATHOLOGY

• Cysts at thecortico-medullaryjunction

• Tubularatrophy and cystic dilatation

• Interstitialinfiltration and fibrosis

• Tubularbasementchanges

Positional cloning of disease genesOrthology analyses

39

47

12 14

(P2)99 100

Positional cloning of disease genesMutational analyses

39

47

12 14

(P2)99 100

Positional cloning of disease genesMutational analyses

39

47

12 14

(P2)99 100

Gene identification strategies

A. Position-independent strategies foridentifying genes

B. Positional cloning of disease genes

C. Combination of both strategies

D. Computational analyses