molecular medicine - 2. key concepts positional cloning in identifying disease alleles (an example...
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Molecular medicine - 2
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Key concepts
Positional cloning in identifying disease alleles
(an example – cystic fibrosis)
Majority of human diseases have polygenic or
multi-factorial risk factors.
Challenges and approaches in the post genomic
era
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Example of identifying a disease allele by positional cloning
Cystic fibrosis
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Pathology ‘Woe to that child which when kissed on the forehead tastes salty. He
is bewitched and soon must die’
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Example of identifying a disease allele by positional cloning
Cystic fibrosis
cystic fibrosis caused by mutations in the CF gene
5% of Caucasians are asymptomatic carriers. Frequency of 1 / 2,500 (~ 30,000)
CF disease locus identified on chromosome 7q 31.2(Kerem 1989; Riordan 1989; Rommens 1989).
Severe autosomal recessive condition among Caucasians.
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CFTR is a Cl- channel (defects result in either a decrease in its Cl- transport capacity or its level of cell surface expression)
CF gene encodes a cystic fibrosis transmembrane conductance regulator (CFTR Cl- channel)
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CFTR function
http://www.infobiogen.fr/services/chromcancer/IntroItems/Images/CFTREnglFig2.jpg
epithelial Cl- transport Cl- transport rate determined by activation of CFTR which in turn depends on its state of phosphorylation.
Acts as a regulator of other channels & transporters e.g CFTR mediates cAMP regulation of amiloride sensitive epithelial Na+ channels (EnaCs)
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Mutations in CFTR
70% of CF patients show a specific deletion F508 deletion in exon 10 (F): NBD-1 domain CFTR misfolding in the ER and targeted for proteosome degradation
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Mutations in CFTR
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Mapping of CF allele
1985 gene for CF linked to enzyme paraoxanase (PON)PON mapped to chromosome 7 and CF mapped to 7q31-32
(random DNA marker D7S15)2 flanking markers established (~2x106bp apart)
proximal MET oncogene and distal D7S8extensive mapping and characterisation around the candidate
region by chromosome walking, chromosome jumping and microdissection (~300kbp cloned)
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CFTR candidate region
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Mapping of CFTR
2 new markers identified – KM19 and XV2c – which showed strong linkage disequilibrium5’ end of gene locatedBovine equivalent of candidate gene isolated from genomic library7 cDNA libraries screened with human clone. 1 cDNA clone identified. Northern blots show 6.5 kb mRNARest of the gene obtained by screening and PCR1989 CFTR gene eventually isolated by mutation screening
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linkage disequilibrium Alleles at 2 or more loci that show a non-random
association are said to be in linkage disequilibrium.Allelic association in cystic fibrosisMarker alleles CF Normal
chromosomes chromosomesX1,K1 3 49X1,K2 147 19X2,K1 8 70X2,K2 8 25
RFLP markers XV2C (X1,X2) and KM19 (K1,K2)
Conclusive evidence defective cAMP-dependent chloride conductance in CFTR-/- cells was restored when CFTR cDNA was transfected and expressed in those cells.
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Letter to Dr. Collins. Courtesy of the National Human Genome Research Institute
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“For any given trait there will be few (if any) large effects, a handful of modest effects, and a substantial number of genes generating small or very small increases in disease risk.”
Nature 447, 661–678 (2007)
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ChallengesSome of the complexities of
human disease traits
• Phenotypic heterogeneity • Phenocopies • Variable expressivity• Incomplete penetrance• Polygenic traits
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Same genotypic mutation causes variable phenotypes
e.g. thalassemias – Caused by mutations in either the or -globin
genes. – Similar genotype can lead to unaffected or severe
phenotypes
Phenotypic heterogeneity
GENOTYPE PHENOTYPE a+ a+ a+ a+ Normal a+ a a+ a+ Silent carrier asymptomatic condition. thalassaemia - 2 a+ a a+ a -thalassaemia trait minor anaemic conditions a+ a+ a a a+ a a a HbH mild – moderate anaemia a a a a Hydrops foetalis foetus survives until around birth
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Many mechanisms contribute to the phenotypic heterogeneity of
thalassaemias
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Disease phenotype is not caused by any known inherited predisposing mutation e.g. BRCA1 mutations
• 33% of women who do not carry BRCA1 mutation develop breast cancer by age 55
Phenocopy
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Variable expressivityExpression of a mutant trait differs in individuals
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Incomplete penetrance
• Positional cloning identified BRCA1 as one gene causing breast cancer.– Only 66% of women who carry BRCA1
mutation develop breast cancer by age 55
• Incomplete penetrance hampers linkage mapping and positional cloning
– when a mutant genotype does not always cause a mutant phenotype
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Two or more genes interact in the expression of phenotype e.g. cancer• QTLs, or quantitative trait loci
– Penetrance / expressivity may vary with number of mutant loci
– Some mutant genes may have large effect– Mutations at some loci may be recessive while
others may be dominant or codominant
Polygenic traits
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Alzheimer’s disease
familial AD – mutations in APP, presenilin-1 and 2Sporadic AD – strong association with APO4, Apolipoprotein 4, which
affects age of onset rather than susceptibility
Sudden cardiac death (SCD)
Affects 5% of people >65 years and 20% of people over 80 has familial (early-onset) or sporadic (late-onset) forms, although
pathologically both are similaretiology of sporadic forms unknown
3 major alleles (APO E2, E3, and E4)
Position
112 158
ApoE2 Cys Cys
ApoE3 Arg Cys
ApoE4 Arg Arg
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Apart from SNPs, structural variants such as CNVs may explain some of these
complexities
• Changes in copy number may directly affect risk factor
• Rearrangements / fusion may alter expression
• CNVs could increase risk of secondary pathogenic rearrangements
• CNVs could indirectly affect environmental interaction leading to different phenotypes
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What approaches should be used in the post-genomic era?
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Mapping complex loci
PAF – population attributable factor:
Fraction of the disease that would be eliminated if the
risk factor were removed
High PAF for single gene conditions (>50% for CF)
Low PAF for complex disease (< 5% for Alzheimer’s)
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Identifying genes involved in complex diseases
Perform family, twin or adoption studies - check for genetic component
Segregation analysis- estimate type and frequency of susceptibility alleles
Linkage analysis- map susceptibility loci
Population association- identify candidate region
Identify DNA sequence variants conferring susceptibility
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Linkage versus Association
Association studies compare the allele frequency of a polymorphic marker, or a set of markers, in unrelated patients (cases) and healthy controls to identify markers that differ significantly between the two groups.
Used to identify common modest-risk disease variants
Higher density of markers needed
e.g. HapMap uses association data
Linkage analyses search for regions of the genome with a higher-than-expected number of shared alleles among affected individuals within a family.
Used to identify rare high-risk disease alleles
<500 markers needed for initial genome scan
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Haplotype Map (HapMap)• Haplotype: specific combination of 2 or more DNA marker alleles situated
close together on the same chromosome (cis markers). E.g. SNPs• HapMap - catalog of common genetic variants in populations• International HapMap Project - identify common haplotypes in four
populations with African, Asian, and European ancestry• provide information to link genetic variants to the risk of disease
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Reading
HMG3 by T Strachan & AP Read : Chapter 14
AND/OR
Genetics by Hartwell (2e) chapter 11 References on Cystic fibrosis: Science (1989) vol 245 pg 1059 by JM Rommens et al (CF mapping)J. Biol Chem (2000) vol 275 No 6 pp 3729 by MH Akabas (CFTR)
Optional Reading on Molecular medicine Nature (May2004) Vol 429 Insight series• human genomics and medicine pp439 (editorial)
Nature Vol 437|27 pp1241-42 October 2005 (HapMap Project)
Nature (Oct 2007) VOLUME 82 NUMBER 4 pp 366-70 (CNVs)