Molecular medicine - 2
Key concepts
Positional cloning in identifying disease alleles
(an example – cystic fibrosis)
Majority of human diseases have polygenic or multifactorial risk factors.
Challenges and approaches in the post genomic era
Example of identifying a disease allele by
positional cloning
Cystic fibrosis
Pathology
‘Woe to that child which when kissed on the forehead tastes salty. He is
bewitched and soon must die’
Example of identifying a disease allele by positional cloning
Cystic fibrosis
Severe autosomal recessive condition among Caucasians.
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).
cystic fibrosis caused by
mutations in the CF gene
CF gene encodes a cystic fibrosis transmembrane
conductance regulator (CFTR Cl- channel)
CFTR is a Cl- channel (defects result in either a
decrease in its Cl- transport capacity or its level of
cell surface expression)
 epithelial Cltransport Cltransport 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)
CFTR function
http://www.infobiogen.fr/services/chromcancer/IntroItems/Images/CFTREnglFig2.jpg
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
Mutations in CFTR
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 D7S8
extensive mapping and characterisation around the candidate region
by chromosome walking, chromosome jumping and microdissection
(~300kbp cloned)
CFTR candidate region
Mapping of CFTR
2 new markers identified – KM19 and XV2c – which showed
strong linkage disequilibrium
5’ end of gene located
Bovine equivalent of candidate gene isolated from genomic
library
7 cDNA libraries screened with human clone. 1 cDNA clone
identified. Northern blots show 6.5 kb mRNA
Rest of the gene obtained by screening and PCR
1989 CFTR gene eventually isolated by mutation screening
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 fibrosis
Marker alleles
X1,K1
X1,K2
X2,K1
X2,K2
CF
chromosomes
3
147
8
8
Normal
chromosomes
49
19
70
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.
Letter to Dr. Collins. Courtesy of the
National Human Genome Research Institute
“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)
Challenges
Some of the complexities of
human disease traits
•
•
•
•
•
Phenotypic heterogeneity
Phenocopies
Variable expressivity
Incomplete penetrance
Polygenic traits
Phenotypic heterogeneity
Same genotypic mutation causes variable
phenotypes
e.g. a/b-thalassemias
– Caused by mutations in either the a or b-globin genes.
– Similar genotype can lead to unaffected or severe
phenotypes
GENOTYPE
a+ a+ a+ a+
a+ a
a+ a+
a+ a
a+ a
a+ a+ a a
a+ a
aa
aa
aa
PHENOTYPE
Normal
Silent carrier
a-thalassaemia trait
asymptomatic condition. a thalassaemia - 2
minor anaemic conditions
HbH
Hydrops foetalis
mild – moderate anaemia
foetus survives until around birth
Many mechanisms contribute to the
phenotypic heterogeneity of thalassaemias
Phenocopy
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
Variable expressivity
Expression of a mutant trait differs in individuals
Incomplete penetrance
– when a mutant genotype does not always cause a
mutant phenotype
• 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
Polygenic traits
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
Alzheimer’s disease
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 similar
etiology of sporadic forms unknown
familial AD – mutations in APP, presenilin-1 and 2
Sporadic AD – strong association with APOe4, Apolipoprotein e4,
which affects age of onset rather than susceptibility
3 major alleles (APO E2, E3, and E4)
Position
112
158
ApoE2
Cys
Cys
ApoE3
Arg
Cys
Sudden
cardiac
death
(SCD)
ApoE4
Arg
Arg
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
What approaches should be used in the
post-genomic era?
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)
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
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 modestrisk 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
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
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)