Download The spectrum of human diseases

Molecular medicine - 2
Example of identifying a monogenic
condition by positional cloning
cystic fibrosis caused by mutations in the
CF gene
Most common severe autosomal recessive condition
among Caucasians.
About 5% of white Caucasians of European descent
are asymptomatic carriers.
Frequency of 1 / 2,500 affecting approximately
30,000 people
In 1985, CF locus was localized on the long arm of
chromosome 7
In 1989, the gene implicated in CF was isolated
(Kerem 1989; Riordan 1989; Rommens 1989).
Pathology
Woe to that child which
when kissed on the
forehead tastes salty. He
is bewitched and soon
must die
CF gene encodes a cystic fibrosis
transmembrane conductance regulator
CFTR is a Cl- channel
(defects result in either a
decrease in its Cltransport 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
F508
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
Mapping of CFTR
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 nonrandom 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)
Letter to Dr. Collins. Courtesy of the
National Human Genome Research Institute
The spectrum of human diseases
Cystic fibrosis
thalassemia
<5%
cancer
Huntington’s
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 Assocciation
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
Direct versus indirect association analysis.
a, In direct association analysis,all functional variants (red arrows) are catalogued and tested for association with disease. A
GeneSNPs image of the CSF2 gene is shown. Genomic features are shown as boxes along the horizontal axis (for example,
blue boxes indicate exons). Polymorphisms are shown as vertical bars below the axis, with the length of the line indicating allele
frequency and colour indicating context (for example, red indicates coding SNPs that change amino acids).
b, For indirect association analysis, all common SNPs are tested for function by assaying a subset of tagSNPs in each gene
(yellow arrows), such that all unassayed SNPs (green arrows) are correlated with one or more tagSNPs. Effects at unassayed
SNPs (green arrows) would be detected through linkage disequilibrium with tagSNPs. Images adapted from GeneSNPs
(http://www.genome.utah.edu/genesnps).
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
Ancient disease loci are
associated with haplotypes
• Start with population genetically isolated for a long
time such as Icelanders or Amish
• Collect DNA samples from subgroup with disease
• Also collect from equal number of people without
disease
• Genotype each individual in subgroups for
haplotypes throughout entire genome
• Look for association between haplotype and disease
phenotype
• Association represents linkage disequilibrium
• If successful, provides high resolution to narrow
parts of chromosomes
Series of closely linked mutations accumulate over time in
the surviving generation derived from a common ancestor
Haplotype analysis provides high
resolution gene mapping
Why is it still so difficult?
Genetic heterogeneity
Mutations at more than one locus cause same
phenotype
e.g. thalassemias
– Caused by mutations in either
the a or b-globin genes.
– Linkage analysis studies
therefore always combine data
from multiple families
Variable expressivity - Expression of a mutant
trait differs from person to person
• Phenocopy
– Disease phenotype is not caused by any
inherited predisposing mutation
– e.g. BRCA1 mutations
• 33% of women who do not carry BRCA1 mutation
develop breast cancer by age 55
Incomplete penetrance
– when a mutant genotype does not
always cause a mutant phenotype
• No environmental factor associated with
likelihood of breast cancer
• 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
– Solution – exclude all nondisease individuals form
analysis
– Requires many more families for study
• Polygenic inheritance
– Two or more genes interact in the expression of
phenotype
• QTLs, or quantitative trait loci
– Unlimited number of transmission patterns for
QTLs
» Discrete traits – penetrance may increase with
number of mutant loci
» Expressivity may vary with number of loci
– Many other factors complicate analysis
» Some mutant genes may have large effect
» Mutations at some loci may be recessive while
others are dominant or codominant
Polygenic inheritance
E.g heart attack or high cholesterol levels
Sudden cardiac death (SCD)
Breast cancer
Common condition – familial or sporadic forms
Although a genetic basis for familial BC identified, the
causes of sporadic disease still unknown
Mutations in 2 loci account for 20-25% of early onset
(<45 years) breast cancer cases due to inherited
factors
– BRCA1: mutations found in 80-90% of families
with both breast and ovarian cancer
– BRCA2: mutations mainly in male breast cancer
familiesSudden cardiac death (SCD)
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
Aetiology 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
Epigenetics – differential imprinting
Prader-Willi syndrome
failure to thrive during infancy,
hyperphagia and obesity during
early childhood, mental retardation,
and behavioural problems
molecular defect involves a ~2 Mb
imprinted domain at 15q11–q13 that
contains both paternally and
maternally expressed genes
Angelman syndrome
births and characteristics include
mental retardation, speech
impairment and behavioural
abnormalities
AS defect lies within the imprinted
domain at 15q11–q13
Genetic causes
Prader-Willi syndrome
Angelman syndrome
70% have a deletion of the
PWS/AS region on their
paternal chromosome 15
70% have a deletion of the PWS/AS
region on their maternal
chromosome 15
25% have maternal
uniparental disomy for
chromosome 15 (the
individual inherited both
chromosomes from the
mother, and none from the
father)
7% have paternal uniparental
disomy for chromosome 15 (the
individual inherited both
chromosomes from the father, and
none from the mother)
5% have an imprinting
defect
<1% have a chromosome
abnormality including the
PWS/AS region
3% have an imprinting defect
11% have a mutation in UBE3A
1% have a chromosome
rearrangement
11% have a unknown genetic cause
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)
•
Mapping complex disease loci in whole genome
studies by CS Carlson et al pp446-452