Example of identifying a monogenic condition by positional cloning

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Transcript Example of identifying a monogenic condition by positional cloning

Molecular medicine 2
CYSTIC FIBROSIS and some other
Cystic fibrosis represents the first genetic
disorder elucidated strictly by positional
Caused by mutations in the CFTR gene (cystic fibrosis
transmembrane conductance regulator) which functions
as a chloride channel and controls the regulation of
other transport pathways
Most common severe autosomal recessive
condition among Caucasians.
About 5% of white Caucasians of European
are asymptomatic carriers.
Frequency of 1 / 2,500 affecting approximately
30,000 people
“Woe to that child which when kissed on the
forehead tastes salty. He is bewitched and
soon must die”
Major symptoms
due to dysfunction of exocrine glands
sweat glands secrete excessive Na &
pancreatic ducts become blocked with
thickened mucus → pancreatic
lungs produce a thickened mucus
All of which give rise to:
Severe malabsorption
recurrent chest infections
sterility in males due to congenital
bilateral aplasia of the vas deferens
CF gene encodes a Cl- channel
called CFTR (cystic fibrosis
transmembrane conductance
member of ATP binding cassette (ABC) membrane transporter
2 homologous halves -1480 amino acids long
each half has 6 transmembrane domains (M1-12) &
1 nucleotide binding domain (NBD) which are linked by
a cytoplasmic regulatory domain (R-domain) that contains
phosphorylation sites
CFTR channel
Minimum channel diameter
– 5.3A
Maximum channel diameter
- 10-13A
Charge selectivity:
R352, cytoplasmic end of M6
Overall structure:
Channel with a large
extracellular vestibule which
narrows towards the
cytoplasmic end where the
anion selectivity filter is
Channel lining is formed by M1,
M3, M6 & M12 segments.
J Biol Chem (2000) vol 275 No 6 pp 3729 by MH Akabas
 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
Regulation of CFTR gating
2 processes control Cl- movement
phosphorylation: necessary to activate the channel. The R domain
contains phosphorylation sites for cAMP-dependent protein kinase
A (PKA), C (PKC) and type II cGMP dependent protein kinases.
CFTR deactivation mediated by phosphatases PP2C & PP2A.
ATP binding & hydrolysis:
Opening / closing of channel controlled by ATP binding & hydrolysis
which occurs in the NBD segment.
The R domain interacts with NBD & regulates their ATP affinity.
In 1985, CF locus was localized on the long arm of
chromosome 7q31.2
In 1989, the gene implicated in CF was isolated
(Kerem 1989; Riordan 1989; Rommens 1989).
CF from gene to product
CF gene encodes a cystic fibrosis
transmembrane conductance regulator
The genetic analysis showed that this gene,
which is responsible for this disorder, contains
24 exons spreading over 250 kb of
chromosome 7 (7q31) and encodes an mRNA
of 6.5 kb.
Genotype- phenotype correlations
Spectrum of CF mutations that affect function
70% of CF patients show a specific deletion F508
single amino acid (F) deletion in exon 10
encodes first portion of NBD-1
Leads to misfolding of CFTR in the
endoplasmic reticulum (ER).
Immature CFTR proteins are then
polyubiquinated & 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 (undermethylated CpG islands was 1 tip-off)
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
Letter to Dr. Collins. Courtesy of the
National Human Genome Research Institute
Examples of diseases identified by
• Direct identification by chromosome
• Pure transcript mapping
– Treacher collins Franschetti Syndrome
• Large scale sequencing and homolog search
Pure transcript mapping
Treacher Collins Franschetti
• Craniofacial development disorder
• autosomal dominant disorder caused by mutations in
‘treacle’ gene TCOF1
• Wise et al. (1997) postulated that the disorder results from
defects in a nucleolar trafficking protein that is critically
required during human craniofacial development. Marsh et
al. (1998) suggested that the disorder results from
aberrant expression of a nucleolar protein. They observed
that mutations in the TCOF1 gene (606847) cause
truncated proteins to be mislocalized within the cell.