molecular medicine 2 cystic fibrosis and some other examples
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Molecular medicine 2 CYSTIC FIBROSIS and some other examples. Cystic fibrosis represents the first genetic disorder elucidated strictly by positional cloning. - PowerPoint PPT PresentationTRANSCRIPT
Molecular medicine 2
CYSTIC FIBROSIS and some other examples
Cystic fibrosis represents the first genetic disorder elucidated strictly by positional
cloning 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 descent are asymptomatic carriers.
Frequency of 1 / 2,500 affecting approximately 30,000 people
Pathology “Woe to that child which when kissed on the forehead tastes salty. He is bewitched and
soon must die”
Major symptomsdue to dysfunction of exocrine glandssweat glands secrete excessive Na & Clpancreatic ducts become blocked with
thickened mucus → pancreatic insufficiency
lungs produce a thickened mucus
All of which give rise to:• Severe malabsorption• steatorrhoea• recurrent chest infections• sterility in males due to congenital
bilateral aplasia of the vas deferens (CBAVD)
CF gene encodes a Cl- channel called CFTR (cystic fibrosis transmembrane conductance regulator)
member of ATP binding cassette (ABC) membrane transporter superfamily
2 homologous halves -1480 amino acids longeach half has 6 transmembrane domains (M1-12) &1 nucleotide binding domain (NBD) which are linked bya cytoplasmic regulatory domain (R-domain) that contains
phosphorylation sites
CFTR channel Minimum channel diameter
– 5.3AMaximum channel diameter
- 10-13ACharge 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 located.
Channel lining is formed by M1, M3, M6 & M12 segments.
J Biol Chem (2000) vol 275 No 6 pp 3729 by MH Akabas
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)
Regulation of CFTR gating
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.
2 processes control Cl- movement
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 D7S8extensive 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 disequilibrium5’ end of gene located (undermethylated CpG islands was 1 tip-off)Bovine 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
Letter to Dr. Collins. Courtesy of the National Human Genome Research Institute
Examples of diseases identified by
• Direct identification by chromosome abnormality
• Pure transcript mapping– Treacher collins Franschetti Syndrome
• Large scale sequencing and homolog search
Pure transcript mapping Treacher Collins Franschetti
Syndrome• Craniofacial development disorder• autosomal dominant disorder caused by mutations in
‘treacle’ gene TCOF1• PATHOGENESIS
• 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.