Molecular medicine 2 CYSTIC FIBROSIS and some other examples

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Molecular medicine 2CYSTIC FIBROSIS and some
other examples
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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

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Pathology
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 Cl pancreatic 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)

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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 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
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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 located. Channel
lining is formed by M1, M3, M6 M12 segments.
J Biol Chem (2000) vol 275 No 6 pp 3729 by MH
Akabas
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CFTR function

• 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)

http//www.infobiogen.fr/services/chromcancer/Intr
oItems/Images/CFTREnglFig2.jpg
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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.
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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).
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CF from gene to product
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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.

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Genotype- phenotype correlations
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Spectrum of CF mutations that affect function
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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
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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).

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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 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

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Letter to Dr. Collins. Courtesy of the National
Human Genome Research Institute
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Examples of diseases identified by

• Direct identification by chromosome abnormality
• Pure transcript mapping
• Treacher collins Franschetti Syndrome
• Large scale sequencing and homolog search

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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.