Zoo blots, exon trapping, computer searches. ... originat

1
Finding Disease Genes
2
Intro

• A more accurate though less snappy title ...
  would be Identifying genetic determinants of
  human phenotypes.
• The problem is, we dont have genes for genetic
  diseases, instead we have genes that produce
  mutant phenotypes when they are mutated in
  specific ways.
• Frequently (usually?) it is not at all obvious
  why mutating a particular gene results in the
  disease phenotype that is observed.
• That being said, the usual method for finding
  disease genes is to collect a number of candidate
  genes and then screen them carefully for the
  presence of mutations in people with the disease
  and the absence of mutations in people who dont
  have the disease.
• complicating factors
• lack of penetrance (people with the mutant
  genotype who dont express it).
• Phenocopies people with mutant phenotype but
  normal genotype
• More than one gene giving the same phenotype

3
General Methods

• Position-independent methods not knowing
  anything about the location of the gene. This
  was once called forward genetics, starting at
  the phenotype, determining which protein was
  involved and then getting to the gene through the
  protein.
• Position-dependent methods starting from the
  approximate location of the gene, to finding the
  gene itself, then translating it to learn about
  the protein and its function. Also called
  positional cloning or reverse genetics. This
  is the method most used today.

4
Position-Independent Methods

• Mostly means identifying and isolating the
  protein product of the gene. Such genes usually
  produce large amounts of well-known and studied
  proteins.
• Gene-specific oligonucleotides hemophilia A
  Factor VIII gene. The most common form of
  hemophilia, X-linked.
• This clotting factor was purified from pig (as a
  protein) long ago, and its N-terminal amino acids
  were sequenced.
• This allowed a group of oligonucleotides to be
  synthesized to match.
• Dealing with degenerate codons too many
  different oligos in a hybridization means very
  low signal.
• use the codons found most frequently in human
  genes for these
• also use a region of peptide that minimizes
  degeneracy.
• These probes were used with colony hybridization
  against a cDNA library.
• Positive clones were re-screened with the
  secondary probe.

5
Antibody Methods

• Antibodies against a known protein. Inject
  purified protein into rabbits (or mice) collect
  the serum from blood Antibodies can be labeled
  and will bind to the protein expressed by cDNA
  cloned into expression vector. A library of
  expressed cDNAs is then screened using the
  antibody.
• Older approach was to use the antibody to isolate
  poly-ribosomes along with mRNA and the growing
  peptide of interest, then clone the mRNA. This
  technique has only worked in a handful of cases.
  The earliest and best known human case is
  phenylketonuria (PKU). PKU is caused by the
  inability to metabolize phenylalanine due to a
  lack of phenylalanine hydroxylase (PAH), which
  converts phenylalanine to tyrosine, leading to
  severe retardation.
• In this case, the original work was done in rats
  phenylalanine hydroxylase (PAH) was purified from
  rat liver, and then antibodies were raised to it.
  Polysomes isolated from rat liver were then
  treated with the antibody, and about 10 of the
  mRNA that precipitated gave PAH when translated
  in vitro. Individual clones were used to
  hybridize to rat liver mRNAs, and a clone that
  hybridized to mRNA that could be translated into
  PAH was selected. This clone was then used to
  find a human PAH cDNA clone from a library.

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Phenylketonuria

• The problem is a buildup of phenylpyruvic acid.
  This compound inhibits pyruvate decarboxylase, an
  essential part of the enzyme complex that
  converts pyruvate into acetyl coenzyme A
  (transition from glycolysis to the Krebs cycle).
  In the brain this is thought to inhibit
  myelination of the nerve fibers, leading to
  retardation.
• Simple treatment low phenylalanine diet. People
  used to be taken off the diet at age 8 or so, but
  disease symptoms sometimes then appear, and
  current recommendations are to remain on the diet
  for life. Homozygous mothers can induce
  retardation in fetus.
• Distribution is worldwide, but common in northern
  Europe, where many different mutant alleles are
  found. Possible heterozygote advantage
  resistance to a fungal toxin that induces
  abortions (?).
• Guthrie test place a drop of blood on a filter
  disk along with Bacillus subtilis bacteria and
  ß-2-thienylalanine, an inhibitor whose action is
  overcome by phenylalanine. Elevated Phe levels
  in the blood cause bacterial growth, which is
  obvious to the naked eye after overnight growth
  on nutrient agar. This test is required in most
  states because PKU has drastic consequences but
  is very easy to treat if caught at birth.

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Position-Dependent Methods

• use recombination mapping and/or somatic cell
  mapping to defined the region of interest as
  tightly as possible. This also helps remove
  phenocopies and people with mutations in other
  genes with similar phenotypes.
• Then get cloned DNA from the entire region and
  map all of the genes (transcribed regions). Zoo
  blots, exon trapping, computer searches.
• Then, look at each gene for mutations in people
  with the disease but not in close relatives.
  Especially helpful are chromosome structural
  variations associated with the phenotype they
  are very easy to detect without having to do a
  lot of sequencing.
• Also, and appropriate pattern of expression
  helps, or a gene that makes sense. Sorting
  through a limited number of candidate genes leads
  to lots of speculation and theories of the day
  about how the mutant phenotype might arise.
• The best final proof restoration of the normal
  phenotype. Transfer the wild type gene to mice
  that show the homologous phenotype and cure it.
  Or tissue culture cells.
• also useful is knocking out the homologous gene
  in mice and generating a phenotype that mimics
  the human disease.

8
Marfan Syndrome

• symptoms
• skeletal long limbs, caved-in chest, spindly
  fingers, long narrow feet, scoliosis (curvature
  of the spine), loose joints
• eye defects nearsighted, lens often misplaced.
• Blood vessels Aortic dissection and rupture,
  sometimes leading to sudden death.
• Abraham Lincoln? Osama bin-Laden?
• Dominant with variable expressivity no clear
  cases of a homozygote known.

9
Cloning Marfan Gene

• Cloning this gene was matter of connecting a
  mapped location with a candidate gene that made
  lots of sense.
• The disease mapped to 15q in pedigrees. Markers
  D15S25 and D15S1 had a lod score 12.1 at theta
  0.00.
• Also nearby on 15q was the fibrillin gene, which
  had previously been cloned as a cDNA from
  connective tissue.
• Fibrillin is a connective tissue protein that
  makes up part of microfibrils, which are elastic
  fibers.
• found RFLPS (a Taq 1 polymorphism) to fibrillin
  with no crossovers between it and MF disease
  gene.
• Also, in situ hybridization using a cDNA probe.
• Also, several Marfans patients with mutations in
  this gene.

10
Fibrillin Gene

• The FBN1 gene is expressed in lens of eye, bones,
  blood vessels.
• There are a large number of different mutations,
  widely distributed throughout the gene, many of
  which are spontaneous, and most of which are
  "private"--only found in one family.
• The gene is about 200 kb long (big), with 65
  exons, coding for 2871 amino acids.
• The protein consists mainly of 43 repeats of a
  calcium-binding domain that resembles epidermal
  growth factor. There are several
  cysteine-cysteine disulfide bridges cross-linking
  each domain.
• There is a closely related gene, FBN2, which has
  mutations giving a marfanoid syndrome.
• Dominant negative phenotype Fibrillin forms
  multi-subunit fibers. In a heterozygote, subunits
  from the good gene combine with subunits from the
  mutated gene, producing a defective fiber.

11
Chronic Granulomatous Disease

• The first successful case of positional cloning
  (1986)
• The phenotype phagocytic cells (macrophages,
  neutrophils, eosinophils) don't work, leading to
  chronic infections (granulomas) at infection
  sites.
• Cells can't make superoxide, which is necessary
  for killing action.
• 2/3 are X-linked, 1/3 autosomal, with a total of
  4 genes giving the same clinical disease.
• Mapped to Xp21 by linkage
• Found a boy with Xp21 deletion had CGD,
  Duchenne Mucular Dystrophy, retinitis
  pigmentosum, other problems.
• Already had this region cloned for DMD (which we
  will talk about next)--which clone has CGD gene?
• This same boy was also used o clone the genes for
  DMD and retinitis pigmentosum

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More CGD

• had tissue culture cells that expressed the
  defect. Used subtractive hybridization
• RNA from mutant cells with cDNA from normal
  cells.
• Then remove all double stranded RNA-DNA hybrids.
• The remaining DNA was highly enriched for cDNA
  from genes expressed in those cells from deletion
  region.
• Southern blot with this cDNA (cloned) with DNA
  from region (on a YAC). clone pERT379 hybridized
• So, pERT379 is cDNA expressed in a normal tissue
  culture line but not in a line from a CGD
  patient, and it is found in the appropriate area
  of the genome.
• Probe a Northern blot with various tissue RNAs.
  Found 5 kb mRNA in RNA from the normal tissue
  culture line and from leukocytes, but not
  fibroblasts, liver or kidney. A good
  distribution.
• Examine CGD patients gene altered or missing in
  4 patients.
• Sequence 486 amino acids, with glycosylation
  sites consistent with membrane-associated
  protein.
• Not homologous to any known protein, but part of
  mitochondrial cytochrome b complex (consistent
  with superoxide generation and previous
  biochemistry).
• Absent in CGD cells but present in normal cells.
  
• In general this subtractive hybridization method
  has only rarely been successful. It si
  complicated and tricky.

13
Duchenne Muscular Dystrophy

• DMD is a progressive muscular disease
  characterized by pseudohypertrophy of the calves
  they swell up but its due to tissue damage and
  not to a buildup of muscle tissue.
• Then progressive skeletal weakness, including a
  waddling gait in walking, and lordosis, a
  forward curvature of the spine.
• Most patients in a wheelchair by age 12, with
  progressive weakness of all muscles.
• Death usually occurs by respiratory failure by 20
  or so. Sometimes death due to weak heart muscles
  or pneumonia due to lack of ability to cough.

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Cloning the DMD Gene

• A heroic effort in the mid 1980s. Two different
  groups succeeded, using different methodologies.
• Kunkel cloning. Starts with the boy with Xp21
  deficiency mentioned above.
• Subtractive DNA hybridization 500x excess of
  DNA from the deletion patient (randomly sheared)
  was melted then mixed with single stranded DNA
  from a normal person that had been cut with the
  restriction enzyme Mbo I.
• The basic idea is that Mbo I leaves a 4 base
  overhang that matches the four base overhang left
  by Bam H1.
• DNA that has one or both strands from the
  randomly sheared DNA will not clone into a Bam
  site.
• Since there is so little DNA with Mbo ends, most
  of it will hybridize with the randomly sheared
  ends.
• However, in the region of the deletion, there is
  no DNA that is randomly sheared, since that came
  from the deletion patient. So, the only DNA
  that can hybridize to from Mbo ends is from
  normal person in the region of the deletion
• Got a series of clones covering the deletion.
• hybridize each clone to Southern blots with DNA
  from the deletion patient and a people with
  various numbers of X chromosomes of X's XY, XX,
  XXX.
• The relevant clones are in the deletion region
  on the X, and so they hybridize appropriately.
• test clones against Southern blot of DMD
  patients. Found pERT 787 was deleted in 5
  patients.
• clone 200 kb around it by chromosome walking.
• probe zoo blot with small pieces of this 200 kb
  to find exons (lt1 of gene!)

15
More DMD

• use exon pieces to identify a 14 kb mRNA (quite
  large 2.2 Mbp for the gene) . Total of 79
  exons. Takes 16 hours to transcribe.
• computer translate to a 4000 AA protein
  dystrophin
• similar to cytoskeletal proteins alpha-actinins
  and spectrin
• located near membrane
• anchors cytoskeleton to surface membrane
• absence possibly makes cells more suseptible to
  tearing?
• Becker MD, a milder form, occurs with milder
  alleles of the same gene several other forms of
  MD as well.
• Many new mutations--because males with it rarely
  survive to reproduce.
• Golden retreiver dogs have a similar syndrome.

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Alternative DMD Cloning

• Worton cloning. Started with 7 girls who had DMD
  but were not homozygous their fathers didn't
  have it. All had translocations with Xp21
  breakpoints. Since Xp21 is the mapped site of the
  DMD gene, these girls probably had the DMD gene
  disrupted by the chromosome break point.
• the normal X in each of these people was
  inactivated in all cells for unknown reasons.
• one patient had a translocation with chromosome
  21 in the ribosomal DNA region. Use cloned
  ribosomal RNA probe to find the junction
  fragment a restriction fragment that spanned the
  chromosome breakpoint, that started in the
  chromosome 21 ribosomal RNA genes and ended in
  the X chromosome DMD gene.
• Somatic cell genetics fuse this persons cells
  with mouse cells. Selective loss of human
  chromosomes occurs.
• Look for a line in which all human chromosomes
  other than the t(X21) have been lost.
• The only human ribosomal RNA genes in this line
  are in the translocation normal human cells have
  ribosomal RNA genes on 4 other chromosomes.
• Searched for abnormal restriction fragment from
  breakpoint region with rDNA probe.
• found a 12 kb Bam fragment where a 5 kb fragment
  was expected.
• It had rDNA on one side and part of DMD gene on
  other side.
• Use DMD side to clone rest of DMD gene.
• Linkage analysis showed that this fragment mapped
  close to the expected location.
• The cloned fragment of the DMD gene was missing
  in 6 out of 107 DMD patients (who presumably had
  deletions for this portion of the gene).

17
Cystic Fibrosis

• The most common fatal genetic disease in the US
  today. It is an autosomal recessive, and it is
  found mostly in people of Northern European
  background.
• Originally called "cystic fibrosis of the
  pancreas
• Affects multiple organs, all of which are
  involved with secretion. Mucus is very thick and
  sticky. Variable symptoms
• lungs thick mucus harbors microorganisms, often
  leading to pneumonia. Also asthma, bronchitis,
  and other lung problems.
• pancreas decreased secretion of digestive
  enzymes lead to malnutrition, intestinal
  blockage, and other digestive problems
• male infertility
• abnormally salty sweat often detectable in
  newborns. A sweat test is the standard method of
  diagnosis for CF.
• Therapy with antibiotics and clearing of lungs
  postural drainage and back slapping. Mucus
  thinning by DNase part of the thickness of mucus
  is due to DNA. High calorie diet supplemental
  pancreatic enzymes.
• Average life span used to be less than 2 years.
  Today people with CF often live into their 20's
  or 30's.
• Possibility of heterozygote advantage resistance
  to cholera and other diarrheal diseases.
  Heterozyogtes secrete less water and chloride in
  response to cholera toxin.
• causative agent of cholera Vibrio cholerae,
  water-borne bacteria
• Cholera was originally endemic to India, but
  starting in the early 1800s a series of cholera
  pandemics began. Many people of the Oregon Trail
  in the 1860s and 1870s died of cholera. Edgar
  Allen Poes story Masque of the Red Death is
  allegedly about a cholera epidemic. Also, Love
  in the Time of Cholera by Gabriel Garcia Marquez.
• Cholera toxin works by opening ion channels in
  the intestine. Ions are released by the cells,
  followed by water. Death is usually from
  dehydration or ion imbalance. Treatment is
  simple keep the patient hydrated and also give
  some salt and sugar. And of course, dont drink
  any more infected water.

18
Cloning the CF Gene

• underlying cause of CF "There are enough
  artifacts in the literature on CF to provide
  material for a PhD thesis on the psychology of
  scientific folly.
• Biochemical work done before the gene was cloned
  pointed to salty skin as a primary clue. Problem
  in chloride ion transport. Chloride can't get out
  of the cell, so water stays in to dilute
  it--can't come out to dilute the mucus.
• Work on cloning the gene started with a search in
  lots of families with many RFLP probes.
• finally found one 15 cM from CF gene on
  chromosome 7. More work led to tightly linked
  markers D7S8 and met (oncogene) flanking it--2
  Mbp apart.(as as later realized)
• no known deletion or rearrangement causes CF.
• Need to clone the region between these genes
• walking by cosmids over 500 kbp
• problem with uncloneable repeated sequences
  needed to jump over them. Cut genomic DNA into
  large fragments, circularize it and clone the
  junction fragments.
• did a zoo blot and found 3 genes conserved.
• RNA expression probed with a Northern blot.. 2
  genes definitely wrong, the third at first was
  not seen in the Northerns, but finally found in
  cDNA isolated from sweat gland cells.
• 113 bp overlap between end of walk and the mRNA!
  Almost didn't walk far enough.
• cloned the rest. total gene 250 kbp, 24 exons,
  1480 AA (long)
• sequence showed trans-membrane protein, a
  chloride ion channel also regulates other ion
  channels.
• Gene named CFTR (cystic fibrosis transmembrane
  conductance regulator)
• 70 of mutants had 3 bp deletion of
  .phenylalanine at position 508, part of the ATP
  binding site.
• Other mutants are very heterogeneous
• Putting the cloned gene (cDNA) into tissue
  culture cells from a CF patient cells restored
  chloride ion transport.

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Waardenburg syndrome

• Waardenburg syndrome. There are several types we
  are discussing type 1 here.
• Different-colored eyes, white forelock, white
  skin patches, deafness.
• Due to partial absence of melanocytes.
  Melanocytes are neural crest cells they
  originate near the developing neural tube and
  migrate laterally down the flanks of the body in
  the embryo.
• Autosomal dominant, but varies in expressivity.

20
Waardenburg

• Critical factor synteny with a mouse gene.
• Mapped is distal 2q, near the gene for placental
  alkaline phosphatase, which had previously been
  assigned to 2q37 the peak lod score was 4.76 at
  a recombination fraction of 0.023.
• A mutation in mice, Splotch, also shows white
  patches and deafness, and maps to a syntenic
  region on mouse chromosome 1.
• Also, the PAX3 gene, mapped as a transcribed DNA
  sequence, was in the proper area in mice. PAX3
  is a transcription factor active in the neural
  crest.
• An unmapped human gene HuP2 showed strong
  sequence identity to PAX3.
• Examine DNA from Waardenburg patients using the
  HuP2 probe 6 out of 17 unrelated patients had
  altered DNA, and 0 out of 50 normal controls had
  altered DNA.
• Since the human HuP2 gene was very similar to the
  mouse gene, it was renamed PAX3.
• The PAX3 protein binds to the promoter DNA for
  the MITF transcription factor and stimulates
  transcription. In turn, the MITF protein
  activates the tyrosinase gene, a critical step in
  the development of melanin pigment. Absence of
  melanin production causes melanocytes to fail to
  completely differentiate.
• Mutations in the MITF gene result in type 2
  Waardenburg syndrome.

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Branchio-oto-renal syndrome

• Critical factor Homology with lower organisms.
• Phenotype Deaf, oddly shaped ears, kidney
  problems (small or missing kidneys), cleft
  palate, cysts on neck.
• Mapped to 8q13. Sequenced 600 kb of DNA, based on
  a patient with a deletion in this region.
• During the sequencing a region was found
  homologous to Drosophila eyes absent (eya) gene
  69 identical and 88 similar amino acids.
• highly conserved in animal evolution, but
  vertebrate and invertebrate.
• Found 7 out of 42 unrelated patients with defects
  in this gene.
• A few patients with eye defects have also been
  found.
• The EYA1 gene seems to be involved in early
  embryonic induction of kidney and ear buds
  similar process in Drosophila.
• The EYA1 protein in a protein tyrosine
  phosphatase it removes (and thus de-activates)
  phosphate groups on other proteins that were
  attached by receptor tyrosine kinases.
• Part of same pathway as PAX3--a regulatory
  cascade.