Download Finding Disease Genes

Finding Disease Genes
Intro
• “A more accurate though less snappy title ... would be ‘Identifying
genetic determinants of human phenotypes’.”
• The problem is, we don’t 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 don’t have the disease.
• complicating factors:
– lack of penetrance (people with the mutant genotype who don’t express
it).
– Phenocopies: people with mutant phenotype but normal genotype
– More than one gene giving the same phenotype
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.
Position-Independent Methods
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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, Xlinked.
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This clotting factor was purified from
pig (as a protein) long ago, and its Nterminal 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.
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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.
Antibody Methods
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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.
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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.
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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.
Phenylketonuria
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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 β-2-thienylalanine, a
growth inhibitor that mimics phenylalanine. This
inhibitor’s action is overcome by large amounts of
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.
Position-Dependent Methods
1. 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.
2. Then get cloned DNA from the entire region and map all of the genes
(transcribed regions). Zoo blots, exon trapping, computer searches.
3. 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.
4. Also, an 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.
5. 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.
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also useful is knocking out the homologous gene in mice and generating a
phenotype that mimics the human disease.
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.
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 Marfan’s patients with mutations in this gene.
Fibrillin Gene
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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 crosslinking each domain.
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There is a closely related gene, FBN2,
which has mutations giving a “marfanoid”
syndrome.
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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.
Chronic Granulomatous Disease
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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.
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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, known by his initials
D.D., with Xp21 deletion: had CGD,
Duchenne Muscular 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 to clone
the genes for DMD and retinitis
pigmentosum
More CGD
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had tissue culture cells that expressed the defect.
Used subtractive hybridization:
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Southern blot with this cDNA (cloned) with DNA from
region (on a YAC).: clone pERT379 hybridized
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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.
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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.
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 is complicated and
tricky.
Duchenne Muscular Dystrophy
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DMD is a progressive muscular
disease characterized by
pseudohypertrophy of the calves:
they swell up but it’s 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.
Cloning the DMD Gene
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A heroic effort in the mid 1980’s. 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.
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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.
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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
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 (<1% of gene!)
More DMD
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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
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similar to cytoskeletal proteins alphaactinins and spectrin
located near membrane
anchors cytoskeleton to surface
membrane
absence possibly makes cells more
susceptible 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 retriever dogs have a similar
syndrome.
Alternative DMD Cloning
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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.
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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 person’s cells with mouse cells. Selective loss of
human chromosomes occurs.
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the normal X in each of these people was inactivated in all cells for unknown reasons.
Look for a line in which all human chromosomes other than the t(X;21) 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.
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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).
Cystic Fibrosis
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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
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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.
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causative agent of cholera: Vibrio cholerae, water-borne
bacteria
Cholera was originally endemic to India, but starting in the
early 1800’s a series of cholera pandemics began. Many
people of the Oregon Trail in the 1860’s and 1870’s died of
cholera. Edgar Allen Poe’s 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, don’t drink any more infected
water.
Cloning the CF Gene
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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.
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Need to clone the region between these genes
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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.
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.
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.
Waardenburg
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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.
Branchio-oto-renal syndrome
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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.
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Found 7 out of 42 unrelated patients with defects in
this gene.
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highly conserved in animal evolution, but vertebrate and
invertebrate.
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.