Positional cloning of the Huntington

1
Positional cloning of the Huntingtons disease
(HD) gene
Mapping and cloning of the HD gene chromosome
walking cDNA libraries Identifying the
disease-causing mutations Studies of the HD
gene identifying orthologous proteins
(BLAST) mouse knockouts (KOs) transgenic
mice Summary of other repeat expansion diseases
2
Goals for the next three lectures
-Try to fill in some gaps -Strengthen the
connections between topics -Some new
information protein similarity (probably today
Monday) knockout mice (probably
Monday) population genetics (Monday?) -Next
Fridays lecture no more than 30 minutes of new
material course evaluations (15-20
minutes) review/problem solving/QS10
3
If I do spend time reviewing topics on Friday it
would be good to know what you need help
with -No more than 1-2 topics (1-2
sentences) -Send to pallanck_at_u.washington.edu -
Need to hear from you before Monday
Solutions to Problem set 6 have been posted on
the course website
Lastly If you feel that an error was made in the
grading of your 2nd midterm exam, send an email
message to Anne Paul summarizing the error, BY
THE END OF THE DAY TODAY.
4
Huntingtons Disease
(reminder from lecture 13)

• A dominant genetic disease affects 8 people
  per/100,000 worldwide
• Symptoms include abnormal body movements
  (chorea), cognitive decline, death
• Symptoms result from neurodegeneration
• Age of onset typically 40s ranges from infancy
  to elderly
• Genetic anticipation (increasing disease severity
  in subsequent generations) often observed
• No cure or treatment

5
Mapping of the Huntingtons disease gene
The informative pedigree
5,000 related individuals from Venezuela
segregating HD Included 100 members
currently affected by HD Included
gt1,000 members with gt25 risk
The markers used
Few markers available so tested random, purified
fragments of human genome Used these random
fragments as probes to conduct Southern blot
analysis to identify RFLPs
On their 12th probe the jackpot! - linkage of
the RFLP to HD!
1983
6
Marker D4S10 shows linkage to HD
40
Results of linkage studies using the probe G8
which recognizes the RFLP marker D4S10
30
20
10
LOD score (Z)
?
-10
-20
Does this result provide significant evidence of
linkage?
-30
-40
7
Where is marker D4S10 located?
Karyotype
?
HD gene 3cM away from D4S10
How to tell?
3x106bp
8
(No Transcript)
9
Narrowing of the HD region
Looking for highly informative recombinants
(haplotypes)
telomere
D4S141
D4S115
D4S111
Y1P18
HD ?
R10
D4S98
Where are the informative recombinants?
D4S43
D4S10
centromere
What Next?
10
Genetic and physical map of the HD region
D4S10
D4S98
HD ?
centromere
telomere
500kb
If 2008
D4S180 AACTGACTTAA
D4S182 CCTAGCTTAGAT
We could also find the genes in this interval
using the UCSC browser
But it was 1992
11
Genetic and physical map of the HD region
D4S10
D4S98
HD ?
centromere
telomere
500kb
D4S180 AACTGACTTAA
D4S182 CCTAGCTTAGAT
How was this done in 1992 (i.e., before the
genome was sequenced)?
12
Chromosome walking (outline)
Make radioactive probes from known sequence
13
Colony hybridization to find the first genomic
DNA clone
genomic DNA clones
X-ray film
14
Colony hybridization (contd)
The colonies you detect must have insert
sequences complementary to your D4S180 probe!
What next?

• Pick one of these clones
• Characterize it (restriction digest, etc.)
• Make a probe from one end of its insert
• Repeat colony hybridization

15
Chromosome walking finding the next clone
Pick one end of the insert PCR amplify the
region Label the PCR fragment with radioactive tag
Amp end
ori end
The goal find the colonies (clones) that
contain this sequence
? overlap your first clone
16
Colony hybridization (contd)
The colonies you detect in the hybridization
could have
- duplicates of your original plasmid - new
plasmids with different (but overlapping) inserts
How could you tell if they were the same as the
original?
Restriction digests or sequencing
17
Assembling a contig
Repeat the process until the clones obtained from
the flanking markers join
insert in original clone
probe
insert in original clone
18
From lecture 13
II. Map location on genome
STS sequence tagged site short, unique genomic
sequencenot present anywhere else in the genome
that can be detected by PCR ID tag for that
portion of genome
Test the BAC by PCR Does it test positive with
PCR primers for STS 24? Does it test positive
with PCR primers for STS62? etc.
Test positive? What does that mean?
19
Genetic and physical map of the HD region
D4S10
D4S98
HD ?
centromere
telomere
500kb
How do we identify the genes in a contig? Which
one is the HD gene?
20
Identifying genes in DNA sequence
Various approaches
...TTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATG
TCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGG... .
..AACTTCTGCTTTCCCGGAGCACTATGCGGATAAAAATATCCAATTACA
GTACTATTATTACCAAAGAATCTGCAGTCCACCGTGAAAAGCCC...
21
Making a cDNA library
cDNA complementary DNA
complementary to mRNA
Start with mRNA from a cell culture or
tissue Copy into DNA using reverse transcriptase
and poly-A tail
One mRNA out of the pool shown here
TTTTTTT-5
22
Genomic vs. cDNA libraries
cDNA library
make cDNA, insert into plasmid, etc.
only mRNA regions (exons) represented frequenc
y of clone proportional to amount of
transcription of the gene
23
Genetic and physical map of the HD region
D4S10
D4S98
HD ?
centromere
telomere
500kb
Which (if any) of these transcripts correspond to
the HD gene?
24
How was the HD gene identified?
Compared sequences from normal and HD individuals
Look for gene alterations specific to diseased
population
Focused on genes that are expressed in the
nervous system
Screened cDNA libraries prepared from normal
brain mRNA
Some potential complications -non-disease
causing (rare) polymorphisms distinguishing the
diseased and normal population -incomplete
penetrance -variable expressivity
-Influence of other genesmany traits
multigenic -Influence of environment -Observation
errors!
25
How was the HD gene identified?
HD ?
IT-15
IT-11
IT-10C3
ADDA
Gene 67 exons gt200 kb mRNA 10,366
bases Protein 3,144 aa 350kDa
26
How was the HD gene identified?
Triplet repeat number
normal
65
50
35
20
5
Further evidence that CAG repeat expansion
mutation is the cause of HD -Two HD patients
with a new mutation (not seen in parents) also
had a repeat expansion. -Length of repeat
correlated with onset and severity.
27
IT-15 is the HD gene (AKA Huntingtin)
HD ?
IT-15
IT-11
IT-10C3
ADDA
non-disease allele
Gene 67 exons gt200 kb mRNA 10,366
bases Protein 3,144 aa 350kDa
28
Why did it take so long to clone the HD gene?
1979-work begins to clone HD 1983-First marker
linked to HD (a lucky break) 1993-HD gene cloned
-There were very few markers for linkage studies
in humans -There were several inconsistencies in
the linkage data -The biology of HD was of
limited help in selecting candidate genes (60
of mRNAs transcribed in the brain) -It is not
easy to identify disease causing mutations!
"We applaud their discovery," adds another
contender, Michael Hayden of the University of
British Columbia, who found himself in the
painful position of having proposed a different
candidate HD gene in Nature the day before the
consortium published their proof-positive results
in Cell. -Virginia Morell (1993) Science 260,
28-30.
29
Repeat instability explains HD genetic
anticipation in HD
CAG repeats tend to expand upon paternal
transmission
30
Why are long CAG repeats unstable?
DNA polymerase
A molecular model
CAGCAGCAGCAGCAGCAG
5
GTCGTCGTCGTCGTCGTCGTC TCGTC
G
3
increases CAG repeat length by 1 CAG
decreases CAG repeat length by 1 CAG
31
Why are only long CAG repeats unstable?
Short repeats often also contain some CAA codons
CAGCAGCAACAGCAGCAGCAACAGCAA
5
3
GTCGTCGTTGTCGTCGTCGTTGTCGTC
3
5
32
How do mutations in Huntingtin cause disease?
HD is a dominant disorder Given what you know
about dominant mutations, provide possible
genetic explanations for the HD phenotype.
-Haploinsufficiency-half the amount of HD gene
product insufficient (like W)? -Dominant
negative-poison subunit (like rab27b)? -Expressed
in wrong place (like Antennapedia) or wrong time
(like lactase) -Protein with a new activity
(like the ABO blood antigens)?
33
How do mutations in Huntingtin cause disease?
HD is a dominant disorder Given what you know
about dominant mutations, provide possible
genetic explanations for the HD phenotype.
-Haploinsufficiency-half the amount of HD gene
product insufficient (like W)?
-Dominant negative-poison subunit (like
rab27b)? -Expressed in wrong place (like
Antennapedia) or wrong time (like
lactase) -Protein with a new activity (like the
ABO blood antigens)?
34
Wolf-Hirschhorn Syndrome (4p-) (The Human
Knockout of the Huntington Locus )

• Microdeletion (contiguous gene deletion)
  syndrome
• Growth retardation, with abnormal facies.
• Cardiac, renal, and genital abnormalities.
• Significantly, basal ganglia is intact no
  movement disorder

Rules out haploinsufficiency as cause of
Huntingtons disease
35
How do mutations in Huntingtin cause disease?
HD is a dominant disorder Given what you know
about dominant mutations, provide possible
genetic explanations for the HD phenotype.
-Haploinsufficiency-half the amount of HD gene
product insufficient (like W)? -Dominant
negative-poison subunit (like rab27b)? -Expressed
in wrong place (like Antennapedia) or wrong time
(like lactase) -Protein with a new activity
(like the ABO blood antigens)?
36
How do mutations in Huntingtin cause disease?
HD is a dominant disorder Given what you know
about dominant mutations, provide possible
genetic explanations for the HD phenotype.
-Haploinsufficiency-half the amount of HD gene
product insufficient (like W)? -Dominant
negative-poison subunit (like rab27b)?
-Expressed in wrong place (like
Antennapedia) or wrong time (like
lactase) -Protein with a new activity (like the
ABO blood antigens)?
37
Does CAG expansion act in a dominant-negative
fashion?
If the repeat expansion in HD acts in a
dominant-negative fashion, a homozygous LoF
mutation should be equivalent
But no homozygous LoF alleles of the HD gene have
been seen in humans!
Perhaps we can create mutations in the mouse HD
gene! But, how do we find the mouse HD gene?
38
Colony hybridization with a human HD probe
ultimately led to the identification of the mouse
HD gene
Human HD protein 3,144 aa Mouse HD protein 3,120
aa
The two proteins match at gt90 of their aas!
If the sequences are conserved, the biological
function is also likely to be conserved
If the biological function is conserved, we can
test whether a mouse bearing a homozygous HD lof
mutation resembles the human disease
Before continuing, lets diverge and consider how
this is done today-in some detail(BLAST) -But
we will focus on using BLAST to find similar
proteins (unlike what you did in QS)
39
Finding the mouse HD gene computationally
We need three things

1. A sequence database.
2. Some way of saying how similar two sequences are.
3. A really fast way of carrying out the similarity
   test.

We have the genome sequences and gene structures
already. Well diverge from HD for a bit and
talk about point 2 now. Point 3 is more
appropriate for a computer course. The method is
called BLAST (basic local alignment search tool).
You should be at least somewhat familiar with
this from QS9.
40
Thinking about protein similarity
Suppose we have the following aligned protein
sequences
PWAVTASCH VYAVQASPH
(human)
(something else)
We can see that both of the something else
sequences appear to be related to the human. But
related to what extent? We need to be
quantitative.
41
Amino acid structures
Hydrophobic
Polar
Charged
phenylalanine F
42
Amino acid frequency
amino acid one-letter frequency percent
alanine A 0.0768 7.68
cysteine C 0.0162 1.62
aspartate D 0.0526 5.26
glutamate E 0.0648 6.48
phenylalanine F 0.0409 4.09
gylcine G 0.0689 6.89
histidine H 0.0225 2.25
isoleucine I 0.0586 5.86
lysine K 0.0596 5.96
leucine L 0.0958 9.58
methionine M 0.0236 2.36
asparagine N 0.0435 4.35
proline P 0.0490 4.90
glutamine Q 0.0394 3.94
arginine R 0.0521 5.21
serine S 0.0700 7.00
threonine T 0.0558 5.58
valine V 0.0663 6.63
tryptophan W 0.0121 1.21
tyrosine Y 0.0315 3.15
    1.0000 100.00
Amino acid frequencies in the entire universe of
known protein sequences.
common
rare
43
log odds calculation
likelihood of seeing amino acid pair in related
protein
log
score
likelihood of seeing amino acid pair at random

• Related proteins taken from BLOCKS database
  (validated related proteins).
• Simply count up how often a particular amino
  acid pair is seen.
• Gives you the numerator likelihood above.

• Gives the denominator likelihood above.

44
Amino acid pair frequencies in related proteins
amino acid one-letter frequency percent
alanine A 0.0768 7.68
cysteine C 0.0162 1.62
aspartate D 0.0526 5.26
glutamate E 0.0648 6.48
phenylalanine F 0.0409 4.09
gylcine G 0.0689 6.89
histidine H 0.0225 2.25
isoleucine I 0.0586 5.86
lysine K 0.0596 5.96
leucine L 0.0958 9.58
methionine M 0.0236 2.36
asparagine N 0.0435 4.35
proline P 0.0490 4.90
glutamine Q 0.0394 3.94
arginine R 0.0521 5.21
serine S 0.0700 7.00
threonine T 0.0558 5.58
valine V 0.0663 6.63
tryptophan W 0.0121 1.21
tyrosine Y 0.0315 3.15
    1.0000 100.00
One block from BLOCKS database
CKS2_XENLAQ91879 NIYYSDKYTDEHFEY CKS1_HUMANP33
551 QIYYSDKYDDEEFEY CKS2_HUMANP33552
QIYYSDKYFDEHYEY CKS2_MOUSEP56390
QIYYSDKYFDEHYEY CKS1_PATVUP41384
QIYYSDKYFDEDFEY CKS1_DROMEQ24152
DIYYSDKYYDEQFEY CKS1_PHYPOP55933
TIQYSEKYYDDKFEY CKS1_LEIMEQ25330
KILYSDKYYDDMFEY O23249
QIQYSEKYFDDTFEY O60191
NIHYSTRYSDDTHEY CKS1_SCHPOP08463
QIHYSPRYADDEYEY CKS1_YEASTP20486
SIHYSPRYSDDNYEY CKS1_CAEELQ17868
DFYYSNKYEDDEFEY
D-D 21 pairs D-E 14 pairs D-P 14 pairs D-T
7 pairs D-N 7 pairs E-E 1 pair E-T
2 pairs E-P 4 pairs T-P 2 pairs T-T
1 pair T-N 1 pair
LOD calcul. (e.g., D-D pair)
One of 29,068 blocks - pair frequencies compiled
from all blocks combined.
log
45
log odds scores (side note)

• Traditionally, we use log base 2 (pedigree LOD
  scores are base 10).
• To make computing fast, scores are usually
  multiplied by 2 and then rounded to nearest
  integer (this is a detail).
• Called half-bit scores (jargon for taking
  twice log base 2).

46
log odds scores (cont.)
likelihood of seeing amino acid pair in related
protein
log
score
likelihood of seeing amino acid pair at random
If amino acid pair seen MORE often than expected
at random?

• odds gt 1, score positive

If amino acid pair seen LESS often than expected
at random?

• odds lt 1, score negative

47
Values from a score matrix (half-bit scores)
one-letter amino acid code
score for alanine (A) - tryptophan (W)
self match scores
48
Amino acid structures
Hydrophobic
Polar
Charged
phenylalanine F
49
Example - similar amino acids get positive scores
Qualitatively, what scores do you expect pairs of
these to have?
I-V
L-V
50
Example - dissimilar amino acids get negative
scores
Qualitatively, what scores do you expect pairs
among these groups to have?
hydrophobic
vs. charged
51
Thinking about protein similarity
Suppose we have the following aligned protein
sequences

• Related to what extent? We want to be
  quantitative.

(Side note - this also indicates the odds of
seeing a match of this quality by chance for the
entire sequence. e.g. bottom match is
. Remember they are half-bit
scores).
52
Getting back to HDfinding the mouse HD gene
A portion of human HD protein sequence (the
query sequence)
MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQPPPPPPPPPP
PQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPLHRPKKELSATKKD
RVNHCLTICENIVAQSVRNSPEFQKLLGIAMELFLLCSDDAESDVRMVAD
ECLNKVIKALMDSNLPRLQLELYKEIKKNGAPRSLRAALWRFAELAHLVR
PQKCRPYLVNLLPCLTRTSKRPEESVQ
summary list of all related proteins (one per
line)
53
Getting back to HDfinding the mouse HD gene
Looking further down on the summary list
Bit score
E value
54
Getting back to HDfinding the mouse HD gene
Portion of Mus musculus HD alignment
amino acid dissimilar
55
  A C D E F G H I K L M N P Q R S T V W Y
A 4 0 -2 1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2
C 0 9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2
D -2 -3 6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3
E 1 -4 2 5 -3 -2 0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2
F -2 -2 -3 -3 6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 3
G 0 -3 -1 -2 -3 6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3
H -2 -3 -1 0 -1 -2 8 -3 -1 -3 -2 1 -2 0 0 -1 -2 -3 -2 2
I -1 -1 -3 -3 0 -4 -3 4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1
K -1 -3 -1 1 -3 -2 -1 -3 5 -2 -1 0 -1 1 2 0 -1 2 -3 -2
L -1 -1 -4 -3 0 -4 -3 2 -2 4 2 -3 -3 -2 -2 -2 -1 1 -2 -1
M -1 -1 -3 -2 0 -3 -2 1 -1 2 5 -2 -2 0 -1 -1 -1 1 -1 -1
N -2 -3 1 0 -3 0 1 -3 0 -3 -2 6 -2 0 0 1 0 -3 -4 -2
P -1 -3 -1 -1 -4 -2 -2 -3 -1 -3 -2 -2 7 -1 -2 -1 -1 -2 -4 -3
Q -1 -3 0 2 -3 -2 0 -3 1 -2 0 0 -1 5 1 0 -1 -2 -2 -1
R -1 -3 -2 0 -3 -2 0 -3 2 -2 -1 0 -2 1 5 -1 -1 -3 -3 -2
S 1 -1 0 0 -2 0 -1 -2 0 -2 -1 1 -1 0 -1 4 1 -2 -3 -2
T 0 -1 -1 -1 -2 -2 -2 -1 -1 -1 -1 0 -1 -1 -1 1 5 0 -2 -2
V 0 -1 -3 -2 -1 -3 -3 3 2 1 1 -3 -2 -2 -3 -2 0 4 -3 -1
W -3 -2 -4 -3 1 -2 -2 -3 -3 -2 -1 -4 -4 -2 -3 -3 -2 -3 11 2
Y -2 -2 -3 -2 3 -3 2 -1 -2 -1 -1 -2 -3 -1 -2 -2 -2 -1 2 7
score (bits) is sum of each aligned residue (x
0.5 because the score table is in half-bits)
Query LTAVGGIGQLT LT GGGQLT Sbjct
LTTPGGLGQLT
56
Does CAG expansion act in a dominant-negative
fashion?
If the repeat expansion in HD acts in a
dominant-negative fashion, a homozygous LoF
mutation should be equivalent
But no homozygous LoF alleles of the HD gene have
been seen in humans!
Perhaps we can create mutations in the mouse HD
gene! But, how do we find the mouse HD gene?
Can do this using an experimental approach (e.g.,
screen a library) or using a computational
approach (e.g., conduct a BLAST search)
Once the mouse HD gene is identified we must
create a recombinant plasmid containing the mouse
HD gene and appropriate markers for generating a
mouse HD mutation (AKA a mouse HD knockout)
57
Studies of HD in animal models a mouse HD KO
Mouse genomic DNA clone bearing HD exons 3-6
Engineering an HD knockout mouse
H
H
X
H
X
3
4
5
6
ampr
ori
58
Studies of HD in animal models a mouse HD KO
gns
3
6
Embryonic stem (ES) cells from an albino (c/c)
strain of mice
11,000
59
Studies of HD in animal models a mouse HD KO
ES cell bearing heterozygous HD KO
60
Studies of HD in animal models a mouse HD KO
Mosaic embryo
Which of these mosaic offspring are most likely
to have the targeted mutation in their germline?
61
Creating the homozygous KO
Mosaic mouse c/c HD-/HD C/C HD/HD
Albino mouse c/c HD/HD
X
The homozygous KO!
62
The phenotypes of the HD KO mice
Phenotypically normal-no brain pathology
c/c HD-/HD
Early embryonic lethal-embryonic developmental
abnormalities
c/c HD-/HD-
The homozygous HD KO displays different symptoms
than the human disease ?HD symptoms do not result
from a lof of the HD gene
63
How do mutations in Huntingtin cause disease?
HD is a dominant disorder Given what you know
about dominant mutations, provide possible
genetic explanations for the HD phenotype.
-Haploinsufficiency-half the amount of HD gene
product insufficient (like W)? -Dominant
negative-poison subunit (like rab27b)? -Expressed
in wrong place (like Antennapedia) or wrong time
(like lactase) -Protein with a new activity
(like the ABO blood antigens)?
What could it be?
64
The HD CAG repeats encode polyglutamine (polyQ)
tracts
(CAG)n
Exons
Promoter
3
4
5
6
1
2
3
etc.
Are proteins bearing long polyglutamine tracts
toxic?
65
Are long polyglutamine (polyQ) tracts toxic?
Evidence in favor -Spinal and Bulbar muscular
atrophy caused by polyQ expansion of androgen
receptor. -proteins with long polyQ repeats fold
abnormally
QQQQQQQQQQQQQQQQQQQQQQQ...
Protein product misfolded conformation

• When length of glutamine tract exceeds a
  certain length threshold ( 35), the
  polyglutamine tract adopts an abnormal
  conformation

66
Creating a mouse with a human HD gene
Creation of a transgenic mouse
Human HD gene
Exons
Promoter
3
4
5
6
1
2
3
etc.
Gene fragment inserts randomly into mouse genome
67
Creating a transgenic mouse (contd)
Surrogate mother
Transgenic offspring? Can be easily tested using
PCR
Phenotypes of HD transgenic mice -tremors,
abnormal gait, learning deficits by 6mos. -brain
polyQ aggregates -cell loss in basal ganglia in
late stages
Confirms protein with new activity (GoF)
mechanism Suggests that polyglutamine expansion
is toxic
68
Are polyQ expansions toxic in a novel context?
polyQ expression is also toxic in flies, yeast,
cell lines, etc.
Do mice develop HD-like pathology?
Phenotypes of HPRT transgenic mice closely
resemble the HD transgenic mice.
suggests that polyQ itself is primarily
responsible for toxicity
69
What have we learned from cloning HD?

• Symptoms result from neurodegeneration
• Age of onset typically 40s ranges from infancy
  to elderly
• Genetic anticipation (increasing disease severity
  in subsequent generations) often observed
• No cure or treatment

But there are several promising strategies on the
horizon
Age of onset correlates with CAG repeat length
can now be predicted (not clear if this is good
or bad)
Genetic anticipation results from repeat length
instability, primarily in paternal germline
Mechanism of neuron death involves intrinsic
toxicity of large polyQ tracts
70
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71
Repeat Expansion Diseases
Fragile X syndrome of mental retardation FRAXE
mental retardation X-linked spinal and bulbar
muscular atrophy Myotonic dystrophy 1 and
2 Huntingtons disease 1 and 2 Dentatorubral
pallidoluysian atrophy Friedreichs
ataxia Oculopharyngeal muscular
dystrophy Myoclonic epilepsy of
Unverricht-Lundborg Spinocerebellar ataxia types
1, 2, 3, 6, 7, 8, 10, 12 17
72
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