1. Positional cloning of the Huntington’s 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 (KO’s)
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. -No more than 1-2 topics (1-2 sentences)
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:
-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 message to Anne Paul summarizing the error, BY THE END OF THE DAY TODAY.

4. Huntington’s Disease
Huntington’s disease results from nerve cell degeneration in the basal ganglia
HD brain
Normal brain
(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 40’s; ranges from infancy to elderly
Genetic anticipation (increasing disease severity in subsequent generations) often observed
No cure or treatment
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5. Mapping of the Huntington’s disease gene
The informative pedigree:
• 5,000 related individuals from Venezuela segregating HD
• Included 100 members currently affected by HD
• Included >1,000 members with >25% 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
She began this work in An extremely high occurrence of HD was found within the 15,000 members of a large group of interrelated families living in fishing villages along the border of Lake Maracaibo in Venezuela. This led to the foundation of the Venezuelan HD project, which characterized these family members clinically and genetically. Most are descendents of a woman who suffered from ‘el mal de San Vito’, the local name for HD, in the early nineteenth century.
On their 12th probe… the jackpot!
- linkage of the RFLP to HD!
1983

6. Marker ‘D4S10’ shows linkage to HD
max = 3cM 40
Results of linkage studies using the probe “G8” which recognizes the RFLP marker D4S10 30 20 10
LOD score (Z) 3 -2 
-10
-20
Does this result provide significant evidence of linkage?
-30
-40

7. ? Where is marker ‘D4S10’ located? FISH Karyotype: telomere
D4S10 (4p16)
centromere
telomere
Karyotype: ?
HD gene ~3cM away from D4S10
Actually radiation hybrid mapping was used initially, but this was later confirmed using FISH.
How to tell?
~3x106bp

8. 1983
1992

9. Narrowing of the HD region
Looking for highly informative recombinants (haplotypes):
derived genotypes
D4S141
D4S115
D4S111
Y1P18
R10
D4S98
D4S43
D4S10 1 C B 2 3 A
(B/C) 5
(C / B)
(B / C)
(1/2)
telomere
D4S141
D4S115
D4S111
HD gene likely to reside here
Y1P18
HD ? HD
R10
D4S98
Where are the informative recombinants?
There were 22 individuals in this pedigree, of which 15 were genotyped. This provided the information to allow derivation of the genotypes of the unknowns. There were questions about where the recombination took place in III-1. It looks like it took place in I-1. Also, there were questions about the terminology (eg., B/C, diamond symbols). Look more closely at this paper next year (1992 Am. J. Hum. Genet. 51, 357). HD
D4S43
D4S10
centromere
What Next?
HD gene likely to reside here

10. HD ? Genetic and physical map of the HD region If 2008:
D4S10
we know the sequence here
D4S180
D4S182
D4S98
and here
HD ?
centromere
What is the DNA sequence in this interval?
~500kb
telomere
If 2008:
A portion of the UCSC Genome browser window:
D4S180:
AACTGACTTAA
D4S182:
CCTAGCTTAGAT
use in a BLAST search
ADDA = alpha-adducin gene.
CCAACTGACTTAAGC…………………….AGCCTAGCTTAGATGC
We could also find the genes in this interval using the UCSC browser
But it was 1992…

11. HD ? Genetic and physical map of the HD region If 2008:
D4S10
we know the sequence here
D4S180
D4S182
D4S98
and here
HD ?
centromere
What is the DNA sequence in this interval?
~500kb
telomere
If 2008:
But it was 1992…
A portion of the UCSC Genome browser window:
D4S180:
AACTGACTTAA
D4S182:
CCTAGCTTAGAT
ADDA = alpha-adducin gene.
How was this done in 1992 (i.e., before the genome was sequenced)?

12. Chromosome walking (outline)
Make radioactive probes from known sequence
Identify D4S180 & D4S182-containing clones in genomic DNA library
Use ends of those clone’s inserts to find other clones with overlapping inserts
Repeat

13. Colony hybridization to find the first genomic DNA clone
genomic DNA clones
replica on filter
release the DNA
bind it to filter
which colonies match up with hyb spots? *
hyb
probe from D4S180 region
X-ray film

14. Colony hybridization (cont’d)
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
colony hyb
The goal — find the colonies (clones) that contain this sequence
 overlap your first clone

16. Colony hybridization (cont’d)
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:
identified using D4S182 probe
identified using D4S180 probe
insert in original clone
a contig
HD gene
probe
insert in original clone
probe
Joining fragments

18. From lecture 13 II. Map location on genome
STS 24 62 17 54 20 9 19 36 4
STS = sequence tagged site… short, unique genomic sequence—not present anywhere else in the genome— that can be detected by PCR… ID tag for that portion of genome
For example:
Which portion of the genome is represented in this BAC’s insert?
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. How do we identify the genes in a contig?
Genetic and physical map of the HD region
D4S10
D4S180
D4S182
D4S98
HD ?
centromere
~500kb
telomere
~40kb each
Cosmid (sort of like a plasmid) contig
ADD1 = alpha-adducin gene.
How do we identify the genes in a contig?
Which one is the HD gene?

20. Identifying genes in DNA sequence
Various approaches…
...TTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGG...
...AACTTCTGCTTTCCCGGAGCACTATGCGGATAAAAATATCCAATTACAGTACTATTATTACCAAAGAATCTGCAGTCCACCGTGAAAAGCCC...
Look for signatures of genes—e.g., promoters
These are things that computers are great at-and some of the things that underlie the UCSC browser
Look for open reading frames
Look for transcribed regions— e.g., make a cDNA library

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’ 5’
AAAAAAA-3’
added by cell during pre-mRNA maturation
insert into plasmid, transform E. coli

22. Genomic vs. cDNA libraries
cDNA library
make cDNA, insert into plasmid, etc.
• only mRNA regions (exons) represented
• frequency of clone proportional to amount of transcription of the gene

23. HD ? Genetic and physical map of the HD region centromere
D4S10
D4S180
D4S182
D4S98
HD ?
centromere
Cosmid (sort of like a plasmid) contig
~500kb
telomere
~40kb each
Used as probes to screen cDNA libraries
ADDA = alpha-adducin gene.
IT-15
IT-11
IT-10C3
ADDA
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
Why wouldn’t all individuals of a genotype show the same phenotype?
~60% of genes are expressed in brain. Also mention-and define expressivity.
-Influence of other genes—many traits multigenic
-Influence of environment
-Observation errors!

25. How was the HD gene identified?
CAG18
IT-15
CAG21
IT-11
IT-10C3
ADDA
Gene: 67 exons; >200 kb
mRNA: 10,366 bases
Protein: 3,144 aa; ~350kDa
A simple PCR test to measure CAG repeat length in IT-15:
GTCn
CAGn
Unique sequences in IT-15 flanking the CAG repeat

26. How was the HD gene identified?
100 90 80 70 60 50 40 30 20
Onset age (years)
CAG repeat length
Correlation of HD age of onset and CAG repeat length
Triplet repeat number
normal HD
42-86 CAG repeats in >150 HD individuals
11-34 CAG repeats in 173 normals 65 50 35
(98% between 11-24) 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)
CAG11-34
IT-11
IT-10C3
ADDA
non-disease allele
Gene: 67 exons; >200 kb
mRNA: 10,366 bases
Protein: 3,144 aa; ~350kDa
CAG42-??
disease allele

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,

29. Repeat instability explains HD genetic anticipation in HD
CAG repeats tend to expand upon paternal transmission:
Too young to show trait
Onset at 2yrs
Onset in early 40’s 65 50 35 20 5
Triplet repeat number 90
120
Expanded CAG repeats are unstable in the paternal germline

30. Why are long CAG repeats unstable?
DNA polymerase
A molecular model:
CAGCAGCAGCAGCAGCAG 5’ 3’
GTCGTCGTCGTCGTCGTCGTC TCGTC G
CAGCAGCAGCAGCAG
GTCGTCGTCGTCGTCGTC TCGTCGTC 5’ 3’ C A G
increases CAG repeat length by 1 CAG
decreases CAG repeat length by 1 CAG T G C
CAGCAGCAGCAGCAGCAG
GTCGTCGTCGTCGTCGTCGTC TC 5’ 3’
OR, less frequently

31. Why are only long CAG repeats unstable?
Short repeats often also contain some CAA codons:
CAGCAGCAACAGCAGCAGCAACAGCAA 5’ 3’ 3’
GTCGTCGTTGTCGTCGTCGTTGTCGTC 5’
CAGCAGCAGCAGCAGCAGCAACAGCAA
GTCGTCGTCGTCGTCGTCGTTGTCGTC 5’ 3’
CAGCAGCAGCAGCAGCAGCAGCAGCAA
GTCGTCGTCGTCGTCGTCGTCGTCGTC 5’ 3’
Prone to expansion?

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)?
Goes back to lecture 4

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.
-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)?
-Haploinsufficiency-half the amount of HD gene product insufficient (like W)?
Goes back to lecture 4

34. Wolf-Hirschhorn Syndrome (4p-)
(The Human “Knockout” of the Huntington Locus )
The most common abnormalties seen include severe to profound mental retardation, microcephaly, seizures, hypotonia, and cleft lip and/or palate. Characteristic facial features, include strabismus, hypertelorism, down-turned "fishlike" mouth, short upper lip and philtrum, small chin, ear tags or pits, and cranial asymmetry. Occasional abnormalities include heart defects, hypospadias, scoliosis, ptosis, fused teeth, hearing loss, delayed bone age, low hairline with webbed neck, and renal anomalies. They are described as happy, loving children.
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 Huntington’s 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.
-Expressed in wrong place (like Antennapedia) or wrong time (like lactase)
-Protein with a new activity (like the ABO blood antigens)?
-Haploinsufficiency-half the amount of HD gene product insufficient (like W)?
-Dominant negative-poison subunit (like rab27b)?

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?
-more mismatches are tolerated if appropriate hybridization conditions are met (salt and temperature). Allows non-identical, but closely-related sequences to hybridize.
okay
Follow this with a slide that shows how they identified mouse HD genomic fragments: give some metrics on this (protein size, degree of identity, similarity, etc.).

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 >90% of their aa’s!
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
Follow this with a slide that shows how they identified mouse HD genomic fragments: give some metrics on this (protein size, degree of identity, similarity, etc.).
Before continuing, let’s 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. doubles in size about every 2 years!
Finding the mouse HD gene computationally
doubles in size about every 2 years!
We need three things:
A sequence database.
Some way of saying how similar two sequences are.
A really fast way of carrying out the similarity test.
We have the genome sequences and gene structures already.
We’ll 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:
amino acid identities
PWAVTASCH
|||||||||
VYAVQASPH
(human)
(something else)
amino acid identities
PWAVTASCH
|||||||||
PWGVHATCW
(human)
(something else)
Made up sequences; just for example. The bars mean that the sequences have been aligned. Show using …XXX… that we are only showing a portion. In most real-life situations we will be comparing longer stretches-this is just for illustrative purposes.
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 41

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 42

43. log odds calculation log At random:
likelihood of seeing amino acid pair in related protein
score =
log
likelihood of seeing amino acid pair at random
likelihood of seeing amino acid pair in related protein:
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.
likelihood of seeing amino acid pair at random:
BLOCKS database maintained by Henikoff lab.
Random: not 1/20 F=frequency of one amino acid X the frequency of the second amino acid X 2 (because can be)
From NCBI BLAST site: A substitution matrix containing values proportional to the probability that amino acid i mutates into amino acid j for all pairs of amino acids. Such matrices are constructed by assembling a large and diverse sample of verified pairwise alignments of amino acids. If the sample is large enough to be statistically significant, the resulting matrices should reflect the true probabilities of mutations occurring through a period of evolution. (See also BLOSUM 62.)
At random:
(the factor of two is because it can be an A-B pair or a B-A pair)
Gives the denominator likelihood above. 43

44. Amino acid pair frequencies in related proteins
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_XENLA|Q NIYYSDKYTDEHFEY
CKS1_HUMAN|P QIYYSDKYDDEEFEY
CKS2_HUMAN|P QIYYSDKYFDEHYEY
CKS2_MOUSE|P QIYYSDKYFDEHYEY
CKS1_PATVU|P QIYYSDKYFDEDFEY
CKS1_DROME|Q DIYYSDKYYDEQFEY
CKS1_PHYPO|P TIQYSEKYYDDKFEY
CKS1_LEIME|Q KILYSDKYYDDMFEY
O QIQYSEKYFDDTFEY
O NIHYSTRYSDDTHEY
CKS1_SCHPO|P QIHYSPRYADDEYEY
CKS1_YEAST|P SIHYSPRYSDDNYEY
CKS1_CAEEL|Q DFYYSNKYEDDEFEY
D-D 21 pairs
D-E 14 pairs
D-P 14 pairs
D-T pairs
D-N 7 pairs
E-E pair
E-T pairs
E-P pairs
T-P pairs
T-T pair
T-N pair
There are many such blocks: these are from a section of corresponding proteins from different organisms. There are thousands of other blocks. Computers do this and they repeat the calculation at each position and for thousands of blocks.
LOD calcul. (e.g., D-D pair):
One of 29,068 blocks - pair frequencies compiled from all blocks combined.
74 (total # of pairs)
21 (# D-D pairs)
log
From aa frequency table
0.05 X 0.05 (f of D-D) 44

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).
Just some definitions; don’t worry about this too much. This is to reduce the round-off error and to increase the computing speed (again don’t worry about this) 45

46. log odds scores (cont.) log
likelihood of seeing amino acid pair in related protein
score =
log
likelihood of seeing amino acid pair at random
If amino acid pair seen MORE often than expected at random?
odds > 1, score positive
If amino acid pair seen LESS often than expected at random?
odds < 1, score negative
Remember:
Log2 1 = 0
Log2 2 = 1
Log2 1/2 = -1

47. Values from a score matrix (half-bit scores)
one-letter amino acid code
score for alanine (A) - tryptophan (W)
This is simply based upon doing the math-not at all relkated to Biochemistry-however, lets look at biochemistry (next slide)
self match scores 47

48. Amino acid structures
Hydrophobic
Polar
Charged
phenylalanine F 48

49. Example - similar amino acids get positive scores
Qualitatively, what scores do you expect pairs of these to have?
I-L
I-V
L-V 49

50. Example - dissimilar amino acids get negative scores
Qualitatively, what scores do you expect pairs among these groups to have?
hydrophobic
vs. charged 50

51. Thinking about protein similarity
Suppose we have the following aligned protein sequences:
PWAVTASCH
|||||||||
VYAVQASPH
(human)
(something else)
PWAVTASCH
|||||||||
PWGVHATCW
(human)
(something else)
Related to what extent? We want to be quantitative.
Adding log odd scores = multiplying probablities.
Top case:
= 20
Bottom case:
= 32
(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 HD…finding the mouse HD gene
# of expected (E) matches (with a score this good from a database of this size) from chance alone
A portion of human HD protein sequence (the “query” sequence):
MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQPPPPPPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPLHRPKKELSATKKDRVNHCLTICENIVAQSVRNSPEFQKLLGIAMELFLLCSDDAESDVRMVADECLNKVIKALMDSNLPRLQLELYKEIKKNGAPRSLRAALWRFAELAHLVRPQKCRPYLVNLLPCLTRTSKRPEESVQ…
BLAST
database of all proteins from human, chimp, dog, mouse, etc.
human
chimp
mouse!
Adding log odd scores = multiplying probablities.
summary list of all related proteins (one per line)

53. Getting back to HD…finding the mouse HD gene
Looking further down on the summary list…
Bit score
E value
zebra fish
sea anemone
fruit fly
Adding log odd scores = multiplying probablities.
Can keep going, but the validity attenuates as you approach E=1

54. Getting back to HD…finding the mouse HD gene
Portion of Mus musculus HD alignment:
amino acid similar
amino acid identical
gap
my query
this match
M. musculus
Adding log odd scores = multiplying probablities.
amino acid dissimilar

55. A C D E F G H I K L M N P Q R S T V W Y 4 -2 1 -1 -3 9 -4 6 2 5 3 8 7 11
notice that this amino acid pair is poorly conserved
Query LTAVGGIGQLT
LT GG+GQLT
Sbjct LTTPGGLGQLT
score (bits) is sum of each aligned residue (x 0.5 because the score table is in half-bits):
= 37 half bits = 18.5 bits 55

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)
Follow this with a slide that shows how they identified mouse HD genomic fragments: give some metrics on this (protein size, degree of identity, similarity, etc.).
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:
Restriction endonuclease sites
etc. H H H X X 3 4 5 6
ampr
ori
Partial digest with H & X H X 3 4 5 6
ampr
ori
neor 4 5
Plasmid bearing HD exons 3-6. Need to have a transition slide to come back to HD. H X 3 6
ampr
ori
Partial digest with H
neor
cut
gns

58. Studies of HD in animal models: a mouse HD KO
gns
neor 3 6
Embryonic stem (ES) cells from an albino (c/c) strain of mice
Neomycin + gancyclovir 3 6
neor
gns 4 5 ES
genome
ES cells die on gancyclovir 3 6
neor
1:1,000 3 6
neor
gns

59. Studies of HD in animal models: a mouse HD KO3 4 5 6 1 2 4…
Blastocyst-stage embryo from a C/C female
ES cell bearing heterozygous HD KO 3 6 1 2 6…
neor
Altered splicing results in frameshift and premature transl. term.

60. Studies of HD in animal models: a mouse HD KO
Mosaic embryo
Place mosaic embryos into surrogate mother
c/c; HD-/HD+
C/C; HD+/HD+
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
c/c; HD-/HD+ OR
c/c; HD+/HD+
C/c; HD+/HD+
genotype (southern blot of blood sample)
c/c; HD-/HD+
c/c; HD+/HD+ X
c/c; HD-/HD+ X
c/c; HD-/HD-
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-
Heterozygotes indistinguishable from littermates in terms of appearance, weight, movement, and behavior. No brain pathology was detected in aged heterozygous mice. The homozygotes were embryonic lethal (very early in embryonic life; before embryonic day 12).
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 1 2 3 3 4 5 6
etc.
AAAAAA
AUG…(CAG)n
M…(Q)n…
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
(CAG)180
Exons
Promoter 1 2 3 3 4 5 6
etc. 1
Promoter
Single-celled mouse embryo
Place embryo into surrogate mother
Gene fragment inserts randomly into mouse genome

67. Can be easily tested using PCR
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?
Insertion of a polyQ tract in the hypoxanthine phosphoribosyltransferase (HPRT) gene
(HPRT) 1 2 3 4
polyQ expression is also toxic in flies, yeast, cell lines, etc.
(CAG)146
Generate transgenic mouse
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 40’s; 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. 1993
Present

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
Huntington’s disease 1 and 2
Dentatorubral pallidoluysian atrophy
Friedreich’s ataxia
Oculopharyngeal muscular dystrophy
Myoclonic epilepsy of Unverricht-Lundborg
Spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 10, 12 & 17