(Huntington’s Disease)
Brain Journal, 2004, Everett & Wood, pp. 2385-2405
Brain Journal, 2004, Everett & Wood, pp. 2385-2405
Different regions of brain
affected by Triple Repeat
Diseases
(Page 413, book)
Isolation Of The Gene Implicated In Spinocerebellar Ataxia Type-1 From Five Primate Species
S.J. Richards, E.B. Whitledge, J.M. Lau, and D.L. Robinson
Department of Biology, Bellarmine University, 2001 Newburg Rd, Louisville, KY 40205
Introduction


Initial SCA-1 PCR
Spinocerebellar Ataxia Type-1 (SCA-1) is a rare, dominantly-inherited,
neurodegenerative disease that results from a tri-nucleotide (CAG)
expansion. Six to thirty-nine CAG repeats occur in the Ataxin-1 gene of
healthy people.3
1
What makes this neurodegenerative disease unique is ‘anticipation’.
As the mutant gene is passed from generation to generation the age of
onset of symptoms decreases as a result of enlargement of the poly-Q
(polyglutamine) region.2
2
3
4
400 bp
300 bp
CAG Repeats In The SCA-1 Gene
of Healthy Animals
Second SCA-1 PCR
Lane:
Lane:
• 100 bp MW ladder
• 100 bp MW ladder
• Human DNA
• Macaque
• Lemur DNA
• Orangutan
• Macaque DNA
• Vervet
Species
The expansion of the poly-Q repeat region occurs during DNA
replication, and can reach as high as 81 CAG repeats in the Ataxin-1
gene of people with SCA-1.

As a result, the protein Ataxin-1 gains a toxic function that results in
the eventual death of the Purkinje cells of the cerebellum and spinal
cord.

The length of the poly-Q region is negatively correlated with the age of
onset.
100 bp
Transformation

After PCR amplification, UA
cloning was used to ligate the
PCR product into a plasmid vector
(Qiagen).
EcoR1
restriction
site
EcoR1
restriction
site
NORMAL Purkinje Cell
ABNORMAL Purkinje
Cell
Objectives
Isolate and sequence the CAG repeat region of the Ataxin-1 gene from five
primate species:
Macaca assamensis (Assamese Macaque)
Cercopithecus aethiops (Vervet Monkey)
Eulemur macaco (Black Lemur)
Pongo pygmaeus (Orangutan)
Lagothrix lagothrica (Woolly Monkey)
Search various DNA databases for other SCA-1 sequences, and use that
information to examine the evolution of the CAG repeat region.
Human
29
Orangutan
24
Chimpanzee
23
Gorilla
22
Orangutan
16
• Wooly Monkey
200 bp
• 100 bp MW ladder

Number of
CAG Repeats

Heat-shock transformation was
then used to incorporate the
plasmid vector into bacterial cells.

Transformants
were
selected
using blue-white screening.

Vector plasmid was purified,
quantified, and a sample was
digested using EcoRI to remove
the insert from the plasmid.
Lemur SCA-1 EcoRI Digestion

Multiple sequence alignments of the CAG repeat region from 14
species were analyzed using the ClustalW program (Vector NTI).
Assamese Macaque
14

This program allows for analysis of this region in an evolutionary
context.
Projected Phylogenic Relationship
Vervet
13
Bonnet Macaque
13
Lemur
4
Dog
4
Lane:
1
6
2
3
4
5
(1) 100 bp MW Ladder
(2) Lemur SCA-1 [3]
(3) Lemur SCA-1 [4]
300 bp
200 bp
(4) Lemur SCA-1 [7]
(5) Lemur SCA-1 [8]
(6) 100 bp MW Ladder
Conclusions
We have successfully isolated the CAG repeat region of SCA-1 from 5 primates.
Methods
Primer design and optimization.
PCR amplification and purification.
Ligation to a plasmid vector.
Heat-shock transformation into competent cells.
Plasmid prep and purification.
Sequencing.
The number of CAG repeats in healthy primates ranged from 2 to 29.
1
2
3
4
5
ClustalW analysis of the CAG repeat region showed fairly predictable
phylogenetic relationships
Woollybetween
Monkeythe species examined.
2
DNA Sequencing

Transformed plasmid samples were sent to the University of Louisville for sequencing.

Sequences were analyzed using Vector NTI (Version 10.0).
The closer the evolutionary relationship a species has to humans, the more
CAG repeats appear in the SCA-1 gene.
House Mouse
2
It appears that a greater number of CAG repeats begin to appear in primates as
the development fine motor skills and dexterity become refined.
PCR
PCR primers were designed using published DNA sequences for the Ataxin-1
gene in both Chimpanzee and Human (NCBI GenBank).
 Forward 5’-ACCTATGCCAGCTTCATCCCATC-3’; TM: 59.0˚ C
 Reverse 5’-GTCATGCAGGTGTAAAGGTCAAGA-3’; T M: 56.8˚ C
DNA was extracted from blood or muscle tissue from healthy animals. Five ng
of DNA template was used for PCR.
References
Zoghbi. 1994. Identification and characterization of the gene causing type 1 spinocerebellar ataxia. Nature 7: 513-520.
Norwegian Mouse
2
Acknowledgements
Chicken
2 their help.
We would like to thank Dr. Roy
Burns and the Louisville Zoo for
2
Orr, H.T., M.Y. Chung, S. Banfi, T.J. Kwiatowski, Jr., A. Servadio, A.L. Beaudet, A.E. McCall, L.A. Duvick, L.P.W. Ranum
and H.Y. Zoghbi. 1993. Expansion of an unstable CAG repeat in spinocerebellar ataxia type 1. Nature 4: 221-226.
3
PCR conditions: initial denaturing at 95 C for 5 min, followed by 35 cycles at
95 for 1:20 min, 52 for 1:43 min, 73 for 1:20 min, and 6 min extension at 73.
Vector DNA
1 Banfi, S., A. Servadio, M.Y. Chung, T.J. Kwiatowski, Jr., A.E. McCall, L.A. Duvick, Y. Shen, E.J. Roth, H.T. Orr and H.Y.
Everett C.M., N.W Wood. 2004. Trinucleotide Repeats and Neurodegenerative Diseases. Brain. 127: 2385-2405.
This project was partially supported by NIH Grant Number P20 RR16481 from the BRIN
Program of the National Center for Research Resources.
We would also like to thank Dr. Ric Devor, Integrated DNA Technologies INC., Coralville, IA.
We would also like to thank Dr. Steven Wilt, Bellarmine University.
Species
Number of CAG Repeats
In SCA-1 Gene
Human
29
Orangutan
24
Chimpanzee
23
Gorilla
22
Orangutan
16
Assamese Macaque
14
Vervet
13
Bonnet Macaque
13
Lemur
4
Dog
4
Woolly Monkey
2
House Mouse
2
Norwegian Mouse
2
Chicken
2
1966
Arlo
Woody Guthrie
(died at 55, in 1967)
Sarah
Huntington's disease is…
…a genetic disease of the central nervous system that produces
speech slurring, involuntary movements, & progressive dementia.
It usually starts between the ages of 30 and 50, and causes death
after about 20 years (usually of pneumonia, choking, or heart failure).
Suicide is common.
Between 100,000-250,000 Americans have it
(or will when they get older).
…one birth in every 10,000 has the disease
It is a dominant mutation which is easily passed on
because people don’t know they have it until later in life.
There is no known cure.
This disease is named after Dr. Huntington (Long Island)
who first diagnosed himself with the disease in 1872.
His father and grandfather both died of the disease.
His distant relative (who first came to America in the 1630’s)
did so after being persecuted in Europe for
consorting with the “devil” and for practicing witchcraft.
It was probably the Huntington’s Disease that caused
people to conclude he was “possessed”.
(At first, people thought Woody Guthrie was an alcoholic….
then schizophrenic….)
?
?
The story of
Nancy Wexler
Nancy Wexler
(Ph.D. in psychology)
Her mother died of Huntington’s
so she may have the disease herself.
In 1979, she saw a film about
a Venezuelan village where
an excessive number of
people had the disease…...
She got federal grant to visit the village and
interview the people.
Once they found out she might
have the disease too, they
eventually grew to trust her (and
answered her prying questions)
She built a pedigree chart of 15,000 Venezuelans and collected
blood from 3,500 of them. This took 13 years….
How did this disease originate in this little village in Venezuela?
In the 1800’s a Portuguese sailor come to the village. Some rumored
he was a drunkard because he always walked as if he was
intoxicated.
Eventually, he married a local woman and had numerous children.
Later, he died of unknown causes......
But his gene for Huntington’s Disease
still survives in this village today (seven generations later)….
Of his 5,000 direct relatives, 250 of them have Huntington’s Disease
(that is 1 out of every 20).
1966
Arlo
Woody Guthrie
(died 1967)
Sarah
The gene was isolated in 1993.
Chromosome 4
Dominant.
The CAG repeats occur in the first exon.
Normal = 6 - 35 repeats
Diseased = 40 – 121 repeats
What is affected
by the mutated
Huntington Protein?
Granular and filamentous
Brain Journal, 2004, Everett & Wood, pp. 2385-2405
And that’s why
Positional Cloning
is important, honey!
Some Human Disease Genes identified by Positional Cloning:
1986 = Duchennes Muscular Dystrophy
1989 = Cystic Firbrosis
1990 = 4 more
1991 = Fragile X Syndrome & 3 others
1992 = Lowe Syndrome & 2 others
1993 = Huntingtons Disease & 11 more
1994 = BRCAI (Breast Cancer), Dwarfism & 11 others
1995 = Alzheimers II, BRCAII & 9 others
1996 = X-linked Myotubular Myopathy & 15 others
1997 = Deafness (DFNAI), Tuberous Sclerosis, Juvenile
Glaucoma & 13 others
1998 = Congenital Night Blindness, Juvenile Parnkinsons Disease
& 10 others
How does the Huntington’s Disease gene
actually cause disease?
A “degenerative
disease”..
..it is 10-20
years before
becoming
fatal.
Apparently, it is a mutation that causes the repetition of the
sequence “CAG”. Whereas a healthy person has 20 or so
repeats (CAGCAGCAGCAGCAG...) people who have this
disease have from 39 to 125 CAG repeats in a row.
The more CAG repeats they have, the earlier the disease shows up.
# of CAG Repeats
Median Age at Onset *
39
66 years
40
59
41
54
42
49
43
44
44
42
45
37
46
36
47
33
48
32
49
28
50
27
Why are these repeats
so harmful?
*Age by which 50% of individuals will be affected
Standard Genetic Code
T
C
T
TTT Phe (F)
TTC "
TTA Leu (L)
TTG "
TCT Ser (S)
TCC "
TCA "
TCG "
TAT Tyr (Y)
TAC "
TAA Stop
TAG Stop
TGT Cys (C)
TGC "
TGA Stop
TGG Trp (W)
C
CTT Leu (L)
CTC "
CTA "
CTG "
CCT Pro (P)
CCC "
CCA "
CCG "
CAT His (H)
CAC "
CAA Gln (Q)
CAG "
CGT Arg (R)
CGC "
CGA "
CGG "
ACT Thr (T)
ACC "
ACA "
ACG "
AAT Asn (N)
AAC "
AAA Lys (K)
AAG "
AGT Ser (S)
AGC "
AGA Arg (R)
AGG "
GCT Ala (A)
GCC "
GCA "
GCG "
GAT Asp (D)
GAC "
GAA Glu (E)
GAG "
GGT Gly (G)
GGC "
GGA "
GGG "
ATT Ile (I)
ATC "
A
ATA "
ATG Met (M)
G
GTT Val (V)
GTC "
GTA "
GTG "
A
G
CAG is
the code
for the
amino acid
Glutamine
Loci is on
Chromosome 4
This CAG gets
repeated up to 125x
“HAP-1” is a protein that
occurs in brain cells. Its
normal function (whatever that is) might be blocked by the Glutamines.
This is why the mutation
is Dominant. Even if an
individual is Hh,
they will have this faulty
protein in their brain.
It doesn’t matter if the
other allele is normal.
It took another 10 years
to discover all of this..
So, maybe extra HAP-1 protein could be delivered to patients
brains, or some other molecule could be added to stop the Glutamines
from attacking it….there is alot of research going on right now.
Basically, there are three molecular approaches that can
be taken once a genetic disease is described at the
biochemical level:
1) develop pharmaceuticals
2) gene therapy
3) early diagnosis
What are the advantages of early diagnosis?
There is a genetic test for
Huntington’s Disease…
It costs $1300.
Only 3% of the people in the
U.S. who are “at risk”
actually take the test….
Why so few do you think?
There are other “Triple Repeat” diseases……
These
Mental genes are sometimes called
Retardation
“Stuttering”
genes…they usually
…X Chromosome
A type of Muscular
affect the neurologicalDystrophy..
system
…Chromosome 19
What does all this have to do with
DNA Replication?
Stuttering genes, and other regions
in the genome that have repeats,
likely became that way because of
mistakes during DNA replication
DNA
Replication
A very quick look..
What does the “S” stand for ??
The “Klenow Fragment”
of DNA Polymerase
Hyperlink to molecular
movies... (1&2 are best)
See places where replication is taking place?
The building block of a new DNA strand
QUESTION: What is the function of the three phosphate groups?
Energy....the last two phosphates break away when the phosphodiester
bond is formed to the 3’ end of the adjacent nucleotide.
QUESTION: If another nucleotide base was going
to be added to this molecule, where would it be added?
DNA Polymerase is a “stupid” enzyme….
It has to be told when & where
to start doing “its thing”...
“Primers” tell the enzyme where to begin
DNA replication.
“priming” before you paint
“priming” a water pump
?
The two new strands grow in the
opposite directions
RNA Primase
DNA (and RNA)
bases are always
added at the 3’ end
of the nucleic acid
chain.
Click Here
for an
animation
Proteins involved in eukaryotic DNA replication:
1. Origin Replication Complex (ORC) = binds to DNA sequences
that represent “initiation sites”....eukaryotes have lots of these
initiation sites available to start replication. They allow the next
two enzymes to do their thing.
2. Helicase = unwinds DNA where the ORC is, separating
the two strands
3. Topoisomerase = prevents original DNA from getting tangled
4. RNA Primase = adds 11 RNA bases near the initiation site
...this tell DNA Polymerase where to start...
Proteins involved in eukaryotic DNA replication
(continued):
5. DNA Polymerase = a large complex of proteins that grab
the appropriate nucleotide triphosphate (one
that is complementary to the DNA strand and
adds it to the 3’ end of the new strand.
6. RNAase = an enzyme that removes RNA primers after
replication. When that’s been done DNA Polymerase
fills in these gaps with the appropriate DNA sequence.
7. DNA Ligase = forms phosphodiester bonds between the
DNA pieces that are not yet connected
(called “nicks”). A nick is when the
phosphodiester bond is broken on one strand
but not the other.
Do cells ever make mistakes in copying DNA?
Absolutely.
Remember that cells can replicate
all of their DNA in hours or even
minutes....so there are bound to be
errors.
There is an error in replication is estimated to occur
about once every 0.1 to 1 billion bases.
But since we have 6 billion bases per cell, that is
between 6 to 60 mistakes per cell.
…although most of them get corrected,
the mistakes that persist represent “mutations”.
For these mutations to be passed on to the next generation the mutation
would have to be carried in the gametes
Fertilized egg
What can happen
if ordinary
somatic cells
obtain new
mutations
of its DNA?
These mutations can show up as:
1) Deletion of a base
2) Addition of a base
3) Repetition of bases (like the CAG Repeat that
causes Huntington’s Disease)
4) Substitution of one base with another
The environment can also induce mutations:
1) Ultraviolet Radiation (sunlight)
2) X-ray Radiation (medical)
3) Chemicals like tobacco smoke, asbestos,
vinyl chloride, alcohol, mustard gas,
creosote
4) Anything that generates free radicals
(charged oxygen ions)
5) Really bad TV Shows, like “Bachelor”...
Standard Genetic Code
T
C
T
TTT Phe (F)
TTC "
TTA Leu (L)
TTG "
TCT Ser (S)
TCC "
TCA "
TCG "
TAT Tyr (Y)
TAC "
TAA Stop
TAG Stop
TGT Cys (C)
TGC "
TGA Stop
TGG Trp (W)
C
CTT Leu (L)
CTC "
CTA "
CTG "
CCT Pro (P)
CCC "
CCA "
CCG "
CAT His (H)
CAC "
CAA Gln (Q)
CAG "
CGT Arg (R)
CGC "
CGA "
CGG "
ACT Thr (T)
ACC "
ACA "
ACG "
AAT Asn (N)
AAC "
AAA Lys (K)
AAG "
AGT Ser (S)
AGC "
AGA Arg (R)
AGG "
GCT Ala (A)
GCC "
GCA "
GCG "
GAT Asp (D)
GAC "
GAA Glu (E)
GAG "
GGT Gly (G)
GGC "
GGA "
GGG "
ATT Ile (I)
ATC "
A
ATA "
ATG Met (M)
G
GTT Val (V)
GTC "
GTA "
GTG "
A
G
Remember
that some
mutations
are going to
be more
important
than others
A change
from GGT
to GGC,
for instance,
would still
yield a glycine
Nature Genetics, 1993
So, Positional Cloning techniques were used to
isolate a 1,200,000 bp piece of Chromosome #6.
Less than 1% of this region actually codes for the
SCA-1 transcript (mRNA).
Globin gene
Healthy
Anemic
Use a probe from for this region
For Sickle Cell Anemia
Probes are valuable
for identifying the
mutations in a
well-characterized gene
A and B are
homologous
chromosomes
Not cut here
You can think
of “B” as “little a”
Southern Blots
(of genomic DNA)
following digestion
with EcoRI enzyme
A and B are
homologous
chromosomes
EcoRI cuts the “A” allele in
half, and Probe 3 allows
you to visualize that. Lets
pretend the “A” allele is
the diseased allele.
Not cut here
You can think
of “B” as “little a”
If you made a
genomic library
of a person with
a RFLP-mapped
disease, you
could use Probe 3
to screen the library.
The other two probes would work too, but be further away from mutation.
that reveals RFLP
Healthy
The RE site for this disease must be here
Diseased
Diseased
Healthy
Diseased
Healthy
So, the hard part
is finding the right
combination of RE
and probe….which
is one reason why
Postional Cloning is
so slow and expensive.
If this band is always
present in people with the
disease then the probe could
be useful in screening a
library.
One way of finding the best probe is:
“Chromosome Walking”
If linkage (by studying pedigree analysis)
can be shown for a disease (that is already cloned),
then begin there,
and “walk”
to the gene of interest.
Linked gene here
Different library made
with different RE
Each time you
make a new probe,
use that to look for
RFLPs in healthy vs.
diseased people.
Different library made
with different RE
Chromosome Walking
If an RFLP can’t be found for the disease of interest (for instance,
point mutations wouldn’t reveal themselves as RFLPs unless
the single mutation was exactly on a RE site) you can look
at transcription.
mRNA can be isolated from healthy vs. sick people (using
Poly-A chromatography) and then ran on a gel, transferred to
a membrane, and probed just like a Southern Blot.
NORTHERN BLOT
If the disease of interest
involves muscle tissue
then this probe might
be important…
especially if it doesn’t
occur in diseased
people.
Northern blot showing the presence of mRNA hybridizing to sadA cDNA in different types
of tissue. 1, Dry seeds; 2, seeds after 16 h of soaking in tap water; 3, shoots 9 d after sowing;
4, cotyledons 14 d after sowing; 5, leaf buds 14 d after sowing; 6, cotyledons 21 d after
sowing; 7, second leaf pairs 21 d after sowing; 8, third leaf pairs 21 d after sowing; 9, fourth
leaf pairs 21 d after sowing; 10, fifth leaf pairs 21 d after sowing; 11, roots from plants grown
in vermiculite 14 d after sowing; 12, roots from plants grown in vermiculite 21 d after
sowing; 13, roots from plants grown in vermiculite 42 d after sowing; 14, stems 21 d after
sowing; 15, tendrils; 16, flowers (white); 17, flowers (purplish); and 18, pods.
Southern Blot
showing “Anticipation”
The length of a centiMorgan (in terms of DNA bases) is
different for each species……
In Humans: 1 cM = 1 million DNA bases (on average)