Brain Journal, 2004, Everett & Wood, pp. 2385-2405

(Huntington’s Disease)

Brain Journal, 2004, Everett & Wood, pp. 2385-2405

Different regions of brainaffected by Triple Repeat

Diseases(Page 413, book)

Vector DNA

1 2 3 4 5

Isolation Of The Gene Implicated In Spinocerebellar Ataxia Type-1 From Five Primate SpeciesIsolation Of The Gene Implicated In Spinocerebellar Ataxia Type-1 From Five Primate Species

MethodsMethods

Projected Phylogenic RelationshipProjected Phylogenic Relationship

CAG Repeats In The SCA-1 GeneCAG Repeats In The SCA-1 Geneof Healthy Animalsof Healthy Animals 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

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

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.

IntroductionIntroduction

ObjectivesObjectives

Primer design and optimization.PCR amplification and purification.Ligation to a plasmid vector.Heat-shock transformation into competent cells.Plasmid prep and purification.Sequencing.

Isolate 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.

NORMAL Purkinje Cell ABNORMAL Purkinje Cell

After PCR amplification, UA cloning was used to ligate the PCR product into a plasmid vector (Qiagen).

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.

EcoR1 restriction site

EcoR1 restriction site

1 2 3 4 5 6

Lane:

(1) 100 bp MW Ladder

(2) Lemur SCA-1 [3]

(3) Lemur SCA-1 [4]

(4) Lemur SCA-1 [7]

(5) Lemur SCA-1 [8]

(6) 100 bp MW Ladder

Lemur SCA-1 EcoRI DigestionLemur SCA-1 EcoRI Digestion

Lane:

• 100 bp MW ladder

• Human DNA

• Lemur DNA

• Macaque DNA

1 2 3 4

Initial SCA-1 PCR Second SCA-1 PCRInitial SCA-1 PCR Second SCA-1 PCR

Lane:

• 100 bp MW ladder

• Macaque

• Orangutan

• Vervet

• Wooly Monkey

• 100 bp MW ladder

S.J. Richards, E.B. Whitledge, J.M. Lau, and D.L. RobinsonS.J. Richards, E.B. Whitledge, J.M. Lau, and D.L. RobinsonDepartment of Biology, Bellarmine University, 2001 Newburg Rd, Louisville, KY 40205Department of Biology, Bellarmine University, 2001 Newburg Rd, Louisville, KY 40205

TransformationTransformation

DNA SequencingDNA Sequencing

Transformed plasmid samples were sent to the University of Louisville for sequencing.

Sequences were analyzed using Vector NTI (Version 10.0).

Multiple sequence alignments of the CAG repeat region from 14 species were analyzed using the ClustalW program (Vector NTI).

This program allows for analysis of this region in an evolutionary context.

PCRPCR 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’; TM: 56.8˚ C

DNA was extracted from blood or muscle tissue from healthy animals. Five ng of DNA template was used for PCR.

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.

Species Number of CAG Repeats

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

ReferencesReferences

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. Zoghbi. 1994. Identification and characterization of the gene causing type 1 spinocerebellar ataxia. Nature 7: 513-520.

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 Everett C.M., N.W Wood. 2004. Trinucleotide Repeats and Neurodegenerative Diseases. Brain. 127: 2385-2405.

AcknowledgementsAcknowledgementsWe would like to thank Dr. Roy Burns and the Louisville Zoo for their help.

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.

We have successfully isolated the CAG repeat region of SCA-1 from 5 primates.

The number of CAG repeats in healthy primates ranged from 2 to 29.

ClustalW analysis of the CAG repeat region showed fairly predictable

phylogenetic relationships between the species examined.

The closer the evolutionary relationship a species has to humans, the more

CAG repeats appear in the SCA-1 gene.

It appears that a greater number of CAG repeats begin to appear in primates as

the development fine motor skills and dexterity become refined.

ConclusionsConclusions

100 bp

200 bp

300 bp400 bp

300 bp200 bp

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

Woody Guthrie (died at 55, in 1967)

Sarah

Arlo

1966

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 ofNancy Wexler

Nancy Wexler (Ph.D. in psychology)

Her mother died of Huntington’sso 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 collectedblood 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 rumoredhe 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 Diseasestill 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).

Woody Guthrie (died 1967)

Sarah

Arlo

1966

The gene was isolated in 1993. Chromosome 4

Dominant.

The CAG repeats occur in the first exon.

Normal = 6 - 35 repeatsDiseased = 40 – 121 repeats

Brain Journal, 2004, Everett & Wood, pp. 2385-2405

What is affectedby the mutated

Huntington Protein?

Granular and filamentous

And that’s why Positional Cloning

is important, honey!

Some Human Disease Genes identified by Positional Cloning:

1986 = Duchennes Muscular Dystrophy1989 = Cystic Firbrosis1990 = 4 more1991 = Fragile X Syndrome & 3 others1992 = Lowe Syndrome & 2 others1993 = Huntingtons Disease & 11 more1994 = BRCAI (Breast Cancer), Dwarfism & 11 others1995 = Alzheimers II, BRCAII & 9 others1996 = X-linked Myotubular Myopathy & 15 others1997 = Deafness (DFNAI), Tuberous Sclerosis, Juvenile Glaucoma & 13 others1998 = Congenital Night Blindness, Juvenile Parnkinsons Disease & 10 others

How does the Huntington’s Disease gene actually cause disease?

A “degenerativedisease”..

..it is 10-20years beforebecoming

fatal.

Apparently, it is a mutation that causes the repetition of thesequence “CAG”. Whereas a healthy person has 20 or so

repeats (CAGCAGCAGCAGCAG...) people who have thisdisease 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

*Age by which 50% of individuals will be affected

Why are these repeatsso harmful?

Standard Genetic CodeT C A G

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 "

A

ATT Ile (I) ATC " ATA " ATG Met (M)

ACT Thr (T) ACC " ACA " ACG "

AAT Asn (N) AAC " AAA Lys (K) AAG "

AGT Ser (S) AGC " AGA Arg (R) AGG "

G

GTT Val (V) GTC " GTA " GTG "

GCT Ala (A) GCC " GCA " GCG "

GAT Asp (D) GAC " GAA Glu (E) GAG "

GGT Gly (G) GGC " GGA " GGG "

CAG isthe code for the

amino acidGlutamine

This CAG getsrepeated 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.

Loci is onChromosome 4

So, maybe extra HAP-1 protein could be delivered to patientsbrains, or some other molecule could be added to stop the Glutaminesfrom attacking it….there is alot of research going on right now.

This is why the mutationis 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..

Basically, there are three molecular approaches that can be taken once a genetic disease is described at thebiochemical level:

1) develop pharmaceuticals2) gene therapy3) early diagnosis

What are the advantages of 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?Why so few do you think?

There are other “Triple Repeat” diseases……

A type of Muscular Dystrophy..

…Chromosome 19

Mental Retardation

…X Chromosome

These genes are sometimes called “Stuttering” genes…they usually

affect the neurological system

What does all this have to do withDNA Replication?

Stuttering genes, and other regionsin the genome that have repeats,

likely became that way because of mistakes during DNA replication

DNAReplication

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?

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.

The building block of a new DNA strand

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 beginDNA replication.

“priming” before you paint

“priming” a water pump

?

The two new strands grow in the opposite directions

RNA Primase

Click Herefor an

animation

DNA (and RNA) bases are always

added at the 3’ endof the nucleic acid

chain.

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 replicateall 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”.

Fertilized egg

For these mutations to be passed on to the next generation the mutationwould have to be carried in the gametes

What can happenif ordinary somatic cellsobtain newmutations

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 CodeT C A G

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 "

A

ATT Ile (I) ATC " ATA " ATG Met (M)

ACT Thr (T) ACC " ACA " ACG "

AAT Asn (N) AAC " AAA Lys (K) AAG "

AGT Ser (S) AGC " AGA Arg (R) AGG "

G

GTT Val (V) GTC " GTA " GTG "

GCT Ala (A) GCC " GCA " GCG "

GAT Asp (D) GAC " GAA Glu (E) GAG "

GGT Gly (G) GGC " GGA " GGG "

Rememberthat somemutations

are going tobe more

important than others

A changefrom GGTto 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).

For Sickle Cell Anemia

Use a probe from for this region

Globin gene

HealthyAnemic

You can thinkof “B” as “little a”

Not cut here

Southern Blots(of genomic DNA) following digestionwith EcoRI enzyme

Probes are valuable for identifying the

mutations in a well-characterized gene

A and B are homologous chromosomes

You can thinkof “B” as “little a”

Not cut here

If you made agenomic libraryof a person witha RFLP-mapped

disease, you could use Probe 3

to screen the library.

EcoRI cuts the “A” allele in half, and Probe 3 allows

you to visualize that. Lets pretend the “A” allele is

the diseased allele.

A and B are homologous chromosomes

The other two probes would work too, but be further away from mutation.

that reveals RFLP

Diseased

Diseased

Diseased

Healthy

Healthy

Healthy

The RE site for this disease must be here

If this band is alwayspresent in people with thedisease then the probe couldbe useful in screening alibrary.

So, the hard partis finding the rightcombination of RE and probe….which is one reason why

Postional Cloning isso slow and expensive.

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.

Different library madewith different RE

Different library madewith different RE

Linked gene here

Each time youmake a new probe,use that to look for

RFLPs in healthy vs.diseased people.

Chromosome Walking

If an RFLP can’t be found for the disease of interest (for instance,point mutations wouldn’t reveal themselves as RFLPs unlessthe 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 interestinvolves muscle tissuethen this probe might

be important…especially if it doesn’t

occur in diseasedpeople.

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)