Huntingtons Disease and its relation to CAG repeats'

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Huntingtons Disease and its relation to CAG
repeats.

• Presenter Yenny Sanchez
• Möncke-Buchner, E., S. Reich, M. Mücke, M.
  Reuter, W. Messer, E.E. Wanker, and D. H. Krüger.
  2002. Counting CAG repeats in the Huntingtons
  disease gene by restriction endonuclease EcoP15I
  cleavage. Nucleic Acid Research 30 e83
• http//nar.oupjournals.org/cgi/content/full/30/16/
  e83

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Presentation Outline

• Introduction
• Overview of Huntington Disease (HD)
• What causes it?
• How is inherited?
• What is the function of the huntingtin protein.
• How can the structure of huntingtin be altered?
• Huntingtin and neurons.
• Primary Article.
• Objective
• Data
• Conclusion
• Application
• Prevalence
• Management

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Overview of Huntington Disease (HD)

• Hereditary degenerative neurological disease that
  leads to dementia.
• Named after George Huntington, who first
  described it in 1872.

DR. GEORGE HUNTINGTON(1850-1916)
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Overview of Huntington Disease (HD)

• Progressive dementia with involuntary movements,
  emotional disturbances, and metal deterioration.
• Disease progresses slowly and death is usually
  due to an intercurrent infection.
• Signs and symptoms develop in middle age, men and
  women are equally likely to develop the disease.

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What Causes HD?

• Genetically programmed degeneration of neurons.
• Specifically affected are cells of the basal
  ganglia.
• Structure that have many important functions,
  including coordinating movement.

1 Cerebrum2 Basal Ganglia3 Brain stem4
Cerebellum
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What Causes HD?

• All the characteristics can ultimately be traced
  back to a small change in the huntingtin gene on
  chromosome 4.
• People who have Huntington disease have a larger
  huntingtin gene. This is due to a repetition in
  the code C-A-G.
• Each C-A-G sequence codes for the amino acid
  glutamine, a protein building block.
• People with HD simply have an increased number of
  these C-A-G repeats toward the beginning of their
  Huntington gene, coding for an excess number of
  glutamines in their huntingtin protein.

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How is HD Inherited?

• Genes are composed of deoxyribonucleic acid
  (DNA), a molecule shaped like a spiral ladder.
• Each rung of the ladder is composed of base
  pairs.
• There are four types of bases adenine (A),
  thymine (T), cytosine (C), and guanine (G).

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How is HD Inherited?

• The gene that produces HD lies on chromosome 4.
• Autosomal dominant disorder.
• Only one copy of the defective gene is necessary
  to produce the disease.
• The genetic defect responsible of HD is a
  repetition in the base pair (CAG) sequences of
  chromosome 4.

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How is HD Inherited?

• Each child of an HD parent has a 50-50 chance of
  inheriting the HD gene.
• If a child does not inherit the HD gene, he or
  she will not develop the disease and cannot pass
  it to subsequent generations.
• A person who inherits the HD gene will develop
  the disease in the middle age.
• A small number of cases are sporadic, occurring
  even though there is no familiar history of the
  disorder.
• These cases are caused by a genetic mutation that
  occurs during sperm development and that brings
  the number of CAG repeats into the range that
  causes the disease.

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What Is the Function of the Huntingtin protein?

• It plays a critical role in nerve cell function.
  Huntingtin regularly interacts with proteins
  found only in the brain.
• People without Huntington have between 9 and 35
  CAG repeats.
• People with Huntington have between 36 and 121
  CAG repeats.
• Excess CAG repeat in the gene changes the shape
  of the protein (huntingtin).
• Each CAG repeat codes for a specific amino acid
  glutamine.
• The extended number of glutamine repeats
  characterizes HD as one of nine polyglutamine
  expansion disorders.
• Diseases which arise from extra copies of the
  CAG codon in certain segments of the DNA. The CAG
  codon codes for the amino acid glutamine.

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How can the structure of huntingtin be altered?

• If there are 41 CAG repeats in a row inside the
  gene, there will be 41 glutamines attached to
  each other in a row inside the huntingtin
  protein.
•  The production of multiple glutamines causes
  mutant huntingtin to fold into a different shape
  than normal.
• Mutant huntingtin interacts with all sorts of
  other proteins that might normally be ignored,
  particularly in the spiny neurons deep inside the
  brain.

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Huntingtin and Neurons

• Excess huntingtin affects nerve cells dendrites.
  It causes dendrites to grow out of control.
• Dendrites are found on every nerve cell and
  extend out from the cell body and are responsible
  for receiving and sending messages to other nerve
  cells.
• New incomplete branches form and other branches
  become contorted, also causes dendrites to swell,
  break off, or disappear altogether.
• This all contributes to nerve cell death.

One method of cell death results from the release
of excess glutamate
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• Counting CAG repeats in the Huntingtons
• disease gene by restriction endonuclease
• EcoP15I cleavage.
• Möncke-Buchner, E., S. Reich, M. Mücke, M.
  Reuter, W. Messer, E.E. Wanker, and D. H. Krüger.
  2002. Counting CAG repeats in the Huntingtons
  disease gene by restriction endonuclease EcoP15I
  cleavage. Nucleic Acid Research 30 e83

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Study Objective

• Möncke-Buchner, et al. were interested in
  studying the numbers of CAG repeats in the HD
  gene by electrophoresis, and the cleavage after
  the insertion of EcoP15I.
• Use of more advanced methods compared to the PCR
  fragments were deletions and insertions of the
  gene occur during the test.
• For example Williams et al. found that
  amplifications products of the IT15 gene were
  greater with the use of capillary electrophoresis
  that using slab gel electrophoresis.

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Fig. 1A - Recognition and cleavage sites of
EcoP15I

• Recognition sequence for EcoP15I consisting of 2
  copies of the asymmetric sequence 5-CAGCAG
  present in opposite strands of the DNA double
  helix. Showing that cleavage occurs 25-27 bp
  downstream of one of these sites.
• This test was done in order to analyze the
  EcoP15I cleavage of HD in exon1.
• Offers new advantages over existing procedures
  for the quantification of CAG repeats. Exhibits
  high-resolution quality, is not hazardous,
  prevents isotopic waste, and is time saving.

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What is EcoP15I?

• Type III restriction-modification enzyme.
• Hetero-oligomeric proteins that behave as
  molecular machines in response to their target
  sequences.
• They translocate DNA in a process dependent on
  the hydrolysis of a nucleoside triphosphate.
• For the ATP-dependent type III restriction and
  modification systems, the collision of
  translocating complexes triggers hydrolysis of
  phosphodiester bonds in unmodified DNA to
  generate double-strand breaks.
• Type I endonucleases break the DNA at unspecified
  sequences remote from the target sequence.
• Type III endonucleases at a fixed position close
  to the target sequence.
• Notable for effective post-translational control
  of their endonuclease activity.

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Fig. 1B - Used DNA substrate at a distance
of 72 bp.

• Cleavage reactions were analyzed by
  polyacrylamide gel electrophoresis.
• EcoP15I cleavage requires the presence of 2
  5-CAGCAG sequences that are located on opposite
  strands of the DNA.
• A single inverse EcoP15I recognition site is
  located on the opposite strand, termed i at 72
  bp downstream of the CAG repeat.
• Arrows indicate the EcoP15I cleavage sites 25-27
  bp downstream of the various recognition sites.

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Fig. 1A and Fig. 1B

• In fig. 1A EcoP15I cleaves 25-27 bp downstream
  of one of the 2 recognition sites.
• In fig. 1B EcoP15I recognition site is located on
  the opposite strand, termed i at 72 bp
  downstream of the CAG repeat.
• In the case of exon1 cleavage can either occur in
  the inverse site i or 25-27 bp downstream of
  any of the (n-1) recognition sequences formed by
  the n CAG repeats.

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Fig. 1C - Calculated length of the DNA
fragments.

• Calculated lengths of EcoP15I cleavage products
  expected for substrates fCAG30, fCAG35, and
  fCAG81.

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Fig. 1D Cleavage patterns

• 10-fold excess of EcoP15I were added.
• Cleavage generated characteristic ladders of DNA
  fragment.
• Besides finding out that EcoP15I cleavages at
  the i site, they also found that multiple DNA
  fragments generated by cleaving within the CAG
  repeat.
• This means that EcoP15I recognizes and cleaves
  the multiple overlapping sites within the CAG
  repeat of HD in exon1 generating ladders of DNA
  fragments of different lengths.

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Fig. 2 Influence of EcoP15I
concentration on cleavage
efficiency and pattern.

• This test was perform in order to determine
  whether the concentration of EcoP15I can
  influence the cleavage efficiency.
• They found that by increasing the enzyme to
  substrate ratio enhanced the efficiency of
  cleavage.
• But as we could see in lane 4 the presence does
  not correspond with the findings.
• CAG repeats proximal to the inverse site were
  preferentially cleaved.

Lane 1 without EcoP15I Lane 2 EcoP15I molar
ration of 1 fmol Lane 3 EcoP15I molar ratio of
10 fmol Lane 4 EcoP15I molar ration of 20 fmol
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ALFexpress DNA Analysis System

• Since the ratio of EcoP15I enzyme to DNA
  substrate was too small to analyze, they used a
  high-resolution method called ALFexpress DNA
  Analysis System to separate and detect EcoP15I
  cleavage fragments.

• ALFexpress DNA Analysis System is used for high
  performance and reliability in DNA analysis.
• Employs a single-dye chemistry format in which
  Cy5 is used to label DNA fragments for detection.
  
• Supported by a wide range of software, sequencing
  and genotyping reagents, and convenient
  application kits.

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Fig. 3 - EcoP15I cleavage using
ALFexpress DNA Analysis System

• As mentioned before EcoP15I cleaved at the
  inverse i recognition site of EcoP15I and at
  the recognition site or 25-27 bp downstream of
  any of the (n-1) recognition sequences formed by
  the n CAG repeats.
• With the use of the ALFexpress DNA Analysis
  System they were able to count 29 DNA fragments
  in the ladder.
• This number corresponds to the 29 overlapping
  EcoP15I recognition sites within the 30 CAG
  repeats of fCAG30.

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Fig. 3 (cont.)

• This demonstrates that all EcoP15I recognition
  sites are recognized and cleaved by EcoP15I.
• They were able to determine the exact CAG repeat
  number.

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Fig. 4 Counting of CAG repeats using
ALFexpress DNA Analysis System

• Test performed in order to determine the repeat
  lengths in the borderline (30-39) and
  pathological range (more than 39).
• They used 3 substrates (fCAG30, fCAG35 and
  fCAG81) to study the cleavage pattern.
• Cleavage by EcoP15I resulted in a ladder of DNA
  fragments.

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Fig. 4 (cont.)

• EcoP15I cleaved at all 3 DNA substrates at the
  inverse EcoP15I recognition site and at each of
  the overlapping EcoP15I recognition sites
  5-CAGCAG within the CAG repeats.
• Concluding this test we can say that EcoP15I
  preferred recognition sites located proximal to
  the inverse recognition site over more distant
  ones.

• This preference is more likely caused by the
  faster formation of the active enzyme DNA complex
  when 2 EcoP15I recognition sites are located
  close to each other.
• Exact quantification of the CAG repeat numbers
  in the normal as well as in the pathological
  range are necessary in order to determine the
  number of glutamine repeats characteristic of HD
  as one of nine polyglutamine expansion disorders.

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Application

• The count of CAG repeats is applicable to the
  analysis of further hereditary neurodegenerative
  diseases caused by expanded CAG repeats.

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Prevalence

• In the United States about 30,000 people have HD.
• Prevalence is 1 in every 10,000 persons.
• At least 150,000 others have a 50 risk of
  developing the disease.
• HD appears to be less common in Japan, China,
  Finland, and African blacks.

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Management

• There is no cure to stop or reverse the curse of
  the disease.
• Medication may be used to treat psychosis,
  depression, or movement problems.
• Antipsychotic drugs may help to alleviate choreic
  movements and may also be used to help control
  hallucinations, delusions, and violent outbursts.
• Tranquilizers can help control anxiety,
  depressions, pathological excitement and sever
  mood swings.

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Questions?