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					   Huntington’s 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
Huntington’s disease gene by restriction endonuclease EcoP15I
cleavage. Nucleic Acid Research 30: e83
             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
Overview of Huntington Disease (HD)

    Hereditary degenerative neurological disease that
     leads to dementia.

        Named after George Huntington, who first described it in

                                     DR. GEORGE HUNTINGTON
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.
                  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 Cerebrum
                                     2 Basal Ganglia
                                     3 Brain stem
                                     4 Cerebellum
                     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.
                How is HD Inherited?

   Genes are composed of
    deoxyribonucleic acid (DNA), a
    molecule shaped like a spiral
   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).
               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.
             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
   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.
         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
   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.
          How can the structure of
           huntingtin be altered?
                                                             U           C           A            G
   If there are 41 CAG repeats in a row inside
    the gene, there will be 41 glutamines
                                                        Phenylalanine Serine    Tyrosisne    Cystine

                                                        Phenylalanine Serine    Tyrosisne    Cystine       C
    attached to each other in a row inside the          Leucine       Serine    STOP         STOP          A
    huntingtin protein.                             U   Leucine       Serine    STOP         Tryptophane   G
    The production of multiple glutamines              Leucine       Proline   Histidine    Arginine      U
    causes mutant huntingtin to fold into a             Leucine       Proline   Histidine    Arginine      C
    different shape than normal.                        Leucine       Proline   Glutamine    Arginine      A
   Mutant huntingtin interacts with all sorts of   C   Leucine       Proline   Glutamine    Arginine      G
    other proteins that might normally be               Isoleucine    Threonine Asparagine   Serine        U
    ignored, particularly in the spiny neurons          Isoleucine    Threonine Asparagine   Serine        C
    deep inside the brain.                              Isoleucine    Threonine Lysine       Arginine      A
                                                    A   Methionine    Threonine Lysine       Arginine      G
                                                        Valine        Alanine   Aspartic acid Glycine      U
                                                        Valine        Alanine   Aspartic acid Glycine      C
                                                        Valine        Alanine   Glutamine    Glycine       A
                                                    G   Valine        Alanine   Glutamine    Glycine       G
          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
Counting CAG repeats in the Huntington’s
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
  Huntington’s disease gene by restriction endonuclease EcoP15I
  cleavage. Nucleic Acid Research 30: e83
                 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.
 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
                   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
   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.
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.
                  Fig. 1A and Fig. 1B

   In fig. 1A EcoP15I cleaves 25-27 bp downstream of one of the 2 recognition
   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.
     Fig. 1C - Calculated length of the
               DNA fragments.

•Calculated lengths of EcoP15I
cleavage products expected for
substrates fCAG30, fCAG35, and
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
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
                                              • They found that by
                                              increasing the enzyme to
                                              substrate ratio enhanced the
                                              efficiency of cleavage.
                                              • But as we could see in lane
   Lane 1 – without EcoP15I
                                              4 the presence does not
   Lane 2 – EcoP15I molar ration of 1 fmol    correspond with the findings.
   Lane 3 – EcoP15I molar ratio of 10 fmol    •CAG repeats proximal to
                                              the inverse site were
   Lane 4 – EcoP15I molar ration of 20 fmol
                                              preferentially cleaved.
    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.
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
                 • This number corresponds to the 29
                 overlapping EcoP15I recognition sites
                 within the 30 CAG repeats of
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.
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.
                            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

• 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.

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

   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.
   There is no cure to stop or reverse the curse of the
    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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