Sicklecell Anemia - A Disease of Diverse Populations

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Sicklecell Anemia - A Disease of Diverse
Populations
Jennie Aizenman Scott Bronson Uwe Hilgert
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Sickle Cell Anemia

• Red blood cells shaped like sickles.

A genetic disorder HBBA, HBBS Severe if both
copies of gene are affected. Benign if only one
copy of gene is affected.
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Why does disease persist?

• HH healthy
• HS benign
• SS severe

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Malaria prevalence
HBS Allele
Mosquito (Anopheles species)
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Genes and Life

• How DNA Encodes Life
•  
• DNA ? RNA ? Protein
• DNA - What are genes?
•  
• Transcription Copying the code
• Translation Reading the code
• Code Table - The Code

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What Are Proteins?

• Polymers of amino acids
• 20 different amino acids
• Consist of core (-NH-CO-) and side group.
• 20 side groups ? 20 characteristics
• Go to http//info.bio.cmu.edu/Courses/Bioche
  mMols/AAViewer/AAVFrameset.htm
• Set left molecule to alanine.
• Set right molecule to tryptophane.
• How do the two amino acids differ?
• Examine glutamate, proline, valine.

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How Do Proteins Work?

• Proteins Structure, Catalysts, Sensors,
• Proteins are polymers (long chains) of amino
  acids.
• Sequence ? Structure ? Function
• Lets look at a protein together.

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Bioinformatics

• DNA and protein sequences are stored in
  databases. Bioinformatics provides the tools to
  handle and analyze these data. Bioinformatics is
  the science of unraveling the information in
  biological molecules utilizing computer
  technology.

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Bioinformatics Structures in Databases

• NCBI National Center for Biotechnology
  Information
• Open browser, go to http//www.ncbi.nlm.nih.gov/
• Find Search Entrez for.
• Change Entrez to Structure.
• Type hemoglobin (after for).
• Click Go.
• Find entry 1KOY, click it, click View 3D
  Structure.

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Hemoglobin

• Oxygen is picked up by iron (Fe)
• which is held by porphyrine (ring),
• which is presented by protein chain,
• of which 4 aggregate in Hb molecule,
• so that together (cooperatively) they influence
  irons affinity to oxygen

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Allosteric Effect - Cooperativity

• http//www.rcsb.org/pdb/molecules/pdb41_2.html
• Hemoglobins 576 amino acids determine its
  ability to bind and release oxygen and serve as
  oxygen transport vehicle in red blood cells.

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Find the Genes

• GenBank database maintained by the National
  Center for Biotechnology Information (NCBI)
• Go to http//www.ncbi.nlm.nih.gov/
• Which organisms can you find mentioned at the
  NCBI homepage?
• What other things catch your attention?

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Roadmaps for Genomes

• Map Viewer tool to locate genes
• Go to http//www.ncbi.nlm.nih.gov/.
• Find Map Viewer, click on it.
• Which genomes can you access?
• Click homo sapiens.
• Answer the questions on your worksheet.

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Search the Map

• Find hemoglobin gene(s)
• Find Search for.
• Into the search window type hemoglobin.
• Click Find.
• Answer the questions in the worksheet.

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Find the Hemoglobin Beta Gene (HBB)

• Click on HBB.
• Answer the questions on the worksheet.

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Find the HBB Nucleotide Sequence

• Go to http//www.ncbi.nlm.nih.gov/.
• Find Search Entrez for.
• Delete Entrez.
• Behind for write hemoglobin homo sapiens.
• Click Go.
• Answer the questions in the worksheet.

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Find and Examine the Nucleotide Sequence

• Click Nucleotide sequence database (GenBank).
• Click Homo sapiens hemoglobin, beta (HBB) mRNA.
• Click on NM_000518.
• Answer the questions on the worksheet.

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Work With the Nucleotide Sequence

• Transfer RNA sequence to database
• Highlight and copy nucleotide 1 through 626.
• Go to http//www.bioservers.org/bioserver/
• Under SequenceServer click Enter.
• Click CREATE SEQUENCE.
• Paste the sequence into the big window.
• Write HBB mRNA into the Name window.
• Click OK.
• This is the RNA sequence, its 626 bp long.

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Find the Gene in the Human DNA Sequence

• How?
• Whats the difference between mRNA and a gene?
• So, how does having mRNA help you find its gene
  in DNA?

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Align mRNA with DNA to Identify Gene Structure

• Copy HBB cDNA from SequenceServer.
• Open a second browser window.
• Go to SIM 4 http//pbil.univ-lyon1.fr/sim4.php.
• Paste the sequence into the cDNA window.
• In SequenceServer, change HBB cDNA to HBB gene.
• Transfer sequence into the lower window of the
  SIM4 tool.
• Click Submit.
• Save result onto desktop and visualize the
  alignment with LalnView.

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How Can Genes Cause Disease?

• What is a genetic disorder?
• An inheritable disorder.
• Discuss healthy vs. carrier vs. disease.
• Beta globin gene HbbA (normal gene/protein)
• Sickle cell gene HbbS (mutated/faulty
  gene/protein)
• Healthy people HbbA HbbA
• Sickle cell trait HbbA HbbS (carrier, sickle
  cell disease)
• Sickle cell anemia HbbS HbbS

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Find the Mutation in the Gene

• How? Whats a mutation?
• Align the sequences in SequenceServer.
• Go to http//www.bioservers.org/.
• Change Classes to Public.
• Find HBB and Sickle Cell Anemia.
• On the left, check the box.
• On the bottom, click OK.

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• Find HBB cDNA, homo sapiens.
• What does cDNA stand for?
• Find the word None, click it.
• Click HBS CDS, homo sapiens.
• What does CDS stand for?
• Find COMPARE.
• Set box next to it to Align Clustal W.
• Click COMPARE WAIT!!

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Find Mutations

• Mutations are changes in sequences.
• Find the differences between the HBB and HBS
  nucleotide sequences.
• Answer the questions in the worksheet.

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Find Mutations

• How?
• Identify what changes the nucleotide differences
  cause within the protein.
• First translate the HBS and HBB coding DNA into
  their respective amino acid sequences.
• How?
• Code Table - The Code

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How do the DNA mutations affect the protein?

• Translate DNA into amino acids.
• Click Open for HBB cDNA, homo sapiens.
• Move the cursor just before the A of the ATG on
  the third line.
• Hit Return/Enter on your keyboard, this moves the
  ATG to the fourth line.
• Highlight and copy the sequence from the ATG to
  the end (dont worry about the stop codon ).
• Click Done.

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How do the DNA mutations affect the protein?

• Translate DNA into aminoacids.
• Open a new browser.
• Open Gene Boy http//www.dnai.org/geneboy/.
• Click Your Sequence.
• Paste the sequence into the workspace.
• Click Save Sequence (should yield 576
  nucleotides).

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How do the DNA mutations affect the protein?

• Translate DNA into amino acids.
• On the Operations panel to the right click
  Transform Sequence.
• Select Amino Acids..
• Highlight the sequence under Reading Frame RF1
  and copy it.
• Open the Word program and paste the amino acid
  sequence into it.
• Place a carriage return at the end of the
  sequence.
• Place a gt sign in front of the sequence,
  followed by the letters HBB.
• Type a carriage return.

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How do the DNA mutations affect the protein?

• Repeat the process for the sickle cell mRNA (HBS
  CDS, homo sapiens) with the following
  modifications
• Use sequence from HBS CDS, homo sapiens instead
  of HBB cDNA, homo sapiens
• copy the entire sequence
• pasting this sequence into GeneBoy should yield
  you 444 amino acids
• write HBS before the sequence instead of HBB.

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How do the DNA mutations affect the protein?

• Align the amino acid sequences for HBB and HBS.
•  Highlight and copy the content of the Word-file.
• Go to http//www.ebi.ac.uk/clustalw/.
• Find Enter or Paste a set of Sequences and
  paste the sequence into the box.
• Click Run.
• WAIT!

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How do the DNA mutations affect the protein?

• The result window shows an alignment of the two
  amino acid sequences.
• Underneath the alignment is a string of stars
  denoting identical amino acids. Find the amino
  acid differences between HBB and HBS. Ignore,
  however, the end where only HBB shows amino
  acids this region is not part of the HBB
  protein. The HBB as well as the HBS proteins end
  with the amino acid sequence AHKYH.
• What are the differences between HBB and HBS?

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Effect of the Glu ? Val mutation on HBB Structure

• How do valine and glutamate differ?
• http//info.bio.cmu.edu/Courses/BiochemMols/AAView
  er/AAVFrameset.htm.
• Set left window to valine.
• Turn the molecule so that the aminoacid-core
  molecules, the red/blue V, is positioned on
  top.
• Set the right window to glutamate position
  red/blue V on top.
• Compare and contrast the two structures.
• Answer the questions in the worksheet.

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Effect of the Glu ? Val mutation on HBB Structure

• How do HBB and HBS differ?
• Open http//www.dnalc.org/bioinformatics/sickle_ce
  ll_module/hbb_cn3d.val.
• Open http//www.dnalc.org/bioinformatics/sickle_ce
  ll_module/hbbs_cn3d.val.
• Close the Message Log windows.
• Align the Cn3D windows side by side.
• Place each Sequence windows underneath its
  structure.

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Effect of the Glu ? Val mutation on HBB Structure

• How do HBB and HBS differ?
• Can you see any difference between HBB and HBS?
• Highlight the differences E in HBB and V in HBS.
• Orient highlighted amino acids in similar
  positions.
• Can you identify differences now?
• Go to Style, Rendering Shortcuts, Worms.
• Zoom in on the highlighted amino acids.
• Go to Style, Rendering Shortcuts, Toggle
  Sidechains.
• Go to Style, Coloring Shortcuts, Structure.
• What difference do you see between HBB and HBS?

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Align the HBS and HBB Proteins

• Go to NCBI at http//www.ncbi.nlm.nih.gov/.
• Find the words Search Entrez for.
• Change Entrez to Structure.
• Into the search window type HBS hit Go.
• Click on 2HBS.
• Click on the term Chain B (find the blue bar ).
• Click on View 3D Structure.
• Click on Open.
• Maximize the Cn3D screen align the Sequence
  screen underneath close the Message Log screen.

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Align the HBS and HBB Proteins

• How different are the two proteins?
• Identify and highlight in both sequences the
  amino acid thats different.
• Can you see a difference now?
• Go to Style, Rendering Shortcuts, click Toggle
  Sidechains. Make sure the V and E in position 6
  of both sequences are highlighted.
• Can you see a difference now?
• Change the highlighting from position 6 to
  position 5 (Proline P).
• Can you see a difference now?

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Summary
Glutamic Acid, E Hydrophilic, Polar
Valine, V Hydrophobic, Non-polar
?
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• Jennies slides

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http//r2d2.dnalc.org/media/a/sickle_cell_big.swf
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http//www.ygyh.org/sickle/causeb.htm
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http//www.rcsb.org/pdb/molecules/pdb41_2.html
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http//www.ygyh.org/sickle/have02.htm
http//www.ygyh.org/sickle/have03.htm
http//www.ygyh.org/sickle/have07.htm
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Geographic Distribution of Hemoglobin S
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Geographic Distribution of Malaria
                                               
                                             
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Malaria Caused by protozoa of the Plasmodium
genus. Overlap of malaria endemic areas
(Plasmodium falciparum) and areas where sickle
cell mutation occurs. Sickle cell mutation
confers protection from Plasmodium
falciparum. Anopheles mosquito
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Malaria infects 300 to 500 million people
worldwide and accounts for over 1 million deaths
annually.
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