Case Studies – ALS and HEMB Purpose: The purpose of this assignment is to make the connection between how a certain mutation/allele of a gene at the DNA level causes physical symptoms at the organism...

Case Studies – ALS and HEMB

Purpose:
The purpose of this assignment is to make the connection between how a certain mutation/allele of a gene at the DNA level causes physical symptoms at the organism level. You will be looking at two different cases studies involving two different genes and two different physical disorders. You will be introduced to a variety of online databases and resources that real scientists use and trust, such as OMIM, UniProt, and Clinicaltrials.gov.

Task:
Use the information from the sites provided to answer the questions. [You will also need some of the information from Chapter 11 in order to answer the questions on protein structure and signal hypothesis, so you may want to skim through that PPT if you get stuck on a question in the assignment.] Please simply send me an e-mail if you get stuck.

Criteria:
You will be graded on correct answers and evidence of correct thought-processes.

The
National Center for Biotechnology Information
(NCBI) is a division of the National Library of Medicine (NLM) at the National Institutes of Health (NIH). The NCBI maintains many enormous databases, including
OMIM, a database that contains thousands of entries for genes and genetic disorders, and is frequently updated by NCBI staff based on published work in the scientific community. Another comprehensive database called
UniProt
contains similar information as OMIM and is supported by a collaboration between the NIH and several European scientific organizations.

Both of these sites provide reliable, up-to-date information on genes, mutant alleles, proteins, and genetic disorders which can be easily accessed by science professionals. Recent publications are cross-referenced.

Today, you will use OMIM and UniProt to research two genetic disorders, and the mutant proteins which cause them. You will then view the 3D protein structures using a simple online resource called
Proteopedia, and look up recent information regarding FDA trial status on
clinicaltrials.gov.

By the end of this activity, you should be able to describe the effect of the common gene mutations on the proteins’ structures and functions, and relate these things to the physiological symptoms of the diseases that are caused by these mutant alleles.

What does the acronym “OMIM” stand for? ______________________________________________

What does “UniProt” stand for? _______________________________________________________

Case Study 1: ALS and SOD1

Activity Instructions:

1. Go to
   OMIM.
   Search OMIM for “FALS” (familial ALS)

1. Scroll down to locate the following entry:


2. #105400, Amyotrophic Lateral Sclerosis 1; ALS1

2. Click on the
   #105400
   to gather background information on ALS, the disease caused by mutant SOD1.

Focus on the Text, Description, and Clinical Management sections. [You will need to scroll down quite a bit to find some of these sections.] Answer the following questions:

1. What does “ALS” stand for?

______________________________________________________________

2. 15 to 20% of cases of familial amyotrophic lateral sclerosis type 1 (“FALS” or “ALS1”) are associated with a mutation in
   what gene?
   
   

______________________________________________________________

3. What is the cytogenetic
   location
   of that gene? _____________________

4. 
   Most
   cases of FALS follow a(n) _____________ _________________ mode of inheritance, although rare cases of ________________ ______________ inheritance have been reported.

5. Amyotrophic lateral sclerosis is a neurodegenerative disorder characterized by the
   
   

death of ________________________________________ in the

_____________________________________ resulting in fatal ________________.

6. How is the famous baseball player Lou Gehrig related to this disease?
   

______________________________________________________________

7. Why is euthanasia (assisted suicide) an issue involved with ALS?
   
   

______________________________________________________________

______________________________________________________________

8. What is the
   title
   of the bestselling book by Mitch Albom, describing the last year in the life of an ALS patient? (Mouse over the name “Albom” to see the book title.)
   

______________________________________________________________

3. Go to the
   UniProt
   page and use the search bar (at the very top) for the gene name (from part (b) above). Click on the entry number for the human version of the gene (entry # P00441).

4. Answer these questions (focus on the Function, Pathology and Biotech, and Interaction sections):

a. What specific reaction (i.e. reactants à products) is catalyzed by superoxide dismutase, when it is functioning normally?

____________________________________________________________

b. This enzyme protects cells against _______________________________________.

c. What 2 metal ions (cofactors) are contained in the enzyme? ______________________

d. The normal SOD1 protein has what type of quaternary structure? _______________

e. When the SOD1 gene contains certain mutations, the mutant protein forms large “fibrillar aggregates” inside of the neurons, which hinders the motor neuron’s ability to function.

Which “natural variants” (ie mutations) cause an increase in fibrillar aggregates? List all the positions (number) and the amino acid changes that result from the mutations. I did the first one for you. [Hint: There are 5 more.]

Natural variants:

Rewritten in standard format:

position 5, AàV A5V

______________________ ________

______________________ ________

______________________ ________

______________________ ________

______________________ ________

The standard way to express a missense mutation is to list the normal amino acid, the amino acid position number (within the whole protein), and the mutant amino acid.

So “position 5, AàV” should be rewritten as “A5V”

Rewrite the other mutations in this standard format.

6. Since the enzyme functions to remove toxic radicals from cells, what would “build up” (or accumulate) in a cell that does not have functional enzymes of this type? (This is not a trick question.)
   

______________________________________________________________

7. What effect does this build-up (see your answer to f) have on cells (especially on motor neurons)?

______________________________________________________________

5. Now we will look at the 3D structure of the normal SOD1 protein
   here
   (http://proteopedia.org/wiki/index.php/Superoxide_Dismutase).

On the right side,
you should see a rotating structure of two SOD1 polypeptides.
Note that you can click and drag on the image to rotate it in 3D.

What secondary structures (α helix and/or β sheet) are visible? How many are there?

(each of the red/orange spiraling ribbons is considered one α helix, and each of the flattened blue ribbons is considered one β sheet. Protein strands that are neither α helix nor β sheet are shown as thinner strings, not flat and ribbon-like. Now answer the question based on the image of normal
SOD1)

________________________________________________________________

6. Stem cells are very promising as a treatment for neurodenerative disorders (ALS, Parkinson’s, etc). Go to
   www.clinicaltrials.gov
   and search for clinical trials regarding amyotrophic lateral sclerosis and stem cell therapies. This is what to type in the search box:

Find the completed study that has results, and click on it.

What is the name of the study?____________________________________________

_____________________________________________________________________

Define autologous._____________________________________________________

For the Primary Outcome, what did they measure, and how?____________________

____________________________________________________________________

____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

The results of this study were published in what 2 journals? Look up the full journal names rather than using the abbreviations.

___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

Case Study 2: HEMB and F9

Activity Instructions:

7. Go to
   OMIM.
   Search OMIM for “HEMB”

Locate the following entry:

#306900. Hemophilia B; HEMB

8. Click on the
   #306900
   to gather background information on HEMB, the disease caused by mutant F9.

Focus on the Text, Description, “Manifesting Females” in Clinical Features, and Inheritance sections. [You will need to scroll down quite a bit to find some of these sections.] Answer the following questions:

a. What does “HEMB” stand for?

______________________________________________________________

b. What are some other names for this disease?

______________________________________________________________

c. Individuals with HEMB have a mutation in the gene called F9 (or “FIX” if using Roman numerals), which encodes what protein? (Hint: Read the information under Text.)

______________________________________________________________

d. What is the chromosomal location of the gene? ________________________________

[So it would be accurate to say that the gene is _____ - linked.]

e. What is the phenotype (symptoms) of hemophilia B? (Hint: you might

click on the reference to hemophilia A, since both diseases have the same symptoms.)

______________________________________________________________

______________________________________________________________

f. Under “Manifesting Females, the following case information is given:

Lascari et al. (1969)
described a daughter of a male with hemophilia B who had an XX karyotype, factor IX level of 5%, and hemarthrosis. The factor IX level in the mother was 100%. The girl was thought to be a manifesting heterozygote with unfortunate lyonization.

The female described above has a father with Hemophilia B and a normal mother. She herself only produces 5% of the normal F9 protein, so she basically has hemophilia as well. That’s her phenotype. What is her genotype?___________________________________________________

Explain what “lyonization” is. When does it occur? How many cells are in the embryo when it occurs? (Hint: the Wikipedia definition is misleading and just not very good. Use a more reliable medical/biological source please.)

______________________________________________________________

______________________________________________________________

______________________________________________________________

What is the probability that any 1 of the cells in the 16-cell embryo will express the normal allele, while the other 15 cells will inactivate the normal allele? (Show your work.) This kind of outcome would still result in hemophilia for that female. (Hint: use the “factorial” equation from Lab 3) ____________________________________________________________

______________________________________________________________

______________________________________________________________

9. Go to the
   UniProt
   page and use the search bar (at the very top) for the protein name (from part (c) above). Use the Roman numeral (IX) instead of the Arabic numeral (9). Click on the entry number for the human version of the gene (entry #P00740).

10. Answer these questions (focus on the Function, Subcellular Location, and Interaction sections):

a. What is the function of this protein, when it is functioning normally?

______________________________________________________________

b. What vitamin is required for the function of this protein?______________________

c. What metal cofactor is required?______________________

d. The subcellular location for this protein is listed as ______________________

e. The normal protein has what type of quaternary structure? _______________

11. Now we will look at the 3D structure of the normal F9 protein
    here
    (http://proteopedia.org/wiki/index.php/Coagulation_factor_IX).

On the right side,
you should see a rotating structure of coagulation factor IX. There are actually two coagulation factors shown, and each is a heterodimer: one heterodimer is made up of pink and yellow subunits, and the other one is made up of green and blue subunits.
Note that you can click and drag on the image to rotate it in 3D.

1. On the left side of the entry, scroll down to the section called “Gene Structure and Expression.” How many intron and exons are in the gene?

______________________________________________________________

2. What does exon 1 encode? _______________________________________________

3. Explain how this relates to the subcellular location described in the answer to question 10d above.

______________________________________________________________

______________________________________________________________

12. Go to
    www.clinicaltrials.gov
    and search for active gene therapy trials regarding hemophilia B. This is what to type in the search box:

a. Pick the first trial listed and click on it. What is the official title of the study you picked? _________________________________________________________________________________________________________________________________________________________________________________

b. List some of the “Outcome Measures” (ie the things that the researchers will be measuring during the study, with regard to the participants) _____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

c. What are the 4 “inclusion criteria” for participants in the study? _____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

Referenced URLs:

www.ncbi.nlm.nih.gov
/
omim

http://proteopedia.org/

www.uniprot.org

www.clinicaltrials.gov