Power Point Presentations

DNA Replication Transcription

Thalassemia

Tazswanas Story

Handout 7

Comparing and contrasting Tazswana’s pre-mRNA intron to the normal intron, answer the following questions (use terms such as 5’ splicing site, 3’ splicing site, etc.)
1. How is Tazswana’s intron similar to the normal β-globin pre-mRNA?
Tazswana’s intron is similar in that they both contain the same starting, close to the 5’ splicing site, and ending, close to the 3’ splicing site, nucleotides GU and CAG.

2. How is Tazswana’s intron different from the normal β-globin pre-mRNA?
Tazswana’s intron is different in that the central nucleotides are different: Tazswana with CAGNNNNGU and normal mRNA with AGNNNNNUU. The nucleotides in bold are also different.

3. Assuming that the cellular splicing machinery will “cut” at the GU 5’ splicing site and the very next 3’ splicing site, CAG, predict how Tazswana’s pre-mRNA will be processed into mRNA. Show the resulting mRNA from this process.
Tazswana’s pre-mRNA will be processed into a normal mRNA if the splicing occurs in the respective GU 5’ Splicing site and CAG 3’ splicing site. The resulting mRNA would be AGG, exon 2 with exon 3 and no intron involved.

4. If the doctors could somehow trick the splicesome (the cell’s machinery that splices pre-mRNA into mRNAs) into ignoring Tazswana’s extra splicing sequence, could she have a normal β-globin mRNA? Explain why or why not.
Yes, if Tazswana’s extra splicing sequence could be ignored and the normal sequence continues, then she could have a normal β-globin mRNA. Ignoring the extra sequence would cause the natural machinery to splice in the correct place making a normal mRNA that would carry out its function normally.

Handout 8—Pre-mRNA Gene Therapy for Tazswana

—What is “alternative splicing” and how does it apply to Tazswana’s case?
—Explain how hybridization (complementary base-pairing) of the DNA oligonucleotides to the cryptic and aberrant pre-mRNA sequences would correct the splicing defect.
—If Tazswana’s 5′ splice site at the correct location (exon2/intron2 boundary) had a point mutation from G to U so that the original 5′splice site read “UU” instead of “GU,” would the gene therapy correct her β-thalassemia? Explain your answer.
—(Optional) Since the oligonucleotides target splice site consensus sequences that are common to all introns, wouldn’t they potentially cause side-effects in many cell types? Discuss within your group and come up with:
—If you were in Tazswana’s family, would you want Tazswana to undergo gene therapy treatment? Explain why or why not.

Answers:


1. Alternative splicing is the RNA splicing variation mechanism in which the exons of the pre-mRNA are separated and reconnected so as to produce alternative ribonucleotide arrangements. These linear combinations then undergo the process of translation where specific and unique sequences of amino acids are specified, resulting in isoform proteins. Alternative splicing facilitates the synthesis of a greater variety of proteins.

2. The hybridization of the DNA oligonucleotides would correct the splicing defect since the attach both exons once again, as if the process never happened. When these oligonucleotides do this, they rewrite the nuclear information so the gene splicing would be executed correctly in the correct spots, specifically the 5’ splice site.

3. The gene therapy would correct Miss X’s beta thalassemia if the 5’ splice site is spliced in the correct location, having a point mutation from G to U so that it can read “UU” instead of “GU”. The gene therapy would basically rewrite the sequence, performing the splicing correctly and eliminating the beta thalassemia gene defect from her genetic code.

4. The oligonucleotides that target splice site consensus sequences will probably not cause any side effects. This process has only been done on a small scale and it proved to have no adverse effects on the subject.

5. If we were in Miss X’s family, we would certainly want her to undergo the gene therapy treatment since it would cure a disease that causes many side effects and a poor quality of life. The gene therapy would cure her from the genetic defect, thus helping her lead a normal life.

Miss X's RNA Test (Handout 6)

Figure 6-1. RNA Hybidization Analysis of Tazswana’s RNA. The grey triangle indicates relative size of nucleic acids: larger RNA molecules migrating higher in the gel than smaller RNA molecules. Lane 1, Mother’s RNA sample. Lane 2, Miss X"s RNA. Lane 3, Miss X's grandmother (see Handout 2: Beta-Globin Inheritance). Lane 4, control patient lacking any thalassemia phenotypes. [Credit: This figure has been modified from Figure 4 in Antonarakis, S.E., et al., β-Thalassemia in American Blacks: novel mutations in the “TATA” box and an acceptor splice site. Proc Natl Acad Sci U S A, 1984. 81(4): p. 1154–8.]

• Compare Lane 2 to Lane 4. In terms of pre-mRNA processing, explain why Tazswana has an extra band. Include drawings of the mRNAs shown in Line 4 versus those in Line 2.
  a. Lane four shows us two exons normally connected without any intron residue. On the other hand, in Lane two we find an extra band showing a much larger mRNA molecule. This extra band indicates that an intron was improperly sliced and connected between the two correct exons. This would make the subject’s mRNA molecule larger than a normal mRNA molecule.
• Predict how the size of her mRNA bands will affect the size and amino acid content of her β-globin protein. a. The size of her mRNA bands will increase the size and amino acid content of her β-globin protein. This is an altered shape of the normal protein and will not function the same way due to a change in shape.
• Tazswana has one normal size β-globin mRNA. Explain why she has severe symptoms and from whom she has inherited the intron mutation. a.The subject has severe symptoms since a splicing defect occurred and a part of an intron remained with the exons making the strip longer. This causes the β-globin protein to function incorrectly because of the altered shape. Thus this protein cannot carry out fully the function that is required of hemoglobin to carry oxygen to the body. She has inherited the intron mutation from both her parents. The father with the splicing defect and the mother the single base substitution in β-globin exon.
• Based on these results discuss Tazswana’s treatment options. a. Tazswana has thalassemia major. The primary treatment is regular blood transfusions, usually every four weeks. In addition to the blood transfusions, doctors recommend injections of Desferal to help the body flush out the extra iron created by the new blood. The injections are given under the skin from a small pump 5 to 7 nights a week. http://www.healthscout.com/ency/68/477/main.html
  b. Splenectomy is the surgical removal of the spleen, which is an organ that is part of the lymphatic system. Splenectomy is sometimes performed if the patient's spleen has become painfully enlarged. http://www.healthatoz.com/healthatoz/Atoz/common/standard/transform.jsp?requestURI=/healthatoz/Atoz/ency/splenectomy.jsp
  c. Bone Marrow Transplant is a procedure that transplants healthy bone marrow into a patient whose bone marrow is not working properly. A bone marrow transplant may be done for several conditions including hereditary blood diseases, hereditary metabolic diseases, hereditary immune deficiencies, and various forms of cancer. http://www.nlm.nih.gov/medlineplus/ency/article/003009.htm
  d. Chalation Therapy: chemical solutions are used in an attempt to prevent or reverse cardiovascular problems. By binding tightly to calcium and other minerals, chelating agents are thought to remove such substances from atherosclerotic plaques. http://www.healthscout.com/ency/68/422/main.html
  e. Alternative Gene Splicing is the coding and non-coding fragments of the gene can be arranged in different ways. When this produces different m-RNA sequences from the same parent gene, the phenomenon is known as Alternative Splicing. The biological consequences of this process can be severe as the same gene leads to formation of different proteins which may be functional, non-functional or malfunctioning. http://www.premierbiosoft.com/bacterial-identification/realtime-PCR/alternative-splicing.html
• Assume the β-globin mRNA in Lane 3 is from Tanzswana’s grandmother. How is it different from the normal mRNA shown in Line 4? Discuss the cause of this difference in terms of the mutation and why Tanzswana’s grandmother is symptom free.
  a. The grandmother’s mRNA is smaller and dimmer than the normal mRNA. This difference is due to the fact her β-globin promoter is removed and not expressed. She is symptom free because she is not a carrier a β-globin error or splicing defect.

• Written on April 9, 2008

Bad News (Handout 5)

The doctor joined the mother for another appointment. This time, it was concluded that Miss X had thalassemia. It is a genetic blood disorder where the red blood cells have an abnormal shape. Miss X's blood cells cannot properly carry oxygen to the body tissues because the abnormal proteins cannot bind oxygen. As a result, Miss X will be physically limited.

Thalassemia has no easy and obvious cure. However, the beta-thalassemia patients are treated with transfusions. Miss X's severe form of thalassemia will cause her to have a severe quality of life and treatment with transfusion would have minimal results. Most likely, Miss X will die in her early teen years.

As for more complex cures, Miss X could try a bone marrow transplant. Also, she may be an excellent candidate for possible experimental gene therapy. If this course is folloed, the doctor would have to test Miss X's RNA. If the tests indicate thats she has an abnormal beta-globin, then Miss X may qualify for the experimental study. This specific change meay be determined by testing Miss X's RNA. Altered mRNA can be detectable using a test called the Northern Blot. The altered mRNA would be larger than the normal one. This larger size is due to incorrect splicing of the beta-globin pre-mRNA. Instead of having a beta-globin mRNA with exons spliced directly together, part of an intron would be retained between the two coding sequences; part of the beta-globin non-coding information would have become part of the code for her beta-globin protein. As a result, the beta-globin protein would not be translated correctly and would cause her symptoms. In this case, a second test would need to be run to verify that Miss X has the specific intron mutations that the gene therapy is designed to correct. By loking at her beta-globin pre-mRNA sequence, one can check this out, making Miss X an excellent candidate for pre-mRNA gene therapy.

(Written on April 8th, 2008)

Miss X's Blood Test (Handout 4) Analysis

Questions

1. Compare the size, shape and color intensity of the red blood cells. Record your observations in the chart below.

2. Thinking about the function of hemoglobin and the shape of Miss X’s red blood cells, explain why Miss X has severe symptoms compared to her mother or a normal person.

The subject has severe symptoms since the function of hemoglobin is to transport oxygen to the body for aerobic respiration, and her red blood cells, are small and weak due to the color and distortion of shape. Compared to the mother and a normal person, the altered shape causes a protein to function improperly or not at all making her symptoms severe.

Written on April 7, 2008

Miss X's Blood Test (Handout 4)

←A C
→B

Panel A: Normal sample

Panel B: Mother's sample

Panel C: Miss X's Sample

Credit: Donald Innes, M.D. Used with permission.

Credit: Copyright © The American Society

for Clinical Laboratory Science.

Used with permission.

Written on April 7, 2008