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