Question 1 (10 marks)
This question is associated with Sections 1.5–1.7. It assesses the module learning outcome KU1.
- Describe the role of each of the following enzymes in genomic DNA replication:
- DNA primase
- DNA polymerase
- DNA topoisomerase
- DNA helicase.
(4 marks)
- Describe the process by which cells respond to the presence of an A–G pairing of two bases in a newly replicated section of DNA in order to restore the correct base sequence.
(4 marks)
- How does the synthesis of the two new strands during DNA replication differ?
(2 marks)
Question 2 (10 marks)
This question is associated with Sections 3.6 and 3.8 and with Activity 3.5. It assesses the module learning outcomes KU1 and KS1.
qPCR and sequence analysis are often used in combination to assess CYP2D6 gene status by determining copy number and DNA variants. An analysis of two parents with the DNA variants present on each of the individual’s chromosomes is shown below:
- Father: *10, *1–*1
- Mother: *4, *10
- Determine all possible CYP2D6genotypes of their children using a Punnett square.
(4 marks)
- Using information from Table 1 (reproduced from Topic 5 Table 3.3), what are the predicted CYP2D6 enzyme activity scores based on the predicted genotypes for each of their possible children?
(2 marks)
| Table 1 CYP2D6 enzyme activity scores for sequence variants (N = copy number of 2 or more). |
| Variant |
Activity score |
Variant |
Activity score |
| *1 |
1 |
*10 |
0.25 |
| *1×N |
>2 |
*11 |
0 |
| *2 |
1 |
*12 |
0 |
| *2×N |
>2 |
*14 |
0.5 |
| *3 |
0 |
*17 |
0.5 |
| *4 |
0 |
*29 |
0.5 |
| *5 |
0 |
*33 |
1 |
| *6 |
0 |
*41 |
0.5 |
| *7 |
0 |
*42 |
0 |
| *8 |
0 |
*53 |
1 |
| *9 |
0.5 |
*62 |
0 |
- Based on the consensus predicted activity scores presented in Table 2, what proportion of their children could potentially be predicted to have an intermediate metaboliser status for nortriptyline, based upon the likely impact of these sequence variants on CYP2D6 enzyme activity? Explain your answer.
(2 marks)
| Table 2 Consensus CYP2D6 metaboliser status activity scores for genotype to phenotype prediction (as of 2019). |
| Metaboliser status |
Consensus activity score |
| Ultra-rapid |
>2.25 |
| Normal |
1.25–2.25 |
| Intermediate |
0.25–1.25 |
| Poor |
0 |
- Which parent is likely to experience greater pain relief from codeine? Explain your answer.
(2 marks)
Question 3 (30 marks)
This question is associated with Sections 3.3, 3.8, 4.5, and 4.7. It assesses the module learning outcomes KU2, CS1, CS2 and KS2.
Michael, a young adult male, is referred from a specialist cardiac clinic for genetic testing to investigate a possible genetic origin for hypertrophic cardiomyopathy. He receives initial counselling about the genetic testing process, a family history is collected, and a blood sample is taken for further genetic investigation. Michael’s mother has no sign or symptoms of the disease and neither do his two younger sisters, who are identical twins. His father is deceased and he is uncertain as to what the cause of his father’s death was. It is explained to him that the investigation of his DNA uses targeted genome sequence analysis of the exons of genes that are known to be associated with hypertrophic cardiomyopathy, and after counselling he consents to his DNA being tested.
- In Topic 5 Activity 3.2 you practised collecting UV absorbance data from a DNA sample. Using the DNA and RNA quantitation laboratory, perform an analysis of the genomic DNA that has been isolated from Michael’s blood sample, which is labelled as DNA3.
- Present your absorbance findings using a table similar to Table 26 in Practical Workbook 2. Comment on the values obtained from the two experimental control samples.
(6 marks)
- Provide an analysis of your absorbance values collected from the undiluted DNA3 sample to confirm that the sample is free of contamination from co-purified protein and therefore suitable for further analysis.
(2 marks)
- A sample of Michael’s DNA is to be sent to a DNA sequencing laboratory for exon sequence analysis. Determine the concentration of the undiluted DNA3 sample using data from the 1 in 2 and 1 in 5 diluted samples. What volume (in μl) of the undiluted DNA would you collect to provide a sample containing 5 μg of DNA? Show how you determined both of these values.
(6 marks)
- (14 marks in total)
- After exon sequence analysis has been performed on DNA3, the findings indicate that Michael carries a mutation in one of his two copies of a gene called MYH7. Mutations in MYH7, which encodes a protein called myosin heavy chain 7, are a known cause of hypertrophic cardiomyopathy and they exhibit a dominant pattern of inheritance. It is likely that Michael’s father also carried the same mutation in MYH7that Michael carries.
- Using the Human gene expression database, research which tissues express the MYH7gene. Provide an image of your findings and the number of MYH7 transcripts present in tissues expressing the gene. What other tissue might be affected by a mutation in the MYH7 gene?
(5 marks)
- One of Michael’s younger sisters, Hannah, is to attend a genetic counselling clinic as she is planning to have children. Since Michael’s investigations, an additional molecular analysis has been performed that confirms that their mother does not carry any mutation in any gene likely to cause hypertrophic cardiomyopathy.
Before Hannah undergoes molecular testing herself, she is to receive counselling to learn more about the genetics of the disorder.
Based upon the information you have, draw a tree for the family using relevant symbols and identify Michael and Hannah on the pedigree.
(5 marks)
- What can you tell Hannah about her likely genotype and whether she may herself be affected by hypertrophic cardiomyopathy?
(3 marks)
- What issues arise from the genetic testing of Hannah with regard to her sister?
(3 marks)
Question 4 (50 marks)
This question is associated with Sections 2.6 and 2.7. It assesses the module learning outcomes CS2, KS2, KS5 and PPS1.
You are tasked with screening a group of individuals to determine their metaboliser status for nortriptyline ahead of a clinical trial in which ultra-rapid metabolisers for nortriptyline are to be excluded. In Activity 2.2 you collected data from the Drug metabolism laboratory and drew a standard curve for nortriptyline.
You should now prepare a standard curve for the metabolite using the following concentrations of 10-hydroxynortriptyline: 5, 10, 20, 40, 60, 70 and 80 nmol l–1.
Collect data from the five individuals in Group 4, use the two standard curves to estimate the concentration of drug and metabolite in each serum sample, and calculate the metabolite ratio for each individual in Group 4 using the guidance provided in Topic 5 Box 2.5. Space to record your data and suitable graph paper is provided in Practical Workbook 2, pages 61–65.
From your study, you should then prepare a written report outlining the study and your findings which answers parts (a)–(d) below. These parts will guide you to the content and style required in this report. A suggested length for each section is provided to help your planning and writing. Note that the total word count for all parts combined is a maximum of 700 words.
You practised writing a short report for TMA 04 using the same section headings, and you may find it helpful to review any feedback your tutor provided as to the style and content of each section.
- Introduction. This should be a brief explanation, written in your own words, of the background to the assays you performed, including:
- the objectives of your study
- the reason why metabolism varies between individuals
- a brief outline of approach you have used
- criteria for assigning metaboliser status.
(6 marks, around 150 words)
- Method. This should be a summary of the experimental aspects of how you collected and processed your data. You should write using the past tense and the text should allow another individual to repeat your study by following the guidance in Practical Workbook 2and obtain similar results. You should include only information on the following:
- how you collected peak area data and how many repetitions you performed
- how you prepared your 10-hydroxynortriptyline standard curve
- how you used your standard curves to determine the concentrations of drug and metabolite in serum samples
- how you calculated the metabolite ratios for nortriptyline.
(8 marks, around 150 words)
- Results. You should present a written description that guides the reader sequentially through your data that is summarised in a series of tables and figures, as detailed below. You should note any data points you discarded due to detector saturation. This written description should be around 300 words.
Your tables and figures should be inserted into your report, and you should refer to each within the written description of the data. Each figure and table should have a title. The following data should be presented:
- Figure 1: your standard curve for 10-hydroxynortriptyline.
- Figure 2: an image of the HPLC trace for individual 4-001, with the markers positioned for collection of the nortriptyline peak area.
- Table 1: your calculated drug and metabolite concentrations for the Group 4 individuals, their metabolite ratio, and metaboliser status – using the format given below in Table 3.
Note: the titles of your figures and the contents of your Table 1 do not count toward the word count total for Question 4.
(30 marks, around 300 words)
| Table 3 Analysis of Group 4 individuals. |
| Individual |
Serum 10-hydroxynortriptyline/nmol l–1 |
Serum nortriptyline/nmol l–1 |
Metabolite ratio |
Metaboliser status |
| Group4-001 |
|
|
|
|
| Group4-002 |
|
|
|
|
| Group4-003 |
|
|
|
|
| Group4-004 |
|
|
|
|
| Group4-005 |
|
|
|
|
- Interpretation. This should be a written description that summarises the interpretation of the data that you have presented as the metabolite ratios and metaboliser status of the Group 4 individuals in your Table 1. Indicate any individual/s that you recommend be excluded from the planned clinical trial because of their metaboliser status.
(6 marks, around 100 words)
Your total word count for parts (a), (b), (c) (excluding table contents and figure titles) and (d) combined should not exceed 700 words.
