A main feature of Huntington's disease (HD) is a loss of neurones from a region of the brain known as the striatum. A study of brain tissue taken from people with HD showed higher than normal (increased) urea concentrations in the anterior striatum and cerebellum regions of the brain. Urea is a toxic waste product of amino acid and protein metabolism. It is produced from ammonia, which is also toxic, in an enzyme-controlled cycle known as the urea cycle. Scientists carried out a study using transgenic (genetically modified) sheep, known as OVT73, to investigate the increased urea concentrations. OVT73 have the mutant allele of the Huntingtin (HTT) gene. At the time of the study, OVT73 were 5 years old and had no detectable loss of striatal neurones or disease symptoms. Samples of tissue were taken from the same region of the anterior striatum in: • six OVT73 (three females and three males) • a control group of non-transgenic sheep.

1. (a)Describe two features of an appropriate control group for this investigation.[1]
2. (b)A whole-genome RNA-sequencing experiment was carried out on the tissue samples using a commercially available diagnostic testing kit. This experiment produced gene expression profiles for comparison. From the hundreds of genes that showed differential gene expression, 24 genes were described as genes of interest because they were involved in urea metabolism or in urea transport out of cells. None of these were genes coding for enzymes in the urea cycle. The 24 genes of interest were identified from RNA sequencing using mRNA transcripts. Explain why it was more appropriate to use RNA sequencing rather than DNA sequencing for the identification of these genes of interest.[2]
3. (c)(i)State which statistical test would have been used to establish the statistical significance between the control and OVT73 data sets. Explain why the test chosen was suitable. statistical test explanation[2]
4. (c)(ii)State the null hypothesis for the investigation.[1]
5. (c)(iii)With reference to Table 2.1, explain the advantage of calculating the ratios.[2]
6. (d)The results of the two RNA-sequencing experiments indicate that there is a difference between OVT73 and the control group in the expression of each of the ten genes shown in Table 2.1. State one feature of the study that contributes to the validity of these results and one feature that would improve the validity of these results. feature that contributes to validity feature to improve validity[2]
7. (e)(i)Complete Table 2.2 by calculating the ratio for the motor cortex.[1]
8. (e)(ii)With reference to Table 2.2, explain how the standard error (SE) values for mean urea concentration are good indicators of the estimates of statistical significance.[1]
9. (e)(iii)SLC14A1 codes for a transport protein that functions in the facilitated diffusion of urea out of cells. Explain the relationship between the results shown in Table 2.2 and the increased gene expression shown by SLC14A1.[1]
10. (f)The results of the study show a relationship between the increased urea concentrations and Huntington's disease, but also highlight the need for further investigation. Some possible causes of increased urea concentrations in cells are: 1. a changed expression in the genes coding for the enzymes of the urea cycle 2. an increase in protein breakdown related to cell death 3. an increase in protein breakdown to provide more energy to a cell that has an increased rate of metabolism. Explain why the results of this study indicate that possible causes 1 and 2 are less likely to be involved in the early stages of HD than possible cause 3.[2]