Bioinformatic Analysis for Gene and Protein abnormalities - Patient Case Study

Task 1: Sequence identification

A patient, herein referred to as ‘Patient X’, aged 42 y, was recently admitted to hospital complaining of fatigue and muscle problems in his legs (rigidity), which is causing him some difficulties in walking. He is worried as this is affecting his work and it seems to be progressively worsening. Upon admittance, he was subjected to a short physical and psychological evaluation, which revealed that in addition to muscle problems, Patient X was having trouble organising his thoughts and his told the doctors that he had recently become a little withdrawn, but prone to random verbal outbursts.

1: Sequence identification    

Identify the sequences in Figures 1 and 2 by BLASTn analysis. Ensure that you demonstrate use of bioinformatics tools by including a legible screen shot with accompanying legend of your analysis and an appropriate description. Ensure you use the settings nucleotide collection nr/nt’ and select ‘somewhat similar sequences (blastn)’.

2: Sequence information  

Provide the following information about the sequences in Figures 1 and 2: corresponding gene name, gene symbol, chromosomal location and exon count of the (full) gene(s).

3: Genomic context      

Look carefully at the genomic context of both identified genes (in the NCBI Gene database), do you see any interesting features up- or downstream of the genes of interest? Take a screenshot of each genomic locus and describe any relevant features in the figure legends.

4: Gene expression   

Looking at the expression data available on the ‘Gene’ database page. Where are the genes, identified in task 1, predominantly expressed? How does their expression relate to their function?

How does the expression of these two genes relate to their normal function? (250 words max)

5: Nucleotide alignment          

Using an alignment tool, identify any mutations in the sequences between the two sets of alleles. Provide evidence that you have used alignment software to conduct this task. You will need to compare the allelic sequences to the known database sequences. Identify and explain any differences you find with reference to literature.

6: Peptide alignment and mutation identification  

It is important to verify if any sequence abnormalities that occur at a nucleotide level will affect the peptide sequence. Use an online translation tool to translate the nucleotide sequences into amino acid sequences. Then align all the amino acid sequences using an online alignment tool. Provide annotated pictures of your alignments, with legends, then identify and summarise your findings, with reference to literature.

7: Further analysis of mutations 

It remains unclear at this stage whether any differences found in the amino acid sequences (in task 6) will be detrimental to the structure/function of the protein(s), so they must be further analysed. Using a combination of the PROVEAN analysis tool, the ExPASy molecular weight calculator and your knowledge about the properties of individual amino acids, determine whether the differences you have the amino acid sequence(s) will affect the protein.

Insert screenshot(s) and legends of PROVEAN analysis (include submission time/date) below:

Provide the length and theoretical molecular weights of the relevant peptides below:

8: Explanation

Using all the information provided to you and collected during your investigation, can you diagnose what the patient may be suffering from? Investigate the evidence, draw together all the data you have collected from all eight tasks in context of the literature and concisely draw your conclusion.  

Provide your explanation (400 words max) below:

9: Primer design  

You decide that it would be useful to design primers to flank the region of variation in the HTT gene that you have been studying above, so that PCR could be employed as a quick diagnosis tool, without sequencing to identify potential sufferers by the length of the amplicons (i.e. the size of the region of variability).

Using online software, or manually, design primers that will only amplify the relevant sequence in the HTT gene. The primers should be specific and meet standard criteria for primer design.