TITLE: Elucidating the roles of SOD3 correlated genes and reactive oxygen species in rare human diseases using a bioinformatic-ontology approach
AUTHORS: Mark Stanworth, Shu-Dong Zhang

[DESCRIPTION]
This is a gene discovery protocol utilising a single seed gene to create correlation lists. These lists are used to identify novel genes in rare diseases.

[STEPS]
SECTION: Software and packages
1. Download and install the required software

 
  

 
  

 
     

SECTION: Expression preparation
2. Download the RAW file for GSE2109
 
  


Extract the CEL files using 7-zip
SECTION: Expression preparation
2.1. Add 'affy' to the R library and normalise the data


 

SECTION: SOD3 correlation data
3. Import the GSE2109_normalised.txt file into excel

Create a new column to calculate the Pearson correlation with the SOD3 probe (205236_at)

 

Create another column calculating the two-tail t-test statistic for the correlations

 

Create another column calculating the two tail t-test p-value
    
 

Create another column calculating the Jarque-Bera normality test statistic

 

    
Create another column calculating the chi-squared p-value for the Jarque-Bera normality test     

 

SECTION: Expression exclusions
4. Apply statistical exclusions:
1. Exclude all expressions with SOD3 correlation p > 2.3x10-5 (Bonferroni corrected alpha 0.05)
2. Exclude all expressions with SOD3 correlation ρ ≤ |0.34|
3. Exclude all expressions failing the Jarque-Bera normality test

SECTION: Gene exclusions
5. Use GSE2109 supplementary file GPL570-9999 to assign gene symbols to expression probes

Update gene names using the HUGO Gene Nomenclature Committee website (https://www.genenames.org/) and note gene class (e.g. protein coding, pseudogene, etc)

Identify optimal gene of duplicate probes by adding 'Jetset' to the R library

 
        
Apply gene exclusions:
1. Exclude expressions with no GPL570-9999 identified gene symbol
1. Exclude suboptimal duplicate gene probes identified by 'Jetset'
2. Exclude non-specific/promiscuous probes, non-coding genes
SECTION: Gene lists
6. Verify robustness of the remaining gene expressions using 'pvclust' in R

 

Incrementally increase the correlation threshold by 0.01 from ρ>|0.34| to ρ>|0.41| 

List 1: Genes with ρ>|0.41|
List 2: Genes with ρ>|0.40|
List 3: Genes with ρ>|0.39|
List 4: Genes with ρ>|0.38|
List 5: Genes with ρ>|0.37|
List 6: Genes with ρ>|0.36|
List 7: Genes with ρ>|0.35|
List 8: Genes with ρ>|0.34|

For all lists, separate genes by correlation direction and denote the daughter lists with superscript '+' for positive and '-' for negative correlations

List 1+: Genes with ρ > 0.41
List 2+: Genes with ρ > 0.40
List 3+: Genes with ρ > 0.39
List 4+: Genes with ρ > 0.38
List 5+: Genes with ρ > 0.37
List 6+: Genes with ρ > 0.36
List 7+: Genes with ρ > 0.35
List 8+: Genes with ρ > 0.34
List 1-: Genes with ρ < -0.41
List 2-: Genes with ρ < -0.40
List 3-: Genes with ρ < -0.39
List 4-: Genes with ρ < -0.38
List 5-: Genes with ρ < -0.37
List 6-: Genes with ρ < -0.36
List 7-: Genes with ρ < -0.35
List 8-: Genes with ρ < -0.34

SECTION: Significant disorders
7. Enter all positive and negative correlation gene lists into Enrichr (maayanlab.cloud), and for each list:

1. In the Diseases / Drugs tab select Orphanet Augmented 2021
2. Copy all significant disorders Name and Adjusted P-value into a spreadsheet. Positive lists gene overlap must contain SOD3 and at two other genes, whereas negative correlation lists do not have to have SOD3 as an overlap gene but must contain 3 list genes in the overlap (hover over the disorder to check)
3. Identify minimum viable positive and negative lists (i.e. the smallest signed gene lists which have a significant overlap with disorder(s)).    
4. Delete from the spreadsheet any disorders from parent lists not in the minimum viable positive and negative lists
5. Identify the gene lists with the greatest overlap and associate the disorder to that list, noting the overlap genes in the spreadsheet
6. Cross reference the Orphanet disorder names (Orphanet: Search by disease name) with the OMIM names (Home - OMIM) noting the causal genes
SECTION: Significant ontologies
8. Individually enter each gene list associated with a disorder into Enrichr (maayanlab.cloud) and add the causal gene(s) for the disorder considered.

1. In the pathways tab, use Elsevier Pathway Collection, from the table note the significant pathways and the adjusted p-value of any pathways containing both a causal gene and a non-overlapping list gene
2. In the ontologies tab note from the Biological Process, Molecular Function, and Human Phenotype tables significant entries and adjusted p-values of any entries containing both a causal gene and a non-overlapping list gene

Associate qualifying entries with the list associated disorders

Note the non-overlapping gene(s) (potentially novel genes) linked to the disorders via the causal gene/list ontologies
SECTION: Literature associations
9. Search the literature for previous works which may lead to potential mechanisms or pathways from the potentially novel genes to disorder presentation. Repeat for superoxide dismutase, superoxide, and hydrogen peroxide. Consider gene aliases / previous symbols in respect of time allocated to the research