MOLECULAR DIAGNOSTICS

ClinVar

• ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/) provides a freely available archive of reports of relationships among medically important variants and phenotypes. 

• ClinVar accessions submissions reporting human variation, interpretations of the relationship of that variation to human health and the evidence supporting each interpretation. 

• The database is tightly coupled with dbSNP and dbVar, which maintain information about the location of variation on human assemblies.

ClinVar was used to document number of pathogenic variants associated with your gene and how many are reviewed by expert panel and professional society.

Fig 1 consists of ClinVar page CYP2C9 gene

• There are 16 allelic variants that could be noticed in total in the database.
• In this they were 4 pathogenic allelic variants.
• They were no reviews by the expert panel or the professional society in the review status filter which we can see in the filter of this database.

 Document how many labs provide tests related to CYP2C9 gene

• NCBI Genetic Test Reference (GTR) 
• The Genetic Testing Registry (GTR) provides a central location for voluntary submission of genetic test information by providers.
• The scope includes the test's purpose, methodology, validity, evidence of the test's usefulness, and laboratory contacts and credentials. 
• The overarching goal of the GTR is to advance the public health and research into the genetic basis of health and disease.

CYP2C9 GENE:

• There are 4 different laboratories offer diagnostic testing related to the disease associated to CYP2C9.

Fig consists of the number of laboratories offering  tests for the disease associated to CYP2C9

• Disease condition associated with my pet gene is "Warfarin response" here we could notice the variant change 430C>T (p.Arg144Cys)  leads to this particular disease.
• Every laboratory has different methods to diagnose the disease. 

DNA STAR to stimulate RFLP:

• RFLP stands for restricted fragment length polymorphism is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated.
• Firstly perform RFLP on natural DNA with out making a variant change and the perform virtual agarose gel simulation using DNA STAR.

Fig represents the Seqbuilder (DNA STAR) module to represent the restriction enzyme (6 cutter) on position 430 in the sequence.

• Gene quest is a module in DNA STAR where we can perform RFLP analysis.
• We observe that there is no restriction enzyme site on the position were there is a variant so we nee d to consider any other restriction enzyme 7 positions adjacent to our variant site so we found  "EarI".
• So, agarose gel  simulation is performed using this enzyme.

Results of RFLP:

Fig represents RFLP summary and agarose gel simulation

• Repeating the same steps for the variant change T<C at position 430.
• Using Edit seq make the variant change in the DNA sequence.

Conclusion:

We can notice that there are restriction sites near the site of your variant but the variant does not affect the digestion of the DNA.