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.

ANTIGENICITY PREDICTION OF EBOLA VIRUS PROTEIN VP40 (GROUP)

• Analysis of ebolavirus protein done by our group Saikrishna, Naushad and Xi.
• We used 3 different patients from NCBI BIO PROJECT module. We tried to analyze that is there any variation in the antigenicity (MHC-I) binding prediction which is done by using IEDB Analysis resource.

 Fig Represents the 3 patients we used to analyze the MHC-I binding prediction.

•  The 3 patients ID's are KM233115.1, KM233091.1, KM233042.1.
• Then we got I got the protein VP40 sequences from these individual patients and did my analysis in IEDB.
• In this I choose one particular allele HLA-0A*01 for all the 3 patients then analyzed to see that is there any change in the MHC-I binding peptide (EPITOPES).
• My team mate Naushad used all the alleles for the viral protein VP24 and Xi  used VP30 and did analysis using DNASTAR.
• You can visualize there blog through the following web link:
• Naushad - http://www.noshiahmad.blogspot.com
• Xi- http://www.missxiwangbioinformatics.wordpress.com 

 Analysis of a particular protein is done using IEDB for individual patients:

1. Patient - KM233115.1 - GenBank: AIG96606.1
2. Patient - KM233091.1 - GenBank: AIG96390.1
3. Patient - KM233042.1 - GenBank: AIG95949.1

                           Fig Represents the patient 1 MHC-I binding prediction.

Fig Represents the patient 2 MHC-I binding prediction.

Fig Represents the patient 3 MHC-I binding prediction

We even used DNASTAR to perform the experiment as my group mates performed but i performed for cross reference using online tool PROPRED-I which is a server used to calculate the MHC-I binding peptides.

• Even in this particular server we notice that "WTDDTPTGS" peptide is a binder but not he best one based on the calculations performed using Log scale values.
  Fig Represents page of Propred-I after submission of sequence
  We can notice that we can see the above mentioned peptide in the following sequence mentioned below
  
   According to the this server best binders for this sequence are :
  
  Fig Represents ranking of peptide binders using Log scale
  
  
  Conclusion: We notice that there is no change in the T cell antigenic determination there is no difference in the three patients with VP-40 gene. We can also see that the binders ranking based on the algorithms used.

ANTIGENICITY OF EBOLA VIRUS VP40 PROTEIN

EBOLA VIRUS

• Ebola, is a disease of humans and other primates caused by Ebola virus. Ebola virus disease (EVD), formerly known as Ebola haemorrhagic fever, is a severe, often fatal illness in humans. 
• The virus is transmitted to people from wild animals and spreads in the human population through human-to-human transmission.

TRANSMISSION

• It is thought that fruit bats of the Pteropodidae family are natural Ebola virus hosts. Ebola is introduced into the human population through close contact with the blood, secretions, organs or other bodily fluids of infected animals such as chimpanzees, gorillas, fruit bats, monkeys, forest antelope and porcupines found ill or dead or in the rainforest. 

VIROLOGY

• Ebolaviruses contain single-stranded, non-infectious RNA genomes. Ebolavirus genomes are approximately 19 kilo base pairs long and contain seven genes in the order 3 UTR-NP-VP35-VP40-GP-VP30-VP24-L-5 UTR.The genomes of the five different ebolaviruses (BDBV, EBOV, RESTV, SUDV and TAFV) differ in sequence and the number and location of gene overlaps. As all filo viruses ebola virions are filamentous particles that may appear in the shape of a shepherd's crook, of a "U" or of a "6," and they may be coiled, toroid or branched. In general, ebolavirions are 80 nanometers (nm) in width and may be as long as 14,000nm.
• In this present blog my emphasis is on VP40 gene of ebolavirus and would like to describe ANTIGENICITY of its protein.  

 STRUCTURE OF EBOLA VP-40 PROTEIN

  

•  Fig Represents 3D Structure of VP-40 protein obtained from PDB Database

FUNCTION

• Promotes virus assembly and budding by interacting with host proteins of the multivesicular body pathway. May facilitate virus budding by interacting with the nucleocapsid and the plasma membrane. 
• Specific interactions with membrane-associated GP and VP24 during the budding process may also occur. The hexamer form seems to be involved in budding.
•  The octamer form binds RNA, and may play a role in genome replication.

 ANTIGENICITY

• Antigenicity is the capacity of a chemical structure (either an antigen or Hapten) to bind specifically with a group of certain products that have adaptive immunity: T cell receptors or antibodies (a.k.a. B cell receptors).
• We can determine antigenicity is a protein sequence in protein sequence using DNA STAR using PROTEAN 3D module.
• We perform this experiment using DNA STAR and we obtain regions in the protein showing high level of antigenicity.
• We can even obtain the B-cell epitope, MHC II epitopes and T cell epitopes.

Fig Represents the output of antigenicity obtained from DNASTAR

Fig Represents the epitope predicted by DNASTAR

• The above figure represents the regions in the protein sequence which exhibits antigenicity. The above mentioned protein is obtained from patient.
• This is done by using NCBI BIO PROJECT module were we can find information regarding the  ebolavirus and its outbreaks on some patients.
•  For the T-cell epitope prediction DNASTAR uses Roth bard-Taylor algorithm which uses amino acid window of 4 or 5 and this predicts the epitopes.
• The epitopes should be int he following re-occurring pattern
• If there are four AA then it should have charged/P, hydrophobic, hydrophobic and polar/G.
• In the above example we could see that there are four amino acids in the figure which are selected G,A,L,R which follows the above mentioned pattern.

IEDB T-CELL EPITOPE PREDICTION

• Every patient has an ID so we obtained a patient with a particular ID KM233090.1 then we analyze VP40 protein.
• We obtain the sequence of VP40 sequence from particular patient then we try to obtain the epitopes from IEDB for one particular HLA allele.
• In this case I choose HLA-A*01 allele for a 9 AA length epitope to be predicted. We can determine epitope to be a good binder if it has low percentile score.
• So by this procedure I found that epitope "WTDDTPTGS" with percentile rank of 0.9.
• The best peptide binder would be the one with the least percentile rank.