bench to bedside (b2b) - omics in medicine - dr s shankar
TRANSCRIPT
Gp Capt S ShankarMD DNB MNAMS FICP FRCP(Glasg)
Professor & Senior Advisor (Medicine) & Cl Immunology
Command Hospital (Air Force), Bangalore
OMICS IN RHEUMATOLOGY
Past Editor in Chief: Indian Journal of Rheumatology 2009-13
Greetings
Patti et al., Nature Reviews, 13, 263-269 (2012)
Molecular machinery that governs
Genotype ---------------------->-------------------------------->-----------------------Phenotype
A snapshot of “The New Genetics”
Systems Biology
Bioinformatics
Epigenetics
To answer questions in rheumatology
MAKING USE OF VARIOUS OMICS?
The himalayan Trekkers
Many start sneezing in Valley A Vs Valley BHypothesis: Linked to some flower pollen…………….which one?
Different ways to answer the question
Gene Association Case/controlGenome wide association studies (GWAS)
Whole genome sequencingEpigentics/EpigenomicsGene Expression/TranscriptomicsProteomic approachMetabolomics
Tool used: Polymerase chain reaction
Candidate Gene association studies
Candidate Gene Association Studies
A candidate gene association study is used when you have an educated guess about a genes involvement in the condition you’re investigating
Candidate gene studies
Reason to suspect : Purple flower is the culprit
Two types of answersSneezing only occurs if purple flowers
present
Second answer commonerSneezing more common in valley A that
has greater number of purple flowers (30%) Vs valley B (10%)
Frontiers in Microbiology. Jul 2015
Genomic Translational Research
Khoury MJ et al (2007) Genetics in Medicine, 9(10):665-674Khoury MJ et al (2012) Am J Public Health 102(1):34-37
Tool used: Microarray
Genome Wide Association Studies (GWAS)
I don’t suspect any flower in particular
I will just catalogue all of them and figure out later
Genome Wide Association Studies
A Genome Wide Association Study (GWAS) is used when you want to investigate the entire genome to determine what polymorphisms are associated with the condition you’re studyingAdvantage: biology of the condition need not be well understood.
Non candidate driven/ Phenotype firstDisadvantage is the large number of subjects required
Over 200 diseases studies, over 1200 studies done
Manhattan plot showing risk loci. By M Kamran Ikram
Genome Wide Association Studies
archive and distribute the results of studies that have investigated the interaction of genotype and phenotype
To recapitulateYou suspect a few
genesCandidate gene studiespharmacogenetics
No clue, but willing to search in a blind fashionGWAS
Technique: Next generation sequencing
Whole genome sequencing
NGS methodology
Let me catalogue it all
I know whom to compare it with
Whole genome sequencing•A 14-year-old boy with SCID, presented 3 times over 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. •Unbiased next generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition.
Our work: Lupus families and MTB diagnostics
Epigenetics
EpigeneticsEpigenetics literally means ‘above’ the genetics.
Has had multiple definitions over time.2008 Cold Spring Harbor Epigenetics meeting:
“An epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.”
Alterations in the DNA sequence = mutations
Valley is same/Similar
Valley A still causes more sneezing despite being similar to BIs it the Rain OR Sunlight OR Difference in Bees?
A B
Some examples
Identical Twins with Different Hair Color
One individual with different eye colour
Individual eye with 2 colours
DNA Methylation & Histone Modifications:The Epigenetic Code
Epigenetic ResearchThe number of
publications in the field has increased dramatically in the last 10 years.
Rheumatoid arthritisMore than 60 sites
identifiedExplains 50% of all
genetic variationMany sites
hypomethylated• Genetic Engineering and Biotechnology
News Feb 1, 2013 (Vol. 33, No. 3)
Question TimeYou wish to check relapse of small cell
carcinoma lung after 3 months of achieving remission. You want to do it by using liquid biopsy, i.e by drawing blood and looking for circulating tumour DNA in blood (EGFR Mutation Test v2) .
What type of study will you chooseA.Candidate gene studyB.GWASC.EpigeneticsD.Whole genome sequencing
Another oneYou see two siblings, both boys, aged 11 and 13
years with lupus, affecting CNS (MR) , Renal, Skin and arthritis. Their 2 sisters are absolutely normal. There is history of consanguinity among parents (first cousins). You think it possibly has a genetic basis. You decide to test the samples from the affected children, sisters, parents and some other cousins to figure it out. Which type of study will you choose
A.Epigenetic studiesB.Whole genome sequencingC.Candidate gene studiesD.GWAS
Each type of cell plays a different tune!
Transcriptomics
Which of the flowers are blooming?
Blooming flowers release pollen
How are any 2 cells different?Why are 2 cells
different?Nerve cell VsSkin cell A
B 1
B2C2
C1
CELL BOTH
ONLY ONE
NOT EXPRESSED
NERVE
A B1 C1
SKIN A B2 C2NERVE
SKIN
How is gene expression studied?Next Gen DNA
sequencing
Real time quantitative PCRqPCR
Microarray
Expression analysis/Transcriptomics studiesTranscriptomic profiling of synovial fluid
mononuclear cells in rheumatoid arthritis and spondyloarthropathy
Mitali Bhattacharjee1,2, Tai-Chung Huang3*, Harrys K. C. Jacob1*$, Derese Getnet3,4, Lavanya Balakrishnan1,5, Luigi Marchionni4,Ghantasala S. Sameer Kumar1,¥, Rajeev Chauhan6, Sachin Srivastava6, Srinivas M. Srikanth1,7, Bipin G. Nair2, T. S. Keshava Prasad1,2,7, Harsha Gowda1, Ramesh Jois8, Akhilesh Pandey3,9,10# and Subramanian Shankar6#
Differential gene expression studiedGene symbol Protein Functional role Fold change
SpA/RADDX3Y DEAD (Asp-Glu-Ala-
Asp) box polypeptide 3, Y-linked
Minor-histocompatability complex
14.2
BMP1 Bone morphogenetic protein-1
Chondrogenesis 5
FAM82B Family with sequence similarity 82, member B
Cell division 2
CACNA2D3 Calcium channel, voltage-dependent, alpha 2/delta
subunit 3
Calcium metabolism 1.6
Proteomics
I am colour blind : But I can analyse the pollen in air and deduce the flower details
I will just catalogue all the proteins and figure out later
What is a proteome?The entire set of
proteins expressed by a genome, cell tissue or organism at a certain time.
Why are 2 cells different?Nerve cell VsSkin cell
A
B 1
B2C2
C1
CELL BOTH
ONLY ONE
NOT EXPRESSED
NERVE
A B1 C1
SKIN A B2 C2
NERVE
SKIN
Sample processing and mass spectrometry
Nature (2014) 509, 575–581
Matrix Assisted Laser Desorption/Ionization
• Introduced by Tanaka, Karas & Hillenkamp (1988)• Analysis of large biomolecules
Desorption
http://www.magnet.fsu.edu/education/tutorials/tools
Irradiation by
Electrospray ionization
• Introduced by Chapman in 1930• John Fenn applied it to study large biomolecules
http://www.lamondlab.com/MSResource/images/lcms
Creating proteome databases
Human tissues used in the study
How about mapping all proteins in the body?
72 Scientists, 46 Indians
Metabolomics
Pollen is just made of some proteins, fats and carbohydrates, I can denature them into simpler stuff
I will just analyse the simple metabolic component and catalogue the
differences
Why Metabolomics?
Making sense of big data
Systems Biology & Bioinformatics
“Science is built up with facts, as a house is with stones. But a collection of facts is no more a science than a heap
of stones is a house.” - Jules Henri Poincaré
A snapshot of “The New Genetics”
Systems Biology
Bioinformatics
Epigenetics
Systems Biology
I know at least 6-7 ways of answering any question using sophisticated
technology
Bioinformatics
Each of the ways throws up a million bit of data. But I have sophisticated
software and trained manpoer to make sense of it
Primary biological databases
Nucleic acidEMBLGenBankDDBJ (DNA Data
Bank of Japan)
Protein
PIR MIPS SWISS-PROT TrEMBL NRL-3D
A network map of Interleukin-10 signaling pathwayRenu Verma · Lavanya Balakrishnan · Kusum
Sharma · Aafaque Ahmad Khan · Jayshree Advani · Harsha Gowda · Srikanth Prasad Tripathy · Mrutyunjay Suar · Akhilesh Pandey · Sheetal Gandotra · T S Keshava Prasad · Subramanian Shankar
Journal of Cell Communication and Signaling
A knowledgebase resource for Interleukin-17 family mediated signaling Jyoti Sharma1,2, Lavanya Balakrishnan1, Keshava K. Datta1,3, Nandini A. Sahasrabuddhe1, Aafaque Ahmad Khan1,3, Apeksha Sahu1,4, Anish Singhal2,5, Derese Getnet6, Rajesh Raju1, Aditi Chatterjee1,2, Harsha Gowda1,3, T.S. Keshava Prasad1,2,4, Subramanian Shankar7* and Akhilesh Pandey6*
Journal of Cell Communication and Signaling
Question TimeYou wish to predict lupus flare in advance. You think
that the perturbations preceding flare can cause changes at genetic level that can manifest in differential protein expression. You decide to follow a group of 300 lupus patients, store their serum every 3 months, so that you can compare any flare specimen to a quiescent specimen. You finally want to identify a reliable biomarker.What type of study will you choose
A.GWASB.Whole genome sequencingC.TranscroiptomicsD.Proteomics
Final thoughtsOmics comprises various aspects of
systems biologyExciting new field : permits to see the
“Unknown”Holds promise in rheumatology research
PathogenesisDiagnosisTreatmentMonitoring
Institute of Bioinformatics
Dr Akhilesh Pandey
Questions