bench to bedside (b2b) - omics in medicine - dr s shankar

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