2013 10 23_dna_for_dummies_v_presented

Wim Van Criekinge

Antwerpen24 oktober 2013

Lab for Bioinformatics and computational genomics

Overview

• Who ? Where ?• Bioinformatics• (Epi)genetics• Technology: Next Gen

Sequencing• Personal Genomics

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biobix.bebioinformatics.be

http://www.bioinformatics.be/

http://www.bioinformatics.be/

All organisms: 1 or more cells

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Cell Theory• All organisms are

composed of one or more cells.

• Cells are the smallest living units of all living organisms.

• Cells arise only by division of a previously existing cell.

Each human cell contains 46 chromosomes (except sperm or egg cells)


Genetic codeDNA


The human genome comprises the information contained in one set of human chromosomes which themselves contain about 3 billion base pairs (bp) of DNA in 46 chromosomes (22 autosome pairs + 2 sex chromosomes). The total length of DNA present in one adult human is calculated by the multiplication of

(length of 1 bp)(number of bp per cell)(number of cells in the body)

DNA: Structure and Function




(0.34 × 10-9 m)(6 × 109)(1013)2.0 × 1013 meters





(0.34 × 10-9 m)(6 × 109)(1013)2.0 × 1013 meters

That is the equivalent of nearly 70 trips from the earth to the sun and back.


Genetic information is stored in the DNA

DNA encodes proteins

“Genes” encode proteins

DNA replication

DNA synthesis and proofreading

Mutations:possible cause of diseases and disfunctionalities


Overview




Microbes are all over us

Summer 2012 Workshop in Biology and Multimedia for High School Teachershttp://commons.wikimedia.org/wiki/

File:Man_shadow_-_upper.png

Thousands of different species on the skin aloneSome thrive on dry patches of the elbow, others thrive in moist environment of armpit

There are millions of microbes per square inch on your body

It is estimated that there are more microbes in your intestine than there are human cells in your body!


Defining Epigenetics

• Reversible changes in gene expression/function

• Without changes in DNA sequence

• Can be inherited from precursor cells

• Epigenetic information is included in the epigenome

• Allows to integrate intrinsic with environmental signals (including diet)

GenomeDNA

Gene Expression

Epigenome

Chromatin

Phenotype


Chromatin is a Key Component of Epigenetic Mechanisms


Chromatin is a Key Component of Epigenetic Mechanisms

• Chromatin organizes genes to be accessible for transcription, replication, and repair

nucleosomehistone

DNA

chromatin

Cellular DNA is packaged into a structure called chromatin

The unit of chromatin is the nucleosome, a complex of a histone tetramer with approx. 125 bp of DNA wound around it


Basic Epigenetic Mechanisms: Post Translational Modifications to Histones and Base Changes in DNA

• Epigenetic modifications of histones and DNA include:– Histone acetylation and methylation, and DNA methylation

HistoneAcetylation

HistoneMethylation

DNA Methylation

MeMeMeMeAc

Me


Geneexpression

• Gene expression (transcription) requires DNA to be physically accessible to transcription factors (TF)

• Epigenetic changes alter the structure of the chromatin, which determines whether DNA is accessible– Open chromatin allows gene expression – Closed chromatin prevents gene expression

Epigenetic Changes can Alter Chromatin Structure and Regulate Gene Expression

Geneexpression

AcAc

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TFTF

ActionableEpigenome


Epigenetics

• Epigenetics is essentially the study of how our environment impacts traits acquired within our lifetimes, altering certain gene expressions which may then be passed on to future generations

• That is, what we do to our own bodies may affect our children & grandchildren more than we thought.

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Epigenetic (meta)information = stem cells


Translational Research towards Personalised Medicine

• DNA diagnostic tests can be used to identify in advance which patients are likely to respond well to a therapy

• The benefits of this approach are to:

– avoid adverse drug reactions

– improve efficacy– adjust the dose to suit the

patient– differentiate a product in a

competitive market– meet future legal or

regulatory requirements


Example: Replication errors

GENETIC

Altered DNA/mRNA/proteins

Altered DNA sequence

Historically, Cancer Was Considered to be Driven Mostly by Genetic Changes

X X

Oncogenesis

Tumor

• Mutations in p53 • Activating mutations in RAS• Mutations or amplifications of the HER-2 gene• Chromosomal translocations in myeloid cells and the

generation of the BCR-ABL fusion protein


Example: Replication errors

GENETIC EPIGENETIC

Example: Chromatin modification errors

Altered DNA/mRNA/proteins

Altered DNA sequence

Altered levels ofmRNA/proteins

Alteredchromatin structure

Recent Evidence Shows that Epigenetic Changes are Also Important in Causing Cancer

X X

Oncogenesis

Tumor


Schuebel et al 2007

Biology uses methylation extensivelyas a “regulatory checkpoint” in (cancer) development

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Methylation of MGMT in GBM

Kaplan-Meier Estimates of Overall Survival in GBM, According to MGMT Promoter Methylation Status

Hegi et al. NEJM 2005, 352(10):997-1003


Cancer Stem Cell Theory: the ‘Root’ of Cancer Growth

Tumor

Epigenetically altered, self-renewing cancer stem cells

Tumor Development and Growth


Gene-specificEpigeneticreprogramming

Personalized Medicine

• The use of diagnostic tests (aka biomarkers) to identify in advance which patients are likely to respond well to a therapy

• The benefits of this approach are to– avoid adverse drug reactions– improve efficacy– adjust the dose to suit the patient– differentiate a product in a competitive market– meet future legal or regulatory requirements

• Potential uses of biomarkers– Risk assessment– Initial/early detection– Prognosis– Prediction/therapy selection– Response assessment– Monitoring for recurrence

Biomarker

First used in 1971 … An objective and « predictive » measure … at the molecular level … of normal and pathogenic processes and responses to therapeutic interventions

Characteristic that is objectively measured and evaluated as an indicator of normal biologic or pathogenic processes or pharmacologic response to a drug

A biomarker is valid if:– It can be measured in a test system with well

established performance characteristics – Evidence for its clinical significance has been

established

Rationale 1:Why now ? Regulatory path becoming more clear

There is more at stake than efficient drug development. FDA « critical path initiative » Pharmacogenomics guideline

Biomarkers are the foundation of « evidence based medicine » - who should be treated, how and with what.

Without Biomarkers advances in targeted therapy will be limited and treatment remain largely emperical. It is imperative that Biomarker development be accelarated along with therapeutics

Why now ?

First and maturing second generation molecular profiling methodologies allow to stratify clinical trial participants to include those most likely to benefit from the drug candidate—and exclude those who likely will not—pharmacogenomics-based

Clinical trials should attain more specific results with smaller numbers of patients. Smaller numbers mean fewer costs (factor 2-10)

An additional benefit for trial participants and internal review boards (IRBs) is that stratification, given the correct biomarker, may reduce or eliminate adverse events.

Molecular Profiling

The study of specific patterns (fingerprints) of proteins, DNA, and/or mRNA and how these patterns correlate with an individual's physical characteristics or symptoms of disease.

Generic Health advice

•Exercise (Hypertrophic Cardiomyopathy)

•Drink your milk (MCM6 Lactose intolarance)

•Eat your green beans (glucose-6-phosphate dehydrogenase Deficiency)

•& your grains (HLA-DQ2 – Celiac disease)

•& your iron (HFE - Hemochromatosis)

•Get more rest (HLA-DR2 - Narcolepsy)

Generic Health advice (UNLESS)


•Drink your milk (MCM6 Lactose intolarance)





Generic Health advice (UNLESS)


•Drink your milk (MCM6 Lactose intolerance)






Overview




Confidential Information | ©2013 MDxHealth Inc. All rights reserved.

Genetics

107 106 105 104 103 102 101 1108109

Full genome bp

GENETIC

Whole-genomesequencing

Enrichment seq(Exome)

PCREnrichment

Targeted Panels

Instrument and Assay providers

CLIA Lab service providers


Overview




Wobblebase Mission

provide tools to both specialists (researchers, bioinformaticians, health care providers) and individual consumers that unlock the power of genomic data to the USER

enable personalized genomics today by simplifying the way we organize, visualize and manage genomic data.

PGM: Personal Genomics Manifesto

Everybody who wants to get his genome sequenced has the human right to do so. No third party can own your genetic data, your genetic data is exclusively yours.

Nobody can be forced to get his genome analyzed or to reveal his genome to a third party.

Your genome should allways be treated as confidential, private information.

People should be advised not to share their identity AND their entire genome on a public forum.

People should be advised to use secure technologies that allow to maximally protect phenotypic and/or genotype data.

People should be able to actively explore, manage and get updated interpretation on their genomic data.

• change the diagnostic/healthcare industry forever by setting a new standard and empowering the user

Wobblebase Mission

Choosing the Red Pill

The Technical Feasibility Argument

The Quality Argument

The Price Argument

The Logistics around the sample on howto manage the data Argument

The Ethical debate

The Privacy/Security concern

Updates are the single moste important feature of

Wobblebase

Notifications

#Rs1805007

Wobblebase

Socialnetworktwitter

Comparison

BioinformaticsAnalysispipelines

UpdatesNotifications

eHealth(fixed

vocabulary)

The Human MicrobiomeChristine Rodriguez, Ph.D.

Harvard Outreach 2012

Summer 2012 Workshop in Biology and Multimedia for High School Teachers

Microbes are all over us

Summer 2012 Workshop in Biology and Multimedia for High School Teachershttp://commons.wikimedia.org/wiki/

File:Man_shadow_-_upper.png

Thousands of different species on the skin aloneSome thrive on dry patches of the elbow, others thrive in moist environment of armpit

There are millions of microbes per square inch on your body

It is estimated that there are more microbes in your intestine than there are human cells in your body!

What is the Human Microbiome?


Microbe: tiny living organism, such as bacterium, fungus, protozoan, or virus

Microbiome: collectively all the microbes in the human body; a community of microbes

Biofilm: a community of microbes that live together on a surface

Microbes in the Human Microbiome include species from each major domain


http://commons.wikimedia.org/wiki/File:Tree_of_life.svg

http://en.wikipedia.org/wiki/File:Grand_prismatic_spring.jpg

http://en.wikipedia.org/wiki/File:SalmonellaNIAID.jpg

http://en.wikipedia.org/wiki/File:Aspergillus_niger_01.jpg

Bacteria

“Extremophile”Archaebacteria

Fungi

http://commons.wikimedia.org/wiki/File:Tree_of_life.svg

What features distinguish the microbial domains?


http://biodidac.bio.uottawa.ca/thumbnails/filedet.htm?File_name=BACT003B&File_type=GIF

http://biodidac.bio.uottawa.ca/thumbnails/filedet.htm?File_name=CELL006B&File_type=GIF

Generalized bacteria and archaebacteria cell

Generalized eukaryotic cell

Bacteria•Have no nucleus or membrane bound organelles•Often sphere (cocci) or rod (bacillus) shape, but others as well

Archeabacteria •Have no nucleus or membrane bound organelles •Can look similar to bacteria or drastically different shapes, such as flat and square•Have some metabolic similarities to eukaryotes

Eukaryotes•Have a true nucleus and membrane bound organelles•Wide variety of shapes. For this presentation, we will focus on fungi•Fungi are unique since they have a cell wall and form spores during reproduction

Microbes are normally found in and on the human body

The following sites are “hotspots” for microbial life


http://nihroadmap.nih.gov/hmp/

Let’s explore these five

regions

Some microbes are native, normally found in the body

Some microbes are introduced, suddenly arriving at a new residence in the body


What’s Happeningin the Nose?

http://commons.wikimedia.org/wiki/File:Human-nose.jpg

There is a delicate balance of microbes that are maintained to keep that environment healthy. Weakened immune systems can throw off that balance and allow the wrong microbes to grow out of control.

Inflammation from viral infection and allergic reactions

Inhaled medicines and oral antibiotics

Cilia and mucous lining trap inhaled microbes

The nose is a primary defender against inhaled pathogens

Nose


The interior lining of the nose contains mucous secreting glands. A wide variety of microbes are normally found there. Here’s a few:

http://commons.wikimedia.org/wiki/File:Human-nose.jpghttp://en.wikipedia.org/wiki/File:Staphylococcus_epidermidis_01.png

• Staphylococcus epidermidis bacteria forms a biofilm that coats the mucosal lining

• Staphylococcus aureus bacteria is fine when kept under control by a protease found in S. epidermidis, but if left to grow out of control, S. aureus can become pathogenic and cause infection

http://en.wikipedia.org/wiki/File:MRSA7820.jpg


Nose

http://commons.wikimedia.org/wiki/File:Human-nose.jpg http://en.wikipedia.org/wiki/File:Aspergillus_fumigatus_Invasive_Disease_Mechanism_Diagram.jpg

• Aspergillus fungal spores are often inhaled through the nose. If the immune system fails to clear these, mold can grow in the lungs

http://en.wikipedia.org/wiki/File:Aspergillus.jpg

•Corneybacterium accolens bacteria is rarely a pathogen, but if it enters the bloodstream due to a torn blood vessel, it can cause serious infections

http://en.wikipedia.org/wiki/File:Corynebacterium_ulcerans_01.jpg


What’s Happening inthe Oral Cavity?

http://en.wikipedia.org/wiki/File:Teeth_by_David_Shankbone.jpg

A wide variety of microbes regularly enter the oral cavity saliva, pH,

temperature, immune system prevent many species from surviving

Oral antibiotics inhibit growth

Brushing and flossing teeth clears some built up biofilm

Symbiosis of the oral microbes that are able to survive these conditions form an elaborate scaffold that lives on the tooth enamel and at the interface with the gums. It forms a barrier for incoming bacteria.

Oral Cavity



The oral cavity has a wide variety of microbes normally found there. Here’s a few:

Fusobacterium sp. bacteria is a larger bacteria that helps form a scaffold for many other bacteria in the oral biofilm

Streptococcus mitis bacteria typically forms a biofilm on the hard enamel surfaces of the teeth. If gums get inflamed, it can enter the bloodstream and cause infection


Oral Cavity


•Prevotella sp. bacteria have natural antibiotic resistance genes. They can attach to epithelial cells or other bacteria and cause larger infections in inflamed areas.

http://microbewiki.kenyon.edu/index.php/File:P_ruminicola.jpg

http://en.wikipedia.org/wiki/File:Candida_albicans_2.jpg

• Candida albicans fungus can cause oral infection known as thrush

http://en.wikipedia.org/wiki/File:Thrush.JPG


What’s Happeningon the Skin?

http://commons.wikimedia.org/wiki/File:Anterior_view_of_male_upper_body,_retouched.jpg

There are several skin environments: oily, dry, moist. Some microbes prefer one over another.

The skin has natural defenses including slightly acidic sweat and antimicrobial peptides.

Microbes hide in crevices to recolonize skin after washing with soap

Antibiotic washes and oral antibiotics disturb normal balance of microbes on the skin

There is a normal balance of microbes on the skin that protect introduced microbes from harming us. Damaged skin gives opportunities for microbes to invade the bloodstream and cause serious illness.


Skin


http://microbewiki.kenyon.edu/index.php/File:Lesionsclosed.jpg

http://microbewiki.kenyon.edu/index.php/File:Lesionsmicro.jpg

• Propionibacterium acnes bacteria colonizes healthy pores, but if pores become clogged, it grows out of control

• Staphylococcus epidermidis bacteria normally colonizes on the skin. But when P. acnes clogs pores, S. epidermidis also grows out of control in the infected pores

• Staphylococcus aureus bacteria can also infect clogged pores like Staph epidermidis. Even worse, many antibiotic resistant strains of Staph aureus make it difficult to treat the infection.


Skin


Trichophyton and Microsporum fungi feast on keratin in the skin and cause ringworm fungal infections

http://en.wikipedia.org/wiki/File:Yeartinfection.JPG


What’s Happeningin the Gut?

http://commons.wikimedia.org/wiki/File:Intestine_and_stomach_-_transparent_-_cut.png

Major barriers for microbes entering the gut:•low pH•Saliva and Bile•Immune system•Finding a place to attach to intestinal wall•Surviving a widely varied diet

For those microbes that manage to colonize the gut:•gut flora perform regular tasks of digestion, vitamin production, many others • Gene transfer between the myriad of species in the gut can generate new

combinations of drug resistant “superbugs”


Gut


Bacteroides thetaiotaomicron bacteria ferments simple carbohydrates in the gut, releasing hydrogen and CO2.

+ carbohydrates

CO2 and H2

Methanobrevibacter smithii archeabacteria consumes hydrogen gas from Bacteroides and produces methane, which is lost from gut as “gas”

CH4 Methane Gas


Gut


http://microbewiki.kenyon.edu/index.php/File:G_reaction1.jpg

Ruminococcus sp. bacteria can be found in significantly high numbers in the gut flora. They break down cellulose in the gut, helping with digestion.

Helicobacter pylori bacteria has a helical shape and colonizes the stomach and upper G.I. tract. It is known to be a major cause of stomach ulcers, although many with H. pylori do not get ulcers.

http://commons.wikimedia.org/wiki/File:Helicobacter_pylori_diagram.png

What’s Happening in the Urogenital Tract?


http://commons.wikimedia.org/wiki/File:Female_Genital_Organs.svg

Urinary system almost sterile due to urea and other chemicals

Urine often flushes out microbes that find their way in

Introducing a catheter into the urethra can introduce microbes directly into the bladder, where a biofilm can grow and cause bladder infection

The vagina has a low pH due to Lactobacillus secreting lactic acid and hydrogen peroxide. Let’s explore the microbiome of this region further.


Urogenital


Lactobacillus and vaginal epithelial cell

http://commons.wikimedia.org/wiki/File:Lactobacillus_sp_01.png

Lactobacillus normally maintain low pH while other species are kept in small numbers in the vagina If Lactobacillus

decreases from antibiotics…

Candida albicans can take over and cause a yeast infection

http://en.wikipedia.org/wiki/File:Candida_albicans_2.jpg

G. vaginalis and vaginal epithelial cell

Gardnerella vaginalis can grow too much and cause bacterial vaginosis.


Urogenital


The urinary tract is normally sterile due to urine flushing out the tract.

But, Escherichia coli from GI tract can infect urinary tract due to poor hygiene and contamination from nearby GI tract opening.

http://commons.wikimedia.org/wiki/File:Pyuria2011.JPG

Urine sample infected with E. coli Urine sample infected with E. coli

http://commons.wikimedia.org/wiki/File:E_choli_Gram.JPG

Interplay Between Medicine and Microbes


Antibiotics

Kills infectious bacteria but also disrupts natural flora. Can result in yeast infections, digestive problems, etc.

http://commons.wikimedia.org/wiki/File:NOVAMOXIN_antibiotic.jpg

http://commons.wikimedia.org/wiki/File:Chemotherapy_bottles_NCI.jpg

Chemotherapy drugs

Gut flora has been shown to modify some drugs during metabolism. This causes many side effects, including upset stomach.

Use of Antimicrobial ProductsHow many do we really need?


http://commons.wikimedia.org/wiki/File:Hands-Clapping.jpg

http://commons.wikimedia.org/wiki/File:Toothpaste.jpg

http://commons.wikimedia.org/wiki/File:Refill_soap.jpg

http://commons.wikimedia.org/wiki/File:Tissue.jpg

http://commons.wikimedia.org/wiki/File:Afwasmiddel.jpg

Products kill germs to reduce infection

But do we need some natural exposure to germs to keep our normal flora around?

Will this allow “superbugs” that can barely survive these treatments to grow and become more prevalent…causing problems for the future?

Is My Gut Microbiome the Same as Yours?


The number and amount of the many different microbes can vary greatly from person to person.


Relative amounts of species

http://en.wikipedia.org/wiki/File:Skin_Microbiome20169-300.jpg

The Microbiome of one person can be different than others in species and relative amounts

Research in the Human Microbiome Project is starting to identify the relative amount of each microbe present at different locations in the body.

So many new questions to answer about the Human Microbiome…


How does the gut flora modify drugs, and how can we minimize side effects?

http://commons.wikimedia.org/wiki/File:Chemotherapy_bottles_NCI.jpg


Why does my gut flora look different than yours? How does that affect obesity, food allergies, and ability to fight disease?

http://commons.wikimedia.org/wiki/File:Hands-Clapping.jpg

Are we making germs more resistant to anitmicrobials? What happens when the germs are resistant to all of the drugs in our arsenal?

What do you want to know?


… $

2013 10 23_dna_for_dummies_v_presented

Education

dna replication

dna synthesis

human cells

bp of dna wound

base pairs bp of dna

computational genomicsdna

computational genomicsoverview

total length of dna