2013 10 23_dna_for_dummies_v_presented

Wim Van Criekinge
Antwerpen
24 oktober 2013

Lab for Bioinformatics and computational genomics

Overview
^[now][transl⎮comput]ational[epi]genomic$

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

biobix
wvcrieki

biobix.be
bioinformatics.be

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EUKARYOTES

PROKARYOTES

All organisms: 1 or more cells


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 code
DNA


DNA: Structure and Function
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)



(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 synthesis and proofreading

Mutations:
possible cause of diseases and disfunctionalities

Microbes are all over us
There are millions of microbes per
square inch on your body
Thousands of different species on the skin alone
Some thrive on dry patches of the elbow, others
thrive in moist environment of armpit

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

http://commons.wikimedia.org/wiki/File:Man_sha
dow_-_upper.png

Summer 2012 Workshop in Biology and
Multimedia for High School Teachers


Defining Epigenetics
Genome
DNA

• Reversible changes in gene
expression/function
• Without changes in DNA
sequence

Chromatin

Epigenome

Gene Expression

Phenotype

• Can be inherited from
precursor cells
• Epigenetic information is
included in the epigenome
• Allows to integrate intrinsic
with environmental signals
(including diet)


Chromatin is a Key Component of Epigenetic Mechanisms


Chromatin is a Key Component of Epigenetic Mechanisms
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
nucleosome

histone
DNA

chromatin

•

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


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

Histone
Acetylation
Ac

Histone
Methylation
Me Me
Me

DNA Methylation


Epigenetic Changes can Alter Chromatin Structure and Regulate Gene Expression

TF

TF

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Gene
expression
•
•

Gene
expression

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


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
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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


Historically, Cancer Was Considered
to be Driven Mostly by Genetic Changes
GENETIC
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Example:
Replication errors
X X

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

Altered
DNA sequence
Altered
DNA/mRNA/proteins

Oncogenesis

Tumor


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

EPIGENETIC

Example:
Chromatin modification errors

Example:
Replication errors
X X

Altered
chromatin structure

Altered
DNA sequence
Altered
DNA/mRNA/proteins

Oncogenesis

Tumor

Altered levels of
mRNA/proteins


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

Schuebel et al 2007

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

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

Tumor
Tumor
Development
and
Growth

Epigenetically
altered, selfrenewing cancer
stem cells


Gene-specific
Epigenetic
reprogramming

Personalized Medicine
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•

•

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—pharmacogenomicsbased
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)

Genetics
Instrument and Assay providers

G
E
N
E
T
I
C

Whole-genome
sequencing

Enrichment seq
(Exome)

Enrichment
Targeted Panels

PCR

bp

Full genome

109

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CLIA Lab service providers

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

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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.

Wobblebase Mission
•

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

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

Notifications
Updates are the single moste
important feature of Wobblebase

Bioinformatics
Analysis
pipelines

Social
network
twitter

Wobblebase

Updates
Notifications

eHealth
(fixed
vocabulary)
Comparison

The Human Microbiome
Christine Rodriguez, Ph.D.
Harvard Outreach 2012


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

“Extremophile”
Archaebacteria

Bacteria

Fungi

http://en.wikipedia.org/wiki/File:Aspergillus_niger_01.jpg
http://en.wikipedia.org/wiki/File:SalmonellaNIAID.jpg
http://en.wikipedia.org/wiki/File:Grand_prismatic_spring.jpg
http://commons.wikimedia.org/wiki/File:Tree_of_life.svg


What features distinguish the
microbial domains?
Bacteria

•Have no nucleus or membrane bound organelles
•Often sphere (cocci) or rod (bacillus) shape, but others as well
Generalized
bacteria and
archaebacteria
cell

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

eneralized eukaryotic cell
http://biodidac.bio.uottawa.ca/thumbnails/filedet.htm?File_name=CELL006B&File_type=GIF
http://biodidac.bio.uottawa.ca/thumbnails/filedet.htm?File_name=BACT003B&File_type=GIF


Microbes are normally found in and
on the human body
The following sites are “hotspots” for microbial life

Some microbes are native,
normally found in the body

Let’s explore
these five
regions

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

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


What’s Happening
in the Nose?
Cilia and mucous
lining trap inhaled
microbes

The nose is a
primary defender
against inhaled
pathogens
Inflammation
from viral
infection and
allergic reactions

Inhaled medicines
and oral antibiotics

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

Nose
The interior lining of the nose contains mucous secreting glands. A wide variety of
microbes are normally found there. Here’s a few:
• 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
http://en.wikipedia.org/wiki/File:Staphylococcus_epidermidis_01.png

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

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

What’s Happening in
the Oral Cavity?
A wide variety
of microbes
regularly enter
the oral cavity

Brushing and flossing teeth
clears some built up biofilm
saliva, pH,
temperature, immune
system prevent many
species from surviving
Oral antibiotics
inhibit growth

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.

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

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.

• Candida albicans fungus can cause oral infection known as
thrush

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


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

What’s Happening
on the Skin?
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.
http://commons.wikimedia.org/wiki/File:Anterior_view_of_male_upper_body,_retouched.jpg

Skin

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


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 Happening
in the Gut?
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”

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


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


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

What’s Happening in the
Urogenital Tract?
Urinary system almost
sterile due to urea and
other chemicals

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

Urine often flushes
out microbes that
find their way in

The vagina has a low pH due to Lactobacillus secreting lactic acid and hydrogen peroxide.
Let’s explore the microbiome of this region further.
http://commons.wikimedia.org/wiki/File:Female_Genital_Organs.svg

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

Candida albicans
can take over and
cause a yeast
infection

If Lactobacillus
decreases from
antibiotics…

Lactobacillus and vaginal epithelial cell

G. vaginalis and vaginal epithelial cell

Gardnerella vaginalis
can grow too much
and cause bacterial
vaginosis.
http://commons.wikimedia.org/wiki/File:Lactobacillus_sp_01.png


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

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

Urine sample infected with E. coli

Urine sample infected with E. coli

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

Interplay Between
Medicine and Microbes

Antibiotics

Chemotherapy drugs

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

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

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


Use of Antimicrobial Products
How many do we really need?

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

Products kill germs
to reduce infection

http://commons.wikimedia.org/wiki/File:Afwasmiddel.jpg
http://commons.wikimedia.org/wiki/File:Tissue.jpg
http://commons.wikimedia.org/wiki/File:Refill_soap.jpg
http://commons.wikimedia.org/wiki/File:Toothpaste.jpg
http://commons.wikimedia.org/wiki/File:Hands-Clapping.jpg


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

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

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

So many new questions to answer
about the Human Microbiome…

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

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

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

What do you want to know?
http://commons.wikimedia.org/wiki/File:Hands-Clapping.jpg
http://commons.wikimedia.org/wiki/File:Chemotherapy_bottles_NCI.jpg



…$

2013 10 23_dna_for_dummies_v_presented

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