Review of Basic Genetic and Bioinformatic Technologies in the Context

Question 1

Central Dogma of What Genomics Measures

Answer 1

Linera flow of information
Genome –> Transcriptome –> Proteome –> Metabolome
DNA –> coding mRNA –> Protein(enzymatic roles and altering of metabolites and cells) –> Metabolites
Aluminium template representing Thymine from Crick and Watson’s model of DNA based on X-ray diffraction photographs by Rosalind Franklin, This explained how genetic information could be copied and passed to future generations –> Nobel Prize in 1962
-idea of how DNA can be copied and replicated

Question 2

Alterations to Central Dogma

Answer 2

1. Central Dogma isn’t the only way information flows
   as DNA –>
   a) coding mRNA
   b) non-coding mRNAs (miRNAs) (micro RNAs)
   -influence stability and degradation of mRNA (translation of coding mRNAs by acting of ribosomes)
   -influence how metabolism occurs on cells
   -Nobel prize awarded of discovering subset of micro RNAs
   -acts on coding mRNAs, Proteins and Metabolites
   Now realising there are alternative ways that information flows through cells
   +
2. Methylation of residues on DNA
3. How different parts of genomes are pulled together in 3D matrix, to influence how genes are used and what turns them on and off

Question 3

Why is the breaking of the central dogma very important

Answer 3

The way that information flows through cells is very important
-and how central dogma has been broken by methylation, imprinting and microRNAs are important for disease

Question 4

DNA genomics

Answer 4

Genomics is focused on DNA part of information flow
Genomics is “how genes are used”
-broad
- how genes expressed, relation to disease, how theyre selected

Question 5

Traditional gene sequencing

Answer 5

Older but still useful technology:
-DNA sequence analysis by Sanger Sequencing (traditionally). Still well used today/
-take a single piece of DNA and sequence it from end to end
–reads genome from template base by base
=Read out
-sequence gene by randomly stopping polymerase, depending on which base it stops at will result in a different coloured floursecent signal
-Further developed by Leroy Hood and coworkers to enable the human genome project

Question 6

Newer gene sequencing Technology

Answer 6

Next Generation Sequencing/ Massively parallel sequencing
-sequence lots of molecules at once
-break up a molecule randomly into lots of different fragments and sequence them all at once/all in parallel
1. Pick Genomic DNA –>
2. cut DNA (with enzymes or random fragmentation (sonication, shattering DNA with sound waves)) –>
3. Add Linkers (little pieces of definied sequence of DNA on ends using enzyme)
-defined sequences=adapters. allow DNA to bind to microscope slide–>
4. Input library –>
5. Flow cell (-defined sequences=adapters. allow DNA to bind to microscope slide)–> In Situ PCR
Then traditional polymerase chain reaction PCR to amplify, build up large number of copies of each individual fragments–>
6. Sequencing –> An image of hundreds of extended molecules
-as amplified, flash of light for base in each position in fragment
-as gradual build up sequence, get a flash of light of base (series of photographs)

Question 7

What is another name for the Newer Genomic Sequencing Technologies

Answer 7

Next Generation Sequencing

• Massively Parallel Sequencing
• massively parallel nature, 100 millions of DNA fragments being sequenced simultaneously
• each flashing light is from different position of microscope of different fragment

Question 8

Use of Next Generation Sequencing

Answer 8

specific piece of DNA

RNA- measure how much each gene is used, by counting number of times each RNA appears in the cell

Question 9

Exome sequencing

Answer 9

Genomes vs Exomes vs Targeted gene “panels”
-Whole Genome (3 billion base pairs)
-Exome = 1% (only 1-2% of the 3 billion base pairs only codes for genes which we know have a clear function of which we understand (e.g. encoding for protein or mRNA))
-exome sequencing of one part of the whole genome
After fragmenting the DNA, capture the DNA fragments corresponding to the exome using baits

Question 10

Whole Genome sequencing

Answer 10

Useful
3 Billion base pairs to a whole genome
can sequence human genome 1000-200genome
-more affordable
-but provides alot of information that we dont know how to use with the current level of scientific knowledge
vs. Exome sequencing is sequencing of the part of genome that we know how to use/know the best
-get much more data on each part of DNA for the same cost and same effort as sequencing whole genome

Question 11

Exome Sequencing mechanism

Answer 11

After fragmentation:
capture the DNA fragments on little beads,
each bead has a piece of complementary DNA that can bind to DNA and pull it away from the 99% that you dont want
=resulting in just having exome sequences that you want
corresponding to the exome using baits (parts of the genome that encode proteins or mRNAs/exome sequences_

Question 12

The recent advance of sequencing technology

Answer 12

Allumina
smaller - and now in big centres and individual research labs - can do several in a week
MinION - sequencing machine that can plug into USB port of laptop
–> increased field use
-e.g. farmers (understand what pathogen is infecting their crops)
-public health nurses what pathogen infecting patient

Question 13

Ease of Next Generation sequencing

Answer 13

Technically not easy to do

• once get all photographs/lights of four bases
• results in knowing the sequence for all the fragments
• A boring but essential job: processing the millions of sequence reads produced by each next generation sequence run (mapping them to the genome - finding out where they belong)
• Sequence Read Alignment

Question 14

Sequence Read Alignment

Answer 14

boring but essential job
Is like a jigsaw puzzle where they give you the cover on the box
-individual DNA Reads are analogous to pieces of jigsaw
-now because of human genome project know sequence of most parts of human genome - use as a reference
-often results in large numbers of reeds that cover any one parts (some pieces are easier to place than others)
-but other pieces are hard to place/align (pieces that look like each other, or pieces with unique features)

Question 15

Variant Calling and Annotation

Answer 15

Identifying and visualising sequence variants
-genome browser used daily to understand
-each little grey lines is a sequence
-2x strands of human genome =sense and antisense strand of human genome
-mutation= green reeds are different from reference = compare to reference to patients germline
=random fragments cover point of mutation and large numbers of different reeds
1. Each reed is a random fragment from Next Generation Sequencing
2. End product of being able to overlay/pile up all different reeds that are randomly produced from fragmenting genome, but can be combined to cover any one base in genome –> then being able to identify mutations not in normal tissue

Question 16

Heterozygous mutation proportion showing mutation

Answer 16

50%
Unless:
1. Two hit hypothesis (lost gene entirely e.g. by deletion and then mutation –> would only see mutant copy) (may have lost one remaining normal chromosome) (mutant reeds take up most of reeds you see)
2. Mosaicism/Heterogeneity
(tumour made up of several parts, only one of which may carry the mutation)
-tumour has several bits, one bit of the tumour may not have the mutation (may only see mutation in 20% of reeds as only in a small proportion of mutation)
-as tumour evolves mutation may occur in new part of the tumour
-mutation may be giving new part of the tumour a competitive advantage, allowing it to grow more rapidly –> slowly the proportion of reeds taken up by this mutation will expand
3. There are normal cells inside tumours. BV, fibroblasts, WBC
-they dont carry mutation
-may compose 20-40% of tumour
=will reduce proportion of reeds carrying the mutation

Question 17

Cost per genome

Answer 17

Costs are falling much quicker than exponentially

• Moore’s Law = exponential increase in number of transistores
• Sudden fall in the last couple of years due to introduction of new technologies
• used in clinical research
• starting to be used in clinical practice

Question 18

Examples of the use of single gene sequencing in clinical practice

Answer 18

1. Clinical Research
   - still focusing on exones as dont understand rest of genome
   - that other components still contributes significantly to disease
   - rapidly helping to start to understand how
2. Early studies - cnacer, paedeatrics
   - germline (born with DNA) or somatic changes (in cancer)
   a) help stratify therapy
   - cancer understand precise combinations of mutations in tumours
   - stratify patients for therapy
   b) explain disease
   - parents knowing what caused child’s disease (diagnosis). can lead to change in plans/prenatal
3. Rapid increase in understanding genome contributions to disease (inc. inherited disease and cancer)

Question 19

Scenario:

A family presents with a high incidence of epithelial ovarian cancer (specific gene sequencing)

Answer 19

-of families with 2 or more cases of epithelial ovarian cancer in first or second degree relatives
~=40% carry BRCA1 mutation
~=10% carry BRCA2 mutation
BRCA2 is a tumour suppressor genes
-encode proteins that help prevent cancer
-involved in sense and repair of double stranded DNA breaks
Germline (inherited) vs Somatic (acquired) BRCA2 gene mutations (or methylation or deletion of second BRCA gene)

Question 20

Sequencing the BRCA2 gene in blood leukocytes of members of the ovarian cancer fmaily

Answer 20

There are hundreds of reported mutations in BRCA2
Some BRCA2 mutations are well known in particular populations/families
However, a large proportion are unclassified and have unknown significance for disease (further knowledge needed to interpret)
These present a challenge to genetic counsellors
This single gene technology is sometimes called “Sanger Sequencing” (sequence one gene at a time)
-but increasingly now done by massively parallel sequencing/next generation sequencing

Question 21

Influence of the Information provided by gene sequencing the BRCA2 gene in blood leukocytes of members of the ovarian cancer fmaily

Answer 21

1. Identify the index case
   - know the mutation that the family carries
   - -> allows for much easier simpler techniques to test for mutation in other family members

Question 22

Will sequencing always identify a ingle mutation in BRCA gene?

Answer 22

No
-Not when there has been a deletion of one copy
-will get a sequence, but no deleted copy being seen (as wont be present)
-can identify that it happened via FISH, or snips
-find heterozygous sNIPs in germline. look for their presence in the sequence,c an look to see if you have lost one copy or not (often need another technique)
Loss of one copy = Loss of heterozygosity (lost ability to see snips) (loss of difference between the 2 inherited alleles)

Question 23

Scenario: sequencing the kras gene to guide targetted therapy
Example: KRAS mutation testing for cetuximab in metastatic colorectal cancer

Answer 23

Gene coding signalling molecule KRAS mutation testing for cetuximab in metastatic colorectal cancer
EGF –> EGFR –> X, Y, Z, KRAS (turns on survival and proliferation and mitotis of cells through many independant pathwyas (including a pathway involving KRAS)) (in a proportion of colon cancers this signalling is a powerful driver of growth of the tumour) –> enhanced cell proliferation and survival
-if can inhibit powerful growth signalling KRAS with drug is a very good treatment
-KRAS encodes for RAS protein
-Cetuximab = monoclonal antibody therapy which inhibits EGFR
-stops singaling cascades, as none are being turned on as receptor is bound/no longer there
-some patients gain/have resistance as have mutation in KRAS (mutant KRAS). allows RAS signalling protein to continue firing signalling of proliferation and survival all but itself
=Activating mutations, turning on RAS signalling functions without requiring EGFR stimulation
–>Can determine whether mutant KRAS resistance to Cetuximab drug is present by sequencing RAS gene in tumours of patients of colorectal cancer (wont benefit from expensive and toxic drug)

Question 24

Scenario: a patient presents with stage 4 melanoma who wanted to investigate all possible treatment options and therapeutic trials

Answer 24

This is the field of GENOMIC MEDICINE:
-the cutting edge between genomic research and clinical practice
-try and identify best treatment
-this field is in clinical trials - not yet routine clinical practice
At a population level we now know a great deal about the genetic changes that drive melanoma
Understanding the multitude of genetic changes in an individual patient’s tumour may allow more sensible stratification of targeted therapies

Question 25

Potential future practice

- what mutations are there in MAP kinase pathway

Answer 25

• what mutations are there in MAP kinase pathway
  -different genes involved in signalling pathway
  -influence other genes
  -analyse mutations to see if patients are particularily well suited to a drug
  How good a candidate is out patient for vemurafenib?
  -acts on mutation in BRAF gene
  Possibly good…
  a) out patient’s tumour has the BRAF mutation that this drug targets
  b) the tumour does not have mutations that activate molecules downstream of BRAF

Question 26

Potential future practice

-Does the tumour have a large number of mutations that can be presented on MHC?

Answer 26

-Does the tumour have a large number of mutations that can be presented on MHC?
e.g. Homozygous deletion of 6p22 to 6p21.3
presence of the RNA
-encodes class one MHC molecules
-bcause no matte r how many mutations there are around the tumour, it will be a good drug predictor of whether the checkpoint molecules work
Checkpoint inhibitor drugs (anti-PDI)??

Question 27

Potential future practice- Blood sequencing

Answer 27

Blood plasma tumour diagnosis
-Detection of circulating tumour DNA in early and late stage human malignancies
-e.g. Neuroblastoma and glioma
-coloured bars on y axis = number of mutant DNA molecules that get out of tumour into blood in every 5 mls of blood
=Liquid biopsies = detect mutations of tumour readily in blood

Question 28

Ethical issues surrounding Genome sequencing

Answer 28

delivering information back to patients

what you do when you’re not sure/dont understand what mutations do

Question 29

Genetic technologies will have a profound impact on medicine by the time you graduate

Answer 29

Medicine is going to become an information science
billions of data points on each individual
Major challenge: Need to develop technologies and scientific understanding to know how to use them

Question 30

2011 survey

Answer 30

Most NZ cancer clinicians expected the Use and Influence of molecular genomic tests to increase over the next ten years
-large influence on clinical decisions

Question 31

Genome sequence data about patients is no longer science fiction

Answer 31

Genomics England (in UK) - cancer, rare and infectious diseases
Baylor College of Madicine - sequence every new patient at Texas Children’s Cancer centre
-paedeatric cancer cases
NZ and Aus have their own clinical exome sequencing trials (from 2013)

Question 32

Massive “Wellness” Studies

Answer 32

Professor Leroy Hoods research group plan to follow 100,00 initially well people continuously for up to 30 years after baseline whole genome sequencing, with the participants having access to their won data
Google X in collab. with Stanford and Duke Uni’s - study 10,000 volunteers with baseline genome sequencing followed by longitudinal profiling of electronic health records, how are people who stay well different
* what is it about people’s genomes that they reach 50-70 without major problems
-why arent they getting cancer/CVD
-how are their genomes interacting with pathogens/drugs

Question 33

This technology is amazing, so why is it not changing medicine more quickly?

Answer 33

Technological limitations remain but can be overcome
Our incomplete molecular understanding of disease is now the major limitation to realising the value of research using genomics and bioinformatics
Technology is waiting for scientific understanding to catch up

Question 34

Conclusion:

Answer 34

The medicine you practice will be synergy between genomic technologies, pathology and traditional clinical acumen