ORGB 423 Lecture Notes - Lecture 8: Cervical Intraepithelial Neoplasia, Exome Sequencing, Cd135
12 Jun 2018
School
Department
Course
Professor
MIMM466 Viral Pathogenesis
2018-03-12 LEC 28 & 29 Genomics of HPV-associated Cancers
Dr. Archambault
From a persistent HPV infection to invasive cancer
• HPV is a Baltimore group 1 virus
• HPV is necessary but not sufficient to develop cervical cancer
o Need additional genetic event (e.g. mutation) infected cell to become cancerous
o Cancer development is a multi-stage process
• Most of the time the immune system clears the infection and you will be asymptomatic
• In individuals where the virus persists, there is a chance of developing cancer
• CIN (cervical intraepithelial neoplasia) stages represent a continuum of cancer progression
Cancer genomics
• The study of the totality of DNA sequence and gene expression differences between tumor cells and normal
host cells
o Aims to understand the genetic basis of tumor cell proliferation and cancer genome evolution under
mutation and selection by the body environment, the immune system, and therapeutic interventions
o Purpose of research: better inform physicians to tailor cancer treatments
Next Generation Sequencing (NGS)
• NGS is also known as high throughput or deep sequencing
• Sequencing the first human genome (~3B bases) cost $2.7B and almost 15 years to complete
• Genome sequencing now costs less than $5k and can be done in a few days
o The data can be analyzed in a few weeks
• The increased speed/throughput and lower cost of current DNA sequencing methods raise the possibility that
genome sequencing will become a standard diagnostic tool in the near future
• A major driver of personalized/precision medicine
Types of genetic alterations detected by NGS
Point mutations
Copy number alterations
Translocation
INDEL (insertions,
deletions)
Homozygous deletions: large deletions of
genes of both copies
Hemizygous deletions: large deletions of
genes on only one chromosome
Gain in copy number: regions that have been
locally amplified
When 2 genes come together and
express a fusion protein with a
dominant oncogenic activity
• Doesn’t usually form a protein
Example: how personalized medicine can transform cancer treatment
1. Sample: The sequencing process began with two small tissue samples taken from Dr. Wartman, abnormal
leukemia cells removed from his bone marrow and normal skin cells.
2. Sequence: The team extracted both DNA and RNA from his cells, giving it two types of genetic material to
test. After a month of work to sequence the material, the team had a large set of results to analyze.
3. Compare: Dr. Wartman’s DNA sequences showed some genetic mutations possibly related to his leukemia,
but none seemed treatable. But RNA sequencing revealed that a normal gene, FLT3, was overactive in his
leukemia cells.
4. Target: The FLT3 gene helps create new white blood cells in the marrow. The cells in Dr. Wartman’s marrow
were covered with an extremely high number of FLT3 receptors, which appeared to be driving the growth of
his leukemia.
5. Treat: A drug called sunitinib, typically used to treat kidney cancer, was known to block FLT3 receptors. Two
weeks after Dr. Wartman began taking the drug, tests revealed that his leukemia was in remission.
o NOTE: Sunitinib inhibits multiple receptor tyrosine kinases (RTKs) needed for cell proliferation or
angiogenesis
Whole Genome vs whole exome sequencing
Whole genome sequencing
Whole exome sequencing
Limitation
Complete sequencing of the tumor/cancer
tissue. The genomic sequence of healthy
tissue from the same patient is used for
comparison.
- Powerful to discover the full range of
genomic alterations.
- Requires high-amount of sequencing.
- Costly
- Sequence only the coding regions
(i.e. exons) of genes.
- Exome constitutes ~ 1-2% of the
complete genome.
- Lower cost and higher throughput
compared with WGS.
Sequence of the genome does
not provide information on
gene expression
RNA Sequencing
• RNA is extracted, converted into DNA by reverse- transcription, and the DNA sequenced.
• mRNAs, miRNAs, other RNAs.
• Provides information on RNA sequence AND abundance
o Mutations.
§ Mutations at splice sites can cause exon skipping, truncated proteins, etc.
o Genes that are expressed and their level of expression.
§ Heat mapping (looking at regions that are either over expressed or under expressed)
o Alternative splicing
o Fusion transcripts (translocation)
• Differential gene expression between tumor and normal tissue is often visualized with a heat map.
Epigenetics: bisulfite sequencing
• DNA methylation on cytosines is a common epigenetic mark.
o DNA methylation occurs at CpG island and represses gene transcription.
o DNA methylation pattern is linked to the level of expression of genes.
• Bisulfite reacts with unmethylated cytosines and deaminates them (CàU)
o Methylated-cytosines are protected; basis of the “bisulfite-sequencing” method.
• Analyze DNA methylation patterns between normal and tumor.
Epigenetics: ChIP-seq
• ChIP = Chromatin Immunoprecipitation.
• ChIP-seq is used to detect DNA sequences that are bound by a given protein, e.g. a transcription factor (TF)
o Cross-link protein to DNA, then sheer DNA
o Use Ab against protein of interest and immunoprecipitate it
o Cross-linked DNA will be immunoprecipitated with it
o Reverse cross-links and sequence that bit of DNA
• Requires an antibody that specifically binds the protein of interest.
• Used for genome-wide identification of transcription factor binding sites.
• Epigenetic studies: Genome wide identification of histone modifications using antibodies that recognize
specific histone modifications.
• E.g. do ChIP assay against acetylated nucleosomes, find that they occur mostly at active promoters
Omics-driven personalized medicine
• Big data analysis: idea that we will be able to use all the info found via data analysis to understand tumors
o Genome, epigenome, transcriptome, proteome, metabolome, microbiome
• Even if we don’t know how to use the data, we should still collect it because it might be useful someday
