Lecture 66

Question 1

What is Global genome analysis?

Answer 1

Assaying all human genes
in one assay has led to advances in genetic testing
(specific gene mutations assayed by large scale
gene sequencing) and molecular profiling (assays
that measure aberrant gene dosage or expression).
This provides the ability to:
1) Assess a patient’s risk of developing a disease and
tailor individual preventative measures
2) Stratify disease status to select treatments op
3) Evaluate impact of genetic variation on response to
medications (pharmacogenetics)

Question 2

What kind of disorder is cancer?

Answer 2

Multifactorial:

1) Cancer is a common disorder
2) Cancer is a genetic disease
3) Hundreds of potential candidate genes

Question 3

What are goals in multifactorial disease?

Answer 3

1) Identify all the genes mutations of which
significantly contribute to disease (gene discovery)
2) Identify predisposing mutations of these genes
that are common in the population
3) Large scale genome sequencing for patients to
assess individual genetic risk for common diseases
4) Gene therapy to “fix” critical defective genes that predispose people to disease

Question 4

How are mutated genes that significantly contribute to disease discovered?

Answer 4

Genome wide DNA microarrays (oligonucleotides representing all human genes
displayed on a glass slide) are available to
identify the subset of mutant candidate genes
driving a particular tumor

Question 5

What are application of DNA microarrays?

Answer 5

1) Comparative Genomic Hybridization (CGH) identifies deleted or duplicated genes which indicates loss or over expression of the gene product – detects CNVs
2) Comparative RNA Hybridization (CRH) directly identifies genes that under or over produce their mRNA – detects functional SNPs that affect mRNA levels

Question 6

What are indications for using a CGH microarray for testing?

Answer 6

1) Unexplained developmental delay or mental retardation
2) Growth abnormality
3) Dysmorphic features
4) Multiple congenital anomalies
5) Cardiovascular malformations

Question 7

Summarize CGH

Answer 7

1) Identifies amplified (overexpressed) and deleted (underexpressed) genes found in common among tumors of a particular type - candidate genes
2) Common dup/del can be used as biomarkers to stratify tumors: metastatic potential, chemotherapy response
3) Does not identify individual genes because deletions and duplications usually involve
multiple genes, so look for functional significance

Question 8

What is the current clinical utility of CGH?

Answer 8

1) Mental retardation
2) Developmental delay
3) Autism
4) Dysmorphic features

Question 9

How is CRH used for a tumor?

Answer 9

1) Tumor sample labeled with Cyanine-5 (red)
2) Normal sample labeled with Cyanine-3 (green)
3) Genes equally active in both samples – no mutation in this gene in the tumor (yellow)
4) Genes not expressed in this
tissue – no signal

Question 10

Common genes that are
candidate genes in the
origination and/or propagation of a certain kind of tumor are:

Answer 10

Targets for designer drugs or gene therapy

Question 11

Candidate genes as biomarkers for stratification of tumors into distinct subcategories:

Answer 11

1) Metastatic vs non-metastatic
2) Response vs poor response to chemotherapy
3) Tumor staging or tissue of origin

Question 12

Summarize CRH

Answer 12

1) Identifies genes with abnormal over or under expression of mRNA in a particular tumor, reflecting an underlying mutation in the gene that alters mRNA production, maturation or turnover
2) Useful in candidate gene identification since each gene is assessed individually. However, it is unlikely that all candidate genes are key contributors to tumorigenesis or propagation, so again look for functional significance
3) Does not identify gene mutations unless the mutation alters steady state levels of mRNA

Question 13

What are current clinical utilities of CRH?

Answer 13

Molecular stratification of tumors

Question 14

What kind of gene mutations are

detectible by microarray studies?

Answer 14

1) Gene deletions or amplifications that decrease or increase gene dosage - CGH
2) Mutations that cause increased or decreased mRNA levels - CRH

Question 15

What is next generation sequencing in clinical DNA diagnostics?

Answer 15

1) Targeted testing
a) Single genes or gene panels
b) No incidental findings
c) No variants of uncertain significance (VOUS)
2) Genome wide scans
a) Targets whole exome or genome with different resolutions
b) Incidental findings likely
c) High rates of VOUS

Question 16

What is gene delivery most effective with?

Answer 16

1) Gene delivery is most efficient using a virus vector: exploits their high degree of infectivity in human cells
2) Removal of essential viral genes disables vector’s disease causing ability and provides space in the viral genome for insertion of therapeutic human genes
3) Vectors of choice: adenovirus and retrovirus

Question 17

How do you prepare a therapeutic gene with a virus?

Answer 17

1) Insert cloned therapeutic gene cDNA into viral genome with essential viral genes deleted
2) Package recombinant viral genome into virus particles in cell culture by transfecting DNA into cells that supply the missing viral proteins needed for replication and packaging of viral particles
3) Harvest high titer defective virus containing the therapeutic gene from the cell culture supernatant

Question 18

Why is cDNA used for therapeutic viral genes rather than genomic DNA?

Answer 18

Introns cause the DNA to be too large to fit into a virus

Question 19

What are advantages and disadvantages of using an adenovirus as a vector for gene therapy?

Answer 19

Advantages:
1) high efficiency infection
2) infects non-growing as well as proliferating cells
3) does not integrate into host genome, therefore low risk of insertional mutagenesis
Disadvantage:
1) transient expression and immune/ inflammatory reactions upon reapplication of therapeutic gene containing virus

Question 20

What are advantages and disadvantages of using an retrovirus as a vector for gene therapy?

Answer 20

Advantages:
1) high efficiency infection
2) infects non-growing cells poorly
3) integrates into the host genome, so can achieve long term expression
Disadvantage:
1) insertional mutagenesis is a significant concern

Question 21

What is therapy for OTCase deficiency?

Answer 21

1) Loss of function disease
2) Normal OTCase cDNA (why cDNA? - genomic cDNA too large) inserted into adenovirus vector to infect liver cells
3) Observed good expression of the normal protein and relief of symptomes initially but expression decreased over time (several months)
4) Problem: reapplication of the construct (necessary for long term gene expression) often caused inflammatory and immunological response

Question 22

What is therapy for ADA deficiency (SCID)?

Answer 22

1) Marrow stem cells from patient
2) Ex vivo infection in culture with retrovirus vector
3) Selection of ADA expressing cells
4) Implantation into patient
5) Successful long term correction and relief of
symptoms
6) Problem: insertional mutagenesis linked to leukemia

Question 23

What is a Lentivirus?

Answer 23

1) subclass of retrovirus able to infect non
2) proliferating and terminally differentiated cells
3) lentivirus vectors are derived from human immunodeficiency virus (HIV-1)
4) pseudotyped viral vectors with different envelope proteins encoded by specific plasmids can target specific tissues e.g. rabies glycoprotein targets virus to neurons, Ebola coat protein to airway epithelium

Question 24

What is a therapy for dominant disorders that have too much or aberrant gene product?

Answer 24

1) shRNA ( short hairpin RNA ) –
mimics natural miRNA
2) Introduce therapeutic shRNA gene via a retrovirus vector
3) Targeted shRNA expression causes decreased cognate mRNA translation (“knock down”) of aberrant or over expressed genes