18- Functional Genome

Question 1

define next generation sequencing

Answer 1

a massive parallel modern sequencing technology for high throughput DNA sequencing - includes WES and WGS

enables the rapid sequencing of DNA/RNA base pairs

Question 2

define whole exome sequencing/WES

Answer 2

type of NGS technique used to capture the sequence of the coding region of the genome - can be used to identify a disease-causing variant

Question 3

define whole genome sequencing/WGS

Answer 3

used to capture the entire genome which isn’t always necessary - most mutations are in protein-coding regions

Question 4

what are the clinical advantages of NGS techniques?

Answer 4

looks for variance in patients, gene expression profiles and disease-causing mutations

pushes healthcare towards personalised medicine

Question 5

describe candidate gene filtering of whole exome sequencing (WES) data

Answer 5

WES can be used to identify a disease-causing variant – less expensive than sequencing an entire genome, majority of mutations are protein-coding

steps to identifying the putative/ disease-causing gene through heuristic candidate gene filtering:

1. start with 3 million SNPs = assume the causal variant is coding
   - ignore structural variants, other forms of genetic variants, non-coding variants, variants in regulatory elements, promoters, enhancers
   - account for indels, insertions, CNVs
2. 15-20,000 coding SNPs = assume the causal variant is non-synonymous, alters the protein sequence
   - synonymous variants are a change in base, not amino acid – exclude
   - use software to divide synonymous and non-synonymous variants based on their predicted effects
3. 7-10,000 coding, non-synonymous SNPs = assume the causal variant has complete penetrance
   - disorder is a monogenic Mendelian disease caused by one gene
   - not caused by a known gene variant in a publicly available database
4. 200-500 novel coding non-synonymous SNPs = assume causal variant has complete detectance
   - determine mode of inheritance of gene – check for co-segregation of genes within families via sanger sequencing

these steps should produce one putative gene as the answer – further filtering techniques through functional tests can be employed
reasons for doubting this is the putative gene

disease-causing variant may be in a non-coding region, poor coverage of exome
functional tests to support that this is the causative gene variant:
1. in vivo/ vitro models to test gene dysfunction
2. understand molecular mechanisms of gene and how it contributed to the disorder
3. see if encoded gene of interest is present in the patient
4. monitor knockdown/ overexpression of gene in model
5. identify how the mutation is functional, how it affects cell development and protein structure/function

Question 6

list the in vitro techniques for investigating putative genes

Answer 6

cell culture
gene knockdown
protein localisation
induced pluripotent stem cells

Question 7

list the in vivo techniques for investigating putative genes

Answer 7

making mutant mice
tissue specific knockout mice through Cre-Lox system
gene knockdown in zebrafish using CRISPR-Cas9
RNA rescue experiments

Question 8

protein localisation as an in vitro technique

Answer 8

affected protein - can see changes in protein localisation and behaviour based on binding and fluorescence

two methods - antibody staining & GFP tagged gene of interest

antibody staining:
- fragmented peptide injected into host, produces an immune response and antibody production against protein by host
- antibodies isolated, purified and used to bind to protein in cell culture
- host-antibody binds to protein, fluorescently labelled antibody binds to host-antibody
- can show differing levels of binding between mutant and normal protein based on fluorescence

GFP tagged:
- cell containing GFP tagged normal gene of interest transfected with CMV promoter
- cell containing GFP tagged MUTATED gene of interest also transfected with CMV promoter
- transcription in both cells
- GFP fluoresces green under UV light, can compare fluorescence between the two types of cells

Question 9

in vitro cell culture technique

Answer 9

• cells removed directly from tissues, cell groups disaggregated by enzymes/ mechanically
• cell groups are grown in cultures in favourable artificial conditions. cells have finite divisions – with chemical treatment they’re immortalised = establishes primary culture
• immortalised cells placed in culture and proliferate
• eventually cells are subdivided, sub-cultured and transferred to new dishes with more room and a fresh growth medium – form the subclone/ cell line. these cells proliferate to a limit
• cells with the highest growth capacity dominate cell populations – creates genotypic and phenotypic uniformity within cell population, less variation means less inconsistency in results.

Question 10

uses of cell culture in vitro studies

Answer 10

metabolic studies

drug screening and vaccines

Question 11

advantages of cell culture

Answer 11

cheap, grow quickly, easily reproducible with consistent results

easily available commercially

less ethical considerations

Question 12

define an induced pluripotent stem cell

Answer 12

derived from adult somatic cells

reprogrammed to de-differentiate into embryonic like stem cells

Question 13

describe the mechanism behind producing an IPSC

Answer 13

non-invasive patient skin biopsy

cells are isolated, cultured and amplified in growth culture

fibroblasts undergo chemical treatment – de-differentiate and become pluripotent stem cells

can be reprogrammed into another cell type the specific genes required for the differentiation process

Question 14

use of IPSCs

Answer 14

personalised medicine

disease modelling - observing the behaviour of any cell line, IPSCs have the patient’s genetic make up

Question 15

two methods of genome editing

Answer 15

gene editing - correcting the mutated gene, restoring protein production and function

homology directed repair - repairing double stranded DNA lesions, a type of gene editing
- restores the pathogenic genetic change causing the disorder

Question 16

the use of model animals in research - why they are used?

Answer 16

help us understand animal and human ill health - better quality of life for domestic and wild animals

good models for understanding human diseases, can use modern technology to genetically manipulate their genomes and recreate an exact disease mutation

Question 17

advantages of mice

Answer 17

• accelerated lifespan, can study their entire life cycle in 2-3 years
• cost-effective, small, easy to transport, reproduce quickly
• can genetically manipulate mice and recreate a disease with the precise mutation = lots of methods and technology suited to mice models
• lots of published literature with mice models = don’t need to repeat experiments already done

Question 18

advantages of zebrafish

Answer 18

• transparent eggs, develop ex utero = can follow embryonic development under a microscope
• females produce 200 eggs on average, more than mice = large numbers good for statistical analysis
• small embryos with fewer cells = easier to transduce individual cell lineages
• can manipulate zebrafish easily
• can administer drugs into the fish medium and see how they affect development = pharmacologically regulated

Question 19

disadvantages of using mice

Answer 19

• less embryos produced compared to zebrafish = 8 babies on average, not enough for statistical analysis
• mice don’t develop ex utero (need to cull the mother)
• experiments can be expensive

Question 20

describe the process of making a mutant mouse

Answer 20

first - construct a targeting vector:
- homology arms = matching sequences that flank the engineered cassette
- complementary to the genomic region sequence you want to modify
- ensure precise localisation of genetic modification
- engineered cassette = will replace the endogenous gene, contains a neomycin resistance gene as a selectable marker to identify positive clones

• targeting vector transduced into pluripotent embryonic stem cells
• homology arms align with complementary sequence of endogenous gene
• homologous recombination occurs = cross-over of engineered cassette and target endogenous region
• recombinant product now has engineered cassette = positive ESC clones can be identified by screening for the neomycin selectable marker
• positive clones then cultured and amplified in growth culture = proliferate to blastocyst stage, implanted into pseudo-pregnant mother mouse = offspring are mutant mice

Question 21

two main methods of gene knockdown in zebrafish

Answer 21

CRISPR-Cas9 system

using antisense morpholinos

Question 22

define a morpholino

Answer 22

a synthetic 25bp oligonucleotide

contains a morpholino sugar backbone instead of ribose (like in DNA)

Question 23

describe the process of gene knockdown in zebrafish using antisense morpholinos

Answer 23

two methods: blocking pre-mRNA splice sites OR preventing translation of mRNA

preventing translation of mRNA
- morpholino binds to translation initiation site located before the translation start codon, prevents ribosome from binding
- no translation = no protein produced = no gene expression

blocking pre-mRNA splice sites
- morpholino binds to pre-mRNA splice sites = introns can’t be removed correctly
- as a result, pre-mRNA transcript is degraded by RNA decay machinery
- no protein produced = no gene expression

Question 24

describe the process of gene knockdown in zebrafish using the CRISPR-Cas9 system

Answer 24

requires guide RNA, Cas9 endonuclease and genomic DNA

guide RNA contains two complementary sequences - one complementary to the target/genomic DNA, another complementary to the binding site of Cas9 endonuclease

genomic DNA must have a PAM sequence - NGG - for Cas9 endonuclease to bind and cleave

1. guide RNA guides Cas9 endonuclease to its complementary sequence = Cas9 binds and forms guide RNA-Cas9 complex
2. Cas9-guide RNA complex scans genomic DNA up and down until it locates a complementary target DNA sequence
3. locates sequence - allows Cas9 endonuclease to cleave genomic DNA, cause a double strand break
4. activates cell’s natural repair mechanisms - two methods:

non-homologous end joining = most effective; imperfect repair mechanism that re-joins broken ends of DNA. can cause small indels and frameshift mutations

homology directed repair = introduces specific mutations by providing a repair template DNA with desired changes. less effective in zebrafish

Question 25

name the various methods for making a gene knockdown/mutant zebrafish

Answer 25

antisense morpholinos

CRISPR-Cas9 system

ENU screen = chemical causing point mutations, allows for screening phenotypes

genome editing = targeted approach

zinc fingers and TALENS = gene editing technique

Question 26

describe the process of RNA rescue experiments using ARCA/ autosomal recessive cerebellar ataxia disorder as an example

Answer 26

used to prove a variant is disease-causing

ARCA is a neurodegenerative disorder, caused by the homozygous mutation of a 3bp deletion in the CHP1 gene - results in cerebellar degeneration

morpholinos used to cause CHP1 gene knockdown in zebrafish - results in excess proliferation of neurons/ hyperplasia, disrupting normal neural development

then inject wild-type RNA from human into morpholino-mediated CHP1 knockdown zebrafish

wild-type RNA serves to restore some functionality, restores crossing over of neurons

RNA rescue with wild-type RNA has a limited degree of rescue due to specificity of CHP1 morpholino = partial rescue restores some functionality but not entirely normal neural development

Question 27

describe how in vitro techniques and gene editing can be used to correct Duchenne muscular dystrophy

Answer 27

Duchenne muscular dystrophy - severe muscular degenerative disorder caused by loss of function of dystrophin gene

dystrophin gene has 79 exons = mutations affecting reading frame result in Duchenne’s

dystrophin mutation causes muscle membrane breakdown when muscle contracts, causes muscle wastage

can use IPSC technology - reprogram fibroblasts into muscle lineage cells
- see dystrophin isn’t expressed with antibody staining, identify the mutation

e.g. lacking exon 44 = use gene editing, cause a frameshift to knock in exon 44. a lot of dystrophin domains have repeat sequences - can at least partially rescue dystrophin gene for dystrophin protein production