Genes and Disease

Question 1

DNA sequencing (Sanger)

Answer 1

1. reaction mixture: primer and DNA template, DNA polymerase, ddNTPs with fluorochromes, dNTPs
2. primer elongation and chain termination
3. capillary gel electrophoresis separation of DNA fragments
4. laser detection of fluorochromes and computational sequence analysis

Question 2

illumina next generation sequencing NGS preparation

Answer 2

ligate linkers
denature then anneal to primers
DNA synthesis
denature and wash
anneal
DNA synthesis
10 x PCR

Question 3

sequencing results of NGS

Answer 3

coloured dots represent cluster of identical DNA fragments
circled coloured dots: colour change reveals which nucleotide was added to DNA fragment in each reaction cycle

Question 4

illumine next generation sequencing NGS process

Answer 4

cut one DNA strand, denature, wash and leave single strand
add new primer then fluorescently labelled dNTPs, one dNTP binds and wash away excess
fluorescent imaging to determine which dNTP bound
chemically remove bound flurophore and wash
repeat until DNA strand is replicated

Question 5

definition of a gene

Answer 5

functional unit of DNA
composed of transcribed regions and regulatory sequences
directs production of polypeptides or RNA’s

Question 6

what percent of genome is protein coding

Answer 6

1-2%

Question 7

what does the other 98-99% of non-coding DNA do

Answer 7

transcribed into non-protein coding RNAs which perform useful functions

Question 8

regulatory RNA to what

Answer 8

long non-coding RNA (lncRNA)
miRNA
siRNA

Question 9

which steps can eukaryotic gene expression be controlled

Answer 9

Question 10

RNAi pathway

Answer 10

Question 11

why is RNAi important

Answer 11

found in all eukaryotes except some fungi
defence against viral infection
protects against transposons and insertional elements
indicates that gene expression is key to evolving complex. organisms

Question 12

missense mutation

Answer 12

genetic alteration in which single base pair substitution (point mutation) alters genetic code
produces amino acid different from usual amino acid at that position
may be functional; and have similar properties
more likely to have phenotypic effect when base pair substitution occurs in first 2 nucleotide positions of codon
number of codons encoding given amino acid vary

Question 13

silent mutation

Answer 13

genetic alteration in which single base pair substitution (point) in protein coding portion of gene alters genetic code
doesn’t result in different amino acid encoded at that position
silent more likely to occur in 3rd nucleotide position of codon
can affect splicing, changes in protein function

Question 14

nonsense mutation

Answer 14

genetic alteration in which single base pair substitution alters genetic code to cause premature termination
truncated protein created is often non-functional or has impaired function
base pair deletion or insertion can result in frame shift, also introduces stop codon
non-stop mutation where stop codon removed

Question 15

hurler syndrome

Answer 15

autosomal recessive
lysosomal storage disease caused by deficient enzyme IDUA
accumulated substances in hurler are heparin sulphate and dermatan sulphate
one of lysosomal enzymes responsible for degradingglycosaminoglycans (GAGs)
Undegraded or partially undegraded GAGs progressively accumulate within tissues and organs
Developmental delay, skeletal, respiratory and cardiac abnormalities

Question 16

Duchenne muscular dystrophy

Answer 16

x linked recessive
Characterised by progressive muscle weakness and atrophy primarily in skeletal muscle
Caused by mutations in dystrophin, a rod-shaped protein that provides a strong mechanical link between the muscle cytoskeleton and the extracellular matrix
Binds cytoskeletal F-actin with its N-terminus and the dystrophin- associated protein complex with its C-terminus
Affects plasma membrane integrity and intracellular Ca2+ concentrations which in turn affects mitochondria and causes eventual cell death

Question 17

hunter syndrome

Answer 17

x linked recessive
Hunter syndrome,ormucopolysaccharidosisII(MPS II), is alysosomal storage diseasecaused by a deficient (or absent)enzyme,iduronate-2-sulfatase(IDS)
The accumulated substrates in Hunter syndrome areheparan sulfateanddermatan sulfate
One of the lysosomal enzymes responsible for degradingglycosaminoglycans (GAGs)
Undegraded or partially undegraded GAGs progressively accumulate within tissues and organs
Clinically related to Hurler Syndrome (MPS I)

Question 18

lysosomal storage disorders

Answer 18

Lysosomal storage diseases (LSDs) are a group of over 70 diseases that are characterized by lysosomal dysfunction, most of which are inherited as autosomal recessive traits.
These disorders are individually rare but collectively affect 1 in 5,000 live births.
LSDs typically present in infancy and childhood, although adult-onset forms also occur.
Most LSDs have a progressive neurodegenerative clinical course, although symptoms in other organ systems are frequent.
LSD-associated genes encode different lysosomal proteins, including lysosomal enzymes and lysosomal membrane proteins.
The lysosome is the key cellular hub for macromolecule catabolism, recycling and signalling, and defects that impair any of these functions cause the accumulation of undigested or partially digested macromolecules in lysosomes (that is, ‘storage’) or impair the transport of molecules, which can result in cellular damage.
Consequently, the cellular pathogenesis of these diseases is complex and is currently incompletely understood.

Question 19

lysosomal storage disorder treatments

Answer 19

nzyme replacement therapy – administration of a recombinant human enzyme to replace the mutated, non-functional one.
Substrate reduction therapy – administration of a small molecule that usually inhibits enzymes in the biosynthetic pathway producing the substrate.
Gene therapy – infection of the patient’s cells with a virus that expresses a functional enzyme or co-factor.
Chaperone therapy – in many cases, the mutation prevents the protein from correctly folding. Chaperones assist in the folding process.
Allogeneic bone marrow transplantation / hematopoietic stem cell transplantation / mesenchymal stem cell transplantation (will accept stem cell transplantation) – the transplanted stem cells produce the required protein.

Question 20

different types of genome editing approaches

Answer 20

rAAV
zinc fingers
CRISPR

Question 21

rAAV

Answer 21

High precision / low thru-put
Any locus, wide cell tropism
Well validated, KI focus

Question 22

zinc fingers

Answer 22

Med precision / med thru-put
Good genome coverage
Well validated / KO Focus

Question 23

CRISPR

Answer 23

New but high potential
Capable of multi-gene targeting
Simple RNA-directed cleavage
Combinable with AAV