Pre-learning for the workshop “Applications of gene

technologies to medicine”
The pre-learning for this workshop has multiple parts. In total the preparation should take one
hour.

Activity Estimated time to complete

1. Brux Amed Case study (Read) 15 minutes
2. Recombinant protein production 20 minutes
Complete three diagrams and
understand the processes
3. CRISPR Cas (Watch videos) 10 minutes
4. Therapeutic cloning (Read) 10 minutes
5. Whole genome sequencing (Read) 5 minutes
Activity 1: Brux Amed Case Study

Below you will find the Brux Amed case study. While reviewing the case study, consider

 the molecular basis of Hunter Syndrome

 different biotechnology techniques are related
 how biotechnology has impacted diagnostic and therapeutic applications.

This activity should take 15 minutes.

Case study information

You are a paediatrician, your next patient is Brux Amed, a three-year-old boy. His Jewish parents are
both present in the consultation. They have five other children, all of whom are healthy (three girls
and two boys).

You have seen Brux multiple times in the last couple of months with recurrent upper respiratory
tract and ear infections. When he was born, he had an umbilical hernia that was treated with
surgery. He is exhibiting a range of symptoms including a swollen abdomen, joint pain and is not
meeting developmental milestones. Brux was referred by his GP because these symptoms are
indicative of a lysosomal storage disorder (Table 1).
Table 1 Mucopolysaccharidosis type II (Hunter syndrome): a clinical review and recommendations for treatment in the era
of enzyme replacement therapy (Wraith et al 2008)

You suspect these symptoms could be caused by a lysosomal storage disorder such as Tay Sachs
Disease, Hunter syndrome or Sanfilippo syndrome.

Lysosomes contain about 50 different degradative enzymes that can hydrolyze proteins, DNA, RNA,
polysaccharides, and lipids. Mutations in the genes that encode these enzymes are responsible for
more than 30 different human genetic diseases, which are collectively called lysosomal storage
diseases because non-degraded material accumulates within the lysosomes of affected individuals.

Therefore, you order a set of urine tests to see if you can rule out any of these three common
lysosomal storage diseases.
Table 2 Common urine test results for three lysosomal storage diseases

Tay Hunter Sanfilippo

Sachs Syndrome Syndrome
Disease
Causative gene(s) HEXA I2S GNS, HGSNAT,
NAGLU, SGSH
Molecule
Glycosaminoglycan - normal elevated slightly elevated
Heparan sulphate
Heparan sulphate normal normal elevated
breakdown products
Glycosaminoglycan - normal elevated normal
Dermatan sulphates
GM2 gangliosides elevated normal normal

Brux’s urinanalysis shows the followings:

Heparan sulphate – elevated

Heparan sulphate breakdown products – normal
Dermatan sulphates - elevated
GM2 gangliosides – normal

Thus you conclude that Brux likely has Hunter syndrome. Hunter syndrome results from a mutation
in the gene IDS that encodes the enzyme Iduronate 2-Sulfatase (I2S) that cells need to break down
heparan and dermatan sulphates and other glycosaminoglycans. When the enzyme is defective or
missing, these sugars build up and can cause developmental delays, organ problems, brain damage,
and early death.

Urine tests are not always accurate. In order to confirm your diagnosis, you order an enzyme assay
for I2S function. You take a blood sample so that the Artemis Laboratory can test the level of I2S
enzymatic activity in Brux’s blood cells using fluorometry. The test results indicate that there is no
functional I2S present.

To confirm the diagnosis, you ask Artemis Laboratory to run polymerase chain reaction (PCR) and
chemical mismatch detection on amplified complementary DNA (cDNA) from Brux. This aims to
detect and characterize deletions and point mutations. Brux’s I2S gene is found to have a mutation
that results in substitution of the amino acid proline at position 120 by arginine (P120R). This
changes the structure of an α-helix in I2S to create major structural deformities in the protein.

You begin enzyme replacement therapy immediately.

Activity 2: Recombinant Protein Production

In the workshop, you will be working in groups to explain the process of recombinant protein
production.

Label the following three diagrams, on different phases of recombinant protein production as
instructed on each page.

This activity should take approximately 20 minutes.

Resources which may assist in completing this task include:

 What is recombinant DNA technology? (sections: “DNA cloning” and “Protein manufacture”)
https://www.britannica.com/science/recombinant-DNA-technology
 Production of genetically engineered insulin and other recombinant proteins used in
therapies [pictures are not embedded but useful]:
https://www.eduhk.hk/biotech/eng/classrm/class_health5.html
 Dated but comprehensive Youtube video on the processes of recombinant DNA technology
Part 1 (6 minutes): https://www.youtube.com/watch?v=6UiKZKFHbMQ

Part 2 (4 minutes): https://www.youtube.com/watch?v=aUuw3erXmN4

Source: https://www.thermofisher.com/au/en/home/life-science/protein-biology/protein-biology-
learning-center/protein-biology-resource-library/pierce-protein-methods/overview-protein-
expression-systems.html#5

a) Fill in the blanks of Figure 1, using the provided phrases.

D)

E)

F)

Figure 1 Cloning DNA into a plasmid (Access Excellence, 1999-2000)

Labels:

Bacteria platted on medium + Insertion of Gene of interest

antibiotic by ligation
Culture Delivery of DNA into bacteria
DNA Purification Recombinant DNA
b) Match the phrases provided below Figure 2 to the steps (1-6) shown in the figure.

4
1
6

3
5

Figure 2 Overview of the process of human Insulin production, as an example of the process where bacteria can
produce large volume of human proteins (HKIEd 2002)

Labels:

Extraction & purification of human insulin Plasmid DNA cut with restriction enzymes
Human insulin-producing gene Recombinant DNA
Introduction of recombinant DNA into a Recombinant DNA multiplying and producing
bacterial cell human recombinant insulin


c). Using lines, indicate the correct order of the steps (Figure 3) required for producing large
quantities of purified therapeutic proteins

Approval from regulatory

authorities for clinical use

Extensive testing to establish clinical

efficacy

Growth of host cells on a

commercial scale

Introduction of gene into host cell

system for expression

Purification of product to meet

standards of regulatory bodies

Figure 3 Production of large quantities of purified therapeutic proteins

Activity 3: CRISPR Cas

In the workshop, you will need an understanding of the principles of CRISPR Cas as tool for gene
editing and the limitations of using it. Please watch the following two short youtube videos on
CRISPR Cas:

Basic principles of CRISPR Cas: https://www.youtube.com/watch?v=MnYppmstxIs (7 min)

Clinical applications of CRISPR Cashttps://www.youtube.com/watch?v=4YKFw2KZA5o (4 min)

Activity 4: Therapeutic cloning

In the workshop you will need to have a basic understanding of therapeutic cloning as a technique.
Consider how Brux or other members of his family may benefit from this technique.
Read Fact Sheet 4: Therapeutic Cloning (Somatic Cell Nuclear Transfer) prepared by the Australian
Stem Cell Centre (2010) may be helpful. (Appendix 1). Refer also to the lecture notes on the basic
principles of therapeutic cloning.

The reading should take approximately 10 minutes.

Activity 5: Genome sequencing

In the workshop, you will participate in an approximately 5-minute group discussion about the use of
genome sequencing and exome sequencing for genetic testing. As pre-learning, perform the
following prior to attending the session:

1. Watch the following short Youtube video (3 minutes) which explains the gist of Whole Genome
GS versus Exome Sequencing https://www.youtube.com/watch?v=s8FbGeOEL9k.

2. Read the short article on “What are whole exome sequencing and whole genome sequencing?”
in Appendix 2. The reading should take approximately 5 minutes.
Appendix 1: Fact Sheet 4: Therapeutic Cloning (Somatic Cell Nuclear
Transfer)
Source: http://www.stemcellfoundation.net.au
Appendix 2: Genome sequencing
What are whole exome sequencing and
whole genome sequencing?
Determining the order of DNA building blocks (nucleotides) in an individual's genetic code,
called DNA sequencing, has advanced the study of genetics and is one technique used to test
for genetic disorders. Two methods, whole exome sequencing and whole genome sequencing,
are increasingly used in healthcare and research to identify genetic variations; both methods
rely on new technologies that allow rapid sequencing of large amounts of DNA. These
approaches are known as next-generation sequencing (or next-gen sequencing).

The original sequencing technology, called Sanger sequencing (named after the scientist who
developed it, Frederick Sanger), was a breakthrough that helped scientists determine the
human genetic code, but it is time-consuming and expensive. The Sanger method has been
automated to make it faster and is still used in laboratories today to sequence short pieces of
DNA, but it would take years to sequence all of a person's DNA (known as the person's
genome). Next-generation sequencing has sped up the process (taking only days to weeks to
sequence a human genome) while reducing the cost.

With next-generation sequencing, it is now feasible to sequence large amounts of DNA, for
instance all the pieces of an individual's DNA that provide instructions for making proteins.
These pieces, called exons, are thought to make up 1 percent of a person's genome. Together,
all the exons in a genome are known as the exome, and the method of sequencing them is
known as whole exome sequencing. This method allows variations in the protein-coding
region of any gene to be identified, rather than in only a select few genes. Because most
known mutations that cause disease occur in exons, whole exome sequencing is thought to be
an efficient method to identify possible disease-causing mutations.

However, researchers have found that DNA variations outside the exons can affect gene
activity and protein production and lead to genetic disorders--variations that whole exome
sequencing would miss. Another method, called whole genome sequencing, determines the
order of all the nucleotides in an individual's DNA and can determine variations in any part
of the genome.

While many more genetic changes can be identified with whole exome and whole genome
sequencing than with select gene sequencing, the significance of much of this information is
unknown. Because not all genetic changes affect health, it is difficult to know whether
identified variants are involved in the condition of interest. Sometimes, an identified variant
is associated with a different genetic disorder that has not yet been diagnosed (these are
called incidental or secondary findings).

In addition to being used in the clinic, whole exome and whole genome sequencing are
valuable methods for researchers. Continued study of exome and genome sequences can help
determine whether new genetic variations are associated with health conditions, which will
aid disease diagnosis in the future.

Source: https://medlineplus.gov/genetics/understanding/testing/sequencing/
Additional Reading (not required for the class)
 Description of Hunter Syndrome and genetic changes involved
o https://medlineplus.gov/genetics/condition/mucopolysaccharidosis-type-ii
o http://disorders.eyes.arizona.edu/disorders/hunter-syndrome-mps-ii
 Methodology
o https://www.ncbi.nlm.nih.gov/gtr/tests/556392/
 Additional information about testing for Lysosomal Storage Disorders
o https://www.ncbi.nlm.nih.gov/gtr/tests/551442/
 Additional information about diagnostic genetic testing by genome sequencing
o https://www.melbournegenomics.org.au/our-work/about-genomics/what-
genomic-test

References

Access Excellence (1999-2000). Cloning into a Plasmid [Infographic]. Retrieved from

http://www.accessexcellence.org/RC/VL/GG/plasmid.html

HKIEd (2002). Human Insulin Production [Infographic]. Retrieved from

https://www.eduhk.hk/biotech/eng/classrm/explain/health3.jpg

Dimitrov, D.S. Therapeutic proteins. Methods Mol Biol. 2012; 899: 1–26.

Wraith, J. E., Scarpa, M., Beck, M., Bodamer, O. A., De Meirleir, L., Guffon, N., … Zeman, J. (2008).
Mucopolysaccharidosis type II (Hunter syndrome): a clinical review and recommendations for
treatment in the era of enzyme replacement therapy. European Journal of Pediatrics, 167(3), 267–
277. http://doi.org/10.1007/s00431-007-0635-4