CELS191 Lecture 25: Investigating the Function of Individual Genes

Lecture 25 Objectives: Investigating the Function of Individual Genes

• Upon the revision of this material, students should be able to:

  • Explain the methodology for obtaining information regarding the function of a gene from its observed phenotype.

  • Outline the utilization of genetic techniques within model organisms to determine gene function.

  • Outline genetic techniques used to establish whether a specific gene variant is pathogenic (disease-causing).

  • Outline the potential applications of gene editing and gene therapy in correcting genetic disorders.

Determining Gene Function from Phenotypes

• Information about gene function is derived by studying phenotypic changes resulting from genetic variation:

  • Study of natural variants: Observing organisms that naturally possess variations in a specific gene reveals the gene's normal role.

  • Creation of variation: If no natural variation exists, researchers can create their own variants.

  • Analysis of both types helps illustrate how specific genetic changes lead to altered phenotypes.

• Natural Variants and Gene Function:

  • The phenotype is the observable characteristic (e.g., polydactyly).

  • The cause is a change/mutation in a gene.

  • Reasoning: If the mutation causes the phenotype, the normal role of the gene is to prevent that phenotype (e.g., the normal function is to regulate the number of digits).

The Scientific Value of Mutants

• Genetic variation is common in the human genome, but most does not impact phenotype.

• Mutations are rare subsets of variation and do not always affect an organism's biological fitness.

• Genetic knowledge gap: Currently, around $4,000$ human genes (approximately $20\%$) have unknown functions.

• Functional conservation: Many of these genes are conserved across species, including mice, fruit flies, and yeast. Creating or finding mutants in these related genes allows for the inference of human gene functions.

Case Study: T.H. Morgan and the White Eye Mutation

• Historical Context: Thomas Hunt Morgan identified the first fruit fly phenotype ($Drosophila$) after more than two years of deliberate breeding.

• Phenotype: White eyes ($white, w$) instead of the wild-type red eyes.

• Significance: This was the first identified example of a sex-linked gene in an animal; it was seen mostly in male flies.

• Biological Function:

  • The normal "white" gene codes for a protein required to transport pigment into the cells of the compound eye.

  • Mutations result in a defective protein and a lack of eye pigment.

  • Human Homologue: The human version of this gene is $\text{ABCG1}$, which produces a protein essential for the transport of lipids and cholesterol into cells.

Case Study: Myotonia Congenita

• An inherited condition observed in goats where muscles fail to relax following a contraction.

• Genetic Cause: A defect in the $\text{CLCN1}$ gene.

• Mechanism: The gene encodes the chloride channel receptor $\text{CLC-1}$. The mutation causes an impairment in chloride ion transport.

• Video Reference: A YouTube video demonstration is available on the CELS191 Aoroa page.

Core Methodologies in Functional Molecular Genetics

• Studying Natural Variants: Analyzing rare, naturally occurring mutations.

• Genetic Screens: Increasing the rate of random mutation (using mutagens like X-rays or chemicals), selecting for a specific phenotype of interest, and sequencing the genome to identify the mutation.

  • Example: The Antennapedia mutation in $\text{Drosophila}$ (legs growing where antennae should be), discovered by Ed Lewis.

• Transgenesis (Genetic Engineering): Copying a gene of interest and inserting it into a different organism.

• Targeted Mutation (Reverse Genetics): Deliberately "breaking" or knocking out a specific gene to observe the resulting consequences.

The Use of Model Organisms

• Model organisms are easily raised in controlled environments and are simple to manipulate genetically.

• Shared genetic heritage allows for comparison between species:

  • Mouse: Diverged $80 \times 10^6\,\text{years}$ ago; contains homologues of almost all human genes.

  • Zebrafish: Diverged $400 \times 10^6\,\text{years}$ ago; contains versions of most human genes.

  • Drosophila (Fruit fly): Diverged $600 \times 10^6\,\text{years}$ ago; contains versions of many human genes.

Transgenesis and Synthetic Biology

• The DNA code is universal, meaning any DNA (including synthetic sequences) can be interpreted by any organism.

• Definition: Transgenesis is the engineering of a multicellular organism by adding "foreign" DNA.

• Structure of a Transgene: Usually consists of a regulatory sequence (promoter) and the gene of interest.

• Transgenesis in Mice:

  • Procedure: DNA is injected into the male pronucleus of a fertilized egg. The egg also contains a female pronucleus, polar bodies, and is surrounded by the zona pellucida. The egg is held by a holding pipette.

  • The egg is re-implanted into a foster mother (specifically into the ovary/oviduct area).

  • The litter is screened for the transgene insertion.

  • Example: Inserting jellyfish Green Fluorescent Protein (GFP) into mice causes them to glow green under specific lighting.

• Applications in Human Health:

  • $1982$: Rat growth hormone gene inserted into mice.

  • $1982$: Human insulin produced by bacteria for Type $1$ diabetes treatment.

  • $1998$: Factor $IX$ for Haemophilia B produced in hamster cells.

  • $2012$: Spider silk for surgical sutures produced in the milk of transgenic goats.

  • $2019$: Brewer’s yeast modified to produce cannabinoids.

Determining if a Gene Variant is Pathogenic

• Clinical Workflow:

  • Sequence the individual's genome.

  • Map the results to a human reference genome.

  • Categorize variations as common variants or novel variants.

  • Use predictive tools to determine if a variant is likely harmful or benign.

  • Validate and test the findings.

• Researchers can use modern genetics to target mutations to a sequence of choice to "break" specific genes in cells or model organisms to confirm their normal function and pathogenicity.

CRISPR-Cas9: Mechanism for Targeted Mutation

• Terminology:

  • CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats.

  • Cas9: CRISPR Associated Protein $9$.

• Origin: The system evolved in bacteria as a mechanism for antiviral defense.

• Components:

  • Cas9 Protein: Contains active sites capable of cutting DNA.

  • Guide RNA (gRNA): An engineered molecule with a sequence complementary to the target gene that "guides" the Cas9 protein to the specific site.

• Process:

  • The Cas9-gRNA complex enters the nucleus.

  • It finds the target sequence in the genome matching the guide RNA.

  • Cas9 creates a double-stranded break (DSB) at the target site.

• Outcomes of DNA Repair:

  • Non-Homologous Repair (Knockout): In the absence of a template, repair enzymes patch the cut, often resulting in errors such as Small Indels (insertions/deletions). This disrupts the gene sequence.

  • Homology-Directed Repair (Gene Editing): If a normal functional template is provided, the repair enzymes use it to "edit" the DNA sequence at the cut site.

• Recognition: The $2020$ Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for this technology.

Somatic Genetic Interventions: Successes and Challenges

• Somatic therapy targets only the affected cells or organs and does not change the germline (it is not passed to the next generation).

• Cystic Fibrosis (Gene Therapy Example):

  • A lethal single-gene autosomal recessive condition (incidence of approximately $1/2500$ live births).

  • Caused by variants in $\text{CFTR}$ (encoding a chloride ion transporter); the common mutation is $\text{CFTR}\Delta\text{F508}$.

  • Manifestations include lung disease and pancreatic insufficiency.

  • Treatment involves delivery of a functional copy of the $\text{CFTR}$ gene to lung epithelial cells via a nebulizer.

  • Plasmids (small, circular, double-stranded bacterial DNA) can carry the human $\text{CFTR}$ transgene for non-integrative therapy to restore function.

• Sickle Cell Disease (Gene Editing Example):

  • Caused by a recessive missense mutation in the $\text{HBB}$ gene on Chromosome $11$.

  • Substitution causes proteins to clump, resulting in sickle-shaped red blood cells.

  • Victoria Gray Treatment: First patient treated via CRISPR. Human fetuses use alpha and gamma chains for hemoglobin ($\text{HbF}$), but a gene called $\text{BCL11A}$ shuts down gamma genes at birth. CRISPR/Cas9 was used to break $\text{BCL11A}$ in extracted bone marrow cells, which were kemudian returned to the patient, enabling the production of $\text{HbF}$.

Germline Genetic Interventions and Ethics

• Germline changes affect the next generation and involve permanent modifications.

• Pre-implantation Genetic Diagnosis (PGD): In families with identified risks, IVF is used to create embryos which are tested before implantation; only healthy embryos are implanted.

• Three Parent Babies: Used when a faulty gene resides on mitochondrial DNA. The nucleus is removed from the biological parent's egg and transplanted into a donor egg (with healthy mitochondria) that has had its own nucleus destroyed. The resulting embryo has nuclear DNA from two parents and mitochondrial DNA from a third donor.

• Ethics and Controversy: The CRISPR "edited" babies allegedly created in $2018$ by He Jianhui (also referred to as Jiankui He) were germline mutations, raising significant ethical concerns.