Human Molecular Genetics - Lab Techniques and Gene Function

Learning Objectives for Human Molecular Genetics

• Gene Function from Phenotypes: Understand how analyzing an organism's observable characteristics (phenotype) provides information regarding the underlying function of a gene.
• Model Organisms in Genetic Research: Outline the utilization of genetic techniques in lab-based model organisms to discover gene roles.
• Identifying Pathogenic Variants: Describe the process of determining if a specific gene variant is the cause of a disease (pathogenic).
• Gene Editing and Therapy: Summarize how modern technologies like CRISPR-Cas9 and gene therapy could be applied to correct genetic disorders.

Information Extraction via Phenotype Analysis

• Natural Variation: By studying organisms that exhibit natural variation for a specific gene, researchers can infer what that gene normally does.
• Synthetic Variation: In instances where no natural variation exists, scientists can create their own variants through laboratory techniques.
• Case Study: Polydactyly:
  • Observation: The phenotype presented is the presence of extra digits.
  • Causal Link: This phenotype results from a specific change in a gene.
  • Inference: Therefore, the normal role of this gene is to prevent the development of extra digits during morphogenesis.

The Value and Conservation of Mutants

• Mutation vs. Variation: While variation in the human genome is widespread, most of it does not affect the phenotype. Mutations are rare subsets of variation that change the phenotype but do not always affect the organism's "fitness."
• The Translation Gap: Approximately $4,000$ human genes (roughly $20\%\text{ of the genome}$) have functions that are currently unknown.
• Genetic Conservation: Many human genes are highly conserved across the animal kingdom, including in fruit flies (Drosophila) and yeast. Creating or finding mutants in these related genes allows for the discovery of human gene functions.

Classic Examples of Rare Mutants and Gene Function

• T.H. Morgan and the White-Eyed Fruit Fly:
  • Discovery: After two years of deliberate breeding, Morgan identified a white-eyed male fly among red-eyed wild-types.
  • Significance: This was the first example of a sex-linked gene identified in an animal.
  • Mechanism: The white ($w$) gene encodes a protein that transports pigment into the compound eye cells. In the mutant, this protein is defective.
  • Human Connection: The human version of this gene is $ABCG1$, which encodes a protein vital for transporting cholesterol and lipids into cells.
• Myotonia Congenita in Goats:
  • Phenotype: The "fainting goat" phenomenon where muscles fail to relax immediately after contraction.
  • Genetics: Caused by a defect in the $CLCN1$ gene.
  • Normal Function: $CLCN1$ encodes the chloride channel receptor $CLC-1$, responsible for chloride ion transport.

Methodologies in Functional Molecular Genetics

• Definition: Functional molecular genetics is the approach used to determine the specific role of a gene through various techniques.
• Key Techniques:
  • Natural Variant Study: Investigating rare individuals with unique phenotypes.
  • Genetic Screens: Increasing the rate of random mutation (using X-rays or chemicals), selecting for a specific phenotype of interest, and sequencing the genome to find the causative mutation.
  • 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 sequence to observe the resulting phenotypic changes.

Model Organisms for Genetic Manipulation

• Criteria for Model Organisms: They must be easily raised in controlled environments and easy to manipulate genetically.
• Mouse (Mus musculus):
  • Divergence: Diverged from humans approximately $80 \text{ million years}$ ago.
  • Context: Possesses homologues (related versions) of almost all human genes.
• Zebrafish (Danio rerio):
  • Divergence: Diverged approximately $400 \text{ million years}$ ago.
  • Context: Contains versions of most human genes.
• Fruit Fly (Drosophila melanogaster):
  • Divergence: Diverged approximately $600 \text{ million years}$ ago.
  • Context: Contains versions of many human genes. Ed Lewis notably discovered the Antennapedia mutation (legs growing where antennae should be) using random mutagenesis in flies.

Transgenesis and Synthetic Biology

• Biological Universality: Because the DNA code is universal, DNA from any source (including synthetic sources) can be utilized by any organism.
• Transgenesis definition: The process of engineering a multicellular organism by introducing "foreign" DNA.
• Mechanisms of Transgenesis: Typically involves a regulatory sequence (promoter) attached to the gene of interest.
• The Green Fluorescent Protein (GFP) Example:
  • Source: Jellyfish (Aequorea victoria).
  • Process in Mice: Injecting the GFP transgene into the male pronucleus of a fertilized egg, followed by re-implantation into a foster mother.
  • Result: Transgenic mice express the jellyfish protein and glow green under specific light.
• Medical Applications of Transgenesis:
  • 1982: Human insulin production in bacteria for Type 1 diabetes treatment.
  • 1982: Rat growth hormone gene inserted into mice to study growth.
  • 1998: Production of Factor IX for Haemophilia B using hamster cells.
  • 2012: Production of spider silk for surgical sutures in the milk of transgenic goats.
  • 2019: Modification of brewer's yeast to produce cannabinoids.

Determining Pathogenicity of Gene Variants

• The Workflow:
  1. Sequence the genome(s) of the affected individual.
  2. Map findings to a human reference genome.
  3. Distinguish between common variants and novel variants.
  4. Predict if a variant is harmful or benign based on its properties.
  5. Validate and test the variant in a model system.

CRISPR-Cas9: Precise Targeted Mutation

• Components:
  • CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats.
  • Cas9: A CRISPR-associated protein (Cas9) that acts as "molecular scissors" to cut DNA.
• Evolutionary Origin: Evolved as an antiviral defense mechanism in bacteria.
• How it works:
  1. Design: Scientists create a "guide RNA" (gRNA) matching the target gene sequence.
  2. Binding: The Cas9-gRNA complex enters the nucleus and finds the matching sequence.
  3. Cleavage: Cas9 creates a double-stranded break at the target site.
• Outcomes of DNA Repair:
  • Knockout (a): In the absence of a template, enzymes repair the cut but often introduce errors called indels (insertions or deletions). This disables the gene.
  • Gene Editing (b): If a repair template is provided, the cell's repair machinery can use it to correctly "edit" or repair the DNA sequence.
• Award: Emmanuelle Charpentier and Jennifer Doudna received the Nobel Prize in Chemistry in 2020 for this discovery.

Correcting Genetic Diseases

• Somatic Gene Therapy: Targets the specific cells or organs affected by the disease. Changes are not passed to the next generation.
  • Example 1: Cystic Fibrosis: Caused by mutations in the $CFTR$ gene (chloride transporter). The variant $\Delta F508$ is specific. Therapy involves delivering a functional copy of the gene via a plasmid in a nebulizer to lung epithelial cells.
  • Example 2: Sickle Cell Disease: Results from a missense mutation in the $HBB$ gene (haemoglobin). Victoria Gray became the first successfully treated patient. CRISPR-Cas9 was used to break the $BCL11A$ gene (which normally suppresses fetal haemoglobin $HbF$ after birth), allowing the bone marrow to produce healthy $HbF$ instead of mutated adult haemoglobin.
• Germline Intervention:
  • Pre-implantation Genetic Diagnosis (PGD): Testing IVF embryos for specific risks before implantation.
  • Three-Parent Babies: Used for mitochondrial disease. The nucleus of the parents' fertilized egg is transferred into a donor egg with healthy mitochondria, resulting in an embryo with nuclear DNA from the biological parents and mitochondrial DNA from the donor.
  • Controversial Germline Editing: In 2018, He Jiankui allegedly created the first CRISPR-edited babies (modifying the germline permanently), which raises significant ethical questions.

Lecture Summary

• Mutations are phenotypic variants that do not always decrease fitness.
• Gene function is often deciphered by studying phenotypes in natural or engineered mutants.
• Model organisms are essential due to high levels of gene conservation with humans.
• Transgenesis allows the production of new proteins or the replacement of defective genes.
• CRISPR-Cas9 provides a way to model disease-causing variants in animals to study disease development.
• While somatic treatments avoid modifying future generations, germline treatments are possible but require heavy ethical consideration.