mbb 304 mt 2

covers lectures 7-11

Non-Coding Regulatory Mutations

Mutations in non-protein coding sequences (promoters, splice sites, UTRs) that alter gene transcription, splicing, or translation efficiency without directly changing a codon sequence.

impacts of regulatory element mutations

1. promoters can be mutated to disrupt transcription factor binding sites to inc or dec gene expression

2. critical mRNA splicing sites can be mutated

3. promoters mutates to enhance transcription factor binding

4. epigenetic regulation of a gene can be altered by a mutation

5. mutations can occur in non-coding RNA

β-Thalassemia Allele Classifications

• β0 alleles produce no functional β-globin

• β+ alleles significantly reduce β-globin synthesis

• β++ alleles cause minor/silent reductions in β-globin expression.

human genome mutation nomenclature

c.301G>A (p.G101R)

• c = coding region

• 301st nucleotide

• G replaces A

• p = protein

• 101st protein, R replaces G

• del = deletion

• ins = insertion

• fs* = frameshifts

B-thalassemia definition

autosomal recessive condition that results from either reduced or absent B-globin gene expression

Developmental Globin Switching

spatial arrangement of hemoglobin genes coincides with developmental timing of expression

• Embryonic: ζ (zeta) and ε (epsilon)

• Fetal (HbF): a (alpha) and y (gamma)

• Adult (HbA): a (alpha) and B (beta)

• Adult2 (HbA2 ): a (alpha) and d (delta)

B-thalassemia - etiology

• start codon mutations - start transcription at wrong place

• promoter mutations - cant initiate open complex to start RNA synthesis

• RNA processing mutations - splicing incorrectly

Promoter Box Mutations (CACCC, CCAAT, TATA)

Point mutations in non-coding promoter elements that disrupt transcription factor and TBP/RNA Polymerase II recruitment, diminishing transcription to cause β+ or β++ phenotypes

• TBP straddles the (T)ATAAAA box with critical Phe residues inserting into the DNA

• TBP bends the DNA, allowing RNA Polymerase II to bind

Start Codon Mutation Effect

Point mutations disrupting the translation initiation codon (ATG to GTG, CTG, ACG, etc.) completely block translation initiation, producing a β0 phenotype.

Conserved Splicing Consensus Signals

Highly conserved pre-mRNA intron sequences required for splicing, located at the 5' splice site (GU), branch point adenine (A), and 3' splice site (AG).

Splice Site Mutation Effect

Mutations at conserved intron boundaries (e.g., IVS1-1 G>A or IVS2-1 G>A) prevent proper intron excision, causing aberrant pre-mRNA splicing and a β0 or severe β+ phenotype.

B-thalassemia - organismal level

symptoms begin ~6 months old

• fetus rely HbF (alpha/gamma)

• adult rely on HbA (alpha/beta) - effects of mutation can be felt

• severity ordered:

  • normal - normal synthesis

  • B trait - slightly reduced

  • B-thalassemia intermedia - markedly reduced

  • B-thalassemia major - no synthesis

β-thalassemia - cellular/molecular level

causes RBC cell death, anemia, iron buildup, jaundice

• symptoms caused by RBC death

• aggregation caused by a-globin accumulation

• aggregates cause

  • membrane damage

  • ineffective RBC maturation

  • reduced survival

  • hemolysis (cell rupture)

structured aggregates vs amorphous aggregates

structured:

• highly ordered, specific conformation

• hair-like filaments or fibrils that tangle

amorphous aggregates

• irregular disordered masses, cluster randomly

• globular clumps

B-thalassemia oxygen transport - tissues vs lungs

• capillaries of body tissues → low [O2], high [CO2]

  • O2 released from hemoglobin and diffuses outwards to meet tissue needs

• capillaries of lungs → high [O2], low [CO2]

  • O2 diffuses inward and binds hemoglobin

B-thalassemia - why fatigue

• bc of low [O2], system switches from Kreb’s cycle → fermentation

• low [O2], low ATP production

• low ATP, lower energy

• lower energy to muscles = fatigue

β-Thalassemia Complications

Systemic consequences of severe anemia including:

• jaundice (elevated bilirubin due to degraded free heme)

• hepatosplenomegaly - enlarged liver/spleen to try produce more RBC

• iron overload (hemochromatosis) - free heme releases iron into blood stream

• chipmunk facies from marrow expansion

B-thalassemia diagnostics

• complete blood count (CBC)

  • popped balloon - due to damaged cell from aggregates

  • target cells - due to aggregates

• hemoglobin electrophoresis

  • detect hemoglobin tetramer variants by charge and molecular weight

  • HbA - adult (alpha/beta)

  • HbA2 - adult2 (alpha/delta) - tries to compensate

  • HbF - fetal (alpha/gamma)

• genetic testing

B-thalassemia - treatment

does not fix problem but treats symptoms

• transfusion-dependent - receives blood every 3-4 weeks

• non-transfusion dependent

  • deferasirox/deferoxamine - binds iron to make it soluble, prevents hemochromatosis

  • hematopoietic stem cell transplant - fixes underlying problem in bone marrow but can cause immuno problems graft vs. host

CACCC box

important regulatory elements for the hemoglobin genes in erythroid cells

Essential vs. Non-Essential Genes

• essential genes - strictly required for basic cellular survival and reproduction

  • disruption of essential genes lead to cell death

  • part of a minimum gene set of a living cell

• non-essential genes can tolerate mutation or loss without immediate single-cell lethality

CRISPR/Cas9 process

• gRNA guides cas9 to cut target DNA sequence

• after dsb, cell’s DNA repair machinery fixes break

CRISPR

clustered regularly interspaced short palindromic repeats

• finds selected sequence in genome based on base complementary

• guideRNA that directs cas9 to target DNA sequence where cas9 makes double stranded break

• og bacterial system has 2nd RNA, TracrRNA, which guides gRNA and Cas9 together

Cas9

CRISPR-associated protein 9

• endonuclease that cuts both strands of DNA

NHEJ vs. HDR DNA Repair

• Non-Homologous End Joining (NHEJ)

  • rapidly rejoins cut DNA ends with error-prone indels (ideal for gene knockouts)

  • often causes ins or del causing frameshifts

• Homology-Directed Repair (HDR)

  • uses a homologous donor template for precise sequence editing

  • supplied HDR can introduce desired sequence change!!

BCL11A Transcription Factor

zinc-finger repressor protein expressed in erythroid lineages after birth

• directly represses fetal γ-globin gene transcription to enforce the switch to adult β-globin

casgevy disrupts an erythroid specific enhancer of BCL11A

Casgevy Mechanism of Action

CRISPR/Cas9 introduces targeted NHEJ indels into the erythroid-specific enhancer of BCL11A

• results in knocking down BCL11A in RBCs to reactivate fetal γ-globin (HbF) and alleviate anemia

allogenic transplantation

medical procedure where healthy blood stem cells collected from DONOR and given to patient

• patients blood stem cells are destroyed by chemotherapeutics and radiation

• healthy blood stem cells extracted from donor

• healthy blood stem cells injected into patient

Casgevy Tissue Specificity

By disrupting an erythroid-specific enhancer rather than the BCL11A promoter or coding sequence, BCL11A expression in the brain and non-hematopoietic tissues remains normal.

Versatility of BCL11A Enhancer Editing

Disrupting the BCL11A enhancer reactivates protective HbF in all patients regardless of their specific underlying HBB gene mutation, eliminating the need for patient-customized guides

• B-thalassemia - due to low B-globin, if less BCL11A, then more y-globin to make healthy HbF

• sickle cell anemia - due to HbS polymer formation, if less BCL11A, increased HbF doesnt participate in polymer formation, which decreases [HbS]

Casgevy Autologous Transplant Steps

• Hematopoietic stem cells are extracted ex vivo

• stem cells edited via Casgevy electroporation

• myeloablative chemotherapy conditioning removes patients existing blood stem cells

• casgevy edited cells re-infused into the patient, and monitored for marrow engraftment

immunogenicity

ability of a substance like a pathogen or therapeutic protein to trigger immune response in body

integration

ability of an encoded DNA sequence to integrate into the genome of a host

nuclear envelopathies (laminopathies)

a group of disorders targeting the nuclear envelope or lamina

Nuclear Lamina Functions

A fibrous meshwork of intermediate filaments (Lamins A, B, C) that:

• maintains nuclear shape

• withstands mechanical stress

• tethers heterochromatin for genome organization

• sequesters transcription factors.

nuclear envelope

• double membrane structure that separates the nucleoplasm from cytoplasm

• membrane is dotted with nuclear pore complexes that facilitate transport between the compartments

lamin A mutation

hutchinson-gilford progeria syndrome (HGPS)

• deletion of 50 amino acids near C-terminus

intermediate filaments - diseases

structures only found in animals, stable, not polarised, expression varies from tissue to tissue

• tumour cells may lose normal morphology but rfetain cytoskeletal proteins characteristic to tissue of origin

  • therefore, by using antibodies against various intermediate filament proteins, we can discover if origin of tumour is epithelial, mesenchymal, or neuronal

CAAX

protein motif found on Lamins and Ras proteins critical for protein localisation

Prelamin A Step 1 (Farnesylation)

Farnesyltransferase (FTase) attaches a 15-carbon farnesyl lipid group to the C-terminal CAAX motif cysteine

• allows membrane association and subcellular targetting

Prelamin A Step 2 (Proteolysis of terminal residues)

ZMPSTE24 protease cleaves the terminal -AAX tripeptide from the farnesylated C-terminus of prelamin A

• exposes cysteine residue for further modification

Prelamin A Step 3 (Carboxymethylation)

Isoprenylcysteine carboxyl methyltransferase (ICMT) catalyses methylation of Lamin A

• slightly enhances hydrophobicity of Lamin A, increasing inner nuclear membrane interactions

Prelamin A Step 4 (proteolysis of 15 terminal residues)

ZMPSTE24 performs a second cleavage, removing the C-terminal 15 amino acids to release soluble, mature Lamin A

• Lamin A can now properly integrate into nuclear lamina

subcellular targeting signals in Lamin A

• nuclear localisation signal (NLS) promotes nuclear import of prelamin A

• farnesylation and carboxymethylation promote membrane association

• removal of farnesylated C-terminal segment permits mature lamin A to incorporate properly into nuclear lamina

hutchinson-gildford progeria syndrome

autosomal dominant, most cases arriving from de novo mutations

• mutation in Lamin A gene that results in defective nuclear lamina formation

• rapid aging, growth failure, hair loss, cardiovascular issues, facial abnormalities, skeletal issues, life span

LMNA c.1824C>T (p.G608G) Mutation

A silent point mutation in Exon 11 of LMNA that creates an active 5' cryptic splice site (CAG/GTGGGC) in prelamin A pre-mRNA

• deletes 150 nucleotides (50 amino acids) from prelamin A mRNA, producing the toxic mutant variant Progerin

Progerin Structural Defect

The 50-amino acid deletion in Progerin removes the second ZMPSTE24 cleavage site (Step 4), preventing final cleavage and causing permanent C-terminal farnesylation.

Progerin Cellular Toxicity

HGPS not caused by absence of Lamin A, but toxic levels of Progerin

• permanently farnesylated Progerin remains anchored to the inner nuclear membrane disrupting lamina assembly

• causes nuclear blebbing, nuclear pore clustering, and heterochromatin loss.

progeria - diagnostics

• physical characteristics visible at age ~2

• genetic testing used for validation

progeria - treatment

1. symptom management + cardiovascular care

2. farnesyltransferase inhibitor - zokinvy (prevents targeting to nuclear envelope, and decreases severity thru no lipids sticking out of intermediate filaments

3. restores normal splicing - blocks splicing site, restores wildtype

4. gene-based therapeutic approaches

ER and golgi apparatus dysfunction

often linked to neurodegenerative disorders, protein trafficking disorders, and ER stress disorders

• RER - starting point of protein biosynthetic pathways

• SER - synthesises lipids, steroid hormones, detoxifies organic compounds, stores Ca+

• golgi - processes + packages proteins, glycosylation for secreted + membrane proteins

vesicle disorders

linked to diabetes + neurological diseases like insulin and neurotransmitter release, and SNAREopathies

• secretory vesicles - material for cell surface packed in secretory vesicles to be fused w membane

• transport vesicles - material moving between compartments in endomembrane system transported in transport vesicles

  • ER → golgi

  • stacks within golgi

endosomopathies

can dysregulate protein + lipid trafficking

• endocytic vesicle - vesicles budded from membrane bc of bulk-phase or receptor mediated endocytosis

• early endosome - primary sorting station to recycle to membrane or degrade

• late endosome - organelle w full complement of acific hydrolases to make a lysosome, but not pH 4-5

Lysosomal Storage Disorder (LSD) Pathway

Deficiency of a lysosomal acid hydrolase prevents substrate degradation

• causes substrate accumulation, endomembrane swelling, cellular toxicity, and tissue-specific dysfunction.

fabry’s disease

A rare genetic disorder caused by mutations in the a-galactosidase A enzyme, which breaks down a glycosphingolipid (globotriaosylceramide)

• cells throughout the body

niemann-pick disease

There are several subclasses (Type A-C). Type A and B are caused by mutations in the enzyme acid sphingomyelinase, which impairs the breakdown of sphingomyelin in various organs

• liver, spleen, and lungs

pompe disease

Also known as glycogen storage disease, as mutations affecting lysosomal acid a-glucosidase impair the breakdown of glycogen

• muscle and heart tissue.

tay-sachs disease

A rare genetic disorder caused by mutations in HEXA, which affect the α-subunit of the B-hexosaminidase A enzyme, which breaks down a glycosphingolipid (GM2 ganglioside)

• brain, spinal cord and nerve cells

tay-sachs disease - organismal symptoms

• life spans depend on age of onset - 4-5yrs, 15yrs, near normal

• seizures, hearing loss, vision loss, difficulty with speech, red spot on the macula

tay-sachs disease - molecular level

mutations in HEXA gene that encodes a-subunit of B-hexosaminidase A leads to neurological symptoms

1. B-hexosaminidase A is a dimeric enzyme composed of one alpha and one beta subunit which cleaves GM2 ganglioside

2. GM2 activator protein (GM2AP) is a lipid transfer protein that transport substrate (GM2 ganglioside) to B-hexosaminidase A (hex A) dimer

3. HexA cleaves GalNAc off of GM2 ganglioside making GM3 ganglioside

4. GM3 ganglioside can be broken down into ceramides, sphingosines to make other lipids

how mutations in a-subunit impairs activity

1. B-subunit binding site

2. GM2AP binding site

3. GM2 binding to catalytic site

4. catalytic activity

Tay-Sachs Residual severity Thresholds

• <0.5% HexA activity causes severe Infantile TSD (onset 3-6 months, early death);

• 5-10% causes Juvenile TSD

• 10-20% causes Adult TSD (slow motor decline with near-normal lifespan).

Ashkenazi Jewish TSD Founder Mutation

A 4-bp (+TATC) frameshift insertion in Exon 11 of HEXA that introduces a premature stop codon, accounting for ~80% of Ashkenazi Jewish Tay-Sachs carriers

French Canadian TSD Founder Mutation

A 7.5 kb deletion encompassing Exon 1 and promoter regions of HEXA, representing the major founder mutation in French Canadian Tay-Sachs carriers

compound heterozygotes

a person who inherits two different mutates alleles for the same gene

Tay-Sachs Diagnostic Tools

• blood test - reduced levels of HexA

• skin fibroblasts - detect reduced enzymatic activity of HexA

• genetic testing - find mutations in alpha-subunit of B-hexosaminidase A

• eye exam - (cherry red spot on retina)

  • fovea (spot) doesnt contain ganglion cells so remains thin, transparent, red

  • surrounding opaque, white retinal ganglion cells fill with GM2 ganglioside and swell

Phenylketonuria (PKU) Molecular Etiology

Mutations in Phenylalanine Hydroxylase (PAH) block conversion of dietary phenylalanine (Phe) to tyrosine, leading to neurotoxic Phe buildup in blood and CNS

wilson’s disease

Copper-transporting ATPase 2 (ATP7B) is mutated, preventing the transport of copper from liver cells into bile, which is then excreted from the body

galactosemia

GALT, GALK1, and GALE enzymes break down galactose. Their mutation leads to toxic galactose and galactose byproducts accumulation in the body

phenylketonuria (PKU) - cellular/molecular level

• low PAH leads to low Tyr levels

• Phe outcompetes Tyr to cross blood brain barrier

• decreased Tyr, Trp levels lead to reduced dopamine, norepinephrine, and serotonine levels

• impairs myelination, brain development, leading to neurological symptoms

phenylketonuria (PKU) - organismal level

autosomal recessive affecting ~58k ppl

• impaired brain development

• intellectual disability

• behavioural problems

• microcephaly (small head)

• epilepsy

• depression, anxiety, phobias

phenylketonuria (PKU) diagnostics

• genetic testing

• routine newborn screening - all babies born in Canada+USA are screened for PKU within 24 hrs of birth

  • high prevalence 1/15,000

  • early diagnosis crucial for preventing severe neurological damage, and intellectual disability associated w PKU

  • existing treatment available

phenylketonuria (PKU) - therapeutics

• Sapropterin (Kuvan) is a synthetic BH4 cofactor tablet that stimulates residual PAH

  • only possible if theres some PAH activity

• Pegvaliase (Palynziq) is a PEGylated enzyme converting Phe to trans-cinnamic acid and ammonia

  • increases solubility, stability, reduced immunogenicity, enhanced circulation time

PKU vs. Tay-Sachs Therapeutic Feasibility

PKU is treatable because phenylalanine is an exogenous dietary substrate manageable outside the brain; Tay-Sachs involves endogenous neural gangliosides behind the blood-brain barrier.

similarities PKU vs Tay-sachs disease

• both involve mutations of enzyme metabolising a substrate (HexA, PAH)

• both due to accumulation of substrate (GM2 ganglioside, Phe)

• both can lead to neurological symptoms

differences PKU vs Tay-sachs disease

• source of substrate very different (dietary vs. cellular)

• site of substrate accumulation very different (blood + body fluids vs. intracellular lysosomes)

• effect of substrate very different (biochemical toxicity high Phe disrupts myelin synthesis vs. mechanical destruction bc GM2 ganglioside build up kills neurons)

gene therapy

medical technique that uses genetic material or modifies genetic material to treat, prevent, or cure diseases

gene replacement

inserts a healthy, functional copy of a missing or mutated gene into cells so they can produce necessary proteins

• would be considered to treat autosomal recessive disorders, though it can also be used in specific autosomal dominant conditions caused by haploinsufficiency

gene editing

uses precision tools like CRISPR-Cas9 to cut, fix, or rewrite specific sequences directly in the DNA

gene silencing

deactivates or "knocks down" a faulty, overactive gene that produces harmful proteins

suicide gene therapy

introduces a toxic or pro-drug-activating gene specifically into cancer cells to trigger cell death

In Vivo vs. Ex Vivo Gene Delivery

• In vivo injects vectors directly into the patient's body

  • cystic fibrosis

  • either express CFTR in appropriate cells (gene replacement) or correct mutant cells (gene editing) in tissues

• Ex vivo harvests patient cells, performs genetic modification in the lab, and re-transplants the corrected cells back into the patient

  • B-thalassemia

  • RBC precursor cells in bone marrow could be modified (gene replacement/editing) to improve B-globin expression

adeno-associated virus

non-integrating virus with low immunogenicity and a 4.8kb genome

• AAV capsids differ in their tissue tropism, and promoters can help restrict transgene expression to particular cell types (in vivo therapeutics)

lentivirus

integrating virus with low immunogenicity and an 8kb genome

• often used for CAR-T therapy (ex vivo therapeutics)

• all four constructs are supplied to the producer cells, providing the components needed to assemble viral vector

• only the TRANSFER vector genome is packaged into the particles used for treatment