Molecular Medicine Exam #1 - Pulmonary and Hematological Diseases

Categories of Gene Mutations

• Point mutations within coding sequences

• Mutations within noncoding sequences

• Deletions and insertions

• Structural alterations in protein-coding genes

• Trinucleotide-repeat mutations

Hereditary disorders:

Transmitted in germline; familial

Congenital

“Born with”

T/F Congenital diseases are not all genetic

True

T/F Not all genetic diseases are congential

True

Transmission patterns for single-gene disorder

• Autosomal dominant

• Autosomal recessive

• X-linked recessive

Cystic fibrosis inheritance pattern

Autosomal recessive

Cystic fibrosis

• caused by mutation in cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for an anion channel (Cl- and bicarb)

• Results in viscous secretions in exocrine glands and in the epithelial lining of the respiratory, gastrointestinal, and reproductive tracts

Cystic Fibrosis carrier frequency

1 in 20 in people of Northern European descent

• most lethal genetic disease that affects people of this descent (1 in 2500 live births)

Chromosome that carries CFTR Gene

Chromosome 7

CFTR domains

• 2 transmembrane domains

• 2 nucleotide-binding domains (NBDs)

• regulatory R domain

CFTR function and binding process

1. Binds to epithelial cells

2. Increases levels of cAMP

3. PKA activated and phosphorylates R domain of CFTR using ATP

4. Opens Cl- ion channel

CFTR Transport throughout the cell

1. Nucleus: Chromosome 7 CFTR gene —> CFTR mRNA

2. ER: Translation and folding

3. Golgi: Processing

4. Cell surface: Function

Cystic Fibrosis pathogenesis - Lumen of sweat duct (exocrine gland)

Mutated CFTR, INCREASES NaCl concentration in sweat

Cystic Fibrosis pathogenesis - Airway (Lining of organ)

• decreased Cl- secretion

• Increased Na+ and H2O reabsorption leading to thick and sticky mucus —> blocks pancreatic ducts, etc

NORMAL CFTR function in lumen of sweat duct

• CFTR opens Cl- channel, allowing for Cl- to go into the lumen from the extracellular space

• CFTR activates ENac, allowing Na+ to enter the cell (augments Na+ absorption)

NORMAL CFTR function in airway

• CFTR opens Cl- channel allowing Cl- to enter the extracellular space from the cell

• CFTR inhibits ENac, preventing Na+ entry into the cell and reduced H2O reabsorption = normal mucus

What does a mutated CFTR gene do in airways

Leads to dehydrated mucus —> defective mucociliary action (crushes cell’s cilia) and the mucus will plug the airways (leads to decreased lung function in some cases)

Describe Class I of CFTR mutation

Class: Protein Production

CFTR defect: No functional CFTR protein

Type of Mutation: Frameshift, nonsense, splice

Drug: kalydeco (ivacaftor)

Describe Class II of CFTR mutation

Class: Protein Processing

CFTR defect: CFTR trafficking/processing

Type of Mutation: Missense, deletion

Drug: Orkambi (Lumacaftor/Ivacaftor) + Trikafta (Elexacaftor + Tezacaftor + Ivacaftor)

Describe Class III of CFTR mutation

Class: Gating

CFTR defect: Defective channel regulation

Type of Mutation: Missense, amino acid change

Drug: Kalydeco (ivacaftor) + Trikafta (elexacaftor + tetracaftor + ivacaftor) + Symdeko (tetracaftor + ivacaftor)

Describe Class IV of CFTR mutation

Class: Conduction

CFTR defect: Decreased channel conductance

Type of Mutation: Missense, amino acid change

Drug: Kalydeco (ivacaftor) + Trikafta (elexacaftor + tetracaftor + ivacaftor) + Symdeko (tezacaftor + ivacaftor)

Describe Class V of CFTR mutation

Class: Insufficient protein

CFTR defect: Reduced synthesis of CFTR

Type of Mutation: Splicing defect, missense

Drug: Kalydeco (ivacaftor) + Trikafta (elexacaftor + tezacaftor + ivacaftor)

Describe Class VI of CFTR mutation

Class: n/a (not included in website)

CFTR defect: Decreased CFTR stability

Type of Mutation: Missense, amino acid change

Drug: Stabilizer

T/F the different types of CFTR mutation classes act as molecular targets dependent on genotypic variability

True

• Each CFTR mutation class affects CFTR differently and provide basis for treatment and modulatory therapy

Describe the relationship between level of CFTR function and severity of symptoms

HIGHER CFTR function = LESS Severe

• inverse relationship

<5% CFTR function symptoms

• Severe chronic sinusitis

• Severe lung disease

• High sweat chloride

• Pancreatic INsufficiency

• Meconium ileus

• Absent vas deferens

<10% - <20% CFTR function symptoms

• Moderate chronic sinusitis

• Variable lung disease

• Intermediate sweat chloride

• Pancreatic sufficiency

• Distal intestinal obstruction syndrome

• Absent vas deferens

50% CFTR function symptoms

• increased rate of chronic sinusitis

• increased rate of lung disease

• increased risk of absent vas deferens

Specific CFTR Gene mutations we have to know:

• F508 deletion (most common)

• Arg117His (milder mutation)

Clinical features of CF

1. Chronic sinopulmonary disease manifested by:

   1. persistent infection with CF pathogens

   2. Constant cough; airway obstruction

   3. Digital clubbing

   4. Nasal Polyps

2. Gastrointestinal and nutritional abnormalities

   1. intestinal: meconium ileus

   2. pancreatic: insufficiency, pancreatitis

   3. Hepatic: jaundice

   4. Nutritional: failure to thrive

3. Salt-loss syndrome

4. Male urogential abnormalities —> obstructive azoospermia

CF pathogens

• Staphylococcus aureus

• Haemophilus influenzae (non-treatable)

• Pseudomona aeruginosa

• Burkholderia cepacia

Teenager with Anosmia

• had chronic inability to smell and nasal congestion

• treated with amoxicillin and corticosteroids that helped temporarily

• nasopharyngoscopy showed complete opacification

  • polyps due to chronic inflammation

• sweat chloride test had a diagnostic positive; genetic sequencing had a (TG)13-5T cystic fibrosis mutation

  • more mild variant

• had sufficient pancreatic function and normal trypsinogen levels

• had sinus surgery, clearance treatments, antibiotics

Describe CFTR Modulator Class: Potentiator and give drug and mutation examples

• helps open the CFTR channel and increases Cl- and bicarb influx

• targets gating, conduction, and insufficient protein mutations (Class III, IV, and V)

• Drug Ex: Ivacaftor, Deutviacaftor, Elexacaftor

• Mutation Ex: F508del

Describe CTFR Modulator Class: Corrector and give drug and mutation examples

• Helps normalize the folding of defective CFTR protein and its movement

• Targets processing mutations (Class II)

• Drug Ex: Vanzacaftor, Elexacaftor, Tezacaftor, Lumacaftor

• Mutation Ex: F508del

Describe CTFR Modulator Class: Amplifier and give drug and mutation examples

• Increases expression of abnormal CFTR mRNA and production of protein

• Targets processing, gating, conduction, insufficient classes (Classes II-V)

• Drug Ex: N/A

• Mutation Ex: F508del

Describe CTFR Modulator Class: Stabilizer and give drug and mutation examples

• Limits removal and degradation of CFTR protein from cell surface

• Targets processing, gating, conduction, insufficient classes (Classes II-V)

• Drug Ex: N/A

• Mutation Ex: F508del, N1303K, I507del, R117H, D115H, R347PE

Gating mutation

• correctly produce and process CFTR protein BUT ion channels do not open properly

• responds to potentiators

• Ex: missense, small deletion mutations

  • F508del

Residual Function mutation

• Retains CFTR function

• Milder phenotype

• usually respond to potentiators

Minimal function mutations

• Negligible function at baseline

• DO NOT respond to CFTR modulators

List the 8 structures in the respiratory pathway from where air enters to alveoli

Nares → nasal cavity → pharynx → larynx → trachea → bronchi → bronchioles → alveoli

Muscles involve in inhalation

• diaphragm and external intercostal muscles

• in labored breathing, muscles of the neck and back may also be involved

Muscles involve in exhalation

• recoil of diaphragm and external intercostal muscles

• internal intercostal muscles and abdominal muscles

Surfactant Purpose

surface tension at the air–liquid interface in the alveoli

• this prevents their collapse

  • Why accumulation of elastase —> elastin breakdown = bad for alveoli

Mathematical relationship between VC, IRV, ERV and TV

VC = IRV + ERV + TV

If CO2 blood levels are too LOW, how does the brain maintain homeostasis

DECREASES Respiratory rate to RAISE CO2 levels

**LOWER RR = LOWER V = MORE CO2 in body

Respiratory. system mechanisms to prevent infection

• vibrissae in the nares (hairs)

• lysozyme in the mucous membranes

• the mucociliary escalator

• macrophages in the lungs

• mucosal IgA antibodies

• mast cells.

Bicarbonate buffer system chemical equation

CO₂ + H₂O ⇌ H₂CO₃ ⇌ HCO₃⁻ + H⁺

How does pH change in respiratory failure

lower pH of the blood

• ventilation slows = less CO2 out = right shift = more H+ ions generated = more acidic

Total lung capacity (TLC)

maximum volume of air in lungs when one inhales completely (usually around 6-7 liters)

Residual Volume (RV)

volume of air remaining in lungs when one exhales completely

Vital capacity (VC)

difference between TLC and RV (max-min)

Tidal Volume (TV)

volume of air inhaled or exhaled in a normal breath

Expiratory reserve volume (ERV)

volume of additional air that can be forcibly exhaled after a normal exhalation

Inspiratory reserve volume (IRV)

volume of additional air that can be forcibly inhaled after a normal inhalation

TLC Lung volume equation

TLC = IRV + TV + ERV + RV

Capacity Lung volume equation

sum of more than 1 lung volume

Kalydeco generic name

(IVACAFTOR)

Kalydeco Class, Mechanism of action, Indication, Adverse effects

Class: potentiator

Mechanism of action:

• binds to CFTR and opens anion channel

• unlocks gate and holds it open

Indication: approved for people with CF aged 1 month and older who have one of 97 mutations

AE:

• Serious: elevated liver enzymes, anaphylaxis, intracranial hypertension, cataracts (cataracts in children and adolescents)

• Common: headache, URI, abdominal pain, diarrhea, nausea, rash, dizziness

Symdeko Class, Mechanism of action, Indication, Adverse effects

Class:

• tezacaftor = corrector

• ivacaftor = potentiator

Mechanism of action:

• tezacaftor: helps the CFTR form the right shape, move to the cell membrane, and stay in the membrane longer

• ivacaftor: opens up the gate and holds it open

Indication:

• Approved for people with CF ages 6 and older with 2 copies of the F508 del mutation AND

• Approved for people with CF ages 6 and older with a single copy of one of 154 mutations

AE:

• Serious: elevated liver enzymes, anaphylaxis, intracranial hypertension, cataracts (cataracts in children and adolescents)

• Common: headache, nausea, sinus congestion, and dizziness

**Fewer side effects, like chest tightness, and fewer drug interactions than Orkambi (lumacaftor/ivacaftor)

Orkambi Class, Mechanism of action, Indication, Adverse effects

Class: modulator

Mechanism of action:

• Lumacaftor: helps the CFTR form the right shape, move to the cell membrane, and stay in the membrane longer (only about 1/3 of the CFTR protein reaches the cell surface with lumacaftor)

• Ivacaftor: opens gate and holds it

Indication: Approved for people with CF aged 1 year and older who have 2 copies of the F508del mutation (F508del/F508 del) in the CFTR gene

AE:

• Serious: elevated liver enzymes, anaphylaxis, intracranial hypertension, cataracts (cataracts in children and adolescents), breathing problems, an increase in BP

• Common: shortness of breath, chest tightness, nausea, diarrhea, fatigue, increase in creatine kinase (CK), rash, URI symptoms, flu or flu-like symptoms, and irregular periods/increase in the amount of menstrual bleeding

Symdeko generic name

tezacaftor/ivacaftor

orkambi generic name

lumacaftor/ivacaftor

Trikafta Class, Mechanism of action, Indication, Adverse effects

Class:

• elexacaftor & tezacaftor = correctors

• ivacaftor = potentiator

Mechanism of action:

• elexacaftor & tezacaftor: help the CFTR form the right shape, move to the cell membrane, and stay in the membrane longer

• ivacaftor: opens the gate and holds it open

Indication: Approved for people with CF ages 2 and older who have at least 1 copy of the F508del mutation or at least 1 copy of 271 mutations

AE:

• Serious: liver damage and liver failure, serious allergic reactions, intracranial hypertension, cataracts (cataracts in children and adolescents)

• Common: headache, URI symptoms, abdominal pain, diarrhea, constipation, rash, increases liver enzymes and bilirubin, increase in CK, flu, inflamed sinuses

Alyftrek Kalydeco Class, Mechanism of action, Indication, Adverse effects

Class:

• Vanzacaftor & tezacaftor = correctors

• Deutivacaftor = potentiator

Mechanism of action:

• Vanzacaftor & tezacaftor = correct the CFTR protein so it forms the right shape and moves to the cell membrane

• Deutivacaftor = binds to defective CFTR and holds open chloride channel; deuterated form of ivacaftor (replace hydrogens with deuterium, a heavier, stable isotope of hydrogen) that has a longer half-life allowing once daily dosing

Indication: people with CF ages 6 & up who are also eligible based on mutations for Trikafta or 31 other rare mutations

AE:drug-induced liver injury and failure, intracranial hypertension, cataracts (in pediatric patients)

Outcomes:

• Improvements in lung function comparable to Trikafta

• Reduction in sweat chloride levels greater than that seen with Trikafta

Trikafta generic name

(ELEXACAFTOR/TEZACAFTOR/IVACAFTOR)

alyftrek generic name

(VANZACAFTOR/TEZACAFTOR/DEUTIVACAFTOR)

Obstructive Lung Disease

characterized by an increase in resistance to airflow due to diffuse airway disease, which may affect any level of the respiratory tract

Restrictive Lung Disease

marked by reduced expansion of lung parenchyma and decreased total lung capacity

1. chest wall disorders

2. chronic interstitial and infiltrative disease

Small Airway disease

• Emphysema (Alveolar wall destruction or overinflammation)

• Chronic Bronchitis (Productive cough, airway inflammation)

Bronchial hyperresponsiveness

Asthma (reversible obstruction)

Chronic Bronchitis Anatomic Site, Major Pathologic Changes, Etiology, Signs/Symptoms

Anatomic Site: Bronchus 

Major Pathologic Changes: Mucous gland hyperplasia, hypersecretion

Etiology: Tobacco smoke, air pollutants 

Signs/Symptoms: Cough, sputum production

Brochiectasis Anatomic Site, Major Pathologic Changes, Etiology, Signs/Symptoms

Anatomic Site: Bronchus

Major Pathologic Changes: Airway dilation and scarring 

Etiology: Persistent or severe infections

Signs/Symptoms: Cough, purulent sputum, fever

Asthma Anatomic Site, Major Pathologic Changes, Etiology, Signs/Symptoms

Anatomic Site: Bronchus

Major Pathologic Changes: Smooth muscle hyperplasia, excess mucus, inflammation

Etiology: Immunologic or undefined causes

Signs/Symptoms: Episodic wheezing, cough, dyspnea

Emphysema Anatomic Site, Major Pathologic Changes, Etiology, Signs/Symptoms

Anatomic Site: Acinus

Major Pathologic Changes: Airspace enlargement; wall destruction

Etiology: Tobacco smoke

Signs/Symptoms: Dyspnea

Small Airways disease/broncholitis Anatomic Site, Major Pathologic Changes, Etiology, Signs/Symptoms

Anatomic Site: Bronchus

Major Pathologic Changes: Inflammatory scarring/obliteration

Etiology: Tobacco smoke, air pollutants, miscellaneous

Signs/Symptoms: Cough, dyspnea

a1 Antitrypsin deficiency

normally inhibits elastase —> mutation leads to the accumulation of elastase —> more elastin break down —> less structural integrity

a1 Antitrypsin deficiency and COPD

• alveolus release neutrophils during inflammation

• neutrophils produce neutrophil elastase to break down elastin (extracellular protein to provide strength)

• a1 Antitrypsin made by hepatocytes gets sent to alveolus via blood

• The mutated glycoprotein will be unable to break down elastase = UNCHECKED

• Elastase will continue to break down elastin —> Alveoli turn into one big cavity because they lost structural integrity —> Pan-acinar EMPHYSEMA (whole acinus lower lobes mostly affected)

a1 Antitrypsin Gene mutation

• located on chromosome 14 on SERPINA1 (serine protease inhibitor clade 1)

• some cause complete absence

• PiZ = misfolded —> stuck in ER —> cell dies

• Normal PiM attributes 50% of function —> 2 normal copies = 100% normal amount of a1 Antitrypsin

• Mutated PiZ attributes ~10% of function

  • PiM + PiZ = ~60% of normal levels a1 Antitrypsin (asymptomatic; safe for nonsmokers)

• PiZ + PiZ = ~15-20% of normal a1 Antitrypsin levels —> a1 Antitrypsin deficiency!!

  • higher risk of lung and liver disease

  • minimal environmental risk factors can prevent individuals from experiencing symptoms

Types of COPD a patient with a1 Antitrypsin deficiency may develop

• Pan-acinar emphysema

• chronic bronchitis

• bronchiectasis

**recall another common cause of COPD is smoking, therefore if a patient smokes and has the mutation, they experience an earlier onset of COPD

a1 Antitrypsin deficiency symptoms

• SOB

• wheezing

• mucus production

• chronic cough

• cirrhosis

• inability to make coagulation factors

• build of toxins —> hepatic encephalopathy

• Portal hypertension —> esophageal varices

• Hepatocellular carcinoma

• Jaundice (Juvenile)

  • minority of infants with PiZZ can develop liver failure

symptoms for SERPINA1 mutation with ABSENT a1 Antitrypsin

ONLY has LUNG-COPD related symptoms:

• SOB

• wheezing

• mucus production

• chronic cough

Diagnosis of a1 Antitrypsin deficiency

• chest x-ray or CT —> look for hyper inflated lungs and damaged tissue

• pulmonary function —> slow exhale

• Blood test —> low a1 Antitrypsin levels

• Cirrhosis via liver ultrasound or biopsy

  • liver cells stained with periodic acid schiff (PAS) —> stains glycoproteins pink (PAS +)

  • Diastase resist since a1 Antitrypsin is stuck in ER and cannot be destroyed

a1 Antitrypsin Therapies

• Augmentation therapy: IV Infusion of normal a1 Antitrypsin

  • slows down progression

• inhalers

• supplemental oxygen

• cirrhosis treatment (Ex: lactulose)

Why are trypsinogen levels high in patients with CF

• mucus produces in patients with CF blocks pancreatic ducts

• mucus traps digestive enzymes like trypsinogen —> leaks into blood

Processing, gating, conduction, or insufficient protein mutations

• class II-V

• A channel is created that retains at least some functional CFTR channel activity either at baseline or following exposure to other classes of CFTR modulators.

Processing mutations

• class II

• CFTR protein is created but misfolds, preventing it from reaching the apical cell surface.

Insufficient protein mutations

• class V

• CFTR protein is created that has normal processing, channel, and gating properties, but the amount of protein present at the cell surface is deficient.

• This can be caused by too little CFTR protein being produced or an increased rate of channel deactivation or removal from the cell surface.

Conduction mutations

• class IV

• CFTR protein is created and moves to the cell surface but with a malformed channel that limits the rate of chloride and bicarbonate movement.

What drug would work best for Class I - CFTR Production Mutations?

Kalydeco (Ivacaftor)

What drug would work best for Class II - CFTR Processing Mutations?

• Corrector + Potentiator

• Orkambi (Lumcaftor/Ivacaftor)

• Trikafta (ELEXACAFTOR/TEZACAFTOR/IVACAFTOR)

What drug would work best for Class III - CFTR Gating and Class IV - Conduction Mutations?

• Potentiators + Correctors

• Kalydeco (Ivacaftor)

• Trikafta (ELEXACAFTOR/TEZACAFTOR/IVACAFTOR)

• Symdeko (TEZACAFTOR/IVACAFTOR)

What drug would work best for Class V - Insufficient CFTR Mutations

• Splicing modulators

• Antisense oligonucleotides

• Kalydeco (Ivacaftor)

• Trikafta (ELEXACAFTOR/TEZACAFTOR/IVACAFTOR)

What drug would work best for Class VI - CFTR Mutations

Stabilizers

Components of Blood

Plasma and Serum

• Leukocytes (WBC

• Platelets

• erthrocytes (RBC)

Plasma

liquid portion of blood minus the cells

consists of:

• water

• blood proteins

• salts

• respiratory gases

• hormones

serum

plasma minus clotting factors

(-) Anti coagulant vial

Serum + blood clot

(+) Anti coagulant vial

• Plasma

• Buffy coat (white blood cells and platelets)

• Red blood cells

Types of WBC

• neutrophil

• lymphocyte

• basophil

• eosinophil

• manocyte