Molecular Mechanisms Of Inherited Disirders

Question 1

Summarize cystic fibrosis

Answer 1

• Common autosomal recessive disorder
• Occurrence: 1 in 2000 in Caucasian’s
• mutation in gebe for ‘cystic fibrosis transmembrane conductance regulatir’
• chloride channel
• ATP Bindibg Cassette (ABC) transporter

Question 2

Describe the structure of CFTR

Answer 2

CFTR Contains:
-Membrane-spanning domains

• ATP binding domains
• Regulatory domain that can be phosphorylated by PKA

Phosphorylation of regulatory domain by PKA activates channel, thus providing regulated Cl- and fluid secretion

Question 3

How is cystic fibrosis regulated intracellular LH?

Answer 3

AC converts ATP to cAMP

cAMP activates PKA

Question 4

What are the clinical features of cystic fibrosis?

Answer 4

Affects exocrine glands

• Malabsorption
• Abnormal sweat electrolytes
• Chronic pancreatitis

Recurrent pulmonary infection: lung abscess, chronic bronchitis, bronchiectasis, honeycomb lung

Obstructive vas deferens(sterility)

Secondary biliary cirrhosis

Meconium ileum (newborn)

Question 5

Describe respiratory in cystic fibrosis

Answer 5

• Mutant CFTR doesn’t transport CL- into airway lumen
• As a result, low Na+ and H2O content of luminal secretions—> thickened and viscid mucus secretions
• Viscid secretions probe for bacterial infections
• Respiratory infections: mortality and morbidity

Question 6

What are the changes in the pancreas in cystic fibrosis ?

Answer 6

• Exocrine pancreatic insufficiency
• Loss of CFTR leads to thicker acinar secretions within duct lumen: duct obstruction and tissue destruction
• Fibrotic tissue and fat replace pancreatic parenchyma- “cystic fibrosis”—> deficiency of pancreatic enzymes
• Nutrient maldigestion and loss of fat in stools (steatorrhea)
  
  • deficiency of fat-soluble vitamins
• Protein malnutrition and growth delay
• Oral enzyme replacement therapy improves nutrition
• Deficient secretion of pancreatic enzymes (lipase, trypsin, chymotrypsin)
  - Normal digestion and nutrition restored by pancreatic enzyme supplements

Question 7

How does cystic fibrosis lead to infertility in cystic fibrosis ?

Answer 7

Infertility in males-lack of vas deferens, known as congenital bilateral absence of vas deferens (CBAVD)

Question 8

How does cystic fibrosis affect intestines?

Answer 8

Meconium ileus and intestinal obstruction neonates

-Due to viscid intestinal secretions

Question 9

What are the changes in sweat glands ?

Answer 9

• In sweat glands, CFTR involved in Reabsorption of NaCl
• In CF very little NaCl is reabsorbed, resulting in high sweat salt content
• High sweat chloride levels- diagnostic test if CF

Question 10

What is the diagnostic test for sweat chloride test for cystic fibrosis ?

Answer 10

• Pilocarpine placed under electrode pad
• Mild electric current passed between electrodes to force pilocarpine into skin, to stimulate sweat glands
• Sweat collected and chloride levels measured
• Elevated sweat chloride levels are diagnostic

Question 11

What is the genotype-phenotype correlation of CFTR mutation?

Answer 11

Autosomal recessive single gene disorder

-CFTR locus on long arm of chromosome 17

• Less than 1000 cystic fibrosis mutations
  
  • Most common (about 70%) is 🔼F508
    - 3-bp deletion
    - Elimination phenylalanine at position 508: 🔼F508 mutation

🔼F508 mutation prevents maturation of protein and reaching plasma membrane (severe phenotype)

-Other CFTR mutations associated with variable severity

Question 12

CFTR mutations characterized by…

Answer 12

allelic heterogeneity

Question 13

What is the ASO test?

Answer 13

Useful of the mutation is known.

Carriers are heterozygous for the mutation, whereas affected children are homozygous for the mutation

-Remember, due to allelic heterogeneity patients may be compound heterozygotes

The most common mutation in cystic fibrosis is a deletion of codon 508 (Phe)

Question 14

What is the molecular defect in sickle cell anemia?

Answer 14

• Point mutation in B-globin gene of hemoglobin
• Glutamic acid replaced by valine at sixth position of B-globin chain
• Acidic amino acid (hydrophilic) replaced by branched chain (hydrophobic) amino acid (MISSENSE)
• Glutamic acid present on exterior of hemoglobin
• Replacement by valine in beta-globin chain creates a hydrophobic pocket on exterior of hemoglobin

Question 15

What happens In sickle cell?

Answer 15

In deoxygenated state, HbS aggregates to form long filaments

HbS Aggregation results in RBC distortion - sickling

Hydroxyurea in management

Question 16

What changes. In deixyy HbS fir sickle cell anemia?

Answer 16

Hydrophobic ‘sticky’ patch in HbS (Glu —> Val)

Question 17

What are the clinical features in sickle cell disease?

Answer 17

• Sickled and distorted RBCs are removed by spleen
• Hemolytic anemia (less lifespan of RBC)
• Splenomegaly
• Sickling (hemolytic) crisis results in excessive removal of Sickled RBC, resulting in worsening of anemia and jaundice (hyperbilirubinemiaa)
• Silent strokes

Question 18

Summarize hemoglobin electrophoresis

Answer 18

• Hemoglobin electrophoresis: HbS (valine) has fewer negative charges than HbA (glutamate)
• HbS moves slower than HbA towards anode (+)

Question 19

Explain ASO test for HbS

Answer 19

ASO test (dot blot test) using a specific probe for HbS

-Heterozygous carriers (one copy of normal allele and one copy of mutant allele). And homozygous affected

Question 20

Describe RFLP test for sickle cell

Answer 20

Mutation for results in loss of restriction site in B globin gene- results in larger fragment being generated

Size of fragment from HbS is larger (moves slower; 1350 bp) compared to fragment from HbA(moves faster; 1150 bp)

Question 21

What are duchenne muscular dystrophy and Becker muscular dystrophy?

Answer 21

• Both disirders are due to mutations in dystrophin gene
• largest gene located on X-chromosome
• DMD is due to almost complete absence of functional dystrophin
• BMD is due to production of abnormal dystrophin or less amounts of dystrophin

Question 22

Explain duchenne muscular dystrophy in detail

Answer 22

• absence kf dystrophin, muscle protein
• X linked recessive protein
• Mkst common muscular dystrophy, affecting 1 in 3,000 boys
• age of onset: 2-6 years
• Death in early 20s due to breathing and heart problems; wheel-chair bound in their teens
• Gowers sign
• males with DMDD have low reproductive fitness (unable to pass the mutation to the next generation

Question 23

Describe Becker’s muscular dystrophy in detail

Answer 23

• Some dystrophin present; truncated dystrophin
• milder form of muscular dystrophy
• X linked recessive, males
• Symptoms similar to DMD but milder
• Age of onset: 20-30 years, milder course of disease
• BMD affects 1 in 30,000 live male births
• Males with BMD typically have normal reproductive fitness

Question 24

What are the mutations in DMD and BMD?

Answer 24

Both DMD and BMD result from mutation in dystrophin gene

• DMD deletions can be distinguished from deletions causing BMD
  
  • Deletions causing DMD cause frameshift mutations
    
    • mutations also involve large deletion of exons
    • NO dystrophin synthesis
• Deletions in BMD are in-frame deletions or promoter mutations
  
  • Some dystrophin, although truncated, is translated
  • Or reduced synthesis of dystrophin

Question 25

How is DMD diagnosed?

Answer 25

Multiplex PCR diagnostics

Simultaneous amplification of many DNA segments in one amplification reaction -this is used to screen for deletions and duplications . Use this slide for histology

Question 26

What are the clinical features of DMD?

Answer 26

Early stages, duchenne and Becker MD affect pectoral muscles, trunk, and upper and lower legs. Difficulty in rising, climbing stairs and maintaining balance

Pseudohypertrophy of calf- due to replacement of muscle tissue with connective tissue and fat in an 8 year old boy with DMD

Question 27

Explain gowers maneuver in boys with DMD

Answer 27

Because of weakened leg muscles, boys with DMD have a distinctive way of rising from the floor, called Gower’s maneuver. First get on hands, and knees, then elevate posterior, then 2alk their hands up the legs to. Raise their upper body

Question 28

Describe the dystrophin gene and protein

Answer 28

-Largest gene- almost 2% of X-chromosome and nearly 0.05% of genome. 79 exons and 8 promoters; 2.2 million base pairs

Mutation hotspots- almost 33% of patients have new mutations

Expressed in smooth, cardiac, and skeletal muscle, with lower levels in brain

Question 29

What is the function of dystrophin?

Answer 29

• Anchors cytoskeleton (actin) of muscle cells to extracellular matrix (basal lamina)
• L8nks actin filaments to proteins of muscle cell membrane

Question 30

What is the dystrophin protein?

Answer 30

Enables muscle to withstand stress of muscle contraction

Loss of dystrophin leads to oxidative injury and myonecrosis

Muscles susceptible to mechanical injury and degeneration

Question 31

Contrast western blot of DMD vs BMD

Answer 31

Histological staining sh9ws an increase in connective tissue between myocytes.

Western blot
-smaller pr9tein s7ze, and may be reduced quantity of dystrophin protein in BMD (In-frame deletion)

-Complete absence 9f dystrophin in DMD (frameshift deletion)

Question 32

Contrast DMD VS. BMD in immunostaining

Answer 32

• Microscopy in BMD and DMD
• Reduced quantity of dystrophin in BMD, and complete absence of dystrophin in DMD
• Increased Connective tissue between myocytes in DMD muscle

Question 33

Aside from immunostaining and western blot, what other tests can diagnose duchenne muscular dystrophy ?

Answer 33

• Serum creatine kinase (MM) levels elevated in patients with muscular dystrophy (indicative of muscle damage)
• Females are carriers and they usually have higher levels of CK-MM in serum
• Some heterozygous females may have clinical expression of the disease due to asymmetric X inactivation. Aka ‘manifesting heterizygotes’ . Many carrier females have adult onset cardiomyopathy