26. Molecular Mechanisms of Inherited Diseases

Question 1

Mechanism of CFTR

Answer 1

PKA phosphorylates regulatory domain which activates channel, thus providing regulated Cl- and fluid secretion.

Made up of membrane-spanning domains, ATP-binding cassette (ABC), Regulatory domain that can be phosphorylated by PKA

Question 2

Repeated bouts of bronchitis (respiratory infections)
Foul smelling, bulky stools (steatorrhea)
Growth records show a decline in rate of growth
Low level of serum proteins and serum albumin (chronic malnutrition)

Answer 2

Case report on cystic fibrosis

Question 3

Cystic Fibrosis affects exocrine glands

Answer 3

Malabsorption
Lungs
Chronic pancreatitis
Abnormal sweat electrolytes (gold standard in diagnosis)

Question 4

Respiratory system in cystic fibrosis

Answer 4

Mutant CFTR does not transport Cl- into airway lumen

As a result, low Na+ and H2O content of luminal secretions —> Thickened and viscid mucus secretions…leads to bacterial infections and mortality and morbidity.

Question 5

Exocrine pancreatic insufficiency
Loss CFTR—> thicker acinar secretions: duct obstruction and tissue destruction
Fat replace pancreatic parenchyma…”cystic fibrosis—> deficiency of pancreatic enzymes
Loss of fat in stools (steatorrhea) (deficiency of fat-soluble vitamins ADEK)
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

Answer 5

Changes in the pancreas in cystic fibrosis

Question 6

Meconium ileus and intestinal obstruction neonates
-due to viscid intestinal secretions
Infertility in males - lack of vas deferens, known as Congenital Bilateral Absence of Vas Deferens (CBAVD)

Answer 6

Other systems involved in cystic fibrosis

Question 7

Sweat chloride test

Answer 7

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

Question 8

Autosomal Recessive single gene disorder
CFTR locus on long arm (q) of Chromosome 7
>1000 cystic fibrosis mutations
Most Common dF508

Answer 8

dF508: 3 bp deletion
Eliminates phenylalanine at position 508.
Malforming occurs, tagged with ubiquiton and destroyed.
d508 mutation prevents maturation of protein and reaching plasma membrane (severe phenotype)
Other CFTR mutations associated with variable severity.
CFTR mutations characterized by allelic heterogeneity

Question 9

Molecular diagnostic test for CFTR

Answer 9

ASO test: useful if mutation is known
Carriers are heterozygous for mutations, whereas affected children are homozygous for mutation.
Remember, due to allelic heterogeneity, pts may be compound heterozygotes

Question 10

Severe lower back and abdominal pain
Lab results: hemoglobin lower than normal
Total bilirubin = 2.3 mg/dL (Jaundice)
Vaso-occlusive sickle cell crisis (hemolytic crisis)
Autosomal recessive disorder

Answer 10

Sickle cell anemia

Question 11

Point mutation in Beta-globin gene of hemoglobin
Glutamic acid (Glu - E) replaced by valine (Val) at sixth position of beta-globin chain.
Hydrophillic amino acid replaced by a Hydrophobic

Answer 11

Molecular defect in sickle cell anemia

Question 12

Replacement by valine in beta-globin chain creates HYDROPHOBIC POCKET on exterior of hemoglobin molecule.

In deoxygenated stated, HbS aggregates to form long filaments

HbS aggregation results in RBC distortion, sickling.

Hydroxyurea in management

Answer 12

Molecular change in sickle cell disease

Question 13

Glu—>Val created hydrophobic “sticky” patch in HbS

Answer 13

Cells start to stick together, create rigidity
Sickled cells get stuck in capillaries and vessels. Blocking vessels thus blocking oxygen supply.

Question 14

Sickled and distorted RBCs removed by spleen.
Hemolytic anemia (less lifespan of RBC)
Splenomegaly
Sickling (hemolytic) crisis results in excessive removal of sickled RBC —> worsening of anemia and Jaundice (hyperbilirubinemia)
Silent strokes (blocked passages in brain)

Answer 14

Clinical features of sickle cell disease

Question 15

Hemolgobin electrophoresis for sickle cell

Answer 15

HbS (valine) has fewer negative charges than HbA (glutamate)

HbS moves slower than HbA towards anode (+)

Question 16

ASO test for HbS

Answer 16

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 17

RFLP test for detection of sickle cell disease and carriers

Answer 17

Mutation results in loss of restriction site on beta-globin gene, resulting in larger fragment being generated

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

Question 18

Both disorders due to mutations in DYSTROPHIN gene (largest gene located on x-chromosome)
Both x-linkedrecessive
DMD due to complete absence of functional dystrophin
BMD due to production of abnormal dystrophin or less amounts of dystrophin

Answer 18

Duchenne Muscular Dystrophy and Becker Muscular Dystrophy

Question 19

Absence of dystrophin, muscle protein
X-linked recessive disorder; Males
Most common muscular dystrophy, affecting 1 in 3,000 boys
Age of onset 2-6 years old
Death in early 20s (breathing and heart problems); wheelchair bound in teens
GOWERS sign (trying to stand up weird)
Low reproductive fitness (unable to pass on mutation to next generation)

Answer 19

Duchenne Muscular dystrophy

Question 20

Some dystrophin present; truncated dystrophin
Milder form of muscular dystrophy
X-linked recessive; Males
Symptom similar to DMD but milder
Age of onset: 20-30 years, Milder course of disease
Affects 1 in 30,000 live male births
Normal reproductive fitness

Answer 20

Becker Muscular dystrophy

Question 21

Deletions causing DMD cause…

Answer 21

FRAMESHIFT mutations
Mutations also involve large deletion of exons
No dystrophin synthesis

Question 22

Deletions in BMD are…

Answer 22

IN-FRAME deletions or promoter mutations
Some dystrophin, although truncated, is translated
Or reduced synthesis of dystrophin
Reading frame still viable

Question 23

DMD diagnostics

Answer 23

Multiplex Polymerase Chain reaction (PCR)
Simultaneous amplification of many DNA segments in one amplification reaction.

Question 24

Clinical features of DMD

Answer 24

Pseudohypertrophy of calf

Due to replacement of muscle tissue with connective tissue and fat.

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

Question 25

DMD
Gowers’ maneuver

Answer 25

1. First get on hands
2. Elevate posterior
3. Then “walk” their hands up the legs
4. To raise their upper body

Question 26

Dystrophin gene and protein

Answer 26

Largest gene
Mutation hotspot: almost 33% of patients have new mutations
Expressed in smooth, cardiac, and skeletal muscle, with lower levels in the brain.

Question 27

Function of dystrophin

Answer 27

Anchors cytoskeleton (actin) of muscle cells to extracellular matrix (basal lamina)

Links actin filaments to proteins of muscle cell membrane.

Question 28

Dystrophin protein

Answer 28

Enables muscles to withstand stress of muscle contractions (shock absorbers)

Loss of dystrophin leads to oxidative injury and myonecrosis

Muscles susceptible to mechanical injury and degeneration

Question 29

Histology and Western blot

Answer 29

Histological staining shows an increase in connective tissue between myocytes

Western Blot: Smaller protein size, and may be reduced quantity of dystrophin protein in BMD (in-frame deletion)

Complete absence of dystrophin in DMD (Frame-shift deletion)

Question 30

DMD vs BMD immunostaining

Answer 30

• Microscopy in BMD and DMD
• Left: Muscle Hematoxylin and eosin staining
• Right: Immunofluorescence microscopy staining with dystrophin specific antibody
• Reduced quantity of dystrophin in BMD, and complete absence of dystrophin in DMD
• Increased Connective tissue between myocytes in DMD muscle

Question 31

Other tests useful in Duchenne Muscular Dystrophy

Answer 31

Serum creatine Kinase (MM) levels elevated in pts 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 heterozygotes’. Many carrier females have adult-onset cardiomyopathy.