Diagnosis And Intervention 08/12/22

Question 1

What types of interventions are there for genetic disorders?

Answer 1

-Diet changes
-Gene therapy
-Medication

Question 2

What are the different levels of intervention?

Answer 2

Levels:

-Mutant gene
-Mutant mRNA
-Mutant protein
-Metabolic or other biochemical dysfunction
-Clinical phenotype
-The family

Question 3

What is the intervention at the mutant gene level?

Answer 3

Modification of the somatic gene type. For example, a hematopoietic stem cell transplantation (HSCT) in B thalassemia.

HSCT uses human leukocyte antigen (HLA)-matched stem cells derived from a donor’s bone marrow, peripheral blood, or umbilical
cord blood. Survival after allogeneic transplantation depends on donor-recipient matching and the graft-versus-host response.

Question 4

What is the intervention at the mutant mRNA level?

Answer 4

Gene therapy, enzyme replacement therapy, and hematopoietic stem cell transplantation. For example, SCID.

Gene therapy:
In 1990, the first clinical trial of gene therapy was attempted in a four-year-old girl with ADA-SCID using infusions of the patient’s T cells treated with a gammaretroviral vector containing normal ADA cDNA. This did
result in the production of endogenous ADA enzyme, but required repeated rounds of T-cell infusions which were not sufficient to remove the requirement for exogenous
ADA enzyme replacement.

Enzyme replacement therpay:
Adenosine deaminase (ADA) is a key enzyme of the purine salvage pathways. Absent or impaired ADA function leads to the accumulation of toxic metabolites. In addition to severe lymphocytopenia affecting T and B lymphocytes and NK cells, neurodevelopmental deficits, sensorineural deafness, and skeletal abnormalities can also
occur. Enzyme replacement therapy can solve this.

Transplant:
Prior to a transplant with stem cells patients require chemotherapy.

Question 5

What is the process for gene therapy?

Answer 5

1) Cells are collected from the patients.
2) Blood stem cells are selected and purified.
3) A working copy of the gene is inserted into the cells.
4) The patient is conditioned to receive treatment.
5) The gene-corrected cells are infused into the patient.

Question 6

What is SCID?

Answer 6

It is a group of inherited disorders characterized by defects in both T and B cell responses. Effects one infant in every 50,000 live births. Individuals with this disorder are severely immunocompromised.

Mutations in the IL2RG gene cause X-linked SCID accounting for 25-40% of all cases. Another form of SCID is caused by a deficiency of the enzyme adenosine deaminase (ADA), normally produced by a gene on chromosome 20. It accounts for
5-10% of the recessive SCID cases. Early detection and treatment are imperative.

Question 7

What is spinal muscular atrophy (SMA) type 1?

Answer 7

SMA affects 1 in 8,000 to 10,000 people worldwide with type 1 accounting for half
of cases.

• Weak respiratory muscles and an
  abnormally bell-shaped chest
• Difficulty feeding and swallowing
• Muscle weakness
• Most individuals die of respiratory failure
  during early childhood

An autosomal recessive disorder that’s caused by a loss of function mutations in the survival of motor neuron 1 (SM1). Presentation can be modified due to copy
number variation (CNV) in SM2.

Question 8

How is SMA type 1 treated with gene therapy at the mutant mRNA level?

Answer 8

Zolgensma is a one off gene therapy with a reported list price of £1.79 million per dose.

Adeno-associated virus (AAV) 9 based gene therapy that deliver a copy functional copy of the SMN1 gene to the nerve cells.

Question 9

How is retinitis pigmentosa treated with gene replacement therapy?

Answer 9

People with inherited retinal dystrophies caused by gene mutations in the 65 kDa retinal pigment epithelium gene (RPE65).

• Retinitis pigmentosa type 20 (RP20)
  Prevalence: 20-30 per 100,000 2% of retinitis pigmentosa linked to RPE65
• Leber’s congenital amaurosis type 2 (LCA2)
  Prevalence: 2-3 per 100,000 6-16% of LCA diagnoses linked to RPE65.

Suggested for patients with confirmed biallelic RPE65 mutations and who have sufficient viable retinal cells. Therefore, gene therapy is only available for select people who fit the criteria.

Question 10

How are genetic and chemical modifiers used in the treatment of sickle cell anaemia?

Answer 10

In the genetic persistence of fetal
haemoglobin hydroxyurea, an inducer of HbF, can be used to reduce the frequency of sickle cell crisis. In another approach CRISPR–Cas9-based genome editing can be used to disrupt the production of the γ-globin inhibitor BCL11A in progenitors of red blood cells increasing fetal heamoglobin levels.

Silencing of the globin (HbF) gene is associated with DNA methylation. The effect of the cytosine analog decitabine (5-aza-2’-deoxycytidine) on the percentage of hemoglobin F (Hb F) in 13 patients with sickle cell disease, compared with their level of Hb F without any treatment. Decitabine is a DNA hypomethylating agent that acts by inhibiting DNA methyl transferase.

Question 11

What is the intervention at the mutant mRNA level?

Answer 11

RNA interference.

Steps:
1) Processing of long dsRNA by RNase III Dicer into small interfering RNA (siRNA) duplexes
2) Loading of one of the siRNA strands on an Argonaute protein possessing endonucleolytic activity
3) Target recognition through siRNA base-pairing
4) Cleavage of the target by the Argonaute’s endonucleolytic activity

Question 12

What does RNA interference do at the mutant mRNA level in hereditary amyloidogenic transthyretin (ATTRv) amyloidosis?

Answer 12

Patisiran is a double-stranded small interfering RNA (siRNA) encapsulated in a lipid nanoparticle for delivery to hepatocytes. It targets the conserved 3′ untranslated regions of both the mutant and wild-type transthyretin
mRNA causing a reduction of all products by degrading and silencing the mRNA. For use in adult patients in the first two stages of nerve damage.

Question 13

What does RNA interference do at the mutant mRNA level in hereditary amyloidogenic transthyretin (ATTRv) amyloidosis?

Answer 13

Hereditary amyloidogenic transthyretin (ATTRv) amyloidosis is an autosomal dominant disease in which abnormal proteins called amyloids build up in tissues around the body over time including around the nerves. It is an adult-onset systemic disease that usually presents as a progressive peripheral neuropathy and is caused by point
mutations in the gene that encodes transthyretin (TTR). Normal transthyretin (TTR) transports vitamin A (retinol) and thyroxine throughout the body.

Clinical presentation is diverse, including length-dependent small-fiber polyneuropathy, all-fiber polyneuropathy, pseudo-chronic inflammatory demyelinating polyneuropathy, upper-limb-onset neuropathy, and motor neuropathy. Half of the patients also have cardiac amyloidosis.

Traditional treatment is a liver transplant for early-stage disease and TTR tetramer stabilizers (Tafamidis approved in EU for Stage 1 hATTR amyloidosis).

Question 14

What does RNA interference do at the mutant mRNA level in Spinal Muscular Atrophy (SMA) Type 1?

Answer 14

Artificially enhancing SM2 production. SM2 has a fault that results in the
skipping of exon 7. Nusinersen is an antisense oligonucleotide that acts as a splice-altering oligonucleotide. It is delivered intrathecally and alters SMN2 pre-mRNA splicing to ensure exon 7 inclusion, boosting the production of the functional protein. Delivered via spinal injection, however, not without side effects.

Question 15

What is the intervention at the mutant protein level?

Answer 15

Either protein replacement or enhancement of residual protein function.

Question 16

How is cystic fibrosis treated at the mutant protein level?

Answer 16

Cystic fibrosis can be caused by different mutations and therefore their underlying cause is different. Depending on the mutation different drugs can be given to help treat the cause (info found in cystic fibrosis flashcards).

Question 17

How does protein replacement therapy work?

Answer 17

Protein therapy can go into five groups:

1) Replacing a protein that is deficient or abnormal
2) Augmenting an existing pathway
3) Providing a novel function or activity
4) Interfering with a molecule or organism
5) Delivering other compounds or proteins

Question 18

What is the intervention at the metabolic or other biochemical
dysfunction level?

Answer 18

This is where disease-specific compensation occurs such as dietary changes or pharmacologic.

Question 19

How is PKU treated at the metabolic or other biochemical dysfunction levels?

Answer 19

PKU has a specific diet to follow to reduce phenyl in the body. PKU can also be treated with drugs. Sapropterin dihydrochloride (Kuvan®) is a synthetic form of BH4. BH4 is a cofactor for phenylalanine hydroxylase
(PAH) and is used to increase residual PAH activity in patients with some PAH activity. It does not replace all dietary restrictions.

Question 20

What other diseases can pharmacologic help with?

Answer 20

Hyperammonemia and CML.

Hyperammonemia is a metabolic condition characterized by elevated
levels of ammonia in the blood resulting in neurological damage. The administration of sodium benzoate diverts ammonia to glycine synthesis.

In CML tyrosine kinase inhibitors are targeted for treatment as this prevents the cells from proliferating.

Question 21

What is the intervention at the clinical phenotype level?

Answer 21

Medical intervention and surgical intervention.

Question 22

How is thalassemia treated at the clinical phenotype level?

Answer 22

Blood transfusion can be done. This can have some negative side effects such as excess iron due to blood transfusions and a swollen belly due to splenomegaly.

Question 23

How is cystic fibrosis treated at the clinical phenotype level?

Answer 23

Medicines for lung problems:
* antibiotics
* mucus thinner
* bronchodilators
* steroid medicine

o Exercise
o Airway clearance techniques
o Dietary and nutritional advice
o Lung transplants

Question 24

What is the intervention at the family level?

Answer 24

Genetic counseling, genetic screening, and presymptomatic diagnosis.

Question 25

How is Down syndrome treated at the family level?

Answer 25

1) Amminocentis testing. Around 10-20ml of fluid is aspirated through the adnominal wall under ultrasound guidance. Usually occurs at around 16 weeks. Most cells in the amniotic fluid have been shed by the fetus
allowing genetic investigations to be undertaken. QF-PCR can be performed on amniotic fluid to detect aneuploidies in chromosomes 13, 18 21, X, and Y. Fluorescently ladled primers are used to analyze up to 5 short tandem repeats pre chromosome. The results indicate a third chromosome 21.

2) Chronic villus sampling. Usually carried out between 11 to 12
weeks. Transabdominal aspiration of the chorionic villus. The tissue is of fetal origin.

3) Cell-free foetal DNA represents extracellular DNA which originates from trophoblastic cells. However, the vast majority of cell-free DNA in maternal blood originates from the mother, with cell-free foetal DNA representing only 3% of the total cell-free circulating DNA in early
pregnancy rising to 6% in late pregnancy. A massive parallel ‘shotgun’ approach is used to detect the over-representation of specific chromosomes.

4) Ultrasound can be used as an indicator of Down’s syndrome. Identifiers can be observed on 2nd-trimester ultrasound. Dilated brain ventricles increased the thickness of the back of the neck, and an abnormal artery to the upper extremities increases the risk by three- to four-fold, and an absent or small nose bone increases the risk by six- to seven-fold. Nuchal thickening, an accumulation of fluid at the base of the skull, is an indicator of potential chromosomal abnormality or cardiac abnormality.

Question 26

How is genetic screening done?

Answer 26

1) The heal prick test:

*Sickle cell disease
* Cystic fibrosis (CF)
* Congenital hypothyroidism (CHT)
Inherited metabolic disorders:
* Glutaric aciduria type 1 (GA1)
* Homocystinuria (HCU)
* Isovaleric acidaemia (IVA)
* Maple syrup urine disease (MSUD)
* Medium-chain acyl-CoA
dehydrogenase deficiency (MCADD)
* Phenylketonuria (PKU)

2)*Population level screening. This is for thalassemia which has a high carrier rate of ~1 in 7 in Greek Cypriots. Five specific mutations account for 98.4% of mutations in Greek Cypriots. Cyprus has one of the highest prevalences of thalassemia in the world. It introduced a successful population-wide prevention programme, based on premarital screening.

3) Pre-symptomatic screening has also possible for Huntington’s disease as the disease is late-onset so a parent may get it after they have reproduced so the children may want to know if they have it before they become ill.