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F Genetic Metabolic Diseases

Q: What is the difference between Wildtype AAV and rAAV (Adeno Associated Virus)?

- Wildetype AAv : Rep mediates integration in specific locus of the human genome but also episomal persistence. - rAAV : no Rep --> no active integration, episomal persistence and random integration.

Q: What is another struggle with AAV vectors (specially regarding cell division)?

AAV does not actively integrate into the host genome. So most vector genomes are lost upon cell division. So the episomal expression will be lost over time. You need an integrating vector to treat children. However, random integration can cause geno-toxicity. * AAV can integrate at double strand breaks, therefore you still can decide where it needs to go.

Q: What is the difference between nonviral and viral vectors?

- Nonviral : naked DNA delivery by injection, liposomes, nanoparticles, and other means. Can be produced in relatively large amounts and are likely to present minimal toxic or immunological problems. Suffer from inefficient gene transfer. - Viral : vectors are derived from viruses with either RNA or DNA genomes and are represented as both integrating and nonintegrating vectors. Integrating vectors hold the promise of lifelong expressiion of the deficient gene product.

Q: What are the diagnostic options for genetic metabolic diseases?

- Metabolite analysis in plasma, urine, tissue etc. - Enzyme measurements in cells, tissues - Mutation analysis in DNA

Q: How doe cells acquire cholesterol?

Cells acquire cholesterol either by de novo synthesis by means of the isoprenoid biosynthesis pathway or via the uptake of low density liptoprotein particles that contain esterified cholesterol.

33 important questions on F Genetic Metabolic Diseases

What are the roles of the liver?

- Central role in many metabolic processes (cholesterol)
- De-toxification of blood
- Production of blood factors (factor VIII and IX)

What is the difference between Wildtype AAV and rAAV (Adeno Associated Virus)?

- Wildetype AAv: Rep mediates integration in specific locus of the human genome but also episomal persistence.
- rAAV: no Rep --> no active integration, episomal persistence and random integration.

What is the struggle usingAAV gene therapy?

AAV gene therapy was used to treat FIX deficiency. It was injected into the hepatic artery. Factor IX Padua was used (this is typical for patients with thrombosis higher activity compared to normal factor IX).

However, the FIX expression went down after 2 months. This is due the memory immune system. Most patients have been infected with AAV before, causing an immune reaction when injected with a rAAV.

Why is blood a hostile environment for virus and viral vectors?

Many humans have already been infected by AAV and therefore have a pre-existing immunity towards AAV. In the blood neutralizing Antibodies bind AAV and block transduction of the hepatocytes.

* The amount of antibodies increases with age, so the change on succes with AAV viral vectors is higher in children.

What is another struggle with AAV vectors (specially regarding cell division)?

AAV does not actively integrate into the host genome. So most vector genomes are lost upon cell division. So the episomal expression will be lost over time.

You need an integrating vector to treat children. However, random integration can cause geno-toxicity.

* AAV can integrate at double strand breaks, therefore you still can decide where it needs to go.

How can targeted integration be achieved?

- CRISPR/Cas
- Homologous repair
- Donor construct

What is the difference between nonviral and viral vectors?

- Nonviral: naked DNA delivery by injection, liposomes, nanoparticles, and other means. Can be produced in relatively large amounts and are likely to present minimal toxic or immunological problems. Suffer from inefficient gene transfer.

- Viral: vectors are derived from viruses with either RNA or DNA genomes and are represented as both integrating and nonintegrating vectors. Integrating vectors hold the promise of lifelong expressiion of the deficient gene product.

What are the diagnostic options for genetic metabolic diseases?

- Metabolite analysis in plasma, urine, tissue etc.
- Enzyme measurements in cells, tissues
- Mutation analysis in DNA

How doe cells acquire cholesterol?

Cells acquire cholesterol either by de novo synthesis by means of the isoprenoid biosynthesis pathway or via the uptake of low density liptoprotein particles that contain esterified cholesterol.

For what is the isoprenoid biosynthetic pathway important?

Cholesterol is synthesized via the isoprenoid biosynthetic pathway, which produces numorous isoprenoids.

Isoprenoids function in processes as cell growth and differentiation, protein glycosylation, signal transduction pathwats and mitochondrial electron transport.

* Mevalonate kinase deficiency (MKD) affects the synthesis of all isoprenoids.

What are the clinical aspects of cholesterol biosynthesis defects?

- Multiple congenital anomalies (incl. Organs)
- Skeletal abnormalities, dysmorphism
- Skin abnormalities
- Developmental retardation
- Psychomotor retardation

What is Hyper-IgD and Period Fever Syndrome (HIDS)?

- HIDS is an autosomal recessive disorder
- Mevalonate kinase Deficiency (MKD)
- Symptoms during fever episode: swollen cervical lymph nodes, splenomegaly, abdominal pain, vomiting, diareea, arthralgia, skin rash (erythematous macules).
- Blood chemistery during fever episodes: granulocytosis and monocytosis, elevates ESR,CRP and SAA, elevated IL-1B, IL-6, TNF-a.
- Fever episodes provoked by vaccination, small infections, stress trauma etc. (sterile inflammation)
- Autoinflammatory disease

What is an autoinflammatory disease?

- Characterized by lifelong recurring, seemingly spontaneous episodes of fever and inflammation (no antibodies as opposed to autoimmune diseases).
- Dysregulation innate immune system
- Associated with increased/prolonged production of pro-inflammatory cytokine IL-1B.

What is the difference and similarity between MKD-HIDS and MKD-MA?

- Both have a mutation in mevalonate Kinase (mevalonate --> 5-phosphomevalonate), but a different mutation in the MK gene
- Both have fever episodes
- MKD-MA is fatal
- Both have an accumulation of mevalonate and therefore mevalonic acid in plasma/urine/tissue during fever episodes.

* MA = Mevalonic Aciduria

How does temperature affect HIDS?

HIDS is due to temperature-sensitive mutation.
- Temperature-sensitive mutation in MK causes temporary block in isoprenoid biosynthesis, which prevents timely availability of isoprenoid end products involved in immune regulation.

During fever: Temperature --> activity MK drops --> upregulation HMG-CoA reductase.

Which isoprenoid is limiting and contributes to the inflammatory phenotype of MKD-HIDS?

Geranylgeranyl-PP. Responsible for IL-1B secretion.
Low Geranylgeranyl-PP leads to low Geranylgeranylation which leads to incorrect activation of Rho-GTPases which leads to the secretion of IL-1B and therefore inflammation (and activation of caspase-1).

What is a possible treatment option for MKD-HIDS patients?

Targeting of the isorpenoid biosynthesis pathway, specially the Geranylgeranyl-PP.

For what are Fatty acids used?

- Fatty acids are 60/70% of the fuel for the heart
- They are also used by the muscles, kidneys, heart and liver after prolonged fasting (when glycogen storage is depleted).
- Liver makes ketone bodies from AcylCoA

Why does fatty acid oxidation require carnitine? And how does carnitine gets into the cell?

- Carnitine is needed to transport Acyl CoA into the mitochondira via acylcarnitine.
- Carnitine gets into the cell with OCTN2 symporter (Na and carnitine)
- It is either obtained from endogenous synthesis (from lysine) or the diet (meat, fish, dairy products).

* OCTN2 is crucial for carnitine uptake into tissues and resportion in the kidneys.

What happens in OCTN2 deficiency?

- Carnitine cannot be taken up into tissue or reabsopted by the kidneys --> low plasma carnitine, high urinary carnitine.
- Primary carnitine (OCTN2) deficiency
- Early onset: acute metabolic decompensation, hypoketotic hypoglycemia, Reye-syndrome, cardiac abnormalities & sudden infant death.
- Later: chronic (cardio)myopathy with/withou muscle weakness --> sudden (cardiac) death.

* All features disappear upon oral carnitine supplementation!

How can a deficiency in the long-chain fatty acid oxidation pathway be treated?

Give the patients medium-chain FA's. (LCFA have a 'longer' pathway).

What are the symptoms of FAO disorders?

- Hypoketotic hypoglycaemia.
- Liver (fatty changes, microvesicular steatosis)
- Heart (acute heart block, progressive cardiomyopathy, arrhytmias, tachycardia).
- Skeletal muscle (rhabdomyolysis)
- Brain (energy deficit)
- Kidneys (renal failure, renal tubular acidosis)
- Eyes (retinopathy)
- Nervous system (neuropathy)

* Retinopathy and Neuropathy only in LCHAD/MTP deficiency.

What is the underlying mechanism behind hypoketotic hypoglycemia in FAO defects?

Markedly increased rate of glucose consumption in all tissues in the absence of a properly functioning beta-oxidation system.

Why is the diagnosis of mitochondrial FAO deficiencies difficult?

1. Lack of clinical signs and symptoms (no external stigmata)
2. Lack of adequate laboratory methods other than FFAs, ketone bodies etc.
--> now acylcarnitine is measures by Mass Spectronomy.

* Each defect has its own Acylcarnitine characteristic.

For what does the neonatal screening screen?

Aminoacids, acylcarnitines (false positives) and galactose.

What is the pathophysiology of mitochondrial FAO disorders?

1. Energy deficiency
2. Toxicity caused by the intra-cellular accumulation of acyl-CoA's, acyl-carnitines and/or other compounds.

What is the treatment for the energy deficiency in mitochondrial FAO disorders?

Patients receive ketone bodies, medium chain triglycerides (if defect in LCFA)

What is the treatment for the toxicity in mitochondrial FAO disorders?

1. Induction of residual enzyme activity (manipulation promotor etc.)
2. Substrate reduction (block influx FA, specially in heart, or inhibition CPT1 --> less acylcarnitines from acylCoA)

What are treatment stragies for mitochondrial FAO disorders?

- Avoidance fasting
- MCT based diet
- Substrate reduction therapy
- Induction residual enzyme activity

What is Adrenoleukodystrophy (ALD)?

- Adrenoleukodystrophy is an inborn error of metabolism. It is X chromosome bound (ABCD1) and affects the brain, spinal cord and adrenal cortex.
- ABCD1 codes for the X-ALD protein called ALDP. It is a ATP-bining casette (ABC) transmembrane trasporter. The nucleotide binding fold is located towards the cytoplasmic surface of the peroxisomal membrane. It imports VLCFAs into peroxisomes.- Leads to adrenal insufficiency, myelopathy and childhood cerebral ALD

Defect ABCD1 --> no B-oxidation by peroxisome --> storage VLCFA

* ALD results in a cerebral inflammatory reaction resulting in loss of myelin.

Why can clinical outcome of ALD not be predicted?

- All ALD males have elevated VLCFA levels
- VLCFA levels (plasma, blood cells, fibroblasts) do not correlate with phenotype
- All ALD patients have a mutation in the ABDD1 gene
- ABCD1 mutations have no predictive value towards clinical outcome (identical mutations are associated with different ALD phenotypes)

Give two reasons why it is rational to screen newborns for ALD

- Adrenal insufficiency: untreated adrenal insufficiency can be life-threatening and it is often discovered only after several hospitalizations. Treatable with hormone replacement therapy.

- Cerebral ALD: untreated cerebral ALD is often fatal. Hematopoietic stem cell transplant is curative, but only in early-stage disease. Patients who are diagnosed on the basis of neurological symptoms are already too late for transplantation.

What are the ISNS general guidelines for neonatal screening?

- Early diagnosis must be a directly beneficial to the neonate
- The benefit is reasonably balanced against financial and other costs
- There is a reliable test suitable for neonatal screening
- There is a satisfactory system in operation to deal with diagnostic testing, counselling, treatment and follow-up of patients identified by the test.
- There must be sufficient public support for the expansion

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Medical Biochemistry and Pathophysiology

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