Urea Cycle Disorders -BioChem Genetics

The Urea Cycle

• 6 Major Enzymatic reactions that occur in the liver within the Hepatocytes: the mitochondria + cytoplasm

• 2 Major functions

  • removal of nitrogenous waste (produced mainly from protein catabolism) → Ammonia incorporated into Urea for Excretion

  • Synthesis of amino acids: Arginine, Ornithine and Citrulline (become ESSENTIAL A.A. in deficiencies of the UREA CYCLE ENZYMES)

• Major presentations of Urea Cycle Disorders:

  • Severe Neonatal Hyperammonemic encephalopathy (exception: Argine deficiency + late/mild onset variants)

• All Autosomal Recessive except for Ornithine transcarboxylase deficiency (OTC):

  • OTC is X-Linked Recessive → only affects Males

The 6 Urea Cycle Disrders

Correspond to the 6 steps of the Urea Metabolic cycle: taken individual relatively uncommon, but all together 1:8-35,000

• N-Acetyl Glutamate Synthetase Deficiency

• Carbamoyl Phosphate Synthetase (CPS1) Deficiency

• ****Ornithine Transcarbamylase (OTC) Deficiency****—> *****MOST COMMON*****

• Arginosuccinic Acid Synthetase I (ASS1) Deficiency → also called Citrullinemia I

• Arginosuccinic Acid Lyase (ASL) Deficiency

• Arginase (ARG) Deficiency

Ammonia (NH3)

• Ammonia is the product of the metabolism/catabolism of protiens/amino acids

  • Normal serum ammonia levels

    • Adults <35 mmol/L

    • Neonates <100 mmol/L (immature liver cells + increased tissue catabolism surrounding delivery)

• Hyperammonemia: happen with great degree with IEMs of Urea Cycle But also seen with other metabolic disorders like Organic Acidemias (excess acid decreased Urea Cycle activity, lesser extent that UCDs)

  • Causes Neuronal excitotoxin increased extracellular glutamate +overexcitation of NMDA receptors→ Cell death and Cerebral Edema

• Clinical Consequence

  • Acute severe elevation: seizures, coma, death

  • Mild chronic elevations: Brain atrophy, cognitive impairment

Classic UCD Presentation: Early

Neonatal Hyperammonemia Encephalopathy

• In utero: protected my maternal urea Cyle activity of liver cells

• At Birth in first 48hours: Ammonia levels rise quickly

  • Decreased feeding w/ vomiting

  • Lethargy

  • Tachypnea (rapid breathing)

  • Seizure activity

• Followed by Rapid

  • Encephalopathy/Coma

  • Respiratory Failure

  • Cerebral Edema and Death

Late-Onset UCD Presentations

• Variable age of onset and severity of Chronic and/or recurrence/Fluctuating symptoms:

  • Headache, Vomiting Ataxia and incoordination

  • Psychiatric/Behavioral disturbance: Delirium, ASD, ADD/ADHD, Manic episodes

  • Cognitive impairment: DD/MR, executive processing defects, early dementia

• Often exacerbated/precipitated by:

  • Fever, Illness, fasting, post-partum, protein load (self restrict protein)

• STILL AT RISK FORM HYPERAMMONEMC ENCEPHALOPATHY:

  • even if previously asymptomatic, can still be fatal

Mutiple Etiologies for Hyperammonemia

Things besides UCDs (MOST COMMON CAUSE) that can also cause the accumulation of excess ammonia:

• Generalized Liver Deiasie (Acute or Chronic)

  • Non-genetic Causes: infections, Toxins, Trauma, Ischemia etc.

  • Genetic Causes

    • Non-IEM: Alpha-1 Antitrypsin (accumulation in liver=chrossis), Alagille syndrome etc

    • Non-UCD IEM

      • Aminoacidopathies: Tyrosinemia Type I

      • Organic Acidemia: elevated Lactic acid which inhibit NAGS

      • Primary Mitochondrial disorders

      • Fatty Acid oxidation Defects: Reye-Like syndrome

      • Carbohydrate Metabolic defects: Galactosemia, Fructosemia

      • Metal Processing defects WILSONS DIEASE, hemochromatosis

  • Primary UCDs

OTC Deficeiny Metabolism

X-Linked Recessive Form

Within the Mitochondria of Liver Cell:

• Carbamyl Phosphate + Ornithine → Citrulline via Ornithine transcarbamylase (OTC) activity: a Block at OTC = decreased Citrulline, increased Arginine and Aringnosuccinate (along the cycle)

  • Ammonia + Orotic Acid also increased (run-off product of carbamyl phosphate NOT becoming citrulline, not usually accumlated)

OTC Deficiency

• Orthine Trancarbamylase (OTC) Deficiency:

  • Most Common UCD

  • X-Linked Recessive Inheritance

  • Incidence 1:56,000 (not increased in any specific group)

Genetic Defect: Orthine Transcarbamylase (OTC) Gene mutation

• High Orotic Acid + Ammonia : Low Cirtrulline+ASS+ARGE

• Female carriers most common, with symptomatic male probands

• Non-carrier females: 3-4% germline mosaicism rate (apprecaible)

Untreated OTC Deficiency

Classic (usually males, only very rarely females)

• At Birth to 24/48 Hours: Asymptomatic

• 2-3 Days old: Progressive hyperammonemia Encephalopathy

  • Poor feeding

  • Vomiting

  • Lethargy

  • Hyperventilation

  • Seizures

• By 1 week Old: Lethal hyperammonemia Encephalopathy

  • Cerebral edema

  • Hypothermia

  • Coma

  • Respiratory failure

  • Death

MILD (Mild mutation males/symptomatic Females - 15%)

• Episodic hyperammonemia Symptoms: ranging form mild to life-threatening

• Chronic symptoms

  • Protein avoidance

  • recurrent headache

  • neuropsychiatric difficulty

Diagnosing OTC Deficiency

Newborn Screening: not done in all states (like NY) but ME, MAN,RI,VT

• LOW CITRULLINE

Diagnostic Evaluation

• Ammonia Level: symptomatic >100, Encephalitic >200

• Blood gas/Metabolic profile/lactic acid levels: exclude other IEMs

• Plasma Amino Acid Profile + Urine Organic Acid Profile

  • High glutamine/alanine + citrulline/ASA/ARG low in blood

  • High Orotic acid in urine

  • ****Allopurinol given to female carriers can expose OTC by preserving Orotic Acid and show Orotic Acidemia****

Confirmatory Testing

• Enzyme activity analysis: needs liver biopsy and not 100% in females due to x-inactivation in the liver

• OTC genotyping via sequencing +del/dup analysis: 60-90% detection

Treating OTC Deficiency

Acute Encephalopathy treatment

• Radpidly remove ammonia: hemodialysis/hemofiltration

• Reduce production: protein cessation 12-24hours, decrease nitrogenous waste

• Prevent Catabolism: high calorie, protein free - IV glucose + lipids and IVE arginine (become essential AA b/c it cannot be synthesized ‘like normal’)

• Provide other ammonia removal agents: IV ammonia scavengers Sodium-Benzoate + Sodium-Phenylbutyrate

Chronic Treatment: after acute encephalopathy has resolved

• Protein restriction + essential AA formula

• Oral cituralline or arginine and ammonia scavenrs (Sodium-Benzoate + Sodium-Phenylbutyrate provide alternative pathways for ammonia excretion)

• Avoid Valproic Acid, fasting, Fever, steroid, protein load

• Liver transplant: only if neurologically intact after EPISODE + poor response to chronic treatment

OTC Deficiency Outcome

Outcome with Treatment: Variable

• Neurocongtive delvepmn depens on intiala hyperammoneicm encephalyopth udration and control of subsquent amonia/glutamate leves

• Ofente ID, ADHA, executive defcits, brain attrophy (even in midl males and symptomatic females)

OTC Carrier Screening + Prenatal Diag

Carrier Screening:

• Molecuelar if mutation known in proband

• Allopurinol Loading test show Orotic Acid elevations

Prenatal implantation: No enzyme/metabolite testing is useful, activity of OTC contained to liver cells

Arginosuccinic Acid Synthetase I (ASS1) Deficiency

A block at the next part of the cycle after OTC: Arginosuccinic Acid Synthetase I

• where citrulline + Aspartate → Arginosuccinate

• Increased Citrulline levels - Called Citrullinonemia

• Increased levels of Ammonia, Orotic Acid (not quite as high as in OTC)

• Decreased Arginaine, Arginosuccinatic Acid

  

Citrullinemia Type I

Arginosuccinic Acid Synthetase I (ASS1) Deficiency

Inheritance Autosomal Recessive

Incidence: 1:200,000 (no increased in any particular group)

Genetic Defect Arginosuccinic Acid Synthetase I (ASS1) Gene

• Increased Citrulline

• Decreased Arginnine,

• High Ammonia and Orotic Acid levels

  ***ANOTHER DEFECT in Citrin Gene (a transporter) causes Citruallenmia type II/III → MILDER***

Untreated Citrullinemia I

Classic

• At birth to 48hours : Asymptomatic

• By 4-7 days old: progressive hyperanomieic encephalopathy

  • Poor feeding

  • Vomiting

  • Lethargy

  • Hyperventilation

  • Seizures

• By 1-2 weeks old: Lethal hyperammonemic Encephalopathy (slightly slower progression than OTC deficieny)

  • Cerebral edema

  • hypothermia

  • coma

  • respiratory failure

  • death

Late-Onset Forms (milder)

• Episodic hyperammonemic symptoms

• Chronic symptoms

  • protein avoidance

  • recurrent headache

  • neuropyshciatric difficulty

  • only a few cases with liver failure

Diagnosing Citrullinemia I

New Born Screening

• Elevated citrulline: but not specfic to Citrullinemia I

  • Citrullinemia II/III (Citrin Deficiency) Arginosuccinic Acid Lyase (ASL) deficiency, Pyruvate Carboxylase deficiency

  • False positive

Confirmatory Testing

• Ammonia Level: symptomatic over 100, encephalopathic over 200 (often 1000s)

• Blood gas/metabolic progile/lactic acid level: exclude metabolic acidosis indicating other IEM

• Plasma Amino Acid + Urine Orgnic acids

  • Plasma: Elevated citrulline/glutatmine/allaine + Decreased Arginosuccinate/Arginine/Ornithine

  • Urine: Increased orotic acid

• ASS1 Activity in Fibroblasts, liver, CVS or aminocytes

ASS1 Genotyping

• gene sequecing + del/dup analysis 96% detection

• genoype/phenotpye correlation not 100%

Treating Citrullinemia I

Acute Encephalopathy treatment

• Rapidly remove ammonia: hemodialysis/hemofiltration

• Reduce production: protein cessation 12-24hours, decrease nitrogenous waste

• Prevent Catabolism: high calorie, protein free - IV glucose + lipids and IVE arginine (become essential AA b/c it cannot be synthesized ‘like normal’)

• Provide other ammonia removal agents: IV ammonia scavengers Sodium-Benzoate + Sodium-Phenylbutyrate

Chronic Treatment: after acute encephalopathy has resolved

• Protein restriction + essential AA formula

• Oral citrulline or arginine and ammonia scavengers (Sodium-Benzoate + Sodium-Phenylbutyrate provide alternative pathways for ammonia excretion)

• Avoid Valproic Acid, fasting, Fever, steroid, protein load

• Liver transplant: only if neurologically intact after EPISODE + poor response to chronic treatment

Citruellemia Type I Outcome

Depends on how quickly the hyperammonemia encephalopathy is dealt with + the control of subsequent ammonia/glutamate levels (LIKE OTC DEFICENCY)

• Even with reatment: Often ID, ADHA, executive deficits, brain atrophy

Citrullinemia Type 1

Carrier Screening: if you know the muration

PRenatal.Preimplation diagonsis

• if mutation is known, it can be detected via screening

• ASS1 enzyme activty can be measured on CVS or Amniocentisis

Arginosuccinic Acid Lyase (ASL) Metabolic Pathway

Arginosucinate → Arginine + Fumarate (by product) via Arginosuccinic Acid Lyase

• Elevated Ammonia, Argininosuccinate, Citrulline (Mild orotic acid)

• Decreased Arginine

Arginosuccinic Acid Lyase (ASL) Deficiency

• Similar to other UCD plus

  • Brittle hair

  • Hypertension

• Inheritance Autosomal Recessive

• Incidence: 1:220,000 (no increased particular population)

  Genetic Defect

  • Arginosuccinate Lyase (ASL) Gene

  • Increased Citrulline and ASA

  • Decreased Arginine, Ammonia

  • Mild to no Orotic Aciduria

Untreated ASL Deficieny

Classic

• At birth to 48hours : Asymptomatic

• By 4-7 days old: progressive hyperanomieic encephalopathy

  • Poor feeding

  • Vomiting

  • Lethargy

  • Hyperventilation

  • Seizures

• By 1-2 weeks old: Lethal hyperammonemic Encephalopathy (slightly slower progression than OTC deficieny)

  • Cerebral edema

  • hypothermia

  • coma

  • respiratory failure

  • death

Late-Onset Forms (milder)

• Episodic hyperammonemic symptoms

• Chronic symptoms

  • protein avoidance

  • recurrent headache

  • neuropyshciatric difficulty

  • ****LIVER FAILURE OFTEN****

  • *****Brittle HAIR****

  • ****Hypertension****

Diagnosing ASL Deficiency

New Born Screening

• Elevated citrulline: but not specific to ASL defeciney

  • Citrullinemia II/III (Citrin Deficiency), Pyruvate Carboxylase deficiency

  • False positive

Confirmatory Testing

• Ammonia Level: symptomatic over 100, encephalopathic over 200 (often 1000s)

• Blood gas/metabolic progile/lactic acid level: exclude metabolic acidosis indicating other IEM

• Plasma Amino Acid + Urine Organic acids

  • Plasma: Elevated citrulline/glutamine/alanine/Arginosuccinate + Decreased Arginine/Ornithine

  • Urine: Increased orotic acid

• ASS1 Activity in Fibroblasts, liver, CVS, amniocytes AND red blood cells

ASL Genotyping

• gene sequencing + del/dup analysis 90% detection

• genotype/phenotype correlation not 100%

Treating ASL Defecieny

Acute Encephalopathy treatment

• Rapidly remove ammonia: hemodialysis/hemofiltration

• Reduce production: protein cessation 12-24hours, decrease nitrogenous waste

• Prevent Catabolism: high calorie, protein free - IV glucose + lipids and IVE arginine (become essential AA b/c it cannot be synthesized ‘like normal’)

• Provide other ammonia removal agents: IV ammonia scavengers Sodium-Benzoate + Sodium-Phenylbutyrate

Chronic Treatment: after acute encephalopathy has resolved

• Protein restriction + essential AA formula

• Oral citrulline or arginine and ammonia scavengers (Sodium-Benzoate + Sodium-Phenylbutyrate provide alternative pathways for ammonia excretion)

• Avoid Valproic Acid, fasting, Fever, steroid, protein load

• Liver transplant: only if neurologically intact after EPISODE + poor response to chronic treatment

ASL Defeciney Outcomes

Depends on how quickly the hyperammonemia encephalopathy is dealt with + the control of subsequent ammonia/glutamate levels (LIKE OTC DEFICENCY)

• Even with treatment: Often ID, ADHA, eventual liver failure and hypertension (UNRELATED TO HYPERAMMONEMIA LEVELS→ even with good control, liver failure and hypertension happen)

• ****Trichorrhexis Nodosa (brittle hair) resolves on Arginine****

ASL Deficiency Screening

Carrier screening: look for specific mutation if found in the proband

Prenatal/preimplantation diagnosis

• If mutation known

• ASL enzyme can be measured on VS/Amniocentesis

• Arginosuccinic acid levels in amniotic fluid when elevated are suggestive of ASL deficiency

Arginase (ARG) Deficiency Metabolism

Last step of the Urea cycle:

• Arginine → Ornithine + Urea (released into urine and expelled) via Arginase activity

• Elevated Arginine, Arginosuccinic acid, Citrulline

• No elevation of Orotic Acid

Arginase (ARG) Deficiency

• ***No neonatal Encephalopathy (in contrast to the other UCDs)***

  Inheritance Autosomal Recessive

  Incidence 1:900,000 (slightly more common in Japan and French Canada)

Genetic Defect

• Arginase (ARG1) Gene

• increased Arginine

• MILD intermittent hyperammonemia

• Occasional mild orotic acid elevation

Untreated ARG Deficiency

No Hyperammonemia Encephalopathy

Neo-natal

• Birth to 1-3 Years old: Asymptomatic

• After 1-3 years old

  • Growth slows

  • Motor/cognitive development slows, regression occurs

  • Spasticity/seizures develop

• Adulthood

  • Severe mental retardation, microcephaly

  • Short stature

  • Severe spasticity and joint contracture

  • Lack of ambulation, bowel, and bladder control

  • Low risk of hyperammonemic encephalopathy

Diagnosing ARGE Deficiency

Newborn Screening

• Elevated Arginine

Confirmatory Testing

• Ammonia levels: Normal or less than 150 umol/L (so usually not encephalopathic)

• Blood gas/metabolic profile/lactic acid level: exclude metabolic acidosis indicating other IEM

• Plasma AA profile + Urine ORgnic Acids

  • Elevated argienien

  • Orotic acid normal or minimially elevated

• Enzyme active of ARG1: measured in Redblood cells, fetal red blood cells (BUT NOT AMNIOCYTE or CHORIONIC VILLI)

ARG1 Genotyping

• gene sequencing + del/dup analysis

Treating ARG Deficiency

• Not typically encephalopathic

  • if it does happen, treat it like the other UCD: dialysis, amonic sacnaer, protien restiction, but NO IV ARGININE: levels already elevated with this condition

• Chronic Treatment: after acute encephalopathy has resolved

  • Protein restriction + essential AA formula

  • ammonia scavengers (Sodium-Benzoate + Sodium-Phenylbutyrate provide alternative pathways for ammonia excretion)

  • Avoid Valproic Acid, fasting, Fever, steroid, protein load

  • Liver transplant: only if neurologically intact after EPISODE + poor response to chronic treatment

ARG Defeicney out come

with treatment, outcome improved but still increased risk of:

• intellecuta diablity,

• short stature

• spascity

• joint ocntracture

• lack of ambulation

• loss of bowl+bladd control

• Liver dysfunciton

ARG Deficiency Diagonsis

• Prenatal/Preimplantation diagnosis

• ARGE enzyme activity only on RBCs: so not via amnio of CVS

  • Possible to test enzyme activity of fetal blood via ubiclial sample, but rarely done

N-Acetyl Glutamate Synthetase (NAGS) Deficiency and Carbamoyl Phosphate Synthetase (CPS1) Deficiency Metabolic pathways

First steps of the Urea Cycle Pathways

• Ammonia + N-acetylglutamate → Carbamoyl Phosphate via Carbamoyl Phosphate Synthetase (CPS1) activity

• Glutamate → N-acetylglutamate via N-Acetyl Glutamate Synthetase (NAGS) activity

• Blocks in NAGS or CPS1 = failure of N-acetylglutmate to combine with ammonia

• SEVER HYPER AMMENIMA with low level of Carbamyl Phosphate + Citrublline, Arginosuccinaic Acid, Arginine, Ornithine

• ****NO OROTIC ACID: no Carbamyl Phospate to convert into it***

NAGS and CPS1 Deficiency

Called the Pre-Orotic UCD: the rarest UCD

Same clinical presentation as OTC/ASS1/ALS

• Neonatal hyperammonemia encephalopathy

• But with NO OROTIC ACIDURIA

Both Autosomal Recessive Inheritance

Genetic Defects

• N-AceytlGlutamate synthetase (NAGS) gene

• Carbamoyl Phosphate Synthetase 1 (CPS1) Gene

NAGS and CPS1 Deficiency Diagnosis

Newborn screening: neither are identified on NBS

Diagnostic Evalution

• Ammonia level: highest of the UCDs, >1000 at diagnosis, Neonatal Encephalopathy

• Blood gas/metabolic profile/lactic acid level: exclude metabolic acidosis indicating other IEM

• Plasma AA and Urine Organic Acids

  • High Glutamine/Alaine

  • Low Citrulline/ASA/arginine

  • ***NO OROTIC ACID***

Confirmatory testing

• Enzyme activity requires liver biopsy

• gene sequencing + del/dup analysis

NAGS/CPS1 Deficiency Treatment

Acute Encephalopathy treatment

• Rapidly remove ammonia: hemodialysis/hemofiltration

• Reduce production: protein cessation 12-24hours, decrease nitrogenous waste

• Prevent Catabolism: high calorie, protein free - IV glucose + lipids and IVE arginine (become essential AA b/c it cannot be synthesized ‘like normal’)

• Provide other ammonia removal agents: IV ammonia scavengers Sodium-Benzoate + Sodium-Phenylbutyrate

Chronic Treatment: after acute encephalopathy has resolved

• Protein restriction + essential AA formula

• Oral citrulline or arginine and ammonia scavengers (Sodium-Benzoate + Sodium-Phenylbutyrate provide alternative pathways for ammonia excretion)

• Avoid Valproic Acid, fasting, Fever, steroid, protein load

• Liver transplant: only if neurologically intact after EPISODE + poor response to chronic treatment

• ***Carglumic Acid: Synheic N-Acytle gluamate (NAG) for NAGS defeciney****

NAGS/CPS1 Deficiency Outcome

Outcome with treatment variabl

• Often intellectual disability, ADHD, executive defects, brain atrophy

NAGS/CPS1 Deficiency Carrier Screening

Molecular: if mutation known in proband or linkage informative

Prenatal/Preimplantation diagnosis:

• No enzyme or metabolite testing is useful