IEM Boards Conditions (All)

Amino Acid Homeostasis

There has to be a balance of our intake (sources) and output (products) of A.As.

• Sources

  • Dietary protein: intake via intestine provides essential and non essential A. As

  • Tissue Catabolism: periods of fasting or metabolic stress trigger breakdown of tissue; largest protein repository is muscle

  • Synthesis: metabolic pathways for non-essential AAs

• Products

  • Anabolism: incorporation into complex proteins

  • Conversion: into Non-essential or metabolic intermediates (Keto or gluccogenesis)

  • Excretion: in urine through conversion of nitrogenous waste to urea

5 Major Aminoacidopathies

When there are defects in the enzymes of the metabolism of Amino Acids, they result in a group of disorders call Aminoacidopathies: (the most common and well understood of the group)

• Phenylketonuria (PKU)

• Tyrosinemia

• Maples Syrup Urine Disease (MSUD)

• Homocystinuria

• Non-Ketonic Hyperglycinemia (NKH) —> not needed for boards

Phenylketonuria (PKU)

Found in individuals with Phenylpyruivic in their urine; had mental retardation

• Inheritance:

  • Autosomal recessive

  • Incidence: 1/10-25,000 (Higher in turkey, Ireland, Japan, Africa)

• Genetic Basis: Always a mutation in Phenylalanine Hydroxylase (PAH) Deficiency

  • Most PKU are compound heterozygotes with >500 mutant alleles (no one allele has overwhelming frequency)

Metoblic Pathway of PKU

• Conversion of Phenylalanine to Tyrosine via PAH enzyme

  • we get all out Phenylalanine from Diet: we do no syntheses ,it is an ESSENTIAL AMINO ACID

• PAH has a cofactor: Tetrahydrobiopterin, necessary for peak PAH activity

  • Insufficient tetrahydrobiopterin (THBP) can also cause accumulation of Phenylalanine as well as other intermediates in the THBP synthesis (and also the recycling) pathway

• A DEfeicny of TYROSINE

Tetrahydrobiopterin (THBP)

• A cofactor that is important in maximizing the activity of some Amino Acid enzymes

  • Phenylalanine —> Tyrosine (affecting the PAH enzyme)

  • Tyrosine —> L-DOPA {—> Dopamine} (affecting the Tyrosine Hydroxylase enzyme)

  • Tryptophan —> 4-OH-Tyrpothan {—> Serotine} (Affecting the Tryptophan Hydroxylase enzyme)

  • Arginine —> Nitric Oxide (Affecting Nitric Oxide Synthase)

• A defiecny of THBP = A defeicny in all these pathways (Overlap with PKU for high Phenylalaine, but also low Dopamine, Seratoin and Nitoric Oxid)

  

Untreated Classic PKU

• At Birth: asymptomatic; moms enzyme activity avoids accumulation of phenylalanine in the fetus

• Neonatal period: take-in of phenylamine via diet; accumulation begins; demonstrate symptoms

  • No fatal neonatal encephalopathy (unlike other metabolic disorders)

  • Fair skin and hair pigmentation: PHE inhibition of tyrosine hydroxylase = Melanin deficiency

  • Urine w musty order: Phenylpyruvic acid in urine

• Infancy onward: intellectual disability and delays manifest

  • irreversible ID: Severe in 50%, moderate-mild in 50%

  • Autism, microcephaly, behavior issue

  • Large % with epilepsy, eczema

Diagnosing PKU

• Newborn Screening: Tandem Mass Spectrometry on heel-prick newborn blood sample, give PHE levels

  • Normal PHE: <120 umol/L (<6 mg/dL)

  • Classic KU >1,200 umol/L (>20 mg/dL)

  • Hyperpheylalaniemia 120-1200 umol/L (2-20 mgdL)

• False negative (<1%): sample collection to early, mishandling or lab error

• Fals positive (frequent): liver disease/immaturity (PAH active mainly in liver), high protein load, sample collection or lab error

• Confirmatory Biochemical testing:

  • Plasma amino acid analysis: we should see Elevated PHE AND elevated PHE/TYR ratio (due to the PHE→TYR BLOCK)

    • (RULE OUT LIVER DIEASE OVERLAP: WE WOULD SEE PHE AND OTHER AAs)

    • THBP testing

• Genotyping

  • PAH gene sequencing: genotype / phenotype correlation incomplete but can predict some treatment response

Treating PKU

• Anyone with PHE >360-600 umol/L ( >6-10 mg/dL)

  • Dietary PHE restriction: Limit dietary protein intake to keep PHE <360umol/L (,6mg/dL) - Classic PKU <1500-250 mg/day PHE intake (not all Hyperpheylalaniemia pts need dietary PHE restriction)

  • Dietary PHE-free protein formulas: PHE-free protein formulas to avoid amino acid deficiency (TYR)

• Outcome with treatment if started by 3 weeks old)

  • Normal IQ

  • Increased risk for subtle Neurocognitive deficits and signs

• Effect of treatment withdrawal (why it is LIFELONG TX)

  • increased ADD/ADHA, depression, anxiety, agoraphobia

  • Progressive demyelination on MRI

  • Decreased IQ scores (5-30 points)

Maternal PKU Embryopathy

• Elevated maternal PHE highly teratogenic (Females with PKU need even MORE dietary control)

  • PHE level in developing fetus is 2x maternal level: (75-90% teratogenicity vs 50% with thalidomide)

  • Goal: Maternal PHE levels <360 umol/l (<6 mg/dL)

• Higher PHE levels correlate with risk for

  • Microcephaly, Mental + Growth retardation, Congenital heart defects

• Offer prenatal ultrasound and echocardiography to look for these issues

Newer PKU Treatments

• Sapropterin Powder

• Phenylalanine Ammonium Lyase SQ injection

• Large Neutral Amino Acids Tablets/GMP

PKU Carrier Detection

If mutations are detected in parents

• Molecular testing of PAH gene in affected individual can facilatate carrier testing: if no mutation identifiable linkage analysis (dup/del anayslsi needed)

• Enzyme analysis not practical as PAH is essentially only exressed in liver (need a liver biopsy which is NOT typically done)

Prenatal implantation diagnosis: Available when molecular testing informative

Tyrosinemia Type I

Also called “Hepatorenal” Tyrosinemia due to progressive liver and kidney failure if untreated : type II and III dont present the same way

• Inheritance: Autosomal Recessive

• Incidence: 1/120,000 (higher in Quebeec)

Genetic defect in: Fumarylacetoacetate Hydrolase (FAH) Deficney

Tyrosinemia Type I Metabolic Pathway

• Phenylalanine → Tyrosine via Phenylalanine Hydroxylase (PAH, PKU remember?)

• Tyrosine → Fumaric Acid + Acetoacetic Acid via 5 step reaction

  • Step 1 Enzyme Block: Tyrosinemia II (Eye irritation, skin changes

  • Step 2 Enzyme Block: Tyrosinemia III (cognitive disability in some but not all)

  • Step 3 Enzyme Bock: Alkaptonuria (Black pee disorder first IBEM identified)

  • Step 4: Fumarylacetoacetic Acid Hydrolase (FAH) Block : Tyrosinemia I

    • High levels of Fumarylacetoaecetic acid accumalte -→ conversion to Succinylacetosectic acid which would normally not accumulate: both Cause TOXICITY AT HIGH LEVELS

Untreated Tyrosinemia I

• At birth: Asymptomatic

• Infancy: Tyrosine, Fumarylacetoaecetic acid (FAA), Succinylacetone (SAT) accumulate

  • Cabbage-like odor in urine

  • Liver dysfunction progression (from FAA)

  • Renal dysfunction progressing to failure

  • Rickets, poor growth

  • Neurologic “crisies”; episodic abdominal +neuropathic pain, weakness, repository failure (from SAT inhibition of porphyrin pathways)

• Adolescence: Death typically by age 10 due to

  • liver failure,

  • respiratory fialure in neurologic crisis,

  • hepatocellular cancer

Diagnosing Tyrosinemia I

Newborn Screening: on heel-prick blood sample

• Elevated Tyrosine and SAT on tandem Mass spect.

  • some false negative: may take time for sufficient elevations to elevations

  • Often false positive: TYR elevation also with liver disease, transient benign or the other forms of tyrosinemia

Confirmatory Biochemical Testing: Plasma amino acid profile

• High TYR in plasma A.As: non-specific to Type I

• High SAT in blood or urine organic acids: Specific to Type I

• Rarely skin fibroblast FAH enzyme activity: PBG in blood (Quebec)

Genotyping

• FAH gene sequencing with del/dup analysis (>95%): cancer be targeted in some populations (French Canadians)

• Genotype: Phenotype correlation no consistent

Treating Tyrosinemia I

Treatment

• Dietary restriction of PHE and TYR

• Dietary supplementation: PHE and TYR-free protein formulas

• Medications : Orfadine - inhibits enzyme prior to FAH (p-HPPD) reducing FAA and SAT

• Liver Transplantation: reserved for liver failure or hepatocellular carcinoma despite treatment

Outcome with treatment:

• with NBS: 90% survival into adulthood with normal growth and function

Screening for Tyrosinemia I

• Carrier Screening

  • Moelcualr tesing for FAH gene if mutation idefineted

  • Linkage analysis if mutation unidentifiable

• Prenatal/Preimplantation diaognsis

  • availbe if moclaur testing infromative

  • if molecualar testing uninformative: SAT acumination can be Measured in the Amniotic fluid when molecular basis in family unknown but not 100% accurate

Branch Chain Keto-Acid Dehydrogenase Deficiency (Maple Syrup Urine Disease)

Urine smells like Maple Syrup / Sweet

• Inheritance Recessive

• Incidence 1:185,000 (increased in Mennonite pop.)

Genetic Basis

• Mutations in genes coding Branch Chain Keto-Acid Dehydrogenase complex subunits: multiple subunits, multiple genes that can cause MSUD)

  • 4 subunits in BCKD complex: E1a, E1b, E2, E3

  • 3 MSUD genes:

    • BCKDHA (E1a)

    • BCKDHB (E1b)

    • DBT (E2)

    • ***DLD (E3) mutations cause different disease***

Branch Chain Amino Acid (BCAA) Metabolism

The Branch Chain Amino Acids: Leucine, Isoleucine, Valine

• These three are converted via multiple steps to energy intermediates Acctly-CoA and Succinyl-CoA

• First Step is via BCAA: When blocked, accumulation of the three branch chain amino acids + their associated keto-acid forms (which are what BCAA actually works with)

• 

Untreated Classic MSUD

• At Birth Asymptomatic

• Within 12-24 hrs after birth: Maple syrup urine and ear wax (from Isoleucine)

  • Dietary intake OR Catabolism in the newborn

• Within 5 Days

  • progressive irritability and decreased feeding

  • progressive encephalopathy/cerebral edema (from leucine: neurotoxic)

• Within 7-10 days after birth

  • massive cerebral edema, coma respiratory failure

  • death within weeks

• Some milder “intermediate” or “intermittent” forms

  • may only experience “decompensation” during catabolic stress

MSUD unlike PKU and Tyrosaenmia I: No threat of neo-natal death + symptom onset longer vs. rapid onset and death in MSUD

Diagnosing MSUD

Newborn Screening: Heel prick blood tandem Mass spec. analysis

• Elevated Leucine and BCAA ratios

  • rarely false negative: early sample collection (before 24hrs), collection or lab error

  • Occasional false positives: other disorders appear positive via elevated LEU (hydroxyprolinemia)

Confirmatory Biochemical Testing

• Plasma AA analysis:

  • Elevated BCAA’s; Leucine, Isoleucine, Valine

  • Allo-Isoleucine presence (only elevated in MSUD)

• Urine organic acid analysis

  • BCAAs present

• Enzyme activity: BCKD enzyme activity measurable n fibroblasts

Genotyping

• BCKD subunit gene sequencing with del/dup analysis: E3 subunit mutations cause distinct syndrome due to its activity in Mutiple metabolic pathways: genotype/phenotype correlation not consistent

• If successful, allows for prenatal/preimplantation genetic diagnosis

Treating MSUD

Preventing Acute decompensation (rapid buildup of BCAAs)

• Decrease catabolism and enhance elimination of Leucine

  • Maximize BCAA-free calories (avoid catabolism)

    • Intravenous infusion/metabolic formula (Dextrose, lipid and BCCA-free nutrient)

    • Supplemental Isoleucine and Valine solutions (avoid deficiency with prolonged treatment - just the precise amount they need, no more)

  • Thiamine supplementation (BCKD coenzyme, increases activity)

  • Hemodialysis in acute severe illness

• Long-term Management

  • Avoid decompensation: fever, fasting, high protein

  • Optimize BCAA-levels: metabolic formula and sing AAs

  • Thiamin: if thiamine responsive

  • Liver transplant for severe poorly responsive forms

• Outcome

  • Normal (20 points lower): BUT ONLY IN THE FIRST 10 years of LIFE

  • After 10days: Severe, irreversible brain damage and/or death

MSUD Carrier Detection

Carrier Screening: can be done if BCKD genes have been found in affected individuals

• linkage analysis if mutations unidentifiable

Prenatal/Preimplantation diagnosis

• Available when molecular testing informative

• Can obtain BCKD enzyme activity on amniocentesis or CVS

Homocystinuria

Deficiency of Cystathionine Beta-Synthase (CBS)

• Inheritance: Autosomal Recessive

• Incidence 1:3350,000 (higher in Qatar, Norway)

Genetic basis: Mutation in Cystathionine Beta-Synthase gene

Onset typically after 3 years old

• No neonatal encephalopathy

• Many cased missed on NBS

Homocystinuria Metabolic Pathway

• Methionine (taken in via diet) → Homocysteine via multiple steps

• Homocysteine → Cysteine via Cystathionine Beta-Synthase (CBS) + B6 (coenzyme)

• Block of CBS = Elevated Homocysteine and decreased Cysteine levels

  • Homocysteine converted back to Methionine (Methionine synthase + B12 coenzyme)

Untreated Homocystinuria

• Marfanoid Skeletal changes

  • Tall and thin, long arms legs fingers

  • Chest pectus

  • High-arched palate

  • Crowded teeth

• Ectopia Lentis (dislocation of lens)

  • Can be first clinical manifestation (typically 3-5 years): different displacement DOWN (vs UP in Marfan’s)

• Neuropsycatric Disease

• Spontaneous Veinous/Arterial Thromboembolism

  • Up to 25% by age 15: casue stroke, death pulmonary embolism

Diagnosing Homocystinuria

Newborn screening: via tandem Mass Spect.

• Elevated Methionine levels

  • false negative: accumulation of MET not high in the first few days

  • false positives: multiple conditions cause elevated MET: liver disease, hypermethioninemia (MET elevated, but not homocysteine)

Biochemical Confirmation

• Plasma AA analysis

  • elevated homocysteine + MET

  • low/absent Cysteine

• Urine: elevated homocysteine (NOT AS SPECIFIC as plasma AA)

• CBS enzyme activity: Skin fibroblasts and hepatocytes

Genetic Confirmation

• CBS gene sequencing and dup/del analysis

• Many mutant alleles, mostly compound heterozygotes

Homocystinuria Treatment

Diet

• Restrict methionine and protein

• MET-free metabolic formula

Medication

• Vitamin B6 (also called Pyridoxine)

  • BBS cofactor challenge (50% B6-responive)

  • Supplement folate and B12 (allow for conversion of homocysteine back to methionine)

• Betaine

  • offers alternative route for homocysteine remethylation: a methyl group donor which lowers homocysteine levels by remethylating homocysteine to methionine

  • used in patients not dramatically B6-repsonive

Outcome

• near normal IQ if treated early and effectively

• Marfan-like complicaitons significantly reduced

Homocystinuria Carrier Detection

Carrier Testing

• If mutation found in affect Individuals

• Linkage analysis if mutation not found on affected individuals

Prenatal/Preimplantation Diagnosis

• CBS enzyme activity assay in amniocytes (not possible on CVS)

• Amniotic fluid homocysteine levels