FINAL Biochem Boards Superset

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

Mitochondria

• Site of oxidation phosphorylation: via the electron transport chain embedded in the inner mito membrane

  • Produce ATP

• The other biochemical processes occur in the Mito:

  • Pyruvate oxidation

  • Krebs Cycle

  • Fatty Acid Beta-Oxidation

Oxidative Phosphorylation

The electron transport chain

• Inner membrane has 5 distinct protein complexes embedded: some encoded by nuclear DNA, some encoded by Mito DNA

• Use NADH + FADH coming form Krebs cycle break down of Actyl-CoA

  • Fatty Acids (generated via F.A. B-Oxidation)

  • Pyruvate (generated via glycolysis)

• Electron transport down the chain of complexes: creates gradient by pumping IN H+ ions

• Complex V uses gradient to Generate ATP as H+ ions move OUT

Why does Mitoondiral diase present with LACTIC ACID and MITO PROLIFERATION?

Lactic Acid

• Glycolysis (break down of 1 glucose molecule) produces: 2 ATP + 2 Pyruvate + NADH

  • NADH: shuttles (H+) into the ETC → LOTS OF ATP MADE → NAD- is produced, ready to ‘shuttle’ a new (H+)

  • Pyruvate: gets converted to Acetyl-CoA → A-CoA gets used in the KREBS CYCLE to make more NADH

  • ATP: just a small amount

• When ETC is not working right due to MITO DIEASE: NADH cannot release it’s (H+), no NAD- is ‘renewed’

  • Pyruvate must react with NADH instead → NAD- + Lactate is produced

  • NAD can be used in glycolysis to make small amount of ATP

  • Lactate builds up → LACTIC ACIDOSIS

Mitochondrial Disorders

Oxidative Phosphorylation/Electron Transport chain dysfunction

Two varieties:

• Secondary Mitochondrial dysfunction: Non-genetic conditions

  • Hypoxemia (inadequate Oxygen for Oxidative Phosphorylation)

  • Medication: valproic acid, HIV meds

  • Toxins: cyanide, rotenone

• Primary Mitochondrial Disease

  • mitochondrial DNA itself or nuclear DNA mutations

General Features of Mitochondrial diease

Mitochondrial disease preferentially affects tissues with the HIGHEST energy demands →

• BRAIN: seizures, ataxia, stroke-like episodes

• MUSCLE: myopathy, hypotonia

• HEART: cardiomyopathy, conduction defects

• EYES: ***PTOSIS*** eye muscle weakness, optic atrophy

• EARS: Hearing loss

Mitochondrial Genome

The Mitochondrial Chromosome: encodes 37 genes

• only 3% of Mito. proteins are encoded by mito DNA

• 97% are encoded by nuclear DNA and imported into mitochondria

Complex 1

46 total proteins

MtDNA encoded: 7

nuDNA: 39

• Leigh Syndrome

• Leukodystrophy

Complex 2

4 proteins: ALL nuDNA ENCDOED

• Leigh Syndrome

• Paraganglioma

• Pheochromocytoma

Complex 3

11 proteins

MtDNA: 1

nuDNA: 10

• Leigh syndrome

• GRACILE syndrome

Complex 4

mtDNa: 3

nuDNA: 10

• Leigh Syndrome

• Hepatopathy

• Cardioencephalomyopathy

• Leukodystrophy/tubulopathy

Complex V

mtDNA: 2

nuDNA: 14

Maternal Inheritance

mtDNA mutations can only be inherited through the mother

• all mito provided by the ovum

• no mito contriubted by the sperm

Heteroplasmy

Mito genomes can differ between mitochondria in a given cell and % of mutant mtDNA can vary in an individual from cell-to-cell and tissue-to-tissue

• Each cell has up to 1000 mitochondria, each with their own copy of the mito genome

• mtDNA mutation rate is 10-20x nuclear DNA mutation rate

Threshold Effect.

• energy requirements vary between tissues

• mtDNA mutation burden varies tissue to tissue (heteroplasmy)

• Tissue specific % mutant mtDNA threshold for disease

• Phenotypic variability results

Example: Brain and Muscle have a lower threshold than Skin and Kidney

Mitochondrial Disorders: Presentation

Can present in almost any way and vary from person to person, but 3 general categories

• “Classic” Mitochondrial diseases: reproducible, multi-organ pattern

• Unexplained multi-organ dysfunction:

  • Hearing loss short stature

  • Diabetes + hypertrophic cardio myopathy

  • ophthalmoplegia +ptosis

• Unexplained single organ syndrome: just hearing loss, epilepsy, GI

Often elevated Lactic acid in Blood or CNA and Mitochondrial proliferation in muscle

Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke like episodes (MELAS)

• Age of Onset: before 40yo (average 5-15)

• Clinical

  • Stroke like episodes + Epilepsy, Dementia

  • Muscle weakness (myopathy), Cardiomyopathy, Lactic Acidosis

  • Hearing-Loss, Retinopathy, Diabetes

• CT/MRI: Infarcts→ but not seen in vasuclar regions: infarct occurs due to region engery insufficney from Mitocondrial

• Etiology: heterogeneous mtDNA mutations (Often mt-t RNA) → VERY dependent on Heteroplasmy with individual

Myoclonic Epilepsy with Ragged Red Fibers (MERRF)

• Adolescent onset

• Clinical manifestations

  • Epilepsy (myoclonic)

  • Muscle weakness (myopathy), Lactic acidosis, Ataxia

  • Encephalopathy, Hearing Loss

• EMG

• EEG:

• Muscle Biopsy: (if done on affected muscle) will show ‘ragged red fibers’ caused by mitochondria proliferation

• Etiology: Single mtDNA-tRNA mutation 80 to 90%

Leber’s Hereditary Optic Neuropathy (LHON)

• Age of onset 20-24 yo

• Clinical:

  • Acute or sub-acute bilateral central vision loss→ Rapid progression to blindness (usually confined to optic nerve)

  • Rarely: heart block, dystonia, MS-like symptoms

• Fundoscopy: early tortuous retinal arteries, followed by optic atrophy

• Etiology: 95% mtDNA “ND” (electron transport subunit) gene mutations MATERNAL INHERITANCE

  • ****4:1 M:F ration → X-linked modifier genes that make females less affected****

Chronic Progressive Ophthalmoplegia (CPEO)

Chronic Progressive Ophthalmoplegia (CPEO)

• External ophthalmoplegia (eye weakness) → can’t look in certain directions

• bilateral ptosis (eyelid drooping)

• mild myopathy (limb weakness)

• Onset ***AFTER*** 20yo (slowly progressive)

Etiology:

• Mainly mtDNA deletions→ can be smaller or larger chunks of mtDNA (smaller =CPEO, larger=KSS)

• Majority are SPONTEOUS

Kearns Sayer Syndrome

<20 years + ophthalmoplegia (ptosis w/ eye-muscle paralysis) + pigmentary retinopathy + HEART BLOCK + Ataxia

• mtDNA Deletions

  • small deletion = CPEO

  • LARGE deletion = KSS

  • Earlier onset than CPEO (before 20yo) and EXTRA systemic mito. disease features (ataxia)

  • *****CARDIAC CONDUCTION DEFECTS → HUGE FEATURE****

• INHERITANCE

  • Single large-scale mtDNA deletion disorders are usually sporadic/de novo rather than showing the classic maternal transmission you associate with many mtDNA point variants.

  • KSS does not usually produce a pedigree full of maternally affected relatives.

Sengers Syndrome

Gene: AGK (dysfunction impairs mitochondrial oxidative phosphorylation and energy production)

• MITOCONDRIAL DIEASE

Inheritance

• Autosomal Recessive

Clinical Features

• Eyes

  • ***CONGENTIAL CATARACTS***

• Cardiac

  • **** Hypertrophic Cardiomyopathy****

• Metabolic

  • Lactic Acidosis

• Muscle

  • Skeletal Muscle Myopathy

[ **Congenital CATARACTS** + **HYPERTROPHIC CARDIOMYOPATHY** + LACTIC ACIDOSIS + skeletal MYOPATHY ]

Subacute Necrotizing Encephalopathy (Leigh Syndrome)

• Large spectrum of 75 genes (mito and nuc. but mostly nuc.) that cause an energy failure in the brain

  • *****USUALLY AUTOSOMAL RECESSIVE****

• 6-12 months onset - death by 3-5 years (25% have later onset or slower forms)

• Clinical: (often abrupt decompensations/regression with infection/fever)

  • Developmental ***REGRESSION***

  • Seizures, Ataxia, Hypotonia, spasticity

  • Ophthalmoplegia, Nystagmus, Optic atrophy

• Diagnostic Testing

  • MRI: ***SYMETRIC LESIONS OF BASAL GANGLIA***

  • Elevated Lactic Acid in blood or Cerebral spinal fluid

  • 10% mtDNA mutation

  • 90% nDNA mutation

• Lower % of mitochondria with the mutant mtDNA→ have NARP instead of Leigh (HETEROPLASMY)

Leigh Etiology

Genetic Heterogeneity

• 10-30% mitochondrial DNA mutations → maternal inheritance

• 90-70% nuclear DNA mutations → Classic Mendelian

  • HETEROPLASMY AFFECT: If a lower # of mito. in a cell have these mutations = Later onset Neuropathy, Ataxia, Retinitis Pigmentosa (NARP)

Pyruvate Dehydrogenase Complex (PDHC) Deficiency: Clinical + Testing

Gene: PDH1

• Failure to convert Pyruvate to Actyl-CoA (via PDH)

• Lactic Acid levels elevated (***PDHC most common cause of Lactic Acidosis***)

Inheritance

• **** X-LINKED****

• Point mutation in NUCLEAR DNA

Clinical Features

Progressive intermittent neurologic deterioration

• hypotonia, seizures, ataxia, ophthalmoplegia, dystonia

• Presents similar to mitochondrial dysfunction

Suggestive Abnormal Tests

• Plasma: increased Lactic Acid + Pyruvate, but normal ratio of Lactic Acid: Pyruvate

  • Distinguished from other Mitochondrial Disease: Pyruvate levels are NOT elevated

• Cerebral Spinal Fluid: increased Lactic Acid

Pyruvate Dehydrogenase Complex (PDHC) Deficiency: Metabolism +Etiology

• Failure to convert Pyruvate to Actyl-CoA (via PDH)

• Lactic Acid levels elevated (PDHC most common cause of Lactic Acidosis)

Etiology: PDHC is a multisubunit complex

• Catalytic components: E1, E2, E3

• Regulatory component: PDH Phosphatase

Confirmation:

• PDHC enzyme activity assay

• Sequencing of

  • E1 → PDHA1 : MOST COMMON , X-Linked (males only)

  • E2 → DLAT, Recessive

Mitodoncrial Diease: Work Up

• Serum levels: increased anion gap + metabolic acidosis

  • Lactic Acid: Pyruvate ratios (>30 Mito. Dis ; <10 PDHC Def.)

• Imaging: brain MRI, Spectroscopy ( LA peaks over brain regions( BasalGang)

  • Basal Ganglia hypodensities: generalized atrphy

  • Hypoplastic corpus callosum if fetal lactic acidosis

• Muscle Biopsy

• Genetic Testing

Mitochondrial Disease: Muscle Biopsy

Allows for:

• Detecting ragged red fibers (mito. proliferation)

• Abnormal mitochondria proliferation

• Detecting enzyme activity of the chain-genes

• Mutational analysis of mitoDNA

Pitfalls:

• need 1 gram of flesh (large amount)

• biopsy of moderately affected muscle

• may not distinguish exact genetic mechanisms

Genetic Testing for Mitochondrial Conditions

mtDNA:

• Leigh Syndrome

• LHON

• MERRF (blood/muscle)

• MELAS (blood/muscle)

• NARP (blood/muscle)

• KSS/CPEO (muscle)

nDNA (all in blood)

• Leigh syndrome

• MNGIE

• Mohr-Tranebjaerg

• Friedreich’s Ataxia

• AR spastic paraparesis

• AD PEO

Mitochondrial Disorders: Treatments

Less evidence for specific treatments that actually improve outcomes

• Trials with Vitamins that optimize Electron Transport chain function:

  • Carnitine

  • Biotin

  • thiamine

  • Riboflavin

• High Fat/ Low Carb diet: low carb→ less glycolysis→ less LA

• Avoid Mito toxic meds

• Reduce LA, control acidosis (dialysis/vent)

Organic Acidemias Background

• Primarily disorders of Amino Acid Catabolism: Mainly

  • Branch Chain Amino Acids (BCAA)

  • Lysine

• Toxicity comes from accumulation of ORGNIC ACIDS not from an A.A. acid accumulating

  • Causes metabolic acidosis with increased “Anion Gap”: Decrease in main anion Bicarbonate (HCO3-)

  • Secondary toxic effects of acidosis

    • Mitochondria→ Lactic acidemia

    • Urea Cycle → Hyperammonemia

    • Bone marrow→ Bone marrow suppression

    • CNS function→ Encephalopathy/Mental retardation

• Major Presentations: Neonatal encephalopathic acidosis, late chronic/intermediate

• All autosomal recessive

Metabolic Acidosis

• Blood pH low due to excess acid (H+) vs Base (HCO3-)

  • normal range pH 7.3-7.45 (measure via Atrial Blood Gas)

  • Normal HCo3- level: 22-26 mEq/L

• Mutiple etiologies for Metabolic Acidosis

  • Lowered HCo3- : loss through GI (diahrria), Renal tubule acidosis, Medications

  • Elevated H+: creation of abnormal acids in blood due to starvation, diabetes; Lactic acidosis due to mitochondrial dysfunction, Organic Acidosis

• Clinical Consequences

  • Neonatal: non-specfic, similar to UCDs presenations,

    • Lethargy, vomting, Tachypena, Hypotonia, Seizures, Coma, Death

  • Adult: Devleopmental Delay, Ataxia, Neurological Deficits, (then the neonatal presenations)

Organic Acidemias

Newborn Screening detects many Organic Acidemias

Organic Acidemias Treatment

• Restrict Dietary Protein disease specific amino acid free formulas

• Prevent Catabolism provide sufficient protein free calories

• Reverse Acidosis ± Hyperammonemia

  • Hemodialysis

  • Ammonia and lactic acid scavengers

    • Sodium bicarbonate, sodium benzoate, phenylbutyrate

• Cofactor therapy for specific Disorders

Propionic Acidemia Metabolism

• Failure of Propionyl-CoA carboxylase

• Step 11 of Isoleucine and Valine metabolism:

• Propionyl-CoA → Methylmalonyl-CoA via Propionyl-CoA carboxylase activity

  • Propinoyl-CoA: the activated mitochondrial form

  • Propionic Acid: free organic acid (interferes with NH3 removal, other stuff)

  • Propinolycarnatine (C3 - what NBS measures): the ‘detox’ / transport form that excess P-CoA gets converted to so it DOESN'T become Propionic Acid

Propionic Acidemia

• Also known as Ketonic hyperglycemia: high level of glycine and ketone bodies

• Autosomal Recessive

• Incidence 1:100,00 (higher in Saudia Arabia and Inuit)

Genetic Defect

• Propinyl-CoA Carboxylase (PCC) alpha or beta subunit genes

  • some genotype/phenotype correlation (null alleles/deletions more severe)

  • BIOTIN cofactor for PCC

• PA accumulation due to PA production from

  • MET/THR/VAL/ISO catabolism,

  • gut bacteria,

  • odd chain FAs

Untreated Propionic Acidemia

Classical Neonatal Encephalopathic Form

• Normal at birth

• Within a few days

  • Poor feeding, lethargy, vomiting hypotonia →encephalopathy, seizures, coma, death

Late-Onset Form

• Developmental delays/regression

• cyclic vomiting

• protein intolerance

• growth impairment

• hypotonia

• metabolic basal ganglia stroke

• cardiomyopathy

• Acute episode of toxic encephalopathy

Rare Cardiac Subtype isolated cardiomyopathy

Diagnosing Propionic Acidosis (PA)

Newborn Screening

• Elevated Propionyl Acylcarnitine (C3) and ratio to other carnitine species (Propionyl-CoA gets combined with Carnatine to try buffer high Prop-CoA levels)

  • other etiologies: Methylmalonic Acidemia, Cobalamin Defects, Maternal B12 Deficiency, False +

Confirmatory Testing

• Atrial Blood Gas: Elevated ammonia, low glucose, high acidosis, increased anion gap

• Complete blood count: suppression of bone marrow→ less blood cells

• Urine Organic Acid Analysis: High 3-OH-proprionate, mthylcitrate, tigly/proprionylglycine but NOT MMA

• Plasma Amino Acid profile:

  • elevated glycine + glutamine, not homocysteine (seen with Cobalamin defects)

• Acyl-Carnitine Profile: Elevated C3 acylcarnitine, not C4-DC unless SUCLA2 deficiency

• PCC enzyme activity: can measure PC enzyme in leukocytes or fibroblasts

PCC Genotyping

• Gene sequencing w del/dup analysis (99% detection rate)

Treating Propionic Acidosis

Acute Acidotic Encephalopathy

• Remove acids and ammonia hemodialysis

  • severe hyperammonia: ammonia scavengers

• Reduce PA production Protein restriction 24-25hr

• Prevent catabolism: glucose and lipids IV

• Enhance PA excretion: IV Carnitine

• Decreased PA production in Gut: Antibiotics (Metronidzole

• Biotin:

Chronic Treatment

• protein restriction and MTVI-free metabolic formula

• Oral Carantine, Biotin, and Antibiotics

• Avoid decompensation

• unresponsive to Tx → liver transplantation

Propionic Acidemia Deficiency Outcome

Treamtnet improves surivial, but invariable there is an affect to some degree

• Neurodevleopmatl disabilty

• metabolic basal ganglia stroke

• seiures

• pancreatisis

• cardiomyopathy

• gorwth impairment

• nuetorpnia, AA defience

• renal failure

• premature ovarian fialure

• hearing and vidual defecits (optic nerve atrphy)

Propionic Acidemia Deficiency Screening

• Carrier Screening:

• PRenatal Diaongis:

  • amontic fluid orgnaic acid measurment possible (some false negatives)

Methylmalonic Acidemia Pathway

• Isoleucine and Valine

• Methlymalonyl-CoA → Succinyl CoA via Methylmalonic-CoA mutase activity

• Methlymalonyl-CoA accumulates

Methylmalonic Acidemia

• Increased Methylmalonic Acid but not homocysteine (other forms of MMA have elevated homocysteine→ not primary MMA, but related to Adenosyl Cobalamin - A )

Genetic Defect: mutation of multiple genes cause similar phenotype

• 60% Methlymalonyl-Co mutase gene mutation (MUT)

• 37% Cobalamin A,B,D2 (MMAA, MMAB, MMADHC)→ the upstream vitamins that will be converted into Adenosyl Cobalamin→ leads to dysfunctional MM-Co mutase

Untreated Methylmolaynic Acdiemia

Infantile Subtype: Most common mut0, cblB mutations

• Normal at Birth

• Within days to weeks: poor feeding, lethargy, vomiting, hypotonia, encephalopathy→ progress to seizures, coma, death

Intermediate phenotype: mut-, cblA, cblD2

• Normal for month to years: fialure to thrive, devleopmental delay, hypotonia, poriten aversion→ risk of carastrophic decompensations

Benign Adult form: typically asymptomatic, can decomapnste

Diagnosing Methylmolaynic Acdiemia

Newborn screening: Elevated Propinoyl Acylcarnitine (and ratios) → but non specific

Confirmatory testing

• Atrial Blood Gas, Ammonia Levels, Completel blood ocunt:

  • Hi AG metabolic acidsosi

  • Elevated ammonia

  • Low gluclose

  • pancytopenia

• Urine Organic Acid: High MMA

• Plasma Amino Acid profile: high glycine + glutamine, no Homocystine (Hcy)

  • CblC/D/F - Hcf + MMA high ;

  • cblD2/E/G - Just hcf High

• Enzyme activity: fibroblasts

Genotyping on genes = 95%

Treating Methylmolonic Acidemia

Treat acute acidotic encephalopathy

• Remove acids+amonia: hemodialysis

• Reduce MMA production: protein restriction

• Prevent catabolism: IV glucose and lipids

• Severe hyperammonemia: Amonia scavengers

• Decrease gut bacteria: Antibotics

• HYDOXYCOBALAMIN (B12) injects: cofactor

Chronic Treatment

• protien restriciton and MTVI-free meatolibc fomumal

• L-Carnitine + OH-B12

• Avoid decompensation

Methylmalonic Acidemia Treatment outcome

• Most patient will have some degree of mental impairment, long term affects

Methylmalonic Acidemia Diagnosis

• Prenatal/Preimplantation:

  • Ammonitic organic acid fluid analysis possible

  • Enzyme activity of CVS and amniocentesis

Iso-valeric Acidemia

• issues with the Isovaleryl-CoA dehygroenase enzyme

• LEUCINE PATHWAY ONLY

• Build up of Isovalryl-CoA (Isovaleric Acid)

• Disorder of Leucine metabolism only (unlike PA or MMA)

Genetic Defect

• IsoValeryl-CoA Dehydrogenase (IVD) Gene Mutation

  • results in increased Isovaleric Acid

  • Sweaty feet odoer is prominent

Untreated Isovaleric Acidemia

Severe Neonatal Onset form

• Normal at birth

• Within day: poor feeding, lethargy, hypotonia, Sweaty feet order → encephalopathy, seizure, coma, death

Mid/Late Onset Form

• unexplained failure to thrive and developmental delay

Benign Adult Form: Typically, asymptomatic but can mildly decompensate

Diagnosing Isovaleric Acidemia

Newborn screening: elevated Isovalerylcarnitine (C5 acylcarnitine) - used to ‘buffer’ Isovaleric Acid that builds up when Acylvaleryl-CoA builds up

Confirmatory testing

• Blood tests:

  • High ammonia

  • Low glucose

  • High metabolic acidosis

• Urine organic acid

  • High IVA

  • High isovaleryl glycine

• Plasma AA levels:

  • High glycine

  • High glutamine

• Enzyme activity: Fibroblast

Genotyping: exact genes unknown

Treating Isovaleric Acidemia

Treat Acute Acidotic encephalopathy

• Remove acids and ammonia: hemodialysis

• Reduce IVA production: protein restriction 24-26hrs

• Prevent Catabolism: IV glucose and lipids

• Enhance IVA excretion: IV carnitine

• If hyper ammonia: ammonia scavengers

• GLYCINE SUPPLMENTAITON-BINDS IVA

Chronic Treatment

• Protein rection and LEUCINE-free metabolic formula

• Oral L-Carnitine and L-Glycine

• Avoid decompensation

Isovaleric Acdiemia Otucome

• Outlook with Treatment is one of the best if treatment done early and effecetively enough

  • can be comepltely asymptomatic as long condition is monitored

  • Leucine tolerance gets better with age

• Even if diaognsis is after neonatal period, and evne with major encaplapthic event in neonatal period—> longer term out look is vairable : CAN BE OK

Isovaleric Acidemia Prenatal Diagnosis

• Ammniotic fluid can be checked for organic acids

Biotinidase Deficiency Pathway

• Biotin is a vital cofactor for of number of different enzymes:

  • ALL ARE CARBOXYLASES

• 3-Methylcrontoyl-CoA carboxylase (Leucine)

• Propinoyl-CoA Carboxylase (Isoleucine and Valine)

• Malonyl-CoA decarboxylase

When there are mutations in the BIOTINADASE gene: Biotin is not properly recycled → these blocks develop

Biotinidase Deficiency (BTD)

• Late multiple Carboxylase Deficiency

• Slightly increased incidence in Hispanic and Middle Easter

Gene Defect: Biotinidase (BTD) gene

• failure to recycle biotin = biotin deficiney

• Biotin co-factor for the carboxylases: cannot combine and make function enzyme

Untreated Biotinidase Deficiency

Affect depends on the residual enzymatic activity when biotin absent

Profound Deficiency (<10% enzyme)

Clinical Features

No acute acidodic encepahlopathy decompensatio events : GRADUAL

• Normal at birth

• Symptoms develop after few months”

  • Developmental delay, seizures, hypotonia, ataxia

  • hearing loss, visual problems,

  • ***alopecia***, ***eczema*** (unique to BTD)

Partial Deficiency (10-30% enzyme)

• intermittent symptoms with stress

Symptoms can be irreversible once present

Diagnosing Biotinadase

Newborn Screening: Elevated C5-OH Acylcarnitine, but not specfic to BTD

Confirmatory Testing

• Blood:

  • High ammonia

  • High acidosis

  • Low gluclose

• Urine Organic Acids: multiple organic acids b/c Bitonaisde affects multiple enzymes → referred to as ‘Multiple Carboxylase Defeicieny’ (MCD) on uOA

  • ( )

• Elevated Hydroxy-Isovalyrl-carnatine (C5-OH)

• Enzyme activity: IMPORTANT STEP: blood sample

  • If Biotinadase activity is normal→ then issue is probably Holocarboxylase Deficiency (presents the same way but is earlier)

Genotyping: sequencing 99% detection

Treating Biotinadase Deficeincy

Rarely severyl acidotic or hyperammonemic

• may occasionally need sodium bi-cabonate (adress acidty)

• may occasionally need amonia scavnerge (adress amonia levels)

• Insitute Biotin therapy immediately

Chronic treatment

• Biotin

• No protien restction

• Avoid raw egg whites (has protein that binds Biotin)

Biotinadase Deficiency outcome

• Extremely great outlook for patients (one of the best for Organic Acidemias)

• As long as treatment is implemented BEFORE the development of severe symptoms

• If detected after symptoms, some are irreversible: optic atrophy, hearing loss, developmental delay can presist

Biotinadase Deficiency Prenatal diagonsis

• Biotinadase enzyme activity can also be measuredin the amniocytes and the amniotic fluid

Glutaric Acidemia Type 1 (GA1) Metabolism

• NOT a Branch Chain Amino Acid metabolism disorder

• Breakdown of LYSINE and TRYPTOPHANE

• Lysine + Tryptophane → Alpha ketoadipic→ Glutyrl-Coa

• Glutyrl-Coa→ Glutaconyl-CoA (shunt to Glutaontic Acid) via Glutaryl-CoA Dehydrogenase activity

Glutaric Acidemia Type 1 (GA1)

• “Cerebral” Organic Acidemia: Often normal

Genetic Defect: Glutaryl-CoA Dehydrogenase (GCDH) gene mutation causing defective Lysine + Tyrptohan metabolism

Glutaric Acidemia Type 1 (GA1) Symptoms

• Often normal at birth or only macrocephalic

• symptoms often begin prior to 2 years of age

  • May start with a Sudden neurologic decompensation: 75% by 14months → fever, illness, metabolic stress

Primary symptoms

• Progressive Macrocephaly

• Acute encephalopathic crisis

  • Hypotonia,

  • Loss of motor skills,

  • Feeding difficulty,

  • SOMETIMES Seizure

• ***Movement Disorder****

  • speciosity

  • dystonia (involuntary movements)

Glutaric Acidemia Type 1 (GA1) Diagnosis

Newboarn screening: Elevated C5-DC (glutaryl) Acylcarnitine

• many False negatives

Confirmatory Testing

• Blood: elvated ammonia, low gluclose, Aciditiy

• Plasma + Urine: C5-DC glutaryl acylcarnitine + glutaric acid

• Enzyme activity: fibroblast

• CT/MRI: Cerberallar atrophy, basal ganlia infact and hemorrhage

Genotyping

Glutaric Acidemia Type 1 (GA1) Treatment

Reverse/Prevent Catabolism When sick: protien free calroeis during metaoblic stress

Dietary Mdofication

• Low LYSINE and TRYOPTHAN

MEdicaitons

• B2 (Riboflavin) is a COFATOR

• Carntine: Binds Glutaric acid and remvoes it

Avoid Valproate (Bind Carnitine)

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