metabolic disorders of proteins

Clinical Biochemistry Metabolic Disorders of Proteins

By: Amir Nader Emami Razavi

April 10, 2023 Total slide. 132 2


What is a metabolic disease?

“Inborn errors of metabolism”

inborn error : an inherited (i.e. genetic) disorder

metabolism : chemical or physical changes in a biological system




Garrod’s hypothesis

product deficiency

substrate excess

toxic metabolite

A

D

B C




Small molecule diseaseCarbohydrate

Protein

Lipid

Nucleic Acids

Organelle diseaseLysosomes

Mitochondria

Peroxisomes

Cytoplasm



How do metabolic diseases present in the neonate ??

Acute life threatening illnessencephalopathy - lethargy, irritability, comavomitingrespiratory distress

Seizures, HypertoniaHepatomegaly (enlarged liver)Hepatic dysfunction / jaundiceOdour, Dysmorphism, FTT (failure to thrive), Hiccoughs



How do you recognize a metabolic disorder ??

Index of suspicioneg “with any full-term infant who has no antecedent maternal fever or PROM (premature rupture of the membranes) and who is sick enough to warrant a blood culture, one should proceed with a few simple lab tests.

Simple laboratory testsGlucose, Electrolytes, Gas, Ketones, BUN (blood urea nitrogen), Creatinine

Lactate, Ammonia, Bilirubin

Amino acids, Organic acids



Inborn Errors of Metabolism

An inherited enzyme deficiency leading to the disruption of normal bodily metabolism

Accumulation of a toxic substrate (compound acted upon by an enzyme in a chemical reaction)

Impaired formation of a product normally produced by the deficient enzyme



Three Types

Type 1: Silent Disorders

Type 2: Acute Metabolic Crises

Type 3: Neurological Deterioration



Type 1: Silent Disorders

Do not manifest life-threatening crises

Untreated could lead to brain damage and developmental disabilities

Example: PKU (Phenylketonuria)



Type 2: Acute Metabolic Crisis

Life threatening in infancy

Children are protected in utero by maternal circulation which provide missing product or remove toxic substance

Example OTC (Urea Cycle Disorders)



Type 3: Progressive Neurological Deterioration

Examples: Tay Sachs disease

Gaucher disease

Metachromatic leukodystrophy

DNA analysis show: mutations



Genetic Basis of

Inherited Disorders

Point mutations,

Insertions, Deletions,

Missense Mutations

and Rearrangements



Generalities of Inherited Disorders

Although each individual IEM is rare, cumulatively they occur ~ 1:5000 live births

Majority of IEM follow an autosomal recessive mode of inheritance



Inborn Errors of Metabolism

Uneventful delivery

Normal birth weight

Non-dysmorphic (no physical findings)

Uneventful days /weeks


Defective Proteins and Disease Defects in Carbohydrate Metabolism Defects in Cholesterol and Lipoprotein

Metabolism Mucopolysaccharide and Glycolipid Disorders Defects in Amino and Organic Acid Metabolism Porphyrias and Bilirubinemias Errors in Fatty Acid Metabolism Defects in Nucleotide Metabolism Disorders in Metal Metabolism and Transport Defects in Peroxisomes Diseases Associated with Defective DNA Repair



Defective Proteins and Disease

Oxygen carrying proteins

Connective tisue proteins

Clotting factors



Diseases Associated with Oxygen Carrying Proteins

Sikle-Cell Anemia

B-Talassemia

A-Talassemia



Diseases Associated with Connective Tissue Proteins

Ehlers-Danlos Type I- Type VIII Ehlers-Danlos with Platelet DysfunctionMarfan's Syndrome Cutis Laxa Occipital Horn Syndrome Cutis Laxa, X-linked Osteogenesis Imperfecta Type I Osteogenesis Imperfecta Type I-C Osteogenesis Imperfecta Silent Type II/III Osteogenesis Imperfecta Type IV Osteogenesis Imperfecta Neonatal Lethal form Osteogenesis Imperfecta progressively deforming



Diseases Associated with Clotting Factor Dysfunction

Afibrinogenemia complete loss of fibrinogen, Factor I Dysfibrinogenemia dysfunctional fibrinogen, Factor I Factor II Disorders Factor III (tissue factor) is the only coagulation factor for which a congenital defect has not been

identified Factor V Deficiency Labile Factor deficiency Factor VII Deficiency Hemophilia A Factor VIII deficiency Hemophilia B Factor IX deficiency Factor X Deficiency Factor XI Deficiency Rosenthal Syndrome, Plasma Thromboplastin Antecedent (PTA) deficiency Factor XII Deficiency Hageman factor deficiency Factor XIII Deficiency Factor V & VIII Combined Deficiency Factor VIII & IX combined Deficiency Factor IX & XI Combined Deficiency Protein C Deficiency Protein S Deficiency Thrombophilia Antithrombin III deficiency Giant Platelet Syndrome platelet glycoprotein Ib deficiency von Willebrand Disease Fletcher Factor Deficiency Prekallikrein deficiency



Defects in Amino Acid Metabolism

Phenylketonuria Type I Tyrosinemia - Tyrosinosis Type II Tyrosinnemia - Richner-Hanhart Syndrome Type III Tyrosinemia Alcaptonuria Homocystinuria Histidinemia Maple Syrup Urine Disease, MSUD

MSUD Type Ib MSUD Type II

Methylmalonic Aciduria Non-ketonic Hyperglycinemia Type I (NKHI)Hyperlysinemia



Syndrome 1Syndrome 1



Case 1Patrick

Birth History: Full Term, 3,620 gm

Uncomplicated Pregnancy, Labor & Delivery

Mother 24 yr old, healthy

No Prenatal exposure to alcohol, drugs, infection, known teratogens

Discharged home on day of life 2



Case 1 (CONTINUED)

Developmental Hx

Rolled over – 3 months

Social smile - 4 m

Stand alone – 14 m

First word – 18 m

Phrases – not yet

Walk alone – 2 yr

Seizure HistoryFirst – 11 mGeneralized, tonic/clonicTotal – 4 seizuresMRI – decreased grey/white differentiation and cortical atrophy



Physical Exam

Growth

Blond hair, blue eyes

Non-dysmorphic child

Neurological exam:Decreased tone, brisk reflexes

Case 1 (Cont)



PatrickNormal

• Abnormal high intensity signal in deep white matter• Leucodystrophy and Cortical atrophy



Case 2

Jeremy newborn male

Full Term: 3,100 gm

Uncomplicated P,L & D

No perinatal infection, no alcohol, no drugs, no known teratogens

Mother - 19 yr old

First Pregnancy

Father -18 yr old

Healthy



Case 2Physical Exam and Labs

Ht & Wt = 70% General exam normal

HC< 5% Neurological exam - normal

Urine Ferric Chloride (FeCl3) is positive



Case 2

Jeremy is now 13 years old and exhibits

Persistent microcephaly

Spasticity

Mental retardation

Coarctation of Aorta



Patient and his Brother•Self selects diet

•low in meat, eggs, cheese•enriched in fruits / vegetables

•Similar pigmentation to his brother

Case 3

Luis (8yo) referred to Developmental Pediatrics clinic

Chief Complaint: Hyperactivity and Learning Disabilities



Case 4

Hannah: 6 month old female

Diagnosed with metabolic disorder on abnormal newborn metabolic screen

Normal growth / development

Normal physical exam

On treatment with metabolic formula



All four cases

Examples of hyperphenylalanemia

Defects in metabolism of phenylalanine

Prototype – PKUElevation of PHE > 20 mg/dl

Normal < 2 mg/dl



PKUClinical Findings

Mousy or musty odorExzemaFair coloring (decreased hair and skin

pigmentation)Behavior Problems

Mental RetardationLose ~ 1 IQ point per week of non-treatment



Phenylalanine Metabolism

Phenylalanine

Essential AA

Major interconversions through tyrosine

PHE

TYR

Body Protein

Melanin

DOPA

NE / EPI

Food Catabolism

50%



Conditionally Essential AA



Essential Amino Acids

Histidine

Isoleucine

Leucine

Lysine

Methionine (and/or cysteine)

Phenylalanine (and/or tyrosine)

Threonine

Tryptophan

Valine



Phenyl lactatePhenylacetatePhenylethylaminePhenylacetyl glutamine

Alternate Disposal Urine

Mousy or mustyodor



PKU

Autosomal Recessive disorder caused by mutation in PAH gene

Newborn screening started in 1963

Incidence: 1 in 15,000

Subtypes and heterogeneity Classic

Moderate and mild

Non-classical or non-PKU hyperphenylalaninemia



PKU

Autosomal Recessive disorder caused by mutation in PAH gene




Moderate and mild

Non-classical or non-PKU hyperphenylalaninemia

% enzyme activity determines clinical severity



PKUAutosomal Recessive disorder caused by mutation in PAH gene




Mild

Hyperphenylalaninemia

% enzyme activity determines clinical severity

(tolerate < 250mg phe/day)

(tolerate 400-600mg phe/day)

(normal diet)



PKUAutosomal Recessive disorder caused by mutation in PAH gene




Moderate

Mild

Hyperphe

Tetrahydrobiopterin (BH4) responsive Hyperphenylalaninemia

• Urine pterins• blood dihydropteridine reductase



BH4 RespondersPAH mutation

62% catalytic

21% regulatory

Allelic pattern1 mild + 1 severe

2 mild

2 severe (rare)

Diet – BH4

without protein restriction



Biological Effects

HyperPhe inhibits transport of large, neutral AA into the brain (as does Leucine)

Inhibition of protein and neurotransmitters

Deficiencies of dopamine, serotonin



Major Neuropathologic changes

1. Hypomyelination (Phe-sensitive oligodendrocytes)

2. White matter degeneration (leucodystrophy)

3. Developmental delay/arrest cerebral cortex

Microcephaly Mental retardation Seizures



Non-Neuro pathology

Hypomelanosis – Why ?



Non-Neuro pathology

HypomelanosisBlond hair, blue eyes, pale

Deficient Tyrosine production (precursor of Melanin)

CardiacCoarctation of the Aorta



Maternal PKU syndrome

First mentioned in literature in 1937

First mentioned as a complication of PKU in 1956

Women with MR and PKU has 3 children, all retarded despite not having PKU

Microcephaly and cardiac defects reported in 1960’s

1983 – MPKUCS begun



Maternal PKU Collaborative Study

Untreated women92% risk of mental retardation

73% risk of microcephaly

40% risk of low birth weight

12% risk of congenital heart diseaseReduced risk if maternal plasma phe levels are normalized pre-conceptually



Maternal PKU syndrome

Dose-Response Relationship Goal: Phe level between 2-6 mg/dl by 8 weeks



The longer it

takes to get Phe

level < 8 mg/dl

the lower the IQ

of the baby



Balancing Metabolic Control

Exposure to normal

PHE intake

Elimination of PHE

from the diet




Exposure to normal PHE intake:

Elevations of PHE

Elevations of PHE-ketones

Deficient TYR, DOPA, NE, EPI

Mental retardation / seizures




Exposure to normal PHE intake:

Elevations of PHE

Elevations of PHE-ketones


Mental retardation / seizures

= Bad




Elimination of PHE from the diet:

Decreases PHE

Decreases PHE-ketones


DEATH from essential AA deficiency




Elimination of PHE from the diet:

Decreases PHE

Decreases PHE-ketones


DEATH from essential AA deficiency

Bad =



Optimal Therapy of PKU

Initiate treatment by 7 days of lifePhenylalanine levelsAge Level Freq of Testing

0-12 months 2-6 mg/dl 1x/week1-12 years Same 2x/month> 12 years 2-15 mg/dl 1x/month

Pregnancy 2-6 mg/dl* 2x/week* 3m before conception



summery

HyperphenylalanemiaAn abnormal lab finding Several defects may result in hyperphe

Specific Dx is criticalPHE restriction in

BH4 deficiency is lethal

Treatment isEffective if begun early and continued for life

Aggressive management during growth and during illness



What about our cases??

Patrick – case 1 Dx ?

3 yr old with developmental delay and seizures…..

Jeremy – case 2

Dx ?

Newborn with microcephaly and + FeCl3

Now mentally retarded

Choices1. Classic PKU – treated or untreated2. Maternal PKU3. Hyperphe



What about our cases??

Patrick – case 1

Classic PKU (mod)

3 yr old with developmental delay and seizures…..

Patrick has permanent disabilities

Jeremy – case 2 Maternal PKU syndrome

Newborn with microcephaly and + FeCl3

Now mentally retardedHe is metabolically normal… but his mother had PKUHis mother wants more children but is not on diet



Our CasesLuis - Case 3

Dx ?

8yo with learning disability and hyperactivity

Hannah - Case 4

Dx ?

6 month old

Normal growth and development

Choices1. Classic PKU – treated or untreated2. Maternal PKU3. Hyperphe



Our CasesLuis - Case 3

Classic PKU (Mexico)

On treatment

His hyperactivity has improved

He will not regain normal intellect

Hannah - Case 4

Classic PKU, treated

Continues to do well on therapy

Growth, development and intellectual situation are normal



Syndrome 2Syndrome 2



Jakob

Jakob was the product of a full term pregnancy

Appeared healthy until day of life nineHospitalized in ICU

At 9 months Jakob is developmentally normal and growing well

However, some times his amino acid levels are dramatically elevated.



MSUD

What is MSUD ?

What odor was the physician asking mom about ?Where else can you smell it ?

Is odor a reliable physical finding ?

What causes neurotoxicity ?

What is the long-term treatment and outcome ?



MSUD

Autosomal RecessiveMutations in branched chain -ketoacid dehydrogenase (BCKDH)

Mitochondrial enzyme complex3 subunits (E1, E2, and E3) encoded by 4 unlinked genes

E1 decarboxylase – heterotetramer ( and subunits)E2 transacylaseE3 dehydrogenase

E1 is thiamine dependent



Maple Syrup Urine Disease

ClassicalNormal newborn, hours to days

Poor feeding and drowsiness

metabolic acidosis, hypoglycemia, cerebral edema, respiratory distress, hiccups, apnea, bradycardia, hypothermia, coma



Clinical Manifestations

Time Symptom/Sign

12-24 hours Maple syrup odor to cerumen

Elevated BCAA

2-3 days Irritability, poor feeding

Ketonuria

4-5 days Encephalopathy (lethargy, apnea, atypical movements

7-10 days Coma and respiratory failure



Metabolic Defect

BCAA amino-transferases

BCKDH- Rate limiting



Branch Chain Amino Acids

Leucine, Isoleucine and ValineComprise ~40% of essential AADuring fasting, ~ 80% of AA released is recycled back into protein synthesis




Transamination and oxidative disposal of leucine occurs in skeletal muscle (50%), kidney (25%) and gut/liver (25%)Nitrogen released is used to form glutamate -> alanine ->

glucose (alanine aminotransferase reaction)

Leucine + -Ketoglutarate -> -Ketoisocaproate and GlutamateGlutamate and Pyruvate -> -Ketoglutarate and AlanineAlanine -> -> -> Glucose




Increase in supply from diet or proteolysis must be met with appropriate increase in anabolic pathway (blocked in disorder)

Most severe biochemical intoxication caused by catabolism of endogenous protein




Defect leads to elevated levels, more pronounced in infants and children due to enhanced rates of growthLeucine accumulation is most toxic



Signs/Symptoms of Acute Toxicity

Ataxia (unsteady, clumsy movements)

Acute dystonia (involuntary muscle contractions)

Mood swings

Nausea, Vomiting, and Anorexia

Hallucinations

Altered level of consciousness

Stroke, coma, and death



Signs/Symptoms of Chronic Toxicity

Mood Disorders – anxiety and depression

Mental retardation

Neurologic deficits (stroke)



Neurotoxicity of Leucine

1. Leucine and KIC intracellular accumulation results in cellular edema





2. Leucine accumulation inhibits entry of other large neutral amino acids






Disrupted monoamine transmitter production

Decreased ‘fast’ neurotransmitter pools – glutamate, GABA, aspartate






3. Metabolites (KIC) induce oxidative injury

Melatonin, Vitamins C and E may be protective



Neurotoxicity of Leucine4. Excess KIC results in consumption of substrates needed

for malate-aspartate shuttle resulting in increased brain lactate and energy failure




KIC + glutamate Leucine + -Ketoglutarate

Increased Aspartate utilization

Decreased functioning of malate-aspartate shuttle

Decreased transfer of reducing equivalent

Energy failure And lactic acidosis




Glutamic Acid is a critical excitatory neurotransmitter

Leucine is trafficked to the brain as a source of –NH2 groups (Leucine-Glutamate cycle)



Neurotoxicity of LeucineElevated Leucine

Accumulation of KIC

drives leucine-glutamate cycle in reverse direction

decreased brain glutamateLEU

2-ketoisocaproate

Isovaleryl-CoA




Elevated Leucine

Altered brain water homeostasis

cell edema



Elevated Leucine

Inhibits entry into the brain of other large,

neutral AA (as in PKU) phenylalanine, tryptophane, methionine, tyrosine,histidine, threonine, and BCAA (L1-NAA-t)

Dystonia and ataxia may arise from acute deficiency of tyrosine and dopamine

Decreased dendritic branching, hypomyelination




MSUD

Goals of Treatment

Restriction of Leucine, Isoleucine, and Valine to maintain post-prandial plasma BCAA near normal level

Supplement free valine and isoleucine

Give glutamine and alanine

Hemodialysis



MSUD

Goals of Treatment

Excessive restrictionGrowth failure

Anemia

Breakdown of mucosa

Immunodeficiency

Dysmyelination, abnormal dendritic branching, microcephaly and mental retardation



Follow-Up Jacob – Age 4 yr

Family unwilling to tolerateContinual stress of life threatening disorder

dietary management, forcing feeds by G-tube when not interested in eating)

Severe limitations on their lives



Liver Transplantation

Liver transplantation results in increase in whole body BCKD activity

Muscle = 50%Kidney = 25%Liver and gut = 25%

Placed on liver transplant list at Pittsburgh and underwent successful liver transplant 3 years agoNow on unrestricted diet



Jacop after liver transplantation



Liver Transplant:

Outcomes

Normalization of BCAA within 6-12 hours

Sustained normalization of BCAA on unrestricted diet (4-18 months f/u)

Strauss KA. Am J Transpl; 2006



Alkaptonuria: a.k.a. Black Urine DiseaseFirst recognized “Inborn Error of Metabolism”

by Archibald Garrod in 1902

Symptoms: Homogentisate in the urine oxidizes to a black color

Also, black deposits in the scleraIn adults, accumulation of depositsin connective tissue leads to arthritis

No effective treatment

Alkaptonuria



Normal urineUrine from patients with alkaptonuria

Symptoms of alkaptonuria



Patients may display painless bluish darkening of the outer ears, nose and whites of the eyes. Longer term arthritis often occurs.



OH

OH

CH2

COOH

HGO

homogentisic acid

CH CH

C

O

CH2

C

O

CH2 COOH

maleylacetoacetic acid

HOOC

Homogentisic acid is an intermediate in the degradation pathway of phenylalanine. The reaction is catalysed by homogentisate dioxygenase (HGO).

A deficiency of HGO causes alkaptonuria.



Defect here causes Type I Tyrosinemia

Defect here causes alkaptonuria

Catabolic pathway for phenylalanine and tyrosine

Homogentisate dioxygenase

Fumarylacetoacetate hydrolase



Tyrosinemia is diagnosed by a blood and urine test. Tyrosinemia is treated by a low protein diet (low in phenylalanine, methionine and tyrosine) and a drug called NTCB.

Tyrosinemia



HOOC

CH CH

C

O

CH2

C

O

CH2 COOH

HOOC

CH CH CH2 COOH

C

O

CH2

C

O

maleylacetoacetic acid

Fumarate + acetoacetate

HOOC - CH2 - CH2 - C - CH2 - C - CH2 -

= =O O

COOH

succinylacetoacetic acid

HOOC - CH2 - CH2 - C - CH2 - C - CH3

= =O O

COOH

Succinylacetonetoxic and mutagenic

homogentisic acid

Deficiency of the enzyme FAAH results in Type I tyrosinemia

FAAH catalyses the last step in the degradation path of phenylalanine and tyrosine.

fumarylacetoacetate

spontaneous

FAAH

Tyrosinemia



The clinical features of the disease fall into two categories:

Acute Chronic

WHAT ARE THE SYMPTOMS OF

TYROSINEMIA?



abnormalities appear in the first month of life

poor weight gain enlarged liver and spleen distended abdomen swelling of the legs increased tendency to

bleeding, particularly nose bleeds Jaundice death from hepatic failure

frequently occurs between three and nine months of age unless a liver transplantation is performed.

Acute tyrosinemia



Homocystinuria

Defective activity of cystathionine synthase



Major phenotypic expression

Ectopia lentis

Vascular occlusive disease

Malar flash

Osteoporosis

Accumulation of homocysteine and methionine



A family of homocystinuria


Albinism



What is Albinism?

A lack of pigment in skin, hair, and eyes.

Albinism is an inherited condition that results from a gene mutation.

Altered genes are unable to exhibit natural pigments that normally occur.

Albinism can occur in nearly all species: animals, plants, or humans.



Albinism in all forms…



Lets take a closer look…What causes Albinism?

Albinism is genetic and is passed on through heredity via the genes.Genes involved are supposed to communicate the pigmentation of eyes, skin, and hair. Albino Individuals have received a recessive gene (aa) from both parents resulting in an incorrect genetic blueprint for pigment.

(Retina of albino)



Albinism



Characteristics of albinism:

Low Vision (20/50 to 20/800) Sensitivity to bright light and glare Rhythmic, involuntary eye movementsAbsent or decreased pigment in the skin and eye and sensitivity to sunburn that could lead to skin cancers or cataracts in later life "Slowness to see" in infancy



Characteristics of albinism:

Farsighted, nearsighted, often with astigmatism Underdevelopment of the central retina Decreased pigment in the retina Inability of the eyes to work together Light colored eyes ranging from lavender to hazel, with the majority being blue



Problems associated with Albinism…

Impaired vision due to the lack of melanin pigment

Skin damage due to Sun

Mild problems with blood clotting

Hearing impairment



Classification & Types of Albinism

Oculocutaneous albinism (OCA): melanin pigment is missing in skin, hair, and eyes.

Ocular albinism (OA): melanin pigment is primarily missing in the eyes and the skin and the hair appear normal.

OCA is more common than OA.



Oculocutaneous Albinism



Genes Associated with Albinism & Pigmentation:

Tyrosinase: major enzyme involved in melanin formation

Location: Chromosome 11

DHICA-oxidase: loose of function of this enzyme leads to albinism

Location: Chromosome 9

Ocular Albinism Gene: role unknown

Location: Chromosome X (primarily Sammy’s fault not Jeff’s)



dopaquinone

dopa

Eumelanins

Mixed-type melanins

Pheomelanins

trichochroms

Tyrosin hydroxylase

Tyrosin hydroxylase

Quinoleimine intermediate

Indole 5,6 quinone



Facts you may not know…

1 in 70 humans carries a recessive gene linked to albinism.If both parents carry an albino gene the chances are 1 in 4 (½ x ½) that offspring will display albinism.If one parent displays albinism, and one does not but carries a recessive gene, the chances of the offspring displaying albinism is 1 in 2 (1 x ½).

Hey want to know something…

Not really, but I suppose you are going to tell me anyway…



Common Myths & Misconceptions

Persons with albinism always have red eyes. Persons with albinism are totally blind. Albinism is contagious. Persons with albinism are the result of evil spirits or wrongdoing. Persons with albinism are retarded or deaf. Albinism results from inbreeding or the mixture of two races. Persons with albinism have magical powers.



In summary…

Albinism is a rather rare recessive mutation that can be witnessed in many multiple species; plant, animal, and human, all with phenotypic similarities.



Albinism in animals



Albinism in films



A family of albinism



Newborn screening



The goal of newborn screening is to eliminate, through early identification and treatment, and improve the quality of life for affected individuals.

Newborn screening



Newborn screening is not just a laboratory service; it is a system of care including, not only testing, but also follow up, definitive diagnosis, treatment, long term management, education and evaluation----

Newborn screening



The goal of newborn screening follow up is to ensure that each affected infant receives the full benefit of early detection and optimal long term treatment and management.

Newborn screening



Follow Up

short term follow up- assure that a definitive diagnostic work-up is done, that the infant really has the disorder and that the infant is started on appropriate treatment

long term follow up- assure that the infant continues to receive appropriate treatment and monitors the long term outcome



THE END

metabolic disorders of proteins

Education

silent disorders type

glycolipid disorders

acute metabolic crises

metabolic diseases present

acute metabolic crisislife

metal metabolism

disease defects

type viiiehlersdanlos