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BIOCHEMICAL GENETICS
Greg Enns, MB, ChB, FAAP
Professor of Pediatrics
Director, Biochemical Genetics Program
Lucile Packard Children’s Hospital
October 22, 2015
Learning Goals


Develop abilities to recognize signs and symptoms
associated with inborn errors of metabolism
Become aware of clues to an underlying inborn error of
metabolism by using simple lab tests
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Lecture Outline

Inborn Errors of Metabolism
◦ Signs and Symptoms
◦ Diagnostic Tests
 Routine labs
 Biochemical profiling
So what exactly is a Biochemical Geneticist anyway?
Inborn Errors of Metabolism
“Metabolic Disorders”
 Inherited disorders of intermediary metabolism

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Autosomal recessive
Autosomal dominant
X-linked
Maternal (mitochondrial) inheritance
Metabolic imbalance most commonly secondary to an enzyme deficiency
 Toxic metabolites
 Deficiency states
 Altered energy production

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Who, What, & How?

Common presentations
◦ Neurologic decompensation
◦ Multi-organ system involvement
◦ Lysosomal storage disorders
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Types of tests
◦ Basic
◦ Complex
◦ Newborn screening

Test interpretations
◦ Common mistakes
◦ Case examples
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Who to Test
Clinical Features

Neurologic symptoms

◦ Cataracts
◦ Pigmentary retinopathy
◦ Altered Mental Status
◦ Developmental regression
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Mental retardation
Psychiatric illness
◦ Autism?
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Failure to thrive
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SIDS
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Non-immune hydrops
Eye findings
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Hepatosplenomegaly
Cardiomyopathy
Bone/joint disease
◦ Contractures
◦ Dysostosis multiplex
◦ Osteonecrosis

Unusual odor
Signs and Symptoms of Inborn Errors of Metabolism
 Appear after an interval of good health (hours,
days) in a usually previously healthy full-term
infant
 Unmasked by “stressors” that lead to
catabolism (e.g. infection, fasting, dehydration,
excessive protein, birth)
 Non-specific (limited response to stress in
neonates)
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Signs and Symptoms of Inborn Errors of Metabolism
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Failure to thrive, feeding difficulties
Respiratory distress
Hypotonia, seizures, lethargy
Hypothermia (especially neonates)
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Signs and Symptoms of Inborn Errors of Metabolism
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Ocular findings
Hepatosplenomegaly
Cardiomyopathy
Renal tubular disease
Abnormal odor
Unusual skin, hair
Dysostosis multiplex
Dysmorphic features
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Signs and Symptoms of Inborn Errors of Metabolism
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
Typically patients who have metabolism disorders display a
constellation of features
Isolated failure to thrive, cardiomyopathy, etc. in the
absence of other organ system involvement or other
clinical features (e.g. developmental delay) would be
unusual
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Signs and Symptoms of Inborn Errors of Metabolism
MAY PRESENT
AT ANY AGE
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Case #1
A 4-day-old boy is taken to the emergency
room on Christmas eve because of poor
feeding and lethargy. He is hypothermic and
is tachypneic. He has elevated (3+) ketones in
his urine and a metabolic acidosis (anion gap
25 with normal lactate). His ammonia is 735
M (normal 5-35).
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Case #1
This presentation is most suggestive of …?
A) Maple syrup urine disease
B) Urea cycle defect
C) Fatty acid oxidation defect
D) Glycogen storage disease
E) Organic acidemia
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Your answer?
E) Organic acidemia
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THE ANION GAP
+
Na
-
[Cl
+
HCO3 ]
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THE ANION GAP
Total serum cations:
Na + UC
Total serum anions:
Cl + HCO3 + UA
So…
NA + UC = UA + Cl + HCO3
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THE ANION GAP
UA – UC = Na+ - [Cl- + HCO3-]
= the anion gap
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Concentrations of “unmeasured” cations and anions(mEq/L)
Unmeasured Anions
Protein
Unmeasured Cations
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PO4
2
SO4
1
Organic acids
5
Total
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K
Ca
Mg
Total
4.5
5.0
1.5
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Metabolic Acidosis with Increased
Anion Gap
Increased Unmeasured Anion:
Methanol, metformin
Uremia
Diabetic ketoacidosis
Paraldehyde, phenformin
Inborn errors of metabolism
Lactic acidemia
Ethanol, ethylene glycol
Salicylates, solvents, strychnine
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Metabolic Acidosis with Increased Anion Gap
Decreased Unmeasured Cation*:
Hypokalemia
Hypocalcemia
Hypomagnesemia
*Possible in theory, but rarely encountered
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Metabolic Acidosis with Normal Anion Gap
•
•
Renal tubular acidosis
Intestinal loss of bicarbonate
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Prototypical Metabolic Pathway
Negative Feedback
TA
A
EAB
EBC
A
B
F
ECD
C
D
G
Cell membrane
A, B, C, D – substrate and products of major pathway
F, G – products of minor pathway
T – transporter ; E - enzymes
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The Laboratory
specimen
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results
Organic Acids
(GC-MS)
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Urine only
Qualitative screen
100’s of compounds
3 hour prep
30 min run
Methylmalonic Acidemia
Ion Abundance
Total Ion Chromatogram
Time (min)
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Case #1
Initial methylmalonic acid (MMA) level 1,200 uM
(nl <0.3 uM)
 Treated with:

◦ High dextrose intravenous fluids + insulin
◦ Intravenous “nitrogen scavenging” medications
 Sodium benzoate + sodium phenylacetate
◦ Intravenous carnitine
◦ Intramuscular hydroxocobalamin

Dialysis is often needed to decrease MMA and
ammonium levels, but avoided in this case
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The Cobalamin Pathway
TC II
TC II
OH-Cbl
OH-Cbl
TC II
Methionine Synthase
E,G
Homocysteine
Methionine
OH-Cbl+3
Me Cbl
F
C,D
+3
OH-Cbl
Cbl+3
Cbl+2
A,H
Cbl+1 B Ado Cbl
L-Methylmalonyl
Succinyl
CoA Methylmalonyl
CoA
CoA Mutase
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Methylmalonic Acidemia
Common signs/symptoms:
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lethargy
intermittent vomiting
dehydration
failure to thrive
respiratory distress
hypotonia
Matsui et al. NEJM 308:857, 1983
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BRAIN INJURY IN ORGANIC ACIDEMIAS
delayed
myelination
caudate and
putamen
hyperintensity
Pediatr Res 40:404-9, 1996
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Case #2
You are called about a 55-year-old male who
became confused and lethargic a few days after
endoscopic sinus surgery to remove nasal polyps
that were causing chronic sinusitis. He had been
discharged home on steroids to prevent swelling.
His wife took him to the emergency room after he
became more confused and disoriented. He
progressed to a coma and was placed on
mechanical ventilation in the intensive care unit.
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Case #2
The ICU physician upon reviewing the initial
laboratory studies noted a respiratory alkalosis and
normal complete blood count, glucose, and liver
enzymes. Cerebrospinal fluid studies and urinalysis
are normal. Blood, urine, and cerebrospinal fluid
cultures are obtained. A urine toxicology screen
was also normal. Initial head CT scan and CXR
were normal.
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Case #2
Your differential diagnosis includes which of the following?
A) Steroid psychosis
B) Occult infection
C) Intoxication
D) Inborn error of metabolism
E) All of the above
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Your answer?
E) All of the above
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Case #2
Although most metabolic studies take days to weeks
to obtain results, you recall that ammonia is a
central respiratory stimulant and urea cycle
disorders often are associated with tachypnea.
Moreover, unlike many metabolic disorders, a
metabolic acidosis is typically not present initially
in urea cycle disorders.
You look at the lab results again and notice the blood
urea nitrogen is low.
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Case #2
So…you check an ammonia (obtained on ice and
sent to the lab quickly) and find an abnormal
elevation.
The ammonia level is 281 M (normal 5-35).
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Case #2
The most likely diagnosis is:
A) Urea cycle disorder
B) Mitochondrial disease
C) Propionic acidemia
D) Medium-chain acyl-CoA dehydrogenase
deficiency
E) Proteus mirabilis urinary tract infection
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Your diagnosis?
A) Urea cycle disorder
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Quantitative Amino Acids
(HPLC)
Plasma, urine, CSF
 40 amino acids (sometimes more)
 10 minute prep
 3 hour run per sample
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Case #2
Metabolic labs:
 glutamine
citrulline 3 uM (8-47)
orotic acid <1 g/mg creatinine (<4.7)
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Urea Cycle Disorders
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Clinical Features
◦ Term birth
◦ ~DOL 2 poor feeding, vomiting, lethargy progressing
to coma, apnea
◦ Seizures may occur with cerebral edema
◦ Often mistaken for sepsis
◦ Later onset with ataxia, altered mental status (postpartum)
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Late-Onset Urea Cycle Disorders
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Behavioral problems
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Agitation
Delirium
Aggression
Confusion
Ataxia
Headache
Hemiparesis
Nassogne et al. JIMD 28:407-14, 452005
Lysosomal Storage Disorders
Family of > 40 disorders; collective prevalence
~1:8,0001
 Enzyme deficiency causes lysosomes to become
engorged1
 Each disease is a consequence of type of substrate
and where it accumulates1
 Progressive accumulation of substrate may result
in irreversible damage2

1. Meikle P et al. JAMA. 1999;281:249-254.
2. Wraith JE et al. J Pediatr. 2004;144:581-588.
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MPS ENZYME BLOCKS
COOH
S-O
O
COOH
O-S
iduronate
sulfatase
COOH
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O
O
O
NAc
NAc
HUNTER (MPS II)
S-O
COOH
S-O
O
O
NAc
-L-iduronidase
S-O
HURLER (MPS I)
O
O
NAc
Lysosomal Storage Disorders
Normal Cell
Abnormal Cell
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CNS Involvement
Significant or severe CNS involvement
(~ 54%)
Mucolipidosis type II / III
2%
Niemann-Pick A
2%
GM 1
Gangliosidosis
2%
No or minimal CNS involvement
(~ 46%)
Sandhoff
2%
Niemann-Pick C
4%
Gaucher type I
13%
Other
2%
Scheie (MPS I)
1%
Hurler/Scheie (MPS I)
4%
Sanfilippo B
4%
Fabry
7%
Tay-Sachs
4%
Hunter Mild
1%
Krabbe
6%
Pompe
5%
Hunter Severe
5%
Sanfilippo A
7%
Morquio
5%
Metachromatic
Leukodystrophy
8%
Adapted from Meikle P et al. JAMA. 1999;281:249-254.
Hurler (MPS I)
4%
Cystinosis
4%
Sanfilippo D
1%
-Mannosidosis
1%
Gaucher type 2 & 3
1%
Niemann-Pick B
2%
Maroteaux-Lamy
3%
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Mucopolysaccharidosis type I
Pathology
-L-iduronidase enzyme deficiency
Accumulation of glycosaminoglycans (GAGs)
Onset
Severe form: first 6 months after birth
Attenuated form: 3 to 8 years of age
Progression
Often life threatening
Severe cases life span < 10 y
Attenuated cases life span ≈ normal
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1. Meikle P et al. JAMA. 1999;281:249-254.
Inheritance
Autosomal Recessive
Prevalence
≈1:90,0001
Disease-ata-Glance
Signs &
Symptoms
Look for unusual symptoms or clusters of more
common symptoms
MPS I
Macrocephaly
Developmental delay
Chronic rhinitis/otitis
Obstructive airway disease
Umbilical/inguinal hernia
Skeletal deformities
Carpal tunnel syndrome
Corneal clouding
Hearing loss
Enlarged tongue
Cardiovascular disease
Hepatosplenomegaly
Joint stiffness
Neufeld EF, Muenzer J. In: Scriver C, Beaudet A, Sly W, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. New York, NY: McGraw-Hill; 2001:3421-3452.
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Disease Progression: Severe MPS I
10 months
12 months
39 months
22 months
34 months
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Disease Progression: Mild MPS I
3 years
4 years
11 years
6 years
8 years
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MPS I
Clinical
Heterogeneity
Attenuated
“Scheie”
MPS I S
Severe
“Hurler-Scheie” MPS
I HS
“Hurler” MPS
IH
Courtesy of Emil Kakkis, MD.
All patients typically have < 1% of normal enzyme levels,
but only MPS l H involves the CNS
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Peripheral Nervous System Involvement
• Carpal tunnel
syndrome
– nerve entrapments/
nerve compressions
• Most patients lack
typical symptoms until severe
compression occurs
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Contractures of the Arm
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Skeletal Abnormalities
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(Clarke LA, 1997) Photo reproduced by permission of Hodder/Arnold Publishers.
Gibbus in MPS I
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Dimethylmethylene Blue Binding Assay For Glycosaminoglycans
Urine + DMB reagent  Blue product
Blank
5 µg/ml
17.5 µg/ml
35 µg/ml
50 µg/ml
Courtesy T. Cowan
Thin-layer chromatography
Normal
MPS I
Hurler
MPS I
Scheie
Dermatan,
Heparan
Dermatan,
Heparan
MPS II
Hunter
Dermatan,
Heparan
MPS III
Sanfilippo
MPS IV
Morquio
MPS VI
MaroteauxLamy
Heparan
Keratan
Dermatan
MPS VII
Sly
Dermatan,
Heparan
Courtesy of T. Cowan
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SUMMARY
High index of suspicion – consider IEM in parallel with more
common conditions
 Presentations are non-specific
 Simple screening tests yield critical diagnostic clues

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Metabolic Disease
Treatment Team
Primary Care
Physician
Ophthalmologist
Surgeon
Otorhinolaryngologist
Pulmonologist
Interventional
Geneticist
Cardiologist
Hematologist
Orthopedist
Nephrologist
Neurologist
Dentist
Palliative Care Specialist
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Anesthesiologist
Gastroenterologist
Genetic Counselor