BIOCHEMICAL GENETICSBIOCHEMICAL GENETICS

Greg Enns, MB, ChB, FAAPProfessor of Pediatrics

Director, Biochemical Genetics ProgramLucile Packard Children’s Hospital

October 22, 2015

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Learning GoalsLearning 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

Lecture OutlineLecture Outline

Inborn Errors of Metabolism◦Signs and Symptoms◦Diagnostic Tests

Routine labs Biochemical profiling

So what exactly is a Biochemical Geneticist anyway?

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Inborn Errors of MetabolismInborn Errors of Metabolism “Metabolic Disorders” Inherited disorders of intermediary metabolism

◦ 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?Who, What, & How? Common presentations

◦ Neurologic decompensation◦ Multi-organ system involvement◦ Lysosomal storage disorders

Types of tests◦ Basic◦ Complex◦ Newborn screening

Test interpretations◦ Common mistakes◦ Case examples

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Who to TestWho to TestClinical FeaturesClinical Features

Neurologic symptoms◦ Altered Mental Status◦ Developmental regression

Mental retardation Psychiatric illness

◦ Autism? Failure to thrive SIDS Non-immune hydrops

Eye findings◦ Cataracts◦ Pigmentary retinopathy

Hepatosplenomegaly Cardiomyopathy Bone/joint disease

◦ Contractures◦ Dysostosis multiplex◦ Osteonecrosis

Unusual odor

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Signs and Symptoms of Inborn Errors of MetabolismSigns 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 MetabolismSigns and Symptoms of Inborn Errors of Metabolism

Failure to thrive, feeding difficultiesRespiratory distressHypotonia, seizures, lethargyHypothermia (especially neonates)

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Signs and Symptoms of Inborn Errors of MetabolismSigns and Symptoms of Inborn Errors of Metabolism

◦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 MetabolismSigns and Symptoms of Inborn Errors of Metabolism

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 MetabolismSigns and Symptoms of Inborn Errors of Metabolism

MAY PRESENTAT ANY AGE

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Case #1Case #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 #1Case #1

This presentation is most suggestive of …?A) Maple syrup urine diseaseB) Urea cycle defectC) Fatty acid oxidation defectD) Glycogen storage diseaseE) Organic acidemia

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Your answer?Your answer?

E) Organic acidemia

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THE ANION GAPTHE ANION GAP

Na+ - [Cl- + HCO3-]

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THE ANION GAPTHE 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 GAPTHE ANION GAP

UA – UC = Na+ - [Cl- + HCO3-]

= the anion gap

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Concentrations of Concentrations of ““unmeasuredunmeasured”” cations and anions( cations and anions(mEq/LmEq/L))

Unmeasured Cations

K 4.5Ca 5.0Mg 1.5Total 11

Unmeasured Anions

Protein 15

PO4 2

SO4 1

Organic acids 5

Total 23

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Metabolic Acidosis with Increased Metabolic Acidosis with Increased Anion GapAnion Gap

Increased Unmeasured Anion:Methanol, metforminUremiaDiabetic ketoacidosisParaldehyde, phenforminInborn errors of metabolismLactic acidemiaEthanol, ethylene glycolSalicylates, solvents, strychnine

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Metabolic Acidosis with Increased Anion GapMetabolic Acidosis with Increased Anion Gap

Decreased Unmeasured Cation*:HypokalemiaHypocalcemiaHypomagnesemia

*Possible in theory, but rarely encountered

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Metabolic Acidosis with Normal Anion GapMetabolic Acidosis with Normal Anion Gap

• Renal tubular acidosis• Intestinal loss of bicarbonate

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A B C DATA EAB EBC ECD

F G

Negative Feedback

A, B, C, D – substrate and products of major pathwayF, G – products of minor pathwayT – transporter ; E - enzymes

Prototypical Metabolic Pathway

Cell membrane

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The Laboratoryspecimen

results

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Organic AcidsOrganic Acids(GC-MS)(GC-MS)

Urine only Qualitative screen 100’s of compounds 3 hour prep 30 min run

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Methylmalonic AcidemiaTotal Ion Chromatogram

Ion

Abu

ndan

ce

Time (min)

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Case #1Case #1Initial 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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TC IIOH-Cbl

TC IIOH-Cbl

OH-Cbl+3 Cbl+3

Me CblHomocysteine Methionine

Methionine Synthase

Cbl+2

L-Methylmalonyl CoA

SuccinylCoA

Ado CblCbl+1

TC II

OH-Cbl+3

MethylmalonylCoA Mutase

F

A,HB

C,D

E,G

The Cobalamin PathwayThe Cobalamin Pathway

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Methylmalonic AcidemiaMethylmalonic Acidemia

Common signs/symptoms:◦ 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

Pediatr Res 40:404-9, 1996

caudate andputamenhyperintensity

delayed myelination

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Case #2Case #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 #2Case #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 #2Case #2

Your differential diagnosis includes which of the following?A) Steroid psychosisB) Occult infectionC) IntoxicationD) Inborn error of metabolismE) All of the above

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Your answer?Your answer?

E) All of the above

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Case #2Case #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 #2Case #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 #2Case #2

The most likely diagnosis is:A) Urea cycle disorderB) Mitochondrial diseaseC) Propionic acidemiaD) Medium-chain acyl-CoA dehydrogenase deficiencyE) Proteus mirabilis urinary tract infection

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Your diagnosis?Your diagnosis?

A) Urea cycle disorder

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Quantitative Amino AcidsQuantitative Amino Acids(HPLC)(HPLC)

Plasma, urine, CSF 40 amino acids (sometimes more) 10 minute prep 3 hour run per sample

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Case #2Case #2

Metabolic labs:

glutaminecitrulline 3 uM (8-47)orotic acid <1 g/mg creatinine (<4.7)

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Urea Cycle DisordersUrea Cycle DisordersClinical 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 (post-

partum)

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Late-Onset Urea Cycle DisordersLate-Onset Urea Cycle DisordersBehavioral problems◦Agitation◦Delirium◦Aggression◦Confusion

AtaxiaHeadacheHemiparesis

Nassogne et al. JIMD 28:407-14, 2005

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Lysosomal Storage DisordersLysosomal Storage DisordersFamily 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 BLOCKSMPS ENZYME BLOCKS

iduronate sulfatase

-L-iduronidaseHURLER (MPS I)

COOH

COOH

S-O

O

NAc

O

COOH

O

NAc

O

S-O

O-S

HUNTER (MPS II)

O

S-O

NAc

O

COOH

O

S-O

NAc

O

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Normal Cell Abnormal Cell

Lysosomal Storage DisordersLysosomal Storage Disorders

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CNS InvolvementCNS Involvement

Metachromatic Leukodystrophy

8%

Sanfilippo A7%

Hunter Severe5%

Krabbe6%

Sandhoff2%

GM 1 Gangliosidosis

2%

Mucolipidosis type II / III2%

Niemann-Pick A2%

Niemann-Pick C4%

Tay-Sachs4%

Sanfilippo B4%

Gaucher type 2 & 31%

Niemann-Pick B2%

Maroteaux-Lamy3%

Cystinosis4%

Morquio5%

Pompe5%

Hurler/Scheie (MPS I)4%

Gaucher type I13% Scheie (MPS I)

1%

Hurler (MPS I)4%

Hunter Mild1%

Fabry7%

Significant or severe CNS involvement(~ 54%)

No or minimal CNS involvement(~ 46%)

Adapted from Meikle P et al. JAMA. 1999;281:249-254.

Other2%

-Mannosidosis

Sanfilippo D

1%

1%

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Mucopolysaccharidosis type IMucopolysaccharidosis type IPathology Inheritance-L-iduronidase enzyme deficiencyAccumulation of glycosaminoglycans (GAGs)

Autosomal Recessive

Onset PrevalenceSevere form: first 6 months after birthAttenuated form: 3 to 8 years of age

≈1:90,0001

ProgressionOften life threateningSevere cases life span < 10 yAttenuated cases life span ≈ normal

Disease-at-a-Glance

Disease-at-a-Glance

1. Meikle P et al. JAMA. 1999;281:249-254.

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MPS I MPS I

Umbilical/inguinal hernia

Cardiovascular disease

Obstructive airway diseaseCorneal clouding

Hepatosplenomegaly

Chronic rhinitis/otitis

Joint stiffness

Developmental delay

Hearing loss

Enlarged tongue

Look for unusual symptoms or clusters of more common symptoms

Carpal tunnel syndrome

Skeletal deformities

Signs &Symptoms

Signs &Symptoms

Macrocephaly

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 IDisease Progression: Severe MPS I

10 months 12 months

22 months 34 months

39 months

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Disease Progression: Mild MPS IDisease Progression: Mild MPS I

3 years 4 years

6 years 8 years

11 years

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“Scheie” MPS I S

“Hurler” MPS I H

All patients typically have < 1% of normal enzyme levels,but only MPS l H involves the CNS

“Hurler-Scheie” MPS I HS

MPS IMPS I Clinical Heterogeneity

Clinical Heterogeneity

SevereAttenuated

Courtesy of Emil Kakkis, MD.

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Peripheral Nervous System InvolvementPeripheral 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 ArmContractures of the Arm

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Skeletal AbnormalitiesSkeletal Abnormalities

(Clarke LA, 1997) Photo reproduced by permission of Hodder/Arnold Publishers.

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Gibbus in MPS IGibbus in MPS I

Dimethylmethylene Blue Binding Assay For Glycosaminoglycans

Blank 5 µg/ml 17.5 µg/ml 35 µg/ml 50 µg/ml

Urine + DMB reagent Blue product

Courtesy T. Cowan

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NormalMPS IHurler

MPS IScheie

MPS IIHunter

MPS IIISanfilippo

MPS IVMorquio

MPS VIMaroteaux-

Lamy

MPS VIISly

Dermatan,Heparan

Dermatan,Heparan

Dermatan,Heparan

Dermatan,Heparan

Heparan Keratan Dermatan

Thin-layer chromatography

Courtesy of T. Cowan

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SUMMARYSUMMARY

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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InterventionalGeneticist

Anesthesiologist

Primary Care Physician

Neurologist

Otorhinolaryngologist

Genetic Counselor

Cardiologist

Gastroenterologist

Surgeon

Pulmonologist

Dentist

Ophthalmologist

Orthopedist

Hematologist

Nephrologist

Palliative Care Specialist

Metabolic DiseaseTreatment Team