Untangling the Spirals of

Metabolic Disease: Primary

Diagnoses and Secondary Effects:
Implications for Treatment
David A. H. Whiteman MD

1909

Archibald Garrod

In his paper, Inborn Errors of

Metabolism, the disease Alkaptonuria
(Ochronosis: Homogentisic Acid
Oxidase Deficiency) is described as
being caused by a gene.
This landmark is usually cited as the
birth of Biochemical Genetics.

CP1022045-49

CP1022045-48

A 2-week-old boy is admitted to the hospital with sepsis

due to Escherichia coli. He is being breastfed and has
been vomiting frequently.
Findings include failure to thrive, lethargy, hypotonia,
jaundice, hepatomegaly, and positive nonglucose reducing
substances in the urine.
Of the following, the MOST likely explanation for these
findings is
A.
B.
C.
D.
E.

galactosemia
glycogen storage disease
lactose intolerance
maple syrup urine disease
urea cycle defect

Classical Galactosemia

Metabolic Cataract and

Glaucoma

Disaccharide Hydrolysis
Maltose + H2O >>>maltase>>> D-Glucose
+
D-Glucose
Lactose + H2O >>>lactase>>> D-Galactose
+
D-Glucose
Sucrose + H2O >>>sucrase>>> D-Fructose
+
D-Glucose

Classical Galactosemia on Diet

Autosomal Recessive Inheritance

Galactosemia Variants
Gal-1-P-UDT

GalKinase

Epimerase

9p13 p21

17q21-q22

1p32 pter

Duarte
Los Angeles
Indian
Rennes

Philadelphia
Negro
Chicago

A 2-day-old female infant is refusing to feed and has

become increasingly lethargic and hypotonic over the past
2 hours. Results of laboratory studies include an arterial
blood gas analysis that reveals a pH of 7.13, PCO2 of 27
mm Hg, and HCO3 of 14 mEq; positive urine ketones;
and an ammonia concentration of 600 mcg/dL. An inborn
error of metabolism is suspected.
Of the following, the MOST appropriate laboratory study
to obtain is
A.
B.
C.
D.
E.

leukocyte lysosomal enzyme activities

plasma long-chain fatty acid levels
serum carnitine level
urine for reducing substances
urine organic acid levels

A 3-month-old infant who has a history of gastroesophageal

reflux has had increasing vomiting for 2 days. This morning
she developed rapid, deep, labored breathing; lethargy; and
shock. Findings include: serum sodium, 144 mEq/L;
potassium, 4.5 mEq/L; chloride, 89 mEq/L; bicarbonate, 5
mEq/L; pH, 7.16; glucose, 48 mg/dL; ammonia, 128 mcmol/L;
and ketonuria.
The MOST likely explanation for these findings is
A.
B.
C.
D.
E.

aminoacidopathy
ethylene glycol poisoning
metoclopramide toxicity
organic acidemia
urea cycle defect

A previously healthy 2-year-old boy is brought to the emergency

department by his mother, who reports that he has had a cold
and fever for the past 2 days. He has been taking only small
amounts of juice and no solid foods. When she tried to arouse
him after his nap today, he was lethargic and unresponsive.
Results of laboratory studies include a glucose concentration of
40 mg/dL, an ammonia level of 200 mcg/dL, and an arterial
blood pH of 7.4.
At this time, the MOST important study to obtain is
A.
B.
C.
D.
E.

plasma acylcarnitine profile

plasma insulin levels
serum acetylsalicylic acid concentration
urine and stool porphyrins
urine organic acids

A Pale Toddler with Recurrent

Abdominal Pain
History
23 year old G2 mother, uncomplicated term pregnancy,
newborn period
Child fed well, with normal growth and
development to 2 years
Intermittent abdominal pain, with fainting and pallor
GI consultation and investigations unremarkable
3 years: Coma at end of day, with URI
Hypoglycemia: 24 mg/dl

A Pale Toddler with Recurrent

Abdominal Pain
Investigation
Carnitine Deficiency: Total 19 Free 11 umol/L
Urine AcylGlcyine Profile:
Increased Hexanoyl-, Octanoyl-, Suberyl-Glycines
DNA Analysis MCAD: A985G (K329E) homozygous
Enzyme Analysis MCAD: 8% Residual Enzyme Activity
compared to controls

A Pale Toddler with Recurrent

Abdominal Pain
Management
Treated with:
Riboflavin 50 mg BID
L-Carnitine 50 mg/kg/day intermittently
Reduced fat diet (maximum of 25% of all calories from fat
Normal growth and development, no residual neurological deficit
Now has muscle cramps with exercise (baseball).
Her older sister (then 7 years of age) is diagnosed on the
basis of DNA analysis, although never symptomatic
Two subsequent uncomplicated pregnancies (no HELLP or AFLP)
one child affected, the second a carrier.

MCAD Deficiency

A Child with Multiple Problems

The Pregnancy

Complicated by abdominal pain, severe nausea and vomiting,

black out spells
Emergency C-section because of maternal hemorrhage
(unknown cause)

A Child with Multiple Problems

The Child
Multiple hospital admissions for vomiting and
dehydration in first year of life
Nissen fundoplication at 18 months of age.
No improvement
Hospitalization at 2 1/2 years for feeding disorder
Episodes of weakness, shaking, decreased activity.
No documented hypoglycemia
Symptoms worse in the morning, and with routine
illness

A Child with Multiple Problems

Diagnostic Investigation
Initial metabolic evaluation negative
Challenge testing:
Hypoglycemia within12 hours of
fasting
Fat beta-oxidation intermediates in urine
Fibroblast assays: reduced Palmitate oxidation and
reduced LCHAD activity

A Child with Multiple Problems

Subsequent Pregnancy

Prenatal diagnosis of LCHAD

Mother developed HELLP syndrome

A Twitchy Floppy Baby

History
Uncomplicated pregnancy and birth: 75th percentile
2 weeks:

Colic

6 weeks:

Muscle twitches
Dramatic Hypotonia
Tires when feeding
Startles easily

8 weeks:

Severe constipation
Growth at 5th percentile

A Twitchy Floppy Baby

Examination
Head circumference and weight below 5th percentile:
length at 10th
Dysmorphic face: telecanthus and prominent forehead,
Flat occiput, ridged sutures
Hypoplastic distal digital phalanges with arch fingerprint forms
Severe hypotonia, no lateralizing neurological signs
Fever of unknown origin (103F max)
Spontaneous twitches of all extremities

A Twitchy Floppy Baby

Investigations
Dysmorphologist: ? FG, ? Schinzel-Gideon Syndromes,
Rule out chromosome anomaly, micro deletions.
Karyotype and multiple FISH micro deletions: Negative
Metabolic Screening: Mild Lactic Acidemia (Max 2.5 umol/L)
EEG: Normal
CK: 107
Carnitine: Total 33 Free 24 umol/L
Skin Fibroblasts:
Modest (55 %) reduction in Palmitate and Myristate Oxidation
LCHAD and LCAT Enzyme activities normal
Mitochondrial DNA deletion/mutation analysis: Unremarkable

A Twitchy Floppy Baby

The Crisis : Diagnosis and Treatment
Anticonvulsants for twitching ineffective
Klonopin
Keppra
Controversy: Seizures vs. Myoclonus
Muscle Biopsy for Enzyme Analysis
NADH Dehydrogenase 37% of controls
Treatment:
L-Carnitine
Vitamins A,B-complex,C,E,K
Ubiquinone (CoEnzyme Q)
Sodium Succinate

RRF

Amino Acids

Glucose

(primarily alanine)

LDH

Pyruvate

NADH + H+
(reduced)
PC

Gluconeogenesis

LACTATE
NAD+

(oxidized)

PDH

Acetyl CoA

CO2 + H2O

TCA Cycle

Pyruvate

Lactate

NADH + H+

NAD+

(reduced form)

(oxidized form)

NADH + H+ + 1/2O2

NAD+ + H2O + energy

Mitochondrial Metabolism

CP1022045-76

Mitochondrial Chromosome

Not a Well Mitochondrion

H+
ATP

ADP

H+
NADH

Succinyl-CoA

H+

H+

NAD+
1/2 O 2 H2 O

SDH
COMPLEX III

COMPLEX I

COMPLEX
IV

CoQ
Cyt C

Primary Hyperlactacidemia

Disorders of Pyruvate Metabolism

Krebs Cycle Defects

Disorders of Gluconeogenesis

Pyruvate carboxylase deficiency

Disorders affecting the pyruvate dehydrogenase
complex (PDH)

Fructose-1,6-diphosphatase deficiency
Glycogen storage disease type I
Phosphoenolpyruvate carboxykinase deficiency

Respiratory Chain Disorders

Secondary to Other Inborn Errors of Metabolism

Organic acid disorders

Fatty acid oxidation disorders

MMBID 8th ed. 2001, chapter 100, p 2289

Zschocke/Hoffmann: Vademecum Metabolicum. Schattauer 1999, p 13

Pedigree Assembly
Pedigree: A graphical representation of a
family, showing relationships among
individuals and medical and demographic
information
Example:

Maternal Inheritance Pedigree

Heteroplasmy can lead to

variable phenotypes in daughter
cells

Disease Map of the

Mitochondrial Genome

Factors that Complicate Analysis

of Traditional Inheritance

Reduced penetrance
Variable expressivity
Pleiotropy
New mutations
Gonadal mosaicism
Genetic heterogeneity

Gonadal (Germline) Mosaicism

One parent is is presumed to have a mixture
of cells: mostly normal, but with at least
some mutant germ cells.
Proportion of mutant gametes depends on
when mutation occurred during mitotic
expansion.
Presents counseling difficulty: What is the
risk in the next pregnancy?

A de novo mutation in a germ

cell lineage produces mosaicism

Case Report
Male infant born at 34 weeks gestational
age to a G3/P1 mother
Maternal serologies were negative
Prenatal course revealed a fetus thought to
be IUGR as well as oligohydramnios
Delivery was via C-section due to fetal
decelerations

Family History
Sibling who died at 25 weeks gestation
secondary to non-immune hydrops
Healthy 18 month old sister
Both parents are in good health and
unrelated

Case Report
Mild respiratory distress postnatally.
ABG showed a pH of 7.33, CO2 was 33,
HC03 was 17
Lactic acid was 10 mM (normal: < 2.2) at
4 hrs of life and increased to 24 mM over
the next 24 hrs
Electrolytes, glucose, NH3 normal
Sepsis work-up initiated

Case Report
2nd day:

Mottled appearance
Blood pressure low
Lactic acid remained at 22 mM
Echocardiogram was normal (anatomy/fxn)

3rd day:
Transfer to Mayo
ABG on arrival: pH 7.13, pC02 99

Physical Examination
Weight and length in the 10-25th percentile.
Head circumference 5-10th percentile
Mild dysmorphic features including short
palpebral fissures, mild micrognathia, long
thumbs
No organomegaly
Muscle hypotonia

Typical Organic Acid Profile

in LCHAD Deficiency
Dicarboxylic
aciduria
3 Hydroxy dicarboxylic
aciduria

*Internal standard

TFP/LCHAD Deficiency
Genes:

- -subunit gene (chromosome: 2p23)

- -subunit gene (chromosome: 2p23)

Incidence:

once considered same as MCAD def. (1:15,000 live births)

Symptoms:
Hypoketotic hypoglycemia
Reye-like syndrome:

hypoglycemia
hyperammonemia
elevated transaminases
brain edema
fatty liver with
microvesicular steatosis
elevated uric acid

Lactic acidosis
Myopathy/rhabdomyolysis

Cardiopathy
hypertrophic/dilated cardiomyopathy
AV-block
ventricular arrhythmias

Retinitis pigmentosa (RP)

Peripheral neuropathy
Cholestasis
Maternal pregnancy complications
Sudden unexplained death

Diagnosis:

TFP/LCHAD
Deficiency

- urine organic acids (hypoketotic dicarboxylic aciduria,

hydroxy dicarboxylic aciduria)

- plasma/blood spot acylcarnitines

- in vitro probe assay in fibroblasts
- molecular genetic analysis (allele frequency of
1528G>C mutation: 87%)

Treatment:
- avoidance of fasting;
- low-fat diet high in complex carbohydrates
- fat primarily as medium chain triglycerides

Elevated Lactic Acid in

MTFP/LCHAD Deficiency
Direct inhibition of mitochondrial oxidative
phosphorylation by 3-hydroxypalmitoyl-CoA
(Ventura J Inher Met Dis 1996)
Long-chain acyl-carnitines may inhibit the
pyruvate dehydrogenase complex (Moore Int J Biochem
1992)

Long-chain acyl-CoA esters inhibit the

mitochondrial ATP/ADP carrier and the
dicarboxylic carrier in vitro (Halperin PNAS 1972)

Molecular Testing
Molecular studies revealed a homozygous 5
bp deletion in exon 4 of the alpha subunit of
the MTFP gene involving base pairs 274 to
278
This novel mutation creates a premature
stop codon
Possible uniparental disomy

Isovaleric acidemia
Defect in breakdown of Isovaleryl-CoA
Product of leucine catabolism

Build up of isovaleric acid

2 clinical manifestations
Severe neonatal presentation
3-6 days
50% mortality

Chronic intermittent type

< 1year
Precipitated by URI or high protein intake
Frequency of episodes decreases with age
Often unveiled when mother stops breast feeding

ISOVALERIC ACIDEMIA
Siblings:
Healthy newborn girl, elevated C5-AcylCarnitine
Increased urine isovalerylglycine
Older brother
? Mild motor delays
Increased Urine Isovalerylglycine
Self treats avoids excessive protein
One parent found to have A282V mutation in
IVCoADehydrogenase gene,
now known to be associated with attenuated disease.
Other parents mutation classical.

MULTIFACTORIAL ?
Siblings:
Boy from a difficult pregnancy with low tone, failure to gain weight,
recurrent illness with fluctuating blood sugars, unusual odor
leg pains, muscle cramps, cardiomyopathy, pancreatic
insufficiency.
Brother with low tone, feeding problems, failure to gain weight,
hypertrophic cardiac septum, cyclical low white blood cells,
gastroesophageal reflux, intermittent lethargy with illness
elevated blood ammonia, fatty liver
Mother with chronic fatigue, neurological symptoms with brain scan
leukodystrophy

MULTIFACTORIAL ?
Both boys:
NBS suggested Glutaric Aciduria II: not confirmed by enzymes
Unusual pattern of fat oxidation in skin cells
Urinary hexanoylglycine: MCAD A985G
Heterozygous for an Unusual new mutation in SPINK1,
(Pancreatitis)
A common variant in CFTR (Cystic Fibrosis)
No mtDNA mutations
Normal Electron Transport Chain Analysis
Some Response to Carnitine, Glycine, Riboflavin and Low Fat

Where Are Genes and What Do They Do?

Chromosome

One Gene
Out of ~3000/chromosome

Nucleus

Location
of Genes

)
Regulatory
Region
46 Total

Cell

B.

mRNA
Gene Expression

(Transfer of genetic information)

Function

DNA

C.
Example of
Genetic
Information
Transfer

Nucleus

Gene Expression
(Transfer of Genetic
Information)

Gene

RNA

Code
C G
for
C G
aa 1
G C
T A Code
T A for
aa 2
G C
A T Code
T A
for
aa 3
G C

Protein

aa 1
aa 2
aa 3
aa 4
aa
aa 5 6

etc.

Double Stranded DNA

Protein

A.

Cell

Protein Coding
Region

DNA

0.00006% of the human genome sequence

121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
1981
2041

AACTGTGTTC
TCTGCCGTTA
GGCAGgttgg
ggagacagag
ttttcccacc
TGGGGATCTG
GAAAGTGCTC
TGCCACACTG
gagtctatgg
taggaagggg
agtgtggaag
cttttgttta
atgccttaac
aaaaaacttt
catattcata
catatttatg
taattttgca
cttatttcta
tgcctctttg
tatttctgca
gctaatagca
ggattattct
tcccacagCT
TCACCCCACC
CCCACAAGTA
TCCCTAAGTC
GCCTAATAAA
tactaaaaag
caaaccttgg
gctaatgcac
ttcttgtaga
ttgttttagc
tcagccttga

ACTAGCAACC
CTGCCCTGTG
tatcaaggtt
aagactcttg
cttagGCTGC
TCCACTCCTG
GGTGCCTTTA
AGTGAGCTGC
gacccttgat
agaagtaaca
tctcaggatc
attcttgctt
attgtgtata
acacagtctg
atctccctac
ggttaaagtg
tttgtaattt
atactttccc
caccattcta
tataaatatt
gctacaatcc
gagtccaagc
CCTGGGCAAC
AGTGCAGGCT
TCACTAAGCT
CAACTACTAA
AAACATTTAT
ggaatgtggg
gaaaatacac
attggcaaca
ggcttgattt
tgtcctcatg
ct

TCAAACAGAC
GGGCAAGGTG
acaagacagg
ggtttctgat
TGGTGGTCTA
ATGCTGTTAT
GTGATGGCCT
ACTGTGACAA
gttttctttc
gggtacagtt
gttttagttt
tctttttttt
acaaaaggaa
cctagtacat
tttattttct
taatgtttta
taaaaaatgc
taatctcttt
aagaataaca
tctgcatata
agctaccatt
taggcccttt
GTGCTGGTCT
GCCTATCAGA
CGCTTTCTTG
ACTGGGGGAT
TTTCATTGCa
aggtcagtgc
tatatcttaa
gcccctgatg
gcaggttaaa
aatgtctttt

ACCATGGTGC
AACGTGGATG
tttaaggaga
aggcactgac
CCCTTGGACC
GGGCAACCCT
GGCTCACCTG
GCTGCACGTG
cccttctttt
tagaatggga
cttttatttg
tcttctccgc
atatctctga
tactatttgg
tttattttta
atatgtgtac
tttcttcttt
ctttcagggc
gtgataattt
aattgtaact
ctgcttttat
tgctaatcat
GTGTGCTGGC
AAGTGGTGGC
CTGTCCAATT
ATTATGAAGG
atgatgtatt
atttaaaaca
actccatgaa
cctatgcctt
gttttgctat
cactacccat

TTTG
ACCTGACTCC
AAGTTGGTGG
ccaatagaaa
tctctctgcc
CAGAGGTTCT
AAGGTGAAGG
GACAACCTCA
GATCCTGAGA
ctatggttaa
aacagacgaa
ctgttcataa
aatttttact
gatacattaa
aatatatgtg
attgatacat
acatattgac
taatatactt
aataatgata
ctgggttaag
gatgtaagag
tttatggttg
gttcatacct
CCATCACTTT
TGGTGTGGCT
TCTATTAAAG
GCCTTGAGCA
taaattattt
taaagaaatg
agaaggtgag
attcatccct
gctgtatttt
ttgcttatcc

CTTCTGACAC
TGAGGAGAAG
TGAGGCCCTG
ctgggcatgt
tattggtcta
TTGAGTCCTT
CTCATGGCAA
AGGGCACCTT
ACTTCAGGgt
gttcatgtca
tgattgcatc
caattgtttt
attatactta
gtaacttaaa
tgcttatttg
aatcattata
caaatcaggg
ttttgtttat
caatgtatca
gcaatagcaa
gtttcatatt
ggataaggct
cttatcttcc
GGCAAAGAAT
AATGCCCTGG
GTTCCTTTGT
TCTGGATTCT
ctgaatattt
atgagctgtt
gctgcaacca
cagaaaagga
acattactta
tgcatctctc

Exons

Identifying differences in gene expression with microarrays

Egg
Normal Tissue

Tadpole
Malignant Tissue

Higher in Egg
/Normal Tissue
Higher in tadpole
/Malignant Tissue
Each spot contains
DNA from a known
cDNA or an EST

From Lehninger

From Lodish et al.