(Published in NEUROLOGY, Feb 12, 2003)

1. Metabolic Testing

While no standard has been set for what constitutes routine testing for an inborn
error of metabolism, the studies most commonly performed on a screening basis include
measures of serum glucose, bicarbonate, lactate, pyruvate, ammonia, creatine kinase, and
amino acids and of urine pH, ketones, and organic acids. Measures of serum long chain
fatty acids, carnitines, and acylcarnitines, of urine mucopolysaccharides, and of
cerebrospinal fluid lactate and amino acids are more often considered to be part of a
second tier of tests, performed when initial screening tests are positive or when there are
specific clinical suspicions. The tests that are frequently available only on a send-out
basis are further described.

Amino Acids

Amino acid profiles can be performed on serum, urine, cerebrospinal fluid, or
amniotic fluid and are available at numerous laboratories throughout the world.
Concentrations of amino acids are determined by high performance liquid
chromatography (HPLC) separation and colorimetric reactions to ninhydryl or other
reagents. Most laboratories require 2-3 milliliters of blood, drawn in a heparin containing
tube (green top) and separated immediately, 5-10 ml of urine without preservatives, or 1
ml of cerebrospinal fluid without preservatives. Samples should be maintained at a
temperature of -20ºC and transported to the laboratory on dry ice by overnight carrier.
Results, including individual amino acid concentrations and an interpretation of the
profile, are usually available within 2-5 days.

Organic Acids

Screening tests for organic acids in the urine are widely available. Uncharged
organic acids are separated by their solubility in organic solvents and then measured by
combination gas chromatography and mass spectrometry. A random sample of 5-20 ml
of urine, without preservatives, should be frozen at -20ºC and shipped on dry ice. Results
are usually available within 2-3 days.

Acylcarnitines

A serum profile of free carnitine and acylcarnitines, evaluating disorders of fatty
acid oxidation, is widely available and most often performed using tandem mass
spectrometry. Blood spot cards at room temperature are sufficient and results are usually
available in 3-5 days.

Mucopolysaccharides

Urine mucopolysaccharide screening tests look for greatly elevated levels of
glycosaminoglycans excreted by patients with mucopolysaccharidoses. The tests are
widely available and most often use dyes which undergo metachromasia in the presence
of these substances. A 5-10 ml random sample of urine is required, however a first void
of the day sample is preferred due its greater concentration. Results are usually available
within 2-5 days.

2. Genetic Testing

Routine karyotype analysis is available from most genetic laboratories. Other
more specific genetic tests may be available on a clinical basis from only a limited
number of laboratories. An excellent source of information regarding common genetic
syndromes, genetic test availability, and individual laboratory sample requirements,
testing procedures, and contact information is available via the internet at the publicly
funded website www.genetests.org
Routine cytogenetic testing to detect aneuploidy, large deletions, and large
rearrangements is widely available and can be performed on blood or tissue samples.
Most laboratories offer high resolution analysis of blood specimens, which examines
DNA banding patterns within cells undergoing the prometaphase stage of mitosis. High
resolution chromosome analysis requires 1-5 ml of blood in a tube with heparin
maintained at room temperature, and provides results within 10 days.

Fragile X

Fragile X syndrome is associated with large expansions of the number of CGG
triplet repeats within the FMR-1 gene on the X chromosome. Testing is widely available
and uses a combination of polymerase chain reaction (PCR) and Southern analysis.
Quantification of the number of CGG repeats identifies large expansions, greater than
200, which are associated with developmental delay as well as smaller expansions, from
44 to 199, as are seen in carriers. Most laboratories require 2-6 ml of blood in a tube with
EDTA (purple-top), which can be stored and sent at room temperature, and provide
results within 2-3 weeks.

Rett Syndrome

Testing for mutations in the MECP2 gene on chromosome Xq28, found in
approximately 80% of patients with clinically diagnosed Rett syndrome, is currently
available on a clinical basis from 11 labs in the world, 6 of which are in the United States.
Testing consists of initial screening using PCR amplification of three exons contained in
the MECP2 gene coding region, followed by denaturing high performance liquid
chromatography (DHPLC) analysis. Positive and negative samples then undergo
automated DNA sequencing. These methods have a sensitivity of 99%. A blood sample
of 2-6 ml in a tube with EDTA (purple-top) should be stored and sent at room
temperature. Results are available within 4-6 weeks.

Subtelomeric Chromosomal Analysis

Subtelomeric chromosomal rearrangements and submicroscopic deletions can be
detected by fluorescence in situ hybridization (FISH) with telomere region-specific
probes, available on a clinical basis in many laboratoreis. Testing generally requires 1-5
ml of blood in a tube with heparin (green-top), stored and sent at room temperature, and
provides results in about 10 days.

Prader-Willi and Angelman Syndromes

Prader-Willi syndrome (PWS) is associated with characteristic facial
dysmorphisms, hypogonadism, developmental delay, feeding problems and hypotonia in
infancy followed by hyperphagia and obesity in childhood. The genetic basis is the
absence of the paternally derived portions of chromosome 15q11-q13. In 70-75% of
cases paternal chromosome contains a deletion within this region. Another 20-25% are
cases of maternal uniparental disomy (UPD) and have received two copies of
chromosome 15 from their mother and none from their father. Approximately 2% of
cases have an abnormality in the imprinting process which causes nonexpression of
paternal genes in the PWS critical region..
Angelman syndrome (AS) has features of characteristic facial dysmorphisms,
developmental delay, speech impairment, ataxia or tremor, and frequent laughter or
smiling. Other frequently associated findings are acquired microcephaly and seizures.
The genetic basis for AS is similar to that of PWS, involving the loss of the maternal
contribution of chromosome 15q11-q13. Approximately 70% of cases are the result of a
maternally derived deletion, 3-5% of cases have paternal UPD, with two copies of
chromosome 15 from their father and none from their mother, and 7-9% of cases have an
abnormality in the imprinting process which causes nonexpression of maternal genes in
the AS critical region. These genetic causes would all exhibit abnormal DNA methylation
patterns. Another 5% of AS patients have point mutations of the UBE3A gene, found
within the 15q11-q13 region, while the genetic basis of 10-14% of AS patients remains
undetermined.
Southern blot analysis to detect abnormal methylation patterns within the
PWS/AS critical region will identify patients with either chromosomal deletions or with
UPD. This will detect nearly all patients with PWS but has a sensitivity of only 80% for
patients with AS. Methylation testing requires 2-6 ml of blood in a tube with EDTA
(purple-top) and 2-3 weeks for results. FISH analysis for PWS or AS, which can
demonstrate the presence of small deletions, insertions, and duplications, requires 1-5 ml
of blood in a tube with heparin (green-top) and 10 days for results. These testing
methods are widely available.
Analysis of the UBE3A gene, via PCR amplification and automated sequencing,
is available from a more limited number of laboratories on a clinical basis for patients
with documented negative methylation testing. This requires 6 ml of blood in a tube with
EDTA (purple-top) and 4 weeks for results.