Frequently Asked Questions About Angelman Syndrome
Angelman Syndrome: Testing 101
T his is an explanation of the genetic tests available for the known causes of Angelman
Syndrome. This is written for parents and lay people who do not have a working
knowledge of genetics or molecular biology but would like to understand the more
technical details of these tests.
T here are numerous tests for Angelman Syndrome. If you are trying to understand the
test results of your child or someone else, it is important to know exactly what test(s)
your child has already had. It is common for parents to know their child was tested for
AS and to remember the geneticist telling them the test was either positive or negative,
but there are several genetic errors which cause AS and each test only detects a certain
number of cases. If a child has already been tested and the results were negative, it is
important to know which tests were conducted to know if AS has truly been ruled out.
Additionally, about one in ten individuals who have all the key symptoms of Angelman
Authors Syndrome have normal or “negative” results on all these tests. These individuals may
receive a “clinical” diagnosis of AS. This means the individual meets the diagnostic
Erin Sheldon. and
criteria of AS but we don’t know what kind of genetic error is causing their symptoms.
Rebecca D Burdine
Remember that all people with AS had “clinical” diagnoses until as recently as the early
1990’s when the first tests were developed to test Chromosome 15 for deletions. Further
research into the genetics of Angelman Syndrome is likely to yield more causes of AS
and, therefore, more tests to confirm the clinical diagnosis.
Reviewed for accuracy
by Wen-Hann Tan,
BMBS Children’s Hospi-
tal Boston, Boston MA
Standard Chromosome Analysis or Cytogenetics Analysis
A ny expectant parent who had chorionic villus sampling (CVS) or amniocentesis
during a pregnancy has had a version of this test. These standard tests look for
obvious changes in chromosome structure and can detect syndromes where extremely
large deletions, rearrangements or duplications occur. For example, in Down Syndrome
an additional fragment of Chromosome 21 is present and can be seen on the karyotype
that is generated with this test. If we think of our chromosomes like a set of encyclopedias,
then the standard chromosome analysis is like lining up the set of encyclopedias in
numerical order and ensuring that there are two of each chromosome with no extra or
missing “volumes” of any chromosome. This is not a detailed test and only rarely reveals
small chromosome errors like the common AS deletions. This standard chromosome
test is useful to rule out syndromes that might appear similar to AS in the young child.
Expectant parents should note that the samples taken for standard karyotype tests can
be used for FISH analysis to detect specific syndromes. Currently, testing for Angelman
Syndrome is not routinely included in prenatal testing because the syndrome is so rare.
This is a karyotype, or map, of the human
chromosomes. You can see each set of
chromosomes matched up from biggest
(chromosome 1) to smallest (chromosome
22). We can think of each chromosome as
Volume 1 a volume in an encyclopedia with two
copies of each chromosome. Note that
No. 2 deletions that cause Angelman Syndrome
Issued May are typically too small to be detected on a
The DNA methylation test
T his test may also be called Southern Hybridization Methylation Specific PCR Assay or Methylation
Specific PCR Test. The key word is “Methylation” and it is the most sensitive diagnostic test for Angelman
Syndrome. This test will positively identify about 80% of individuals with Angelman Syndrome. Methylation
refers to a chemical “tag” added to DNA and can be used to identify whether the DNA was contributed by
the mother or the father. Where this tag is added occurs in a distinct pattern if it came from the mother and
a different distinct pattern if it came from the father. The pattern of this tag can be examined to determine
which parent contributed the DNA. The DNA methylation test will determine if an individual has one of
each pattern (one from each parent) or an Angelman Syndrome pattern (where only the pattern from the
father is present.) Using our encyclopedia analogy, DNA is like the pages inside the encyclopedia volumes.
The methylation test is like opening the two “volumes” of chromosome 15 and looking up the page of
the specific “chapter” contributed by each parent to see which parent’s pattern is visible on the page.
See the illustration below for one way scientists determine which “chapters” are present in an individual’s
chromosomes. If this test reveals that the distinct maternal pattern is missing from the special chapter,
the individual being tested has Angelman Syndrome and further testing is needed to determine if this is
caused by a deletion, UPD or an ICD.
This is one way scientists can determine which methylation patterns are present on Chromosome 15. There are other ways
this test can be done on a technical level, but this example is to show you what a result might look like. Step 1 - a blood
sample is taken from the individual to be tested. The DNA from the blood cells is isolated. Step 2 - The DNA from step
one contains all of the individual’s DNA, including both copies of chromosome 15. Methyl groups are chemical tags added
to DNA. These methyl group “tags” exist on each “volume” of Chromosome 15 where we would expect to see the distinct
pattern associated with only the mother or only the father. The places on the DNA where methyl groups are added depend
on which parent the chromosome came from. Note in the diagram that the maternal copy has a different number and
different placement of methyl groups (shown as purple ovals) compared to the paternal copy. The DNA sample is cut into
smaller pieces with enzymes. An enzyme is a protein that acts like a scissor and cuts DNA only in specific regions. In the
maternal copy, one specific site is blocked by the methyl group and can’t be accessed by the scissor. The paternal copy can be
cut because this area is not hidden from the enzyme. Step 3 - The DNA pieces generated in Step 2 are separated by size on an
agarose gel. An agarose gel is a lot like a piece of Jello that has hardened. DNA can be added to the top of the gel and forced
to move through the gel by applying electricity to the gel. DNA has a negative charge and will move towards a positive
electric source. Since the DNA pieces have to find their way around the Jello particles, smaller pieces move faster than
larger ones, thus smaller pieces move closer to the bottom of the gel. Note in step 2 that the paternal copy will be cut while
the maternal copy will not be cut, thus the paternal copy will produce a smaller piece of DNA. Researchers can then use a
procedure known as a “southern blot” to visualize only the DNA pieces from the Angelman/Prader-Willi area of Chromosome
15. Representative results are visible on the illustration above. In Lane 1 and Lane 6, both the maternal copy (larger black
band) and paternal copy (smaller black band) are present on the gel, thus these individuals have correct methylation on
their copies of Chromosome 15. In Lanes 2 and 3, these individuals only have the paternal methylation pattern present,
thus these individuals are missing the maternal methylation pattern and have Angelman Syndrome (AS). For comparison,
the individuals in Lanes 4 and 5 have only the maternal methylation pattern and are missing the paternal pattern. These
individuals have Prader-Willi Syndrome (PWS).
The FISH test
T he FISH stands for “fluorescent in situ hybridization”. This test determines if part of Chromosome 15
is physically missing from the individual. This test is like counting the pages of both “volumes” of
Chromosome 15 and detecting where there are missing pages in the volume. If there are too few pages in
one of the volumes it as though a section has been torn out, and we know that some of the “pages” have
been deleted. This test cannot tell us which parent’s chromosome is missing pages so a FISH test needs
to be performed along with the DNA methylation test to confirm the individual has Angelman Syndrome
and not Prader-Willi Syndrome, a disorder caused by missing a “chapter” from the father’s chromosome
rather than the mother’s.
If an individual has a positive methylation test for AS and a positive FISH test for loss of Chromosome
15, then we know the person has Angelman Syndrome caused by a deletion. This is the most common
cause of AS and about 70% of individuals with AS have this deletion. These deletions can occur randomly
months before the mother was even born. A woman’s eggs form when she herself is a fetus at about five
months gestation. The chromosomes in her developing eggs are rapidly duplicating and separating as
the eggs multiply, and errors in chromosome structure can easily occur at this stage. These cell errors
are common and usually harmless. Most eggs containing errors, if fertilized, fail to develop and do not
result in pregnancy. Some deletions are harmless and can result in a perfectly typical baby. Deletions are
a problem in the small number of cases where the paternal chromosome that will later fertilize the egg
cannot compensate for the missing information. This is what occurs in Angelman Syndrome. Chromosome
deletions are relatively common and there is no evidence that anything specific caused it or could have
prevented it; it is simply random.
If the FISH test is positive but the methylation test is negative for Angelman Syndrome, then the individual
has Prader-Willi Syndrome, a distinctly different disorder caused by the same mechanism of “imprinting”.
In these individuals, the deletion occurred on the paternal chromosome so it is like a “chapter” is missing
from the father’s volume for which the mother’s chromosome can’t compensate. In the methylation test,
these individuals will show only the methylation pattern from the mother and are missing the pattern
from the father.
This is an actual picture of chromosomes that have been tested using FISH. To
perform this test for AS, a probe for the UBE3A gene is generated. The probe is a
sequence that will find its exact match on Chromosome 15 and bind only to the
UBE3A gene. The probe is also “tagged” with a fluorescent molecule so that it can be
seen on the chromosomes under a microscope. The picture above shows a positive
FISH test result for Angelman Syndrome. In this test two probes were tagged with
florescent molecules; one probe is tagged in green and one in red. A common gene
on a different chromosome was tagged with the green fluorescent molecule; the
two green dots confirm there are two copies of this gene present in the sample,
showing that both chromosomes have the gene that the green tag was testing for.
The probe made to find UBE3A was tagged with a red fluorescent molecule but
there is only one red dot in this picture. This is an abnormal result and means that
one copy of UBE3A is missing from this individual’s DNA. This lab result can’t tell us
if the one chromosome with the red tag is from the maternal or paternal copy of
Chromosome 15 but it is a positive result indicating that this person has a deletion
on one of their two Chromosome 15s. The DNA methylation test is needed to
confirm whether this individual has Angelman Syndrome or Prader-Willi Syndrome.
PCR Assay to detect uniparental disomy (UPD) and imprinting center defects (ICD)
If an individual has a positive methylation test for Angelman Syndrome, but a negative FISH test, then they
either have UPD or ICD. A PCR (polymerase chain reaction) test is then used to determine if the individual
has two copies of the father’s chromosome 15 (UPD) or whether the individual has one chromosome from
each parent, but with incorrect methylation (ICD). This test requires a blood sample from the parents
as well as the individual so that genetic information specific to each parent can be searched for in the
child’s chromosomes. It is like taking down both “volumes” of Chromosome 15 and searching for sections
contributed by each specific parent. It looks for information that would be present in both volumes, as
well as information that only the father’s “volume” and only the mother’s “volume” would contain. If the
test reveals that both “volumes” were inherited from the father and neither was inherited from the mother,
then the test is positive for Angelman Syndrome caused by UPD. UPD is a random occurrence at the time
of conception where the egg loses the mother’s copy of Chromosome 15 and the father’s copy duplicates
itself to compensate for this absence. Nothing is known to cause it or to prevent it, it is simply random.
If the PCR Assay shows that both parents contributed a “volume” 15 just as nature intended, this result
indicates that the individual must have an ICD. In individuals with an ICD, even though the individual
inherited a copy of Chromosome 15 from the mother, and UBE3A is present, the methylation pattern is
not properly established. An ICD is like having an error in the “table of contents” of the mother’s 15th
chromosome. The volume is complete but the cells can’t find the information the brain needs because of
a “typo” in the table of contents. The ICD can be further examined to understand the nature of this “typo.”
The typo can be a tiny deletion, as though a section of the table of contents was erased, or a mutation
as though the pages were “renumbered” or scrambled so that the cells in the brain can’t find the pages
they need. Some cases of ICD are hereditary, meaning that the mother had this error in her own “table of
contents”, and further testing of the mother is indicated to see if she carries this error. If this “typo” was
random and is not present in the mother’s own “volume”, then her chances of having another child with
AS are very low. But if the mother herself has this “typo”, then her chances of having another child with the
same typo are at least 50%. To understand how a mother can carry this “typo” and not have any symptoms
of AS herself, remember that if the mother inherited this typo from her father, it would have been silent in
her and would not have caused her any problems. For more examples, look at the genetic scenarios under
A bout 20% of people who have all the symptoms of Angelman Syndrome will get negative results
on all of the tests listed so far. The next test is to sequence their UBE3A gene directly. Sequencing
is like opening up the Chromosome 15 inherited from the mother and looking at the UBE3A “chapter”
and carefully spell-checking each word. This is more complicated than it sounds. Each of us have slight
differences in how the “words” of our DNA are spelled, but these differences do not change the meaning
of the information and do not affect how we learn or develop. For example, the word “color” can also
be spelled “colour” and both are correct. Changes that occur in DNA that do not appear to affect gene
function are called polymorphisms; these are harmless differences that naturally occur between people.
If an individual has a change in the sequence of their UBE3A gene, it is important to determine if this
change is a harmless polymorphism, or an actual mutation that affects the gene’s ability to function that
has caused Angelman Syndrome in the person being tested.
T o understand how this test works, you need to understand how your DNA is used to produce proteins
such as UBE3A. DNA sequence is made of nucleotides that come in 4 types: A (adenine), C (cytosine), G
(guanine), and T (thymine). These four “letters” are strung together in different combinations to produce
chromosomes. DNA is “double stranded” and looks like a twisted ladder. The nucleotides make the “rungs”
of the ladder by binding to each other. A binds to T. C binds to G.
T he sequence of A, C, G, and Ts on your chromosomes can be read by your cells just like you can read a
book. When a sequence provides the instructions needed to produce a protein, we call this sequence
a gene. Thus the UBE3A gene is a sequence of A, C, G, and Ts on Chromosome 15 that tell the cell how
to make the UBE3A protein. To make the protein, your cell “reads” the DNA and produces a copy of
the important information in a strand of RNA. In RNA the sequence is the same as the DNA, but the T
nucleotide is replaced with U (uracil). You can think of RNA as an important page in the encyclopedia
that you photocopy in order to take away to another location. The cell then “reads” RNA by looking at
nucleotides in groups of three. These three letter words are referred to as the genetic code and tell the
cell which amino acids should be placed next to each other in order to make a protein that is functional.
This figure illustrates how DNA provides the sequence information needed to make proteins. DNA is double stranded and
made up of the four nucleotides A, C, G, and T. Note that in the two strands, A always pairs with T and C always pairs with G.
The cell can copy DNA sequence needed to make a protein into a single strand molecule called RNA. Note that the RNA in
this example is exactly the same as the top strand of DNA except that the T nucleotide is replaced with U. The RNA is read by
the cell as three letter “words”. Each “word” tells the cell which amino acid should be added together in what order to make
a functional protein. In the example above, the “word” CUU tells the cell to add amino acid Leucine (L) next to amino acid
Valine (V) which was encoded by the “word” GUG.
T here are many areas in all our genes that can have random DNA differences or misspellings which
don’t affect anything about how we learn or develop. Some of these differences are natural variations
(like “color” and “colour”) while others are misspellings that are too minor to affect the meaning of the
sentence. If we looked carefully at the DNA or “text” in each of our chromosomes, we’d find all kinds of
misspelled “words”. Not all differences in DNA sequence are created equal, just like a single misspelling
in a sentence can be insignificant or can completely change the meaning of the sentence. It is difficult to
simply look at the UBE3A gene and know how significant any one misspelling is. To understand the types
of differences that can occur, and what they might mean, please look at the following diagram where we
compare sequences and changes with sentences from a book.
O n the left is our sequence example and the protein it would encode. On the right is our encyclopedia
example and sentences. In the first example of a sequence change (#1), this change is a polymorphism
and does not affect the protein that is made. You can see that both the “words” ACC and ACG mean
Threonine to the cell. So although there is a change, it doesn’t affect the protein. In our encyclopedia
analogy, the sentence is slightly changed, but the meaning is exactly the same.
I n the second example of a sequence change (#2), this change did alter the meaning of the sequence and
changed the protein. In this case, the word meaning Leucine (L) has changed to mean the word Proline
(P). This can significantly alter the protein and make it non-functional or less functional. In our sentence
analogy you can see that one word has changed and now the sentence fails to make sense.
I n the third example (#3), a change in the sequence tells the cell to stop making the protein prematurely.
Normally the “stop” sequence, in this case UAA, is only found at the very end of the protein. This sequence
change has added this stop instruction too early and the whole protein is not made. In our analogy, it is
like putting the final period early in the sentence which stops the sentence too soon and it no longer
I n the fourth example (#4), a change in sequence has occurred where an extra nucleotide was added into
the sequence. Since we know that cells read sequence in three letter words, this shifts the words and
makes new ones from the original code. The same thing can occur if deletions of one or more nucleotides
is found. Any additions or deletions that change the three letter word spacing will likely make a non-
functional protein. In our sentence analogy, the spacing has changed and now the words no longer make
I n example #1, this would not be a change that causes Angelman Syndrome. In example #3, this change
would most likely cause Angelman Syndrome as proteins that aren’t fully made are usually non-functional.
Similarly, example #4 would be confirmation of an Angelman Syndrome diagnosis.
T he changes in example #2 are more tricky to determine if they are harmful or not. In the example
provided, this change would likely cause Angelman Syndrome, because scientists know that placing the
amino acid Proline (P) into proteins in the incorrect place often causes proteins to become non-functional.
But other amino acid changes aren’t of huge consequence. For example, changing the amino acid alanine
(A) for threonine (T) isn’t usually harmful in protein function. So how do we decide if the change is harmful
(switching “encodes” for “explodes” in our sentence) or a polymorphism (switching “encodes” for “makes” in
our sentence which wouldn’t change the meaning)? First, the UBE3A gene would need to be sequenced
in the parents and/or in any available family members. To understand how this type of analysis would tell
us whether the change causes Angelman Syndrome, examine these scenarios below. In all these cases, a
mutation or “misspelling” is discovered in a child’s UBE3A gene and the parent’s and/or siblings are then
- Neither parent has the same error in their UBE3A gene. If the child meets the clinical criteria of
Angelman Syndrome then we would assume he or she has AS caused by a random UBE3A
mutation and this test result would likely be positive for AS.
- The mother’s UBE3A gene is normal but the father has the same mutation as the child. Because
the father’s UBE3A is “silent” on his child’s chromosome, we assume this mutation is a harmless
polymorphism. This test result would be negative for AS.
- The mother’s UBE3A has the same mutation as the child’s, but her other children also have the
same mutation but do not meet the clinical criteria for Angelman Syndrome. We assume this
mutation is a harmless polymorphism. This test would be negative for AS.