Genetic Testing for Angelman Syndrome 101This 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.
If you want to jump to a specific test, please use the links below.
Standard Chromosome Analysis or Cytogenetics Analysis
The DNA methylation test
The FISH test
PCR Assay to detect uniparental disomy (UPD) and imprinting center defects (ICD)
There 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 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 criteria of AS but we don't know what kind of genetic error is causing their symptoms. 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.
Standard Chromosome Analysis or Cytogenetics AnalysisAny 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.
The DNA methylation testThis 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.
The FISH testThe 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.
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 UBE3A sequencing.
UBE3A SequencingAbout 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.
To 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.
The 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.
There 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.
On 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.
In 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.
In 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 makes sense.
In 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 sense.
In 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.
The 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 tested:
- 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.
- The mother's UBE3A gene has the same mutation as the child's but the father's UBE3A is normal. The mother's parents are tested and the maternal grandmother also has this mutation. We assume this is a harmless polymorphism because the mother inherited the mutation from her mother but does not herself have AS. This test result would be negative for AS.
- The mother's UBE3A gene has the same mutation as the child's but the father's is normal. The mother's parents are tested and her father has the same mutation. We assume the child has AS caused by an inherited UBE3A gene mutation which originated with the maternal grandfather. All of his children have a 50% chance of carrying this gene mutation but it would not cause AS in his own children since his UBE3A is not expressed in his offspring. However, all of his daughters could have children with AS as this mutated copy will be inherited by their children from their mother. The affected child's mother is assumed to have a 50% chance of having more children with AS. UBE3A gene testing is indicated for all of the maternal grandfather's children to see if they inherited this gene mutation too.
- As more and more individuals with UBE3A mutations are discovered, we will also find that multiple individuals have the same sequence changes in UBE3A. If multiple unrelated individuals who fit the clinical criteria for Angelman Syndrome have the same sequence change, and this change is not present in individuals who do not have AS, it is likely this change affects the function of UBE3A. This test result would be positive for AS.
Clinical DiagnosisAny individual who meets the diagnostic criteria for Angelman Syndrome may receive a clinical diagnosis of AS. The tests described above will have normal (negative) results for a certain number of individuals who appear to have AS. Until further research explains more about how UBE3A functions to affect speech, movement and learning, these individuals and their families will not know the mechanism that is causing the symptoms of AS. Because some of these cases are hereditary, it is worthwhile for any individual who meets the clinical criteria of Angelman Syndrome to undergo the full spectrum of testing so that family members will have the most information possible about whether they are at risk of having more family members with this disorder.
Differential DiagnosisAny individual who has had negative results on the above tests could have a syndrome or disorder that closely mimics the symptoms of Angelman Syndrome. Doctors should investigate whether the individual could have any of the following disorders instead of AS. The most useful test at this stage might involve CGH Microarray analysis (also called Genomic Microarray or Chromosomal Microarray). This test can search for missing or extra chromosomal material in regions that are known to be affected in a variety of disorders. While this test will not find every change in a person's genome, it can evaluate areas that are involved in greater than 200 known disorders at once. The microarray is similar to scanning hundreds of specific volumes in the set of encylopedias to look for missing pages, extra pages, or pages that are under- expressed and appear "washed out". This is a relatively new test and is constantly evolving to test for more disorders. Currently, the test is expensive and is not covered by all insurance companies. It is important to realize that this test evaluates only selected regions of the genome. The test does not cover everything that could be wrong with an individual, and it will not identify changes in regions that are not evaluated.
This article was authored by Rebecca D Burdine PhD and Erin Sheldon
Reviewed for accuracy by Wen-Hann Tan, BMBS Children's Hospital Boston, Boston MA
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