The UBE3A Gene and its Role in Angelman Syndrome

 By: Livija Medne, MS, CGC  - April 2000

 

The Children's Hospital of Philadelphia

Clinical Genetics Center, Suite 9S20
34th Street and Civic Center Boulevard
Philadelphia, PA 19104
phone:    215-590-2920
fax:         215-590-3298

Angelman syndrome (AS) is a complex neurological disorder that is caused by various genetic rnechanisms. Although the mechanisms vary in their etiology and recurrence risks, all cause the absence of proper expression of the disease gene called UBE3A from the maternally inherited chromosome 15.

BASIC GENETICS  

Every cell in the human body (except red blood cells called erythrocytes) contains all the genetic material, called DNA (short for deoxyribonucleic acid), that makes up the estimated 100,000 human genes. DNA strands are found in the chromosomes of the nucleus as a double helix, in which two DNA molecules are held together to form a duplex. DNA is a very large molecule and is composed of a sugar and phosphate backbone with I of 4 (adenine, guanine, thymine, cytosine) bases attached to it (figure 1 ). Four bases provide the variation in the human DNA. Each gene has its unique base pair sequence. Three base pairs to­gether encode an amino acid and the variation in the combination leads to 20 different amino acids. A chain of amino acids makes up a protein which is the gene product, and a functional unit. This is just like three letters (bases) making up a word (amino acid) and a chain of words making up a sentence (protein). A specific change in the appropriate DNA base pair sequence is a mutation. A mutation in a gene leads to a production of an abnormal protein or causes a protein not to be made at all. 

DNA makes up the backbone of a chromosome and at certain stages in the life of a cell we can best visualize T C, G. it under a microscope. It is at this specific stage the chromosomes can be visualized and photographed to make a karyotype. There are two copies of each chromosome or 23 pairs, as one chromosome of each pair comes from the mother (maternal) and the other from the father (paternal). The first 22 pairs are called autosomes. The 23 rd pair makes up sex chromosomes as they determine one's gender. Women typically have two X chromosomes (46,XX) but men have one X and one Y chromosome (46,XY).

 GENETICS OF AS

Angelman syndrome was known as a distinct clinical entity before the genetics were fully understood. It has taken years of research to elucidate the different genetic mechanisms that can lead to AS. There are 4 major genetic mechanisms that cause Angelman syndrome (figure 2):

1. Chromosome 15q11 -q13 deletion (a very small piece missing) accounts for 65-75% of AS cases and has a less than 1% recurrence risk. It was first observed on high resolution chromosome analysis that some patients with AS had a very small piece missing from the long (q) arm of chromo some 15 between bands q 11-13. This led to the development of the FISH (fluorescence in-situ hybridization) test to readily diagnose this common deletion from the maternally derived chromosome 15.

2. Paternal uniparental disomy (UPD) accounts for 3-5% of AS cases and has less than I% recurrence. Patients with UPD have two paternal copies of chromosome 15 and no maternal copy of chromosome 15. These observations suggest that each copy of chromosome 15 is marked with "a label" (an imprint) for its parental origin. This is thought to regulate expression of genes on each chromosome 15. Thus AS represents a loss of functionally important imprinted genes on chromosome 15 that are only expressed from the maternal chromosome 15.

3. Imprinting center (IC) mutations account for 7-9% of AS cases, and can have significant recurrence. The imprinting center acts as the 'switch' that turns on the maternal copy of the UBE3A gene and turns off the paternal copy in certain tissues of the central nervous system. If there is a mutation in the IC, it cannot perform its 'switch' function. If the IC mutation occurs sporadically in the affected individual, the recurrence risk is less than 1%. However, if the patient's mother carries the IC mutation on her own paternally inherited chromosome 15, there is a 50% risk of recurrence. 

4. UBE3A mutations account for 6-20% of AS cases. If it happens sporadically in the affected individual, the recurrence risk is less than 1%. However, if the patient's mother carries the UBE3A mutation on her own paternally inherited chromosome 15, there is a 50% recurrence risk. Let's talk more about the UBE3A gene.

 THE UBE3A GENE

In 1996/1997, the laboratories of Dr. Joseph Wagstaff from Children's Hospital in Boston and Harvard School of Medicine and Dr. Arthur Beaudet from Baylor College of Medicine found a single gene on chromosome l5q called UBE3A that caused Angelman syndrome (figure 3). They showed that some patients with AS have mutations in the UBE3A gene. The gene encodes a protein called E6-AP ubiquitin protein ligase (also known as ubiquitin ligase 3). The exact mechanism of how the deficiency of this protein causes the clinical features of AS is not completely understood. However, it is known that E6-AP acts as an enzyme necessary for normal protein turnover within cells. This may suggest that the clinical findings are due to failure to degrade various proteins, accumulation of which may be deleterious to an individual.

What makes the UBE3A gene unique, is that it demonstrates tissue specific imprinting. The gene is expressed from maternal and paternal alleles in all tissues (organs) except specific parts of the central nervous system. UBE3A is imprinted in the human brain with the paternal copy of the gene being naturally silenced. In other words, in the brain the UBE3A is only expressed from the maternal copy. If this does not happen due to a mutation or deletion of UBE3A, the enzyme is not made and it is thought that certain proteins are not degraded in the brain. Recent animal studies have shown that the gene is preferentially expressed from the maternal allele with silencing of the paternal allele in the hippocampus and cerebellum in mice brains. The tissue specific imprinting tits the clinical presentation of AS since affected individuals have various neurologic problems and complications, but do not have involvement of other organ systems.

As mentioned above, UBE3A is naturally silenced on the paternally inherited copy in certain parts of the brain. Therefore, if a UBE3A mutation is inherited from the father, the person is unaffected as the paternal copy is not expressed. If the carrier of the UBE3A mutation is a male, he has a 50% chance of passing on the mutation, but is not at risk of having children with AS. Again, it is because the paternally inherited copy of the UBE3A gene is naturally silenced in the brain. if the carrier of the UBE3A mutation is a female, she also has a 50% chance of passing on the mutation. However, in this case if the mutation is passed on, the child will have Angelman syndrome. This is due to the fact that the maternal copy of the UBE3A gene has to function in the brain as the paternal copy is naturally silenced.

Figure 3 Genetic map of 15q11-q13 region.  cen- centromere (constriction on a chromosome that separates the short [p] and the long [q] arms of a chromosome); tel – telomere (end of a chromosome).The jagged lines indicate the two common centromeric breakpoints and one telomeric breakpoint.  The distance between a centromeric breakpoint and the telomeric breakpoint represents the deleted DNA in the common deletion.  Circles in gray indicate genes implicated in Prader-Willi syndrome (PWS).  The black circle represents the UBE3A – the disease gene in Angelman syndrome (AS).  The white circles represent other genes.  IC – imprinting center.

TESTING Testing for UBE3A is indicated if a patient clinically has AS and other mechanisms (15q 11-13 deletion, paternal UPD, IC mutations) have been ruled out. If a mutation is identified, the affected patient's mother needs to be tested to determine whether she carries the same mutation on her own paternally inherited chromosome 15. This is very important to determine accurate recurrence risks (table 1).

Currently testing is offered by three laboratories on a clinical or research basis. Clinical testing means that the specimen is examined and results are reported for the purpose of diagnosis, prevention and/or treatment in the care of an individual patient. There is a specific turnaround time for clinical tests that depends on the complexity of testing. There is a charge for clinical tests which also varies according to the test complexity. The bill for the test should be submitted to an insurance company for reimbursement. The laboratories offering clinical testing must be CLIA (Clinical Laboratory Improvement Act/ Amendment) approved which requires that certain quality and proficiency standards are met. Research testing means that the samples are examined for the purpose of better understanding of a condition, or for development of a clinical test. The laboratory is not required to issue a test result, although in general many laboratories will provide a verbal or written result if a mutation is identified. There is no set turn-around time for research tests. There is no fee for testing done on a research basis as the cost is covered by the researcher. A laboratory may deny participation in research if the patient or family does not meet the goals of the research project or if the laboratory has sufficient samples. Research laboratories are not subject to CLIA approval.

Clinical testing for UBE3A mutations is offered by two laboratories in the United States:

1. Baylor College of Medicine DNA Diagnostic Laboratory. The turn-around time is 4 weeks. The cost is $1800 for the index case and $250 for each additional family member to test for the familial mutation. For information on how to arrange the test, ask your health care provider to call 713-798-6536.

2. University of Chicago Genetics Services. The turn-around time is 4 weeks and the cost is $2000 for the index case. For information on how to arrange the test, ask your health care provider to call 1-888-824-3637.

Both laboratories use the same technique to identify UBE3A mutations. Testing is done by PCR amplification of the ten exams (building blocks) contained in the UBE3A gene coding region. PCR (polymerase chain reaction) is a technique used to produce mul­tiple copies of a specific DNA segment. Once enough DNA is produced, it is followed by automated direct sequencing of the ampli­fication products. This identifies the exact sequence of the base pairs in the UBE3A gene (figure 4c). The accuracy of this test is greater than 99%.

Research testing for UBE3A mutations is offered by Dr. Joseph Wagstaff's laboratory. His laboratory first screens the samples by single-strand conformation polymorphism (SSCP) (figure 4). If a shift in the DNA migration pattern is identified, the corresponding region of the UBE3A gene is sequenced to look for the specific mutation. Dr. Wagstaff will be at the Children's Hospital in Boston, MA until April 30, 2000. He can be reached there by calling 617-355-8043; e-mail: wagstaffgal.tch.harvard.edu. Dr. Wagstaff will be at the University of Virginia Health Center in Charlottesville, VA after May 1, 2000. The telephone number there is 804-924-2508.

The genetics of AS is a complex and evolving field. In the past few years we have been able to identify the various genetic mechanisms that cause AS. Studies are underway now to correlate the clinical features of the patients with their specific genetic mechanism of AS. The early data suggest that the prognosis does not differ significantly between the patients with different genetic mechanisms. However, knowing the specific genetic mechanism of AS is critical for accurate recurrence risk counseling. Availability of the UBE3A gene testing will now make diagnosis and recurrence risk counseling much more accurate.   Contact Livija: Livija Medne, MS, CGC Board Certified Genetic Counselor Clinical Genetics Center, Suite 9S20 Division of Human Genetics and Molecular Biology and Divison of Neurology The Children's Hospital of Philadelphia 34th Street and Civic Center Boulevard Philadelphia, PA 19104 phone:    215-590-2920 fax:         215-590-3298

Figure 4 SSCP helps establish a difference in the DNA base sequence due to a mutation.  Using special enzymes, DNA of the gene tested is ‘cut’ into smaller pieces and loaded in a special gel.  An electrical current is applied to the gel to cause DNA migration or movement down the gel.  A single base pair change will change the conformation (spatial arrangement) of DNA (4a) which in turn will lead to a different migration pattern (4a & 4b).  The part of the gene that showed abnormal migration is sequenced by a special computer.  A mutation is identified when the patient’s DNA base pair sequence differs from the normal standards.  Figure 4c shows the presence of base pair C substituting for the normal A.  This constitutes a mutation.

service@asclepius.com

Pages provided as a public service by: Harold Anderson
Revised: October 08, 2004 .