Charles A. Williams, M.D. 7/4/97
This one-day session provided an exciting update on several areas of AS research. I have reviewed the presentations given by the invited speakers and also summarized the presented abstracts.
Dr. Art Beaudet (Baylor College of Medicine) reviewed their group's mutation analyses on the ubiquitin protein ligase E3A (UBE3A) gene which appears to be the causative AS gene. UBE3A is an enzymatic component of a complex protein degradation system. termed the ubiquitin-proteasome pathway. Ubiquitin is a small protein molecule that can be attached to proteins via this pathway, thereby causing them to be degraded. However, we currently do not know what brain proteins the UBE3A enzyme is normally supposed to be degrading (see New England Journal of Medicine, 335:25, 1897-1905, 1996 for a review of ubiquitin-proteasome pathway).
Beaudet's lab has studied a group of "triple-non" AS individuals meaning that they were non-deletion, non-UPD and had no imprinting defect; they were thus FISH and methylation test negative. By sequencing all coding regions of UBE3A they were able to find mutations in only 20%. They have identified 12 AS patients with mutations and 10 of those mutations were truncating type (frameshift or nonsense DNA changes) that are predicted to disrupt the UBE3A protein by severely shortening it. He presented several theories as to how those "triple-non" patients without identifiable UBE3A mutations may still have abnormalities in the UBE3A gene, the most likely one involving abnormalities in upstream regulatory elements (i.e., a promoter). These regulatory regions however have not yet been characterized. UBE3A-negative persons can be termed "quadruple-non" and this group still comprises about 15% of all AS individuals. Current UBE3A mutation testing is being done only on a research basis and is not commercially available.
Beaudet then presented mouse data demonstrating that the UBE3A gene is
differentially expressed in brain. Paternal UPD mice, disomic for the corresponding AS
gene region (plus some additional regions), have diminished UBE3A protein in the
hippocampal, cerebellar perkinjie and olfactory areas (protein detected by in situ
hybridization). These observations have some relevance to humans especially regarding the
cerebellar ataxia in AS. The question of whether there is any olfactory dysfunction in AS
was raised but not answered.
Dr. Mark Lalande (Children's Hospital, Boston) reported on studies evaluating UBE3A.
In
humans, they have studied UBE3A expression in brain using RT-PCR. There are 5
isoforms (different variants) of UBE3A RNA. In brain, all isoforms appear reduced
in AS deletions compared to PWS deletions, again evidence that UBE3A is functioning
differently in maternal versus paternal derived deletions.
Lalande also reported on his studies involving chromosome pairing. He has demonstrated that the 15q11-13 regions appear to come in closer association (during the late S phase of the cell cycle) than do other regions of the chromosomes 15. This does not happen in chromosomes with 15q11-13 deletions or in chromosomes from AS and PWS individuals that test methylation positive. Preliminary data on 2 UBE3A mutation patients also fail to show closer pairing. Closer pairing has been seen in other imprinted chromosome regions (i.e., 11p15) but the significance to this pairing phenomenon is unknown.
Dr. Richard Olson (Departments of Pharmacology and Neurology, UCLA) reported on studies
involving epilepsy in 20 AS individuals (9 deletion, 4 UPD, 5 imprint, 2 UBE3A mutation)
and compared seizures in the 9 with deletions to the 11 others. Seizure types appear to be
similar combinations of atypical absence, myoclonia, tonic spasm and drop seizures) but
frequency and severity appeared greater in the deletion group. He hypothesized that the
deletion group has more seizure dysfirriction because of the deletion of GABA receptor
genes, particularly GABRB3. This gene maps close to UBE3A in the 15q11-13 region.
It encodes a type A receptor which is a ligand-gated chloride channel protein. There are
at least 17 GABA receptor genes and their protein products combine in groups of 5
(pentamers) to form the GABA receptors. In brain the most common combination is a1B2Y5
. In humans, postmortem analysis of AS deletion brain tissue revealed more decreased
benzodiazepine binding (a measure of GABAA receptors) than
expected if GABRB3 was not imprinted. The role of GABRB3 in the epilepsy in AS is thus
under further study.
Dr. Joseph Wagstaff (Children's Hospital, Boston) reviewed their work in characterizing
a
chromosomal inversion that was pivotal in isolating the UBE3A gene (Tatsuya Kishino
was a crucial investigator in this effort). They have now studied sporadic and familial
cases of AS that are Non-Deletion, non-UPD and non-Imprinting (termed NDUI patients,
similar to Beaudet's "triple-non" class) and found 4 different mutations (1
mutation in 2 brothers) accounting for an approximate 20% detection rate. The 4 mutations
were all predicted to lead to a non-flinctional UBE3A protein due to truncations.
Based on the initial results of UBE3A analysis, about 50% of the identified
mutations appear to be inherited (when. inherited, they were always from the mother), some
occurring when there was only 1 affected child in the family.
Dr. Wagstaff presented the results of their melatonin study (supported in part by ASF research funds) involving 13 AS individuals (12 deletion, 1 UPD). AS individuals appeared to have normal to low-normal serum melatonin secretion patterns. Administration of 0.3 mg melatonin before sleep appeared to improve sleep based on measures of total body movements (quantified by actigraph recording). He concluded that melatonin may be helpful in some AS children. If melatonin is used, physicians should prescribe 0.3 mg (do not use high dose melatonin) 30 minutes to 1 hour before bedtime and do not give in the middle of the night if child awakens.
Dr. Rob Nicholls (Case Western Reserve) presented additional mouse data. Maternal UPD mice fail to suck and die and paternal UPD ones have growth delay, but UPD in the mouse is complicated since chromosome 7 (a part of which is equivalent to our chromosome 15) has other imprinted regions outside its corresponding AS/PWS region. A mouse mutant (mutant P30PUb) has a microdeletion encompassing the UBE3A gene but this deletion appears to be clinically similarly expressed when passed through maternal or paternal germ lines. They evaluated brain RNA expression in mutant P30PUb mice with maternal inheritance and no RNA is expressed in the hippocampal area, consistent with UBE3A being imprinted.
Nicholls has studied 8 AS families with imprinting center mutations and feels that they clinically resemble typical AS deletion individuals. He reviewed how the imprinting center (IC) must function to switch on and off genes residing in the 15q11-13 region. Mutations in the IC appear to "fix" the imprinting switch so that it can not be changed in subsequent generations, The IC lies about 3-500,000 base pairs from UBE3A and the IC transcribes RNA but does not make any protein. The IC appears to have a bipartite structure; PWS IC mutations cluster in one region that is physically separate from AS IC imprinting deletions. The AS critical region in the IC has been narrowed to about 2,000 base pairs. All identified mutations thus far have been deletions but about 50% of individuals with IC abnormalities have no identifiable deletion/mutation, even when the entire IC is sequenced. Worldwide, there are about 15 known IC deletion and about 15-20 apparent non-deletion individuals with IC abnormalities. Mutation testing for IC patients is currently only being performed in research labs.
In the afternoon abstract session Dr. Anne Moncla (Medical Genetic Department,
Marseille) presented. a study of 89 AS patients. She observed a less severe phenotype in
those with UPD, imprinting mutations and those with genetically negative tests. Dr. Tim
Delorey (Departments of Pharmacology and Neurology, UCLA) reported on their development of
a mouse knockout lacking GABRB3. In the homozygous state, epilepsy, cleft palate and
behavioral abnormalities were observed. The heterozygote mutant does not have an obvious
phenotype and the heterozygote has not been passed specifically through maternal and
paternal generations. Dr. Rob Nicholls presented data on the development of a
methylation-PCR method for rapid diagnosis of those AS and PWS patients with methylation
abnormalities. This method, when further validated, may be applicable to population-based
screening programs. Dr. Laura Laan. (Department of Neurology, Leiden) compared 12
genetically negative AS individuals to 29 having either a deletion or UPD; there were
minor differences but she concluded that all had essentially identical phenotypes. Dr.
Robert Buckley (Center for Human Development and Disabilities, University of Washington)
reported on 9 newly diagnosed adults living in a residential care facility containing 225
residents. He felt that the true incidence of AS has been
underestimated. Dr. Bernard Dan (Neurology Department, Brussels) reported on the clinical
and EEG findings in 5 AS cases (2 deletion, 1 UPD, 1 IC mutation, 1 with negative studies)
concluding that deletion cases were more affected. He also presented data obtained from
computer-assisted kinematic analysis of 6 AS children compared to 6 normal and 6 with
cerebral palsy (spastic diplegia type). Leg flexion was analyzed from the standing
position and distinctive aspects of AS movements could be identified, such as decreased
knee flexion compared to normal or CP children. One other paper was presented by Dr. Dan.
and three other individuals submitted but did not present their abstracts. Space does
not allow for their review here.
Charles A. Williams, M.D.
Chairperson, ASF Scientific Advisory Committee
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