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 The
contribution to human diseases of the genetic mechanism called imprinting
has been discovered only during the past decade. Imprinting is not consistent
with our previous understanding of how genes are expressed. Prior belief
was that each gene in a cell had 2 alleles, one on the maternal and the
other on the paternal chromosome. When a gene was turned on, both alleles
were transcribed equally and functional protein was produced from both
chromosomes.
For the majority of genes, this is exactly what happens. However, when
there is genetic imprinting, just 1 of the 2 alleles is expressed, while
the other is silenced or "imprinted." Whether the allele is transcribed
or not depends on whether it lies on the chromosome derived from the father
or that derived from the mother. Angelman and Prader-Willi syndromes are
2 illnesses that exemplify this mechanism. The molecular basis for Prader-Willi
was discussed in the last column. The molecular basis for Angelman will
serve as the focus for the present column. I will review the clinical
symptoms before turning to a discussion of the genetic basis for the disorder.
Individuals with Angelman syndrome (AS) have severe motor and intellectual
retardation. They are often hypotonic at birth, develop epilepsy soon
thereafter, and rarely develop speech. They have unusual facies characterized
by a large mandible and an open-mouthed expression. Additional features
include an abnormal gait and puppet-like movements of their limbs. They
are often described as "happy" children because of their frequent smiles
and laughter. Several additional features include a facility for protruding
their tongues, abnormal skin pigmentation, and a characteristic abnormal
EEG discharge pattern.
Approximately 10 years ago, high-resolution cytogenetic studies of patients
with Angelman and Prader-Willi syndromes were performed. The initial studies
suggested that the same chromosomal band was deleted in both disorders
(15q11.2). The deletions appeared indistinguishable by cytogenetic or
molecular genetic methods. This was surprising as the 2 disorders have
very distinct symptoms. Closer inspection, however, revealed that the
deletions in patients with AS were somewhat larger, were more variable
in size, and sometimes included bands 15q12 and part of 15q13.
It was then discovered that the deletions found in individuals with AS
are usually derived from the maternal chromosome. This is in contrast
to Prader-Willi syndrome, in which the deletions derive from the paternal
chromosome. More recently, it has been demonstrated that although the
deleted regions are very close to each other, they are in fact clearly
separated. This separation of the affected chromosomal segments suggested
a mechanism by which the paradox could be resolved. It was proposed that
on chromosomes that came from one's father, a segment of the DNA is imprinted
whereas a nearby region is not. The reverse is true on the chromosome
that came from one's mother, with the first segment of DNA being transcribed
while the nearby region is silenced.
This is indeed the case. In AS, the relevant region on chromosome 15q
is normally imprinted on the paternal chromosome while it is expressed
on the maternally derived chromosome. The normally expressed maternal
copy is often deleted, and one might expect the allele on the paternal
chromosome to compensate and produce functional protein. However, the
paternal copy is unable to do so as it remains imprinted.
In Prader-Willi syndrome, a segment of DNA very near to the Angelman region
is imprinted, but in the opposite direction. In this case, a group of
genes on chromosome 15q are imprinted on the maternally derived chromosome
and are expressed on the paternally derived chromosome. When a deletion
occurs on the paternal chromosome, the relevant maternal genes are unable
to compensate. The genes for these 2 disorders lie very close to each
other, and it turns out that relatively large deletions occur that span
both regions. Which disorder occurs depends on whether the deletion lies
on the paternal or the maternal chromosome. (Fig.
1)
For the majority of cases with AS, the deletion of the actively transcribed
segment on the maternal chromosome 15q12 is responsible for the disorder.
However, several other genetic mechanisms have recently been discovered.
Very rarely, both copies of a chromosome come from a single parent, a
condition known as uniparental disomy. This genetic abnormality, in combination
with imprinting, will lead to the disorder. Thus, AS sometimes occurs
as a result of uniparental disomy of the father's chromosomes. When paternal
disomy occurs, the affected individual still has 2 alleles of all genes.
Both derive from the father, however, and remain silenced with no production
of functional protein.
A third mechanism that has been discovered in AS is due to a mutation
within the "imprinting center." This region on chromosome 15 regulates
which segments of DNA are imprinted. It appears that there is one center
that imprints both genes within the Angelman region and within the Prader-Willi
region. Occasionally, mutations or structural abnormalities occur within
this imprinting center. When they are present, the imprinting center is
unable to function properly, and 1 of the 2 syndromes will result, depending
on whether the maternal or paternal chromosome has been affected.
It was discovered recently that a fourth mechanism, a mutation in a single
gene (UBE3A), can also lead to AS. This discovery underscores an important
distinction between Angelman and Prader-Willi syndromes. Prader-Willi
appears to result from abnormalities in several genes. No family has yet
been discovered in which mutations of a single gene lead to the PWS phenotype.
To summarize, in about 70% of the cases, AS occurs because of a deletion
occurring on the maternal chromosome at 15q11-q13. In about 2% of the
cases, paternal uniparental disomy is the genetic mechanism, while in
approximately 3% of cases a mutation in the imprinting center occurs.
In the remaining 25% of cases, it appears that the molecular basis is
mutations of the single gene, UBE3A.
What is the normal function of the UBE3A protein, and how does its absence
lead to disease? Many proteins within cells are long-lived. Examples of
such proteins are cytoskeletal protein required for the structural integrity
of the cell. Other proteins, however, are short-lived and must be rapidly
removed and degraded. Examples of such proteins are transcription factors,
receptors, and proteins involved in signal transduction pathways. It is
important to have a quick turnover of these proteins, which regulate many
basic cellular functions. This is analogous to what happens at the synaptic
cleft, where neurotransmitters must be quickly removed to allow for the
rapid and repeated signals necessary for sustained synaptic transmissions.
In addition, it is important to remove cellular proteins that are damaged
or not folded correctly, as these structural abnormalities may interfere
with the normal functioning of biochemical pathways. Cells have evolved
an intricate system for the removal of these proteins. Proteins that are
to be targeted for degradation are covalently linked to a 76 amino acid
protein called ubiquitin. Ubiquitin was originally named because it is
expressed in most cells of our bodies as well as in most organisms. It
is highly conserved from yeast and Drosophila to humans, and only a few
amino acid changes exist between any 2 homologues.
Several steps must occur to ensure the orderly removal of proteins destined
for destruction. The concerted actions of 3 enzymes (E1, E2, and E3) are
required. In most organisms, there is a single E1, but many different
E2 and E3 isoforms. In the first step of the process, the E1 enzyme activates
ubiquitin. Activated ubiquitin is passed on to a ubiquitin-conjugating
protein (E2). E3 is required for the last step in which ubiquitin is attached
to the protein targeted for destruction. In many cases, long chains of
ubiquitin are attached to the protein. It is the final step that involves
the protein, UBE3A.
The addition of ubiquitin results in the rapid destruction of the targeted
protein by a cellular organelle termed the proteasome The proteasome recognizes
proteins that have stretches of ubiquitin attached and engulfs them. A
series of proteases within the proteasome complex proteolytically cleaves
the ubiquitinated proteins to single amino acids that are reused by the
cell. In the absence of UBE3A, many unwanted proteins cannot be degraded
and many cellular functions become compromised. Although there appear
to be specific proteins that UBE3A targets, these are still unknown. It
is likely that severe disturbances in normal cellular function occur within
neurons, and these disturbances are then reflected in abnormal cortical
functioning and severe mental retardation.
An added twist to the imprinting story was recently discovered. UBE3A
is imprinted only within the brain. This means that throughout much of
the rest of the body, both alleles are expressed whether they derive from
the mother's or the father's chromosome. This discovery suggests yet another
level of control over the expression pattern for various proteins. It
is likely that as the molecular bases for other childhood disorders are
discovered, more genes will be found to be imprinted within the CNS or
perhaps within specific subregions of the brain.
A final point is worth mentioning. One of the genes that lies within the
deleted region in affected individuals is a critical component of the
receptor that binds to the inhibitory neurotransmitter, [gamma]-aminobutyric
acid (GABA). This gene encodes for a subunit of the GABAA receptor
and was initially thought to be the gene responsible for AS. It is indeed
responsible for some of the symptoms. For example, these individuals often
have significant EEG abnormalities and clinical epilepsy, and it is now
thought that the GABAA receptor is responsible for these symptoms.
A mouse model that lacks this particular gene was recently developed that
has some of the same abnormal discharge patterns seen in subjects with
AS. However, the mutation does not explain the majority of symptoms. Moreover,
the finding of point mutations in the UBE3A gene alone indicates that
this gene is responsible for the core symptoms in AS, while additional
symptoms may be caused by the additional mutations of nearby genes. This
is another example of a contiguous gene syndrome in which several genes
are deleted and cause the observed phenotype in affected individuals.
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