Angelman_Syndrome-2.jpg
Wide jaws and space between the teeth is common for children with Angelman Syndrome.

Angelman syndrome (AS) is a neurodevelopmental disorder, which causes retardation, speech deficits, seizures, tremulous movement of the limbs, characteristic facial features and unusually friendly behaviour in the affected individuals.[1] Epilepsy and sleeping disorders are also observed in the majority of individuals.[2] The patients with Angelmen syndrome fail to develop language as well as communicate their basic needs and require great amount of care and therapy, since they are not able to sustain themselves. [2] The syndrome is diagnosed at about the age of 12 months when the first symptoms appear, including motor skills deficit, as well as mental retardation, which becomes more obvious later on.[1] The cause for this syndrome is most often a deletion or a mutation on the maternal allele of the UBE3A gene, located on chromosome 15.[1] Since the gene is paternally imprinted in the majority of neurons, a mutation of the maternal copy is sufficient to alter the phenotype of the progeny.[2] The UBE3A gene codes for the ubiquitin protein ligase E3A, which has proven to be essential for proper neurodevelopment.[1] There is currently no treatment that can effectively reverse the symptoms associated with the syndrome in people.[2] However recent studies have shown that topoisomerase inhibitors can unsilence the imprinted copy of the UBE3A gene and reverse the signs of the Angelmen syndrome in miceand hopefully will help the development of new more effective therapies for the affected patients.[1]

1. Diagnosis of Angelman syndrome


Angelman syndrome (AS) is a severe neurodevelopmental disorder, affecting approximately 1 in 12,000-20,000 births.[2] [3] The affected individuals are fully dependent on their caregivers, since even after therapy they are unable to perform basic everyday tasks, such as dressing and feeding themselves [2] ,[3] . The first symptoms of the syndrome appear in infancy and include global developmental delays as well as seizures [3]. At that stage Angelman syndrome is challenging to diagnose without genetic testing since many of its features are also common in other neurodevelopmental disorders [3] . Often at such early age, AS is mistaken for Prader-Willi syndrome due to the similarity in symptoms and the fact that both are often caused by a deletion in the same chromosomal domain [3] . Later on (usually around the age of 3) children with the syndrome present unique for the syndrome abnormalities such as overly happy and friendly behavior, the absence of speech, balance and movement deficits, tremulous movement of the limbs and characteristic physical appearance [1] , [2] . Often the children with AS have body weight in the 25th percentile and head circumference in the 3rd percentile[4] .

EEG patterns are characteristic, making EEG good diagnostic tool,even when genetic testing is not available. [3] ,[5] ,[6] , Irregular delta waves, epileptic discharges and irregular theta waves are common in the affected individuals [6] ;however in a small fraction of patients, genetically confirmed to have AS, the abnormal EEG patterns are absent.[3] Even though MRI is one of the most common diagnostic tools for many other conditions, it is not useful for detecting AS. [3] In some cases MRI reveals atrophy, mild dysmyelination or hypomyelination, however the findings are not sufficient to diagnose the syndrome. [3]

In a study conducted in the Hospital Universitario Miguel Servet in Zaragoza (Spain) over the span of 21 years, the diagnosed with the syndrome females were twice as many as the diagnosed males, suggesting that females are more likely to express the syndrome, however the reasons behind the statistic are not clear.[4]
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Characteristic Angelman EEG. Triphasic delta wave activity arrowed.


2. Genetic causes: Disruption of the maternal allele of Ube3a
Angelman syndrome is a genetic disorder and is caused by the disrupted function of the UBE3A gene which codes for ubiquitin protein ligase E3A and improper regulation of the imprinting process of genes at chromosome 15q11–q13. [1] ,[7] It has been estimated that in about 85-90% of cases [4] , [1] the syndrome is caused by a deletion or mutation in the 15q11.2–q13 region which contain the Ube3a gene and is crucial for proper neuronal development. [1] In some rare cases the syndrome is caused by imprinting defects, affecting the transcription in that region. [7] In less than 10% of all individuals with AS, the cause is not known as the Ube3a locus appears normal. [3]

There are some differences in the severity of AS symptoms, depending on genotype. [3] In the cases where AS has resulted from deletion of the Ube3a locus the symptoms are generally more prominent than the symptoms of those with a mutated maternal copy of the gene. [7] Those with large deletion in the range of 5-7Mb exhibit seizures, severe motor deficits, feeding difficulties and language impairment. [3] , [1] In the cases of uniparental disomy or an imprinting defect, AS individuals have healthier muscle tone, develop speech better and have less frequent seizures.[3]At an earlier age these children appear to develop normally both physically and mentally, which may delay the diagnosis of AS. [5]

Unbalanced 10;15 translocation has been shown to be another possible cause for Angelman syndrome.[5] This suggests that not only molecular cytogenetic analyses are important for prenatal screening. Screening for chromosomal rearrangement, such as karyotyping, is also useful in detecting chromosomal abnormalities, which may be familial, making genetic counseling highly recommended. [5]


2.1. The Ube3a gene


The Ube3a gene codes for an ubiquitin-protein ligase which is involved in the degradation of ubiquitin, which is important in neuronal development.[5] Angelman syndrome is often a result of the loss of function of the protein[1] . AS is not the only neurodevelopmental disorder that is caused by malfunction of the Ube3a gene. The lack of functioning Ube3a protein has also been associated with autism spectrum disorder, suggesting that the protein has an important and unique role in the proper development of the nervous system.[7]
Ube3a also interacts with abnormal the spindle-like microcephaly-associated protein (ASPM); an important gene, closely associated with proper neurodevelopment.[8] Microcephaly is a condition where the head circumference of a patient is more than two standard deviations smaller than the average head circumference for that age and it often associated with mental retardation [8] ,[9] . As a result of the interaction between ASPM and Ube3a, in more than 80% of AS cases postnatal microcephaly is also observed. [8] ,[10]

The 15q11-13 locus has also been associated with some cases of Autism Spectrum Disorder, where it duplicated or triplicated, affecting the expression of other genes related to Autism. In a study the concentration of Ube3a was tripled in mice neurons, looking at its effect on traits such as social interaction, communication skills and stereotypic behaviour[11] ; all of which are impaired for both AS and autism but in opposite ways[1] , [2] ,[11] . For example, while autistic children are asocial, children with AS exhibit overly happy and friendly behaviour. [1],[11] In this experiment, when the Ube3a concentration was increased, glutaminergic synaptic transmission was suppressed by reducing presynaptic release of neurotransmitter, reduced synaptic glutamate concentration and decreased postsynaptic AP coupling.[11] These findings imply that overexpressed Ube3a protein can have an effect on autism traits and reduces excitatory synaptic transmission. [11]

2.2. Genomic imprinting


Angelman syndrome was one of the first conditions linked to genomic imprinting.[4] Imprinting is a genetic mechanism where only one allele (either the maternal or paternal allele) of a certain gene is expressed and the other allele is silenced and is not transcribed in a given cell. [1],[4] The Ube3A gene is paternally imprinted in some cells, although in most somatic cells both copies are expressed.[1],[2] ,[7] In the brain mainly the maternal copy is expressed and the paternal copy of the gene is silenced. [7] Even though the gene is haplo-sufficient, a disruption of the maternal copy would result in loss of function of the Ube3a protein in most neurons, regardless of the presence of a normal paternal allele.[2] ,[7] ,[9]
The imprinting of the locus associated with AS, which includes the Ube3a gene and the imprinting of the locus associated with Prader-Willi (PWS) are controlled by mutually dependent imprinting centers.[7] According to the currently accepted model the PWS imprinting center activates the paternal allele and the AS imprinting center inactivates the PWS imprinting center on the maternal allele.[9] Therefore the AS imprinting center inhibits gene expression of the paternal allele. The paternal imprinting of Ube3a is also regulated by a paternal non-coding antisense transcript (Ube3a-ATS). [9] The expression of Ube3a-ATS is brain specific, so the paternal copy is active and transcribed in the rest of the body. [9]

2.3. Genetic testing


There are several different methods used to diagnose Angelman syndrome. Identifying the syndrome and establishing whether it was a case of deletion, mutation or rearrangement, are very important, in order for the family to receive proper genetic counseling and be informed about the risks of AS if planning to have children.[3]
The first test usually performed to diagnose AS is DNA methylation analysis.[3] Methylation analysis is able to detect deletion of 15q11.2-q13, uniparental disomy or an imprinting defect in that region and thus confirm the disorder[3],[12] . In individuals, not affected by the syndrome, Southern blot analysis, as well as methylation specific polymerase chain reaction assays detect one methylated SNRPN allele (maternal) and one unmethylated allele (paternal).[3],[12] In AS patients the test only detects one unmethylated allele and the maternal methylated allele is missing, indicating a deletion, imprinting defect or uniparental disomy.[3] In order to identify what was the genetic mechanism, causing AS for the given individual, further testing is required.[3]
After the methylation analysis detects AS, the next test performed is FISH or array comparative genomic hybridization analysis, in order to detect deletions.[3] When no deletions are found analysis of DNA polymorphisms on chromosome 15 can be used to determine if this is a case of uniparent disomy or an imprinting center defect.[9]
If the patient exhibits the signs of AS but there are no methylation nomalies detected UBE3A sequence analysis are performed, in order to check for mutations.[3]


3. Genetic treatment under development: Topoisomerase inhibitors as Ube3a activators

Currently there is no cure for Angelman Syndrome.[1]In spite of the physical therapy and the speech exercises administered, symptoms such as seizures and low muscle tone are hard to manage.[2] ,[3] New evidence suggests that gene therapy might be able to improve or eliminate the symptoms of AS. In a recent study, done by Dr. Hsien-Sung Huang and his team managed to successfully treat mice with AS to completely recovery from the disorder.[1] The gene therapy is based on activating the paternal wild type copy of the Ube3a gene which is normally paternally imprinted in neurons, in order to compensate for the mutated or deleted maternal allele and restore Ube3a protein function.[1] This is hypothesized to promote normal neurodevelopment and functioning of the nervous system later in life.[1],[13]

3.1. Topoisomerase I and II inhibitors as activators of the silenced paternal Ube3a allele

Ube3a paternal imprinting is regulated by topoisomerases I and II, which when inhibited no longer silence the Ube3a gene.[1] Topoisomerase I is important for coordinating various DNA processes which include the relaxation of DNA supercoiling, transcription, recombination, damage signaling, genomic imprinting and replication.[9] The versatility of the protein makes it and its inhibitors a primary target for investigating possible genetic therapy not only for neurodevelopmental disorders, but also other genetic conditions and cancers. [12]
In recent study topoisomerase inhibitors, including topotecan and irinotecan, were identified and used in mice to activate the imprinted paternal allele.[1] When administered in Ube3a-null mice, topotecan was able to upregulate the transcription and translation of functional Ube3a by downregulating the expression of Ube3a antisense transcript, which overlaps.[1]


3.2. Topotecan as an upregulator of Ube3a

Drugs such as topotecan and irinotecan are used as topoisomerase I inhibitors and are derived from the compound camptothecin. [1],[13] However, many cells are resistant to the drugs at the levels of transport, drug action, metabolism, signaling and most importantly repairing the lesions in the DNA which are consequences of the drug action.[12] These drawbacks need to be taken into consideration when trying to improve the efficacy of such drugs. Topotecan, as well as other topoisomerase inhibitors, links DNA to topoisomerases to form stable complexes which are separable from free topoisomerase enzymes.[11] That way topotecan significantly reduces the amount of free topoisomerase I in neurons so it unsilences the paternal Ube3a allele.[1]
Topotecan also downregulates the expression of the Ube3a antisense transcript.[1] Ube3a is repressed in cis by a large antisense transcript that eventually silences the paternal allele of Ube3a.[1]That indicates that topotecan can be used to activate the imprinted Ube3a allele by blocking transcription of imprinted antisense RNA, without specifically affecting the methylation of the imprinting centre.[1],[13] The paternal Ube3a expression in some groups of neurons lasted more than 12 weeks after the treatment, suggesting the effect of topoisomerase inhibitors on gene expression can be long term and giving hopes for the effective development of such treatment in human. [13]


3.3. Effect on neuronal structure after treatment with topoisomerase inhibitors in mice

In the study where topotecan was used to reverse AS in mice, the expression of the paternal Ube3a allele was observed in the nervous system, where it is normally imprinted at sites such as the neocortex, hippocampus, cerebellum, striatum, and spinal cord.[1], [12] The effect of the drug were directly proportional to the amount that was injected.[1]
As a control the mice were only injected in one hemisphere and not in the other, resulting in paternal Ube3a being unsilenced in the treated hemisphere and almost no effect on the untreated hemisphere, suggesting that the topoisomerase inhibitor topotecan is indeed effective in activating the paternal copy of the Ube3a gene throughout the nervous system.[1] ,[2] ,[13]


3.4. Changes in synaptic plasticity due to impairment of Ube3a

In order to study in more detail how Ube3a depletion affects the brain, synaptic development in Ube3a-null mice was studied and more specifically dendritic spines density since Ube3a is localized mainly in the postsynaptic compartments and the nucleus.[2]

In AS mice the dendritic spine density and length are reduced in neurons such as pyramidal neurons in CA1 of the hippocampus, the neurons in layers III-V of the cortex and purkinje neurons in the cerebellum.[2] The affected areas are thought to be important for cognitive processes and motor function in mice and a defect there, would result in symptoms similar to the AS symptoms in human.[2] This shows the importance of Ube3a for regulating excitatory postsynaptic development and function in neurons. For example contextual fear conditioning was impaired in these mice.[2] It was shown that the induction threshold for NMDAR dependent LTP increases at CA1 hippocampal synapses when Ube3a function is impaired.[14] Some brain areas were not affected by the loss of function of the Ube3a protein, which implies that dendrtic spine density is dependent on Ube3a only in some neuronal populations.[2] ,[14]


4. Relation to human brain morphology

Animal models are often used in science, due to the highly phylogenetically preserved systems and mechanisms[2] ,[14] Mice models have proven to be an effective tool when studying AS, since mice possess a chromosomal region that is similar to the human 15q11-q13 where Ube3a and other orthogolous genes, are also imprinted.[2] In neither mice nor human is the Ube3apromoter region methylated which excludes the Ube3a promoter as a possible mechanism for maternal expression in both species.[2]
Ube3a is paternally imprinted in both species, which is important for the relevance of the studies mentioned above.[2] Another important similarity is that the paternal Ube3a allele expression is silenced by a large antisense RNA transcript, so imprinting is established using similar mechanisms in mice.[2] Mice models rely on the loss of UBE3A in neurons in the central nervous system and the fact that mice exhibit several AS-relevant phenotypes, such as motor deficits and microcephaly. [2] , [15]

Since mice were shown to have similar activity dependent regulation of their axon initial segments as human, they were used in studies, looking at the alterations of resting potentials, action potential amplitudes and threshold potentials in the pyramidal neurons in the hipocampal CA1 area in AS mice.[15] Evidence was found for abnormalities of the intrinsic membrane properties and changes specific to the axon initial segment in AS, which is important in studying the pathology of the disorder and developing treatments.[15]

These similarities make experiments, conducted on mice, useful in developing genetic and other treatments that could eventually be used in human. Successfully activating the Ube3a paternal allele using topoisomerase inhibitors is a great step forward towards the discovery of a drug that could potentially treat or cure Angelman syndrome.

  1. ^ Huang, H., Allen, J.A., Mabb, A., King, I.F., Miryala, J., et al. (2011). Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons. Nature, 481, 185-189.
  2. ^ Mabb, A.M., Judson, M.C., Zylka, M.J., Philpot, B.D. (2011). Angelman syndrome: insights into genomic imprinting and neurodevelopmental phenotypes. Trends in Neurosciences, 34, 293-303.
  3. ^ Williams, C. A. (2005). Neurological aspects of the Angelman syndrome. Brain and Development, 27 (2), pp. 88-94.
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  10. ^ Smith, E.Y., Futtner, C.R., Chamberlain, S.J., Johnstone, K.A., Resnick, J.L. (2011). Transcription is required to establish maternal imprinting at the Prader-Willi syndrome and Angelman syndrome locus. PLoS Genet, 7(12), e1002422.
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