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Suchanuch Putaprapasri 1009

Angelmans syndrome

Background

Angelman syndrome (AS) is a genetic disorder effecting the human nervous

system. This disorder occur from inactive UBE3A gene which are transfer from both

of the parent. Both of UBE3A copies are turned on, or make active, in many tissue of

the body. But in humans brain, there is only the copy from mother that is active.

This is cause by genomic imprinting phenomenon. So if the mother copy of the gene

is inactive or lost due to gene mutation (about or chromosomal change, the brain

will not work properly in some cases. About 70 percent of Angelman syndrome

occur when a segment of the mother copy of UBE3A is deleted and about 11

percent for Angelman syndrome to occur from mutation in UBE3A gene. However,

some cases has also report Angelman syndrome happened in MTHFR, GABRB3

gene.

Most cases of Angelman syndrome are not inherited. These genetic changes

occur as random events during the formation of reproductive cells or in early

embryonic stage. Affected people typically have no history of the disorder in their

family. Rarely, a genetic change responsible for Angelman syndrome can be

inherited.

Characteristic of Angelman syndrome are delayed development, intellectual

disability, severe speech impairment, and problems with movement and balance or

ataxia. Most affected children also have recurrent seizures (epilepsy) and a small
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head size (microcephaly).Children with Angelman syndrome typically are happy with

frequent smiling, laughter, hyperactivity and fascination with water. Most affected

children also have difficulty sleeping and need less sleep than usual. However, at

birth, these people usually have normal prenatal and birth history. Moreover,

Angelman syndrome has no major birth defect. As people grow up, some problems

get better. Other common features include unusually fair skin with light-

colored hair and an abnormal side-to-side curvature of the spine (scoliosis). The life

expectancy of people with this condition appears to be nearly normal. To conclude,

the four major causes of Angelman syndrome are the imprinting mechanisms of

maternal chromosome, the target proteins of UBE3A, the noncoding antisense

transcript in UBE3A imprinting and the contribution of other genes such as methyl-

binding CpG-binding protein 2 and gamma-aminobutyric acid A receptor, subunit

beta3 to the phenotype of Angelman syndrome.

In deeper details, Angelman syndrome mainly related to epigenetics due to

demethylation or failed to methylate the chromosome 15q on the maternal allele.

Imprinting defect is the main cause for this. Which meant there are problem in

genomic imprinting. However if demethylation occur on 15q paternal allele, this will

cause a different disorder, Prader-Willi. So in this testing, we are analyzing for

abnormal methylation and UBE3A sequence. Abnormal methylation can be deletion

of maternal chromosome 15q, which is UBE3A or imprinting defect of the same

maternal chromosome. For the method of analyzing, karyotype and chromosomal

microarray can be performed.

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A picture example of genes that Angelman syndrome and Prader-Willi can occur.

Blue boxes represent paternal genes and red boxes are maternal genes. The black

boxes are biallelical genes and arrow heads represent the orientation of

transcription.

MLPA (Multiplex Ligation-dependent Probe Amplification) is a PCR method, which

can be perform at any laboratories. It can detects abnormal copy numbers of up to

50 different genomic DNA or RNA sequences, which is able to distinguish sequences

differing in only one nucleotide. This method is designed for detecting deletion or

duplications of gene sequences. The results will be out within 24 hours. There are

four main steps: DNA denaturation and hybridization, ligation reaction, PCR reaction

then electrophoresis.
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Preimplantation genetic diagnosis (PGD) may be an option for those who has

Angelman syndrome causes from UBE3A pathogenic variants or IC deletions (we are

interested in IC deletions or the imprinting defect). But in this report will be focusing

more on MLPA.

Objectives

The main purpose of this work is to give knowledge about Angelman

syndrome, a non-lethal disease but cause a major effect to those who has this

disease. I may not be able to give the treatment to cure the root problem, this

disease, but treatments for improving the symptoms are available. Other main

purpose for this work is to identify how Angelman syndrome is an epigenetics, how

to test it and what cause it to be epigenetic.

The final objective is to show that Angelman syndrome is an epigenetic.

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Question

What are the causes of Angelman syndrome that are epigenetics and how to

identify it?

Hypothesis

If the patient has Angelman syndrome but their parent or twin didnt have the

disorder, then Angelman syndrome is an epigenetics.

Treatment

Unfortunately there is no cure for Angelman syndrome but there are available

treatment depending on the signs and symptoms. Possible treatment and medicine

for this disorder are

Anti-seizure medication - for control seizures

Physical therapy for helping with walking and movement problems

Communication therapy - sign language and picture communication

Behavior therapy - help overcome hyperactivity, a short attention span and

to aid in development
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Material& Method

1. Minimum 100 Patients with Angelman syndrome (Cant be a newborn, since

the newborn still have normal phenotype. Sometimes, development can be
delay too.). Find a person whose parents are normal and if possible, a normal
twin to show that this disease is an epigenetic disease. These people should
consists of people with all causes of Angelman syndrome. Bring their mom
too.

2. 3mL-5mL blood collected in an EDTA (lavender-top) tube

3. EDTA (royal blue-top)

4. ACD solution B (yellow-top)
5. Sodium heparin (green-top)
Materials number 3, 4 and 5 are to be use in MLPA.
6. Deads human brain (optional, for more accurate data since UBE3A gene
deletion does not impact much if it isnt the deletion in the brain DNA.
Newborn brain are unacceptable.)
7. Multiplex ligation probe amplification (MLPA)
8. Preimplantation genetic diagnosis (PGD)
9. Southern Blob (DNA Blob)
10.Refrigerator
11.DNA size marker
12.X-ray film (optional)
13.CN2097 medicine

Procedure

1. Find DNA methylation level of the patient by MLPA (including PCR and gel

electrophoresis after DNA denaturation and hybridization, ligation reaction) or

PGD method.
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Leave the specimen at room temperature for 8 days and refrigerated

for 8 days. However the specimen cant be frozen.

This process will determine if the person has Angelman syndrome or

Prader-Willi or not. But this process wont tell the cause of the disorder,

so we will proceed to the next step.

2. If DNA methylation analysis is normal, proceed on testing UBE3A or by using

multi-gene panel.

3. After the testing of UBE3A. If a pathogenic variant is not

identified, gene deletion/duplication analysis can be considered.

4. Multi-gene panel testing that use UBE3A or MTHFR, GABRB3 gene may be

considered in individuals who have features of AS but

normal DNA methylation analysis.

5. If single-gene testing and use of a multi-gene panel has not confirmed a

diagnosis of AS, more comprehensive genomic testing can be used if wish,

but just only the first four step that Ive mention is enough for analyzing the

disorder and it causes.

6. Compare the results got from the patients and use it to analyze with their

mother, since Angelman syndrome have many causes but all are related to

genes from mother. Now, we will run down to only 2 causes for Angelman

syndrome.
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7. Pick the patients with has Angelman syndrome from imprinting defects and

UBE3A deletion. Collect their data. Now were going to confirm that these are

really epigenetics.

8. To determine, we need to look at two things,if a 15q11-13 deletion was

transmitted from a female with Angelman syndrome to her child and if the

UBE3A gene was functionally imprinted in.

9. The patients will be genotyped by microsatellite analysis. Then DNA

methylation imprints will be analyzed by Southern blot analysis and

methylation-specific PCR.

10.The UBE3A will not be imprinted and this will support our hypothesis.

Guideline for the results of finding the causes of Angleman Syndrome

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Here is a clearer explanation of the results you might get from testing the

methylation level. In here, we are looking for the Deletion and IC deletion

(imprinting).

Some other Molecular genetic testing methods

Southern Blot

Southern blotting is the process of transferring DNA fragments from an

electrophoresis gel to a supporting membrane, then the membrane carries a semi-

permanent reproduction of the DNA banding pattern of the gel which we will be able

to read and analyze the data from it.

Example of a way of reading Southern Blot.

lane 1 = DNA size markers

lane 2 and 5 = normal control DNA sample
lane 3 & 6 = AS patient
lane 4 & 7 = query AS patient.
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Treating the Angelman Syndrome

A newly discover medicine, CN2097, believed to be able to protect neurons

under conditions of stroke and in disease states such as multiple sclerosis, help in

correcting the signaling defects in Angelman syndrome. However, CN2097 can

breaks down easily so the patient might need to take the medication often. But like

some other disease, example diabetes, patient wont be cure for it but the medicine

given make the patient better and healthier, even though the diabetes wont be

gone. For CN2097, we could try it in different ages of people and see if it can cure

and help patient live a better life after the Angelman syndrome start showing its

symptoms, which can be around 5 months in normal cases. The control variable in

this experiment would be the patient with Angelman syndrome but no intake of

CN2097 medicine. Then other variables is the ages of patient who has Angelman

syndrome and these will be the one who intake the CN2097 medicine. However,

using a new medicine on human can be dangerous, by using mouse would be

better. In mouse, we will see how much a mouse, representing a patient, at different

age can intake the CN2097. Since this is a new medicine, the scientists already

identify that the short-term effects arent dangerous but the long term isnt clear

yet. So now we got two things to consider in testing in a mouse, amount of CN2097

intake and how long. Mouse gender can also be used as a variable in this

experiment, though might not be really affect but we should just make sure that

every gender didnt really have problem with the CN2097. Then we could proceed to

human. Record how people at different ages react to the CN2097 medicine.
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Bibiography

Aditi I Dagli, MD, Jennifer Mueller, MS, CGC, and Charles A Williams, MD
(2015, May 14). Angelman syndrome. Retrieved from
https://www.ncbi.nlm.nih.gov/books/NBK1144/

Juan E Castillo-Fernandez, Tim D Spector, and Jordana T Bell (2014 July 31).
Epigenetics of discordant monozygotic twins: implications for disease. Retrieved
from https://www.ncbi.nlm.nih.gov/pm c/articles/PMC4254430/
Prader-Willi/Angelman Syndrome, Molecular Analysis (n.d.). Retrieved from
http://www.mayomed icallaboratories.com/test-
catalog/Clinical+and+Interpretive/35535

Lalande M1, Calciano MA.(2007 April). Retrieved from

https://www.ncbi.nlm.nih.gov/pubmed/ 17347796

SALSA MLPA P336 UBE3A probemix (n.d.). Retrieved from

https://mlpa.com/WebForms/WebFormProd uctDetails.aspx?
Tag=_tz2fAPIAupKyMjaDF-E-t9bmuxqlhe_Lgqfk8Hkjuss.&ProductOID=_H8yF8M-JC24.

MLPA - an introduction (n.d.). Retrieved from

https://mlpa.com/WebForms/WebFormMain.aspx?
Tag=zjCZBtdOUyAt3KF3EwRZhNWLtcfv9pVl/tHJIM
%5Cfa9FWO8KMqctOGIoqYwxaGF9Y

Prader-Willi/Angelman Syndrome, DNA Methylation Analysis (n.d.). Retrieved

from http://www.ques tdiagnostics.com/testcenter/BUOrderInfo.action?
tc=11369&labCode=SJC

Simon C Ramsden, Jill Clayton-Smith, Rachael Birch and Karin Buiting( 2010
May 10). Practice guidelines for the molecular analysis of Prader-Willi and Angelman
syndromes. Retrieved from http://bmcmedgen
et.biomedcentral.com/articles/10.1186/1471-2350-11-70

Monaghan KG, Wiktor A, Van Dyke DL.(2002 December). Diagnostic testing

for Prader-Willi syndrome and Angelman syndrome: a cost comparison. Retrieved
from https://www.ncbi.nlm.nih.gov/pubmed/ 12509717
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Molecular Analysis of Angelman Syndrome (n.d.). Retrieved from

http://www.compgene.com/as.htm

Girardet A, Moncla A, Hamamah S, Claustres M. (2005 December). Strategies

for preimplantation genetic diagnosis of Angelman syndrome caused by mutations
in the UBE3A gene. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/15901461.

Lossie AC., Driscoll DJ. (1999 October). Transmission of Angelman syndrome

by an affected mother. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/11258627

Pre-implantation genetic diagnosis (PGD) (2014 April 1). Retrieved from

http://www.hfea.gov.uk/pre implantation-genetic-diagnosis.html

Angelman Syndrome Foundation-Funded Research Identifies

Miscommunication of Neurons, New Drug Compound that May Lead to Treatments
(n.d.). Retrieved from https://www.angelman.org/research-discovers-new-clues-for-
potential-treatments-of-angelman-syndrome/

Treatment of stroke using a novel PDZ binding peptidomimetic drug (n.d.)

Retrived from https://www.sbir.gov/sbirsearch/detail/369027