Angelman Syndrome by MS-MLPA
- Summary and Pricing
- Clinical Features and Genetics
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Differential diagnoses with several overlapping features with AS include Mowat-Wilson syndrome (Test #1567), X-linked Christianson syndrome (Test #562 and Test #600), Rett syndrome (Test #1455 and Test #600), Pitt-Hopkins syndrome (Tests #1523, #1522 and #600), 2q23.1 deletion syndrome (Test #1321), and Phelan-McDermid syndrome (Test #600).
AS is caused by absence of a functional UBE3A gene on the maternally inherited chromosome. Loss of maternally expressed genes at 15q11.2-13 can arise from several different genetic mechanisms. Approximately 65-75% of the AS patients have a recurrent deletion of the 15q11.2-13 on the maternally inherited chromosome, ~3-7% have a paternal uniparental disomy (UPD) of chromosome 15, 3% have imprinting defect (ID) i.e., mutations within the imprinting control region that establishes a paternal methylation pattern on the maternal chromosome (approximately 10-15% of the AS individuals with ID have a very small deletion in the AS IC), and 5-10% of AS patients have point mutations in the maternally inherited UBE3A gene (Test #574) (Williams et al. 2010; Dagli et al. 2012).
Around 10% of the patients with a clinical diagnosis of AS have no discernable abnormality at the 15q11.2-q13 region. Some of these patients include mosaic cases of imprinting defects, but a majority result from unidentified defects in UBE3A or mutations in other genes.
Alternatively, absence or loss of expression of paternally expressed genes at 15q11.2-13 lead to PWS (Test #2056).
At PreventionGenetics we use commercially available methylation-specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) for the diagnosis of PWS and AS (Procter et al. 2006). This method combines both DNA methylation analysis and copy-number analysis across the entire PWS/AS region. MS-MLPA contains 32 dosage-sensitive probes (for copy number detection) specific for the 15q11.2 region, and five probes determine the DNA methylation status at differentially methylated sites in 15q11.2. Additionally, the dosage-sensitive probes cover SNORD116, one of the snoRNA gene clusters in the PWS/AS region (Ramsden et al. 2010).
The advantages of MS-MLPA method include;
1) Combined detection of both copy-number and methylation status.
2) Can differentiate between PWS caused by paternal deletion or UPD/Imprinting defect.
3) Detection of methylation status at five distinct differentially methylated regions (instead of one locus).
4) Detection of small deletions encompassing IC and SNORD116 gene cluster.
5) Detects more than 78% of the AS cases.
AS patients with normal methylation analysis of the 15q11.2-q13 region should reflex to UBE3A gene sequencing (Test #574) followed if necessary by UBE3A deletion/duplication analysis via aCGH (Test #600).
Indications for Test
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- Genetic Counselor Team - email@example.com
- Srirangan Sampath, PhD, FACMG - firstname.lastname@example.org
- Dagli A, Buiting K, Williams CA. 2012. Molecular and Clinical Aspects of Angelman Syndrome. Mol Syndromol 2: 100–112. PubMed ID: 22670133
- Lossie A, Whitney M, Amidon D, Dong H, Chen P, Theriaque D, Hutson A, Nicholls R, Zori R, Williams C, Driscoll D. 2001. Distinct phenotypes distinguish the molecular classes of Angelman syndrome. J Med Genet 38: 834–845. PubMed ID: 11748306
- Nygren AOH, Ameziane N, Duarte HMB, Vijzelaar RNCP, Waisfisz Q, Hess CJ, Schouten JP, Errami A. 2005. Methylation-Specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 33: e128. PubMed ID: 16106041
- Procter M, Chou L-S, Tang W, Jama M, Mao R. 2006. Molecular Diagnosis of Prader–Willi and Angelman Syndromes by Methylation-Specific Melting Analysis and Methylation-Specific Multiplex Ligation-Dependent Probe Amplification. Clinical Chemistry 52: 1276–1283. PubMed ID: 16690734
- Ramsden SC, Clayton-Smith J, Birch R, Buiting K. 2010. Practice guidelines for the molecular analysis of Prader-Willi and Angelman syndromes. BMC Med Genet 11: 70. PubMed ID: 20459762
- Williams CA, Driscoll DJ, Dagli AI. 2010. Clinical and genetic aspects of Angelman syndrome. Genet Med 12: 385–395. PubMed ID: 20445456
Methylation-specific Multiplex Ligation-dependent Probe Amplification
Multiplex Ligation-dependent Probe Amplification (MLPA) is a semi-quantitative technique that is used to determine the relative copy number of up to 60 DNA sequences in a single multiplex PCR-based reaction. It is based on amplification of up to 60 probes, each of which detects a specific complementary DNA sequence of approximately 60 bp in length (often exons in genes of interest). Briefly, each MLPA probe is made up of two half-probes that hybridize immediately adjacent to each other at the target DNA. These adjacent probes are then ligated into one single probe before being amplified in a PCR reaction. Multiplexing is achieved by different probes varying in sizes ranging from 150-500 bp, that are all amplified using a common PCR primer pair. One of the PCR primers is fluorescently labelled enabling separation and detection of the amplification products in a capillary electrophoresis instrument. The peaks heights of the amplification products of the target DNA sequence is then compared to the peak heights in various reference DNA samples. A deletion or a duplication is inferred from the relative decrease or increase in peak height respectively.
A modified MLPA technique termed Methylation-specific MLPA (MS-MLPA) is used to detect both the copy number and methylation status of up to 50 DNA sequences in a single multiplex PCR-based reaction. The basic principle of MS-MLPA is very similar to MLPA except that the target DNA sequences recognized by the MS-MLPA probes contain restriction sites for enzymes such a HhaI or HpaII that are sensitive to cytosine methylation of one CpG site in their recognition sequence. When target DNA is digested with these enzymes a probe amplification product will only be obtained if the CpG site is methylated. The level of methylation is determined by the ratio of the relative peak area for each target probe from digested vs undigested DNA sample.
MS-MLPA is a robust method that is widely used for the clinical diagnosis of several genetic imprinting disorders like Prader-Willi syndrome /Angelman syndrome, Beckwith-Widemann syndrome, Russell-Silver syndrome, Lynch syndrome and Albright hereditary osteodystrophy. MS-MLPA has several advantages over other assays such as MS-PCR based on bisulphite sequencing, southern blotting, and methylation analysis including PCR following restriction digestion with methylation sensitive enzyme. MS-MLPA investigates methylation status at multiple loci, thereby reducing the risk for false positive or false negative results due single nucleotide polymorphisms (SNPs) at the probe binding sequence.
Both MLPA and MS-MLPA will not detect point mutations in sequences recognized by the probes. In addition it will not detect inversions, balanced translocations or copy number changes that lie outside the sequence detected by the MLPA probes.
MLPA probes are sensitive to changes within the sequence detected by the probe. A single nucleotide change (such as SNPs or pathogenic mutations) very close to the probe ligation site can prevent ligation of the two oligonucleotide probes. In addition, sequence changes further from the ligation site can affect probe binding and hence decrease probe signal mimicking a deletion.
MLPA is sensitive to DNA characteristics such as impurities, method used for DNA isolation, salt concentrations in solution, and degree of DNA degradation. The effect of these characteristics can be minimized by using the same DNA extraction methods for all samples analyzed by this method and by matching the test and control DNA from the same source.
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- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.