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Angelman Syndrome by MS-MLPA

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  • Clinical Features and Genetics
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TEST METHODS

MS-MLPA

Test Code TestIndividual Gene PriceCPT Code Copy CPT Codes
2056 UBE3A$540.00 81331 Add to Order
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Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity
Approximately 78% of AS cases will be detected by this assay. Because of simultaneous detection of both copy number and methylation status, MS-MLPA is able to differentiate between AS caused by maternal deletion and those caused by paternal UPD or ID. If no deletion is present, DNA polymorphism analysis will be necessary to differentiate between maternal UPD and ID. This method cannot detect AS caused other molecular mechanisms, including UBE3A point mutations.

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Clinical Features
Angelman syndrome (AS) is a neurodevelopmental disorder characterized by severe developmental delay, intellectual disability, speech impairment, seizures and characteristic behavior with an inappropriate happy demeanor with easily provoked laughter, short attention span, smiling and excitability. Individuals with AS plateau at a developmental level of 24 to 30 months, and cognitive performance usually shows severe functional impairment. Most children with AS have reduced need for sleep, and frequent awakening at night is common (Lossie et al. 2001). Fifty percent of the children develop microcephaly (Occipital Frontal Circumference < 2SD) by the age of 12 months. All children with AS have some component of hyperactivity with seemingly ceaseless activity. Language impairment is severe with most individuals lacking speech entirely, but a few develop single-word vocabularies. A large subset of children with AS qualify for a comorbid diagnosis of autism. A subset of AS patients also have hypopigmentation of the skin, hair and eyes, attributable to haploinsufficiency of OCA2 gene (due to maternal deletion) located close to UBE3A gene. The prevalence of AS is one in 12,000-20,000.

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).
Genetics
The AS and related Prader-Willi syndrome (PWS) region is localized to a 5-6 Mb genomic region on the proximal long arm of chromosome 15 (15q11.2-q13). This region contains several genes that are differentially expressed depending on whether the region is inherited from the father or the mother, i.e., some genes in this regions are expressed only from the paternal chromosome and some expressed only from the maternal chromosome. This differential gene expression is achieved by differential methylation (imprinting) patterns on the paternal and maternal chromosomes. The PWS paternally-only expressed region contains five protein coding genes (MKRN3, MAGEL2, NDN, NPAP1, and SNURF-SNRPN), one ORF (C15orf2), and a family of small nucleolar RNA (snoRNA) genes. The AS maternally-only expressed region contains one gene UBE3A. Deletion mapping in PWS/AS patients identified two small regions of deletion overlap (SRO) that define two critical imprinting centers (IC). PWS-SRO is a 4.3 kb region that lies at the 5’ end of the bicistronic SNURF-SNRPN, and has CpG islands encompassing the promoter, exon 1 and intron 1 of SNURF-SNRPN. AS-SRO is 880bp in size and maps 35 kb proximal to the SNURF-SNRPN exon 1.

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).
Testing Strategy
Methylation analysis (Nygren et al. 2005) of the PWS/AS IC is by far the most efficient starting point for a genetic diagnosis of AS based on a clinical suspicion alone, as it can be used for all three classes of molecular defects (deletion, UPD and Imprinting defect). Additionally, methylation analysis differentiates between PWS and AS for patients with 15q11.2-q13 deletion without the need for the analysis of parental samples. Methylation analysis makes use of the differentially methylated maternal and paternal chromosome region at 15q11.2-q13 to identify maternal-only, paternal-only or bi-parental inheritance.

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
Indications for testing include confirmation of clinical diagnosis of AS and reflex testing after a positive deletion test using CMA or FISH.

Gene

Official Gene Symbol OMIM ID
UBE3A 601623
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Angelman Syndrome 105830

Related Tests

Name
2q23.1 Microdeletion Syndrome and Autism Spectrum Disorder via the MBD5 Gene
Angelman Syndrome via the UBE3A Gene
Autosomal-Recessive Intellectual Disability via the NRXN1 Gene
Christianson Type X-Linked Mental Retardation via the SLC9A6 Gene
Cortical Dysplasia-Focal Epilepsy Syndrome via the CNTNAP2 Gene
Mowat-Wilson Syndrome via the ZEB2 Gene
Phelan-Mcdermid Syndrome, 22q13 Deletion Syndrome, or Autism Spectrum Disorder via the SHANK3 Gene
Prader-Willi Syndrome by MS-MLPA
Rett Syndrome via the MECP2 Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • 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
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TEST METHODS

Methylation-specific Multiplex Ligation-dependent Probe Amplification

Test Procedure

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.

Analytical Validity

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.

Analytical Limitations

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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Ordering Options


myPrevent - Online Ordering
  • 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.
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.

SPECIMEN TYPES
WHOLE BLOOD

(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.

DNA

(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.

CELL CULTURE

(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.
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