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Prader-Willi Syndrome by MS-MLPA

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

MS-MLPA

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
2056 SNRPN$540.00 81331 Add to Order
Targeted Testing

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Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity
Approximately 99% of PWS 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 PWS caused by paternal deletion and those caused by maternal UPD or ID. If no deletion is present, DNA polymorphism analysis will be necessary to differentiate between maternal UPD and ID. However, MS-MLPA will pick up some IC deletions and deletions encompassing the SNORD116 gene cluster.

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Clinical Features
Prader-Willi syndrome (PWS) is a multisystem disorder characterized by severe hypotonia, global developmental delay, feeding difficulties and failure to thrive in the newborn period, followed by hyperphagia (excessive eating) and development of morbid obesity in late infancy and early childhood. In addition, there is delay in the development of motor milestones and language, with all individuals showing some degree of cognitive impairment. Behavioral problems such as temper tantrums, stubbornness, manipulative behavior and obsessive-compulsive features are common in early childhood, and psychosis in adolescents and adults (Gunay-Aygun et al. 2001). Older male and female patients manifest hypogonadism (genital hypoplasia, incomplete pubertal development and infertility). Characteristic physical features include short stature, strabismus, almond-shaped palpebral fissures, narrow nasal bridge, thin upper lip with down-turned mouth, and scoliosis. Hypopigmentation of hair, eyes, and skin are seen in a third of the affected individuals. PWS has an estimated incidence of 1 in 15,000-25,000 live births.
Genetics
The PWS and related Angelman syndrome (AS) 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 intron1 of SNURF-SNRPN. AS-SRO is 880bp in size and maps 35 kb proximal to the SNURF-SNRPN exon 1. The CpG islands are unmethylated in the expressed chromosomes and methylated in the unexpressed chromosomes (Cassidy et al. 2012).

PWS is caused by loss of function of five coding genes on paternally inherited chromosome 15q11.2-q13. Loss of paternally expressed genes at 15q11.2-13 can arise from several different genetic mechanisms. Approximately 75-80% of the PWS patients have a recurrent deletion of the 15q11.2-13 on the paternally inherited chromosome, ~20-25% have a maternal uniparental disomy (UPD) of chromosome 15, < 5% have imprinting defect (ID) i.e., mutations within the imprinting control region that establishes a maternal methylation pattern on the paternal chromosome (approximately 10-15% of the PWS individuals with ID have a very small deletion in the PWS IC), and < 1% have a structural chromosome abnormality (e.g., translocation) involving 15q11.2-q13 .

Absence or loss of expression of maternally expressed UBE3A gene leads to Angelman Syndrome (Test Code #574).
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 PWS 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.

At PreventionGenetics we use commercially available Methylation-specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) for the detection of PWS (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 for determining the DNA methylation status at differentially methylated sites in 15q11.2. Additionally, the dosage-sensitive probes cover the SNORD116 (one of snoRNA gene clusters in PWS/AS region) gene cluster (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 99% of the PWS cases.
Indications for Test
Indications for testing include confirmation of clinical diagnosis of PWS and reflex testing after a positive deletion test using CMA or FISH.

Gene

Official Gene Symbol OMIM ID
SNRPN 182279
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Prader-Willi Syndrome 176270

Related Tests

Name
Angelman Syndrome by MS-MLPA
Angelman Syndrome via the UBE3A Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Cassidy SB, Schwartz S, Miller JL, Driscoll DJ. 2012. Prader-Willi syndrome. Genet Med 14: 10–26 PubMed ID: 22237428
  • Gunay-Aygun M, Schwartz S, Heeger S, O’Riordan MA, Cassidy SB. 2001. The Changing Purpose of Prader-Willi Syndrome Clinical Diagnostic Criteria and Proposed Revised Criteria. Pediatrics 108: e92–e92. PubMed ID: 11694676
  • 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
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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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