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Quantitative analysis of SNRPN [correction of SRNPN] gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome

Record type: Article

Background: Angelman syndrome (AS) and Prader–Willi syndrome (PWS) are 2 distinct neurodevelopmental disorders caused primarily by deficiency of specific parental contributions at an imprinted domain within the chromosomal region 15q11.2-13. In most cases, lack of paternal contribution leads to PWS either by paternal deletion (70%) or maternal uniparental disomy (UPD; 30%). Most cases of AS result from the lack of a maternal contribution from this same region by maternal deletion (70%) or by paternal UPD (5%). Analysis of allelic methylation differences at the small nuclear ribonucleoprotein polypeptide N (SNRPN) locus can differentiate the maternally and paternally inherited chromosome 15 and can be used as a diagnostic test for AS and PWS.
Methods: Sodium bisulfite–treated genomic DNA was PCR-amplified for the SNRPN gene. We used pyrosequencing to individually quantify the resulting artificial C/T sequence variation at CpG sites. Anonymized DNA samples from PWS patients (n = 40), AS patients (n = 31), and controls (n = 81) were analyzed in a blinded fashion with 2 PCR and 3 pyrosequencing reactions. We compared results from the pyrosequencing assays with those obtained with a commonly used methylation-specific PCR (MS-PCR) diagnostic protocol.
Results: The pyrosequencing assays had a sensitivity and specificity of 100% and provided quantification of methylation at 12 CpG sites within the SNRPN locus. The resulting diagnoses were 100% concordant with those obtained from the MS-PCR protocol.
Conclusions: Pyrosequencing is a rapid and robust method for quantitative methylation analysis of the SNRPN locus and can be used as a diagnostic test for PWS and AS.

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Citation

White, Helen E., Durston, Victoria J., Harvey, John F. and Cross, Nicholas C. (2006) Quantitative analysis of SNRPN [correction of SRNPN] gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome Clinical Chemistry, 52, (6), pp. 1005-1013. (doi:10.1373/clinchem.2005.065086).

More information

Published date: 8 March 2006
Keywords: pair 15, male, female, cost-benefit analysis, autoantigens, uniparental disomy, dna, genomic imprinting, humans, diagnosis, ribonucleoproteins, polymerase chain reaction

Identifiers

Local EPrints ID: 60420
URI: http://eprints.soton.ac.uk/id/eprint/60420
PURE UUID: e02534ba-c6f5-4126-b0af-d1a4aaf4149b
ORCID for Nicholas C. Cross: ORCID iD orcid.org/0000-0001-5481-2555

Catalogue record

Date deposited: 08 Sep 2008
Last modified: 17 Jul 2017 14:23

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