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


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), 1005-1013. (doi:10.1373/clinchem.2005.065086).

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Description/Abstract

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.

Item Type: Article
ISSNs: 0009-9147 (print)
Related URLs:
Keywords: pair 15, male, female, cost-benefit analysis, autoantigens, uniparental disomy, dna, genomic imprinting, humans, diagnosis, ribonucleoproteins, polymerase chain reaction
Subjects: R Medicine
R Medicine > RM Therapeutics. Pharmacology
Q Science > QR Microbiology
Divisions: University Structure - Pre August 2011 > School of Medicine > Developmental Origins of Health and Disease
University Structure - Pre August 2011 > School of Medicine
University Structure - Pre August 2011 > School of Medicine > Human Genetics
ePrint ID: 60420
Date Deposited: 08 Sep 2008
Last Modified: 27 Mar 2014 18:42
URI: http://eprints.soton.ac.uk/id/eprint/60420

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