White, Helen E., Durston, Victoria J., Harvey, John F. and Cross, Nicholas C.
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), . (doi:10.1373/clinchem.2005.065086).
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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.
|Digital Object Identifier (DOI):
||pair 15, male, female, cost-benefit analysis, autoantigens, uniparental disomy, dna, genomic imprinting, humans, diagnosis, ribonucleoproteins, polymerase chain reaction
|8 March 2006||Published|
||08 Sep 2008
||16 Apr 2017 17:32
|Further Information:||Google Scholar|
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