Received on December 14, 2005
Accepted on March 8, 2006

Molecular Diagnostics and Genetics

Quantitative Analysis of SRNPN Gene Methylation by Pyrosequencing as a Diagnostic Test for Prader-Willi Syndrome and Angelman Syndrome

¹ National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Odstock, Salisbury, Wiltshire, United Kingdom

^* To whom correspondence should be addressed. E-mail: H.E.White{at}soton.ac.uk.

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.