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This Article Full Text Full Text (PDF) 065086.Suppl Data All Versions of this Article: clinchem.2005.065086v1 52/6/1005    most recent Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by White, H. E. Articles by Cross, N. C.P. Search for Related Content PubMed PubMed Citation Articles by White, H. E. Articles by Cross, N. C.P. Related Collections Molecular Diagnostics and Genetics

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

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

^aAddress correspondence to this author at: National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Salisbury, Wiltshire, SP2 8BJ, United Kingdom. Fax 44-1722-338095; 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.

The following articles in journals at HighWire Press have cited this article:

				H. E. White, V. J. Hall, and N. C.P. Cross Methylation-Sensitive High-Resolution Melting-Curve Analysis of the SNRPN Gene as a Diagnostic Screen for Prader-Willi and Angelman Syndromes Clin. Chem., November 1, 2007; 53(11): 1960 - 1962. [Abstract] [Full Text] [PDF]

				E. Dejeux, V. Audard, C. Cavard, I. G. Gut, B. Terris, and J. Tost Rapid Identification of Promoter Hypermethylation in Hepatocellular Carcinoma by Pyrosequencing of Etiologically Homogeneous Sample Pools J. Mol. Diagn., September 1, 2007; 9(4): 510 - 520. [Abstract] [Full Text] [PDF]