Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman syndromes.
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. Lack of paternal contribution results in PWS either by paternal deletion (approximately 70%) or maternal uniparental disomy (UPD) (approximately 25%). Most cases of AS result from the lack of a maternal contribution from this same region, by maternal deletion (70%) or paternal UPD (approximately 5%). Analysis of allelic methylation differences at the small nuclear ribonucleoprotein polypeptide N (SNRPN) locus differentiates the maternally and paternally inherited chromosome 15 and can be used as a diagnostic test for AS and PWS.


METHODS
Methylation-sensitive high-resolution melting-curve analysis (MS-HRM) using the DNA binding dye EvaGreen was used to analyze methylation differences at the SNRPN locus in anonymized DNA samples from individuals with PWS (n = 39) or AS (n = 31) and from healthy control individuals (n = 95). Results from the MS-HRM assay were compared to those obtained by use of a methylation-specific PCR (MSP) protocol that is used commonly in diagnostic practice.


RESULTS
With the MS-HRM assay 97.6% of samples were unambiguously assigned to the 3 diagnostic categories (AS, PWS, normal) by use of automated calling with an 80% confidence percentage threshold, and the failure rate was 0.6%. One PWS sample showed a discordant result for the MS-HRM assay compared to MSP data.


CONCLUSIONS
MS-HRM is a simple, rapid, and robust method for screening methylation differences at the SNRPN locus and could be used as a diagnostic screen for PWS and AS.
White
Helen E
HE
National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Odstock, Salisbury, Wiltshire, United Kingdom. H.E.White@soton.ac.uk
Hall
Victoria J
VJ
Cross
Nicholas C P
NC
D017204:Angelman Syndrome / Q000175:diagnosis / Q000235:genetics*; D001324:Autoantigens / Q000235:genetics*; D002884:Chromosomes, Human, Pair 15; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D011218:Prader-Willi Syndrome / Q000175:diagnosis / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics*; D044366:Transition Temperature; D055517:snRNP Core Proteins
D001324:Autoantigens; D017411:Ribonucleoproteins, Small Nuclear; C526417:SNRPN protein, human; D055517:snRNP Core Proteins; D004247:DNA