Quantitative analysis of SNRPN(correction of SRNPN) gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome.
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 (approximately 70%) or maternal uniparental disomy (UPD; approximately 30%). Most cases of AS result from the lack of a maternal contribution from this same region by maternal deletion (approximately 70%) or by paternal UPD (approximately 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.
National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Odstock, Salisbury, Wiltshire, United Kingdom. H.E.White@soton.ac.uk
Helen E
HE
White
Victoria J
VJ
Durston
John F
JF
Harvey
Nicholas C P
NC
Cross
D017204:Angelman Syndrome / Q000175:diagnosis* / Q000235:genetics; D001324:Autoantigens / Q000235:genetics* / Q000378:metabolism; D002884:Chromosomes, Human, Pair 15; D003362:Cost-Benefit Analysis; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D018392:Genomic Imprinting; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D016133:Polymerase Chain Reaction; D011218:Prader-Willi Syndrome / Q000175:diagnosis* / Q000235:genetics; D015203:Reproducibility of Results; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013447:Sulfites; D055517:snRNP Core Proteins
D001324:Autoantigens; D007202:Indicators and Reagents; D017411:Ribonucleoproteins, Small Nuclear; C526417:SNRPN protein, human; D013447:Sulfites; D055517:snRNP Core Proteins; C042345:hydrogen sulfite