Quantitative analysis of SNRPN [correction of SRNPN] gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome
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 (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.
pair 15, male, female, cost-benefit analysis, autoantigens, uniparental disomy, dna, genomic imprinting, humans, diagnosis, ribonucleoproteins, polymerase chain reaction
1005-1013
White, Helen E.
2181c0b9-fc3b-407e-95eb-3510524603e5
Durston, Victoria J.
7336d597-984a-4ad4-8be4-886b22705061
Harvey, John F.
b27b83e2-c681-4a87-9ce9-7686fc1bba36
Cross, Nicholas C.
f87650da-b908-4a34-b31b-d62c5f186fe4
8 March 2006
White, Helen E.
2181c0b9-fc3b-407e-95eb-3510524603e5
Durston, Victoria J.
7336d597-984a-4ad4-8be4-886b22705061
Harvey, John F.
b27b83e2-c681-4a87-9ce9-7686fc1bba36
Cross, Nicholas C.
f87650da-b908-4a34-b31b-d62c5f186fe4
White, Helen E., Durston, Victoria J., Harvey, John F. and Cross, Nicholas C.
(2006)
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).
Abstract
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.
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Published date: 8 March 2006
Keywords:
pair 15, male, female, cost-benefit analysis, autoantigens, uniparental disomy, dna, genomic imprinting, humans, diagnosis, ribonucleoproteins, polymerase chain reaction
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Local EPrints ID: 60420
URI: http://eprints.soton.ac.uk/id/eprint/60420
PURE UUID: e02534ba-c6f5-4126-b0af-d1a4aaf4149b
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Date deposited: 08 Sep 2008
Last modified: 16 Mar 2024 03:23
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Author:
Helen E. White
Author:
Victoria J. Durston
Author:
John F. Harvey
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