TY - JOUR
T1 - Validation of a multiplex methylation-sensitive PCR assay for the diagnosis of Prader-Willi and Angelman's syndromes
AU - Buller, A.
AU - Pandya, A.
AU - Jackson-Cook, C.
AU - Bodurtha, J.
AU - Tekin, M.
AU - Wilkinson, D. S.
AU - Garrett, C. T.
AU - Ferreira-Gonzalez, A.
PY - 2000/1/1
Y1 - 2000/1/1
N2 - Background: Prader-Willi (PWS) and Angelman's (AS) syndromes are two distinct clinical entities caused by alterations in an identical but differentially methylated region of DNA on chromosome 15q. Highly complex laboratory tests are required for diagnosis because the disorders are caused by several genetic mechanisms. Methylation-specific PCR (MSPCR) is a relatively, simple alternative method to detect the methylation status of the PWS/AS region. Methods and Results: DNA was treated with sodium bisulfite, with alterations to the published method in which a neutralization step after the alkali treatment of the modified DNA enabled the use of the modified product directly in the PCR, eliminating the need for ethanol precipitation. Multiplex MSPCR using primers to methylated and unmethylated DNA was optimized to yield equal amplification efficiency for both products. Complete concordance was observed during the clinical validation of 40 previously characterized samples, except for one patient with mosaic AS detected by fluorescence in situ hybridization. Conclusion: We have developed and validated a multiplex MSPCR assay with alterations of the original published protocol that is technically robust and reproducible and can be used as a screening assay to detect PWS and AS.
AB - Background: Prader-Willi (PWS) and Angelman's (AS) syndromes are two distinct clinical entities caused by alterations in an identical but differentially methylated region of DNA on chromosome 15q. Highly complex laboratory tests are required for diagnosis because the disorders are caused by several genetic mechanisms. Methylation-specific PCR (MSPCR) is a relatively, simple alternative method to detect the methylation status of the PWS/AS region. Methods and Results: DNA was treated with sodium bisulfite, with alterations to the published method in which a neutralization step after the alkali treatment of the modified DNA enabled the use of the modified product directly in the PCR, eliminating the need for ethanol precipitation. Multiplex MSPCR using primers to methylated and unmethylated DNA was optimized to yield equal amplification efficiency for both products. Complete concordance was observed during the clinical validation of 40 previously characterized samples, except for one patient with mosaic AS detected by fluorescence in situ hybridization. Conclusion: We have developed and validated a multiplex MSPCR assay with alterations of the original published protocol that is technically robust and reproducible and can be used as a screening assay to detect PWS and AS.
KW - Angelman's syndrome
KW - Methylation analysis
KW - Multiplex PCR
KW - Prader-Willi syndrome
KW - Sodium-bisulfite modification
UR - http://www.scopus.com/inward/record.url?scp=0033785853&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033785853&partnerID=8YFLogxK
U2 - 10.1054/modi.2000.9729
DO - 10.1054/modi.2000.9729
M3 - Article
C2 - 11070159
AN - SCOPUS:0033785853
SN - 1177-1062
VL - 5
SP - 239
EP - 243
JO - Molecular Diagnosis and Therapy
JF - Molecular Diagnosis and Therapy
IS - 3
ER -