Evaluation of a novel assay for detection of the fetal marker RASSF1A: facilitating improved diagnostic reliability of noninvasive prenatal diagnosis
Evaluation of a novel assay for detection of the fetal marker RASSF1A: facilitating improved diagnostic reliability of noninvasive prenatal diagnosis
Background
Analysis of cell free fetal (cff) DNA in maternal plasma is used routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, fetal rhesus D status and some single gene disorders. True positive results rely on detection of the fetal target being analysed. No amplification of the target may be interpreted either as a true negative result or a false negative result due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A promoter has been reported as a universal fetal marker to confirm the presence of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated maternal sequences are digested leaving hypermethylated fetal sequences detectable. Complete digestion of maternal sequences is required to eliminate false positive results.
Methods
cfDNA was extracted from maternal plasma (n = 90) and digested with methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A, SRY and DYS14 was performed by real-time PCR.
Results
Hypermethylated RASSF1A was amplified for 79 samples (88%) indicating the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of these samples were male at delivery, five had inconclusive results for SRY analysis and one sample had no amplifiable SRY.
Conclusion
Use of this assay for the detection of hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.
e45073-[5pp]
Oudejans, Cees
90263014-7b8f-47ea-afd5-f2c8f4ace601
White, Helen E.
2181c0b9-fc3b-407e-95eb-3510524603e5
Dent, Carolyn L.
2cf3d5ab-bf6f-4ae0-8c07-f99bdd1be226
Hall, Victoria J.
a60eea2a-3fb9-460e-a845-692f8970e68a
Crolla, John A.
c5f23751-8de9-4a55-9cc5-ca2fb635769c
Chitty, Lyn S.
1c097cb0-0e7a-48e0-bc8f-e0bdbf67f16e
14 September 2012
Oudejans, Cees
90263014-7b8f-47ea-afd5-f2c8f4ace601
White, Helen E.
2181c0b9-fc3b-407e-95eb-3510524603e5
Dent, Carolyn L.
2cf3d5ab-bf6f-4ae0-8c07-f99bdd1be226
Hall, Victoria J.
a60eea2a-3fb9-460e-a845-692f8970e68a
Crolla, John A.
c5f23751-8de9-4a55-9cc5-ca2fb635769c
Chitty, Lyn S.
1c097cb0-0e7a-48e0-bc8f-e0bdbf67f16e
Oudejans, Cees, White, Helen E., Dent, Carolyn L., Hall, Victoria J., Crolla, John A. and Chitty, Lyn S.
(2012)
Evaluation of a novel assay for detection of the fetal marker RASSF1A: facilitating improved diagnostic reliability of noninvasive prenatal diagnosis.
PLoS ONE, 7 (9), .
(doi:10.1371/journal.pone.0045073).
(PMID:23024794)
Abstract
Background
Analysis of cell free fetal (cff) DNA in maternal plasma is used routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, fetal rhesus D status and some single gene disorders. True positive results rely on detection of the fetal target being analysed. No amplification of the target may be interpreted either as a true negative result or a false negative result due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A promoter has been reported as a universal fetal marker to confirm the presence of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated maternal sequences are digested leaving hypermethylated fetal sequences detectable. Complete digestion of maternal sequences is required to eliminate false positive results.
Methods
cfDNA was extracted from maternal plasma (n = 90) and digested with methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A, SRY and DYS14 was performed by real-time PCR.
Results
Hypermethylated RASSF1A was amplified for 79 samples (88%) indicating the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of these samples were male at delivery, five had inconclusive results for SRY analysis and one sample had no amplifiable SRY.
Conclusion
Use of this assay for the detection of hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.
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Published date: 14 September 2012
Organisations:
Human Development & Health
Identifiers
Local EPrints ID: 343501
URI: http://eprints.soton.ac.uk/id/eprint/343501
ISSN: 1932-6203
PURE UUID: da5b58a7-05ba-42dd-ac75-b95702700d0a
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Date deposited: 08 Oct 2012 11:18
Last modified: 14 Mar 2024 12:04
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Contributors
Author:
Cees Oudejans
Author:
Helen E. White
Author:
Carolyn L. Dent
Author:
Victoria J. Hall
Author:
John A. Crolla
Author:
Lyn S. Chitty
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