Hypermethylated RASSF1A in Maternal Plasma: A Universal Fetal DNA Marker that Improves the Reliability of Noninvasive Prenatal Diagnosis

Chan, K. C. Allen; Ding, Chunming; Gerovassili, Ageliki; Yeung, Sze-Wan; Chiu, Rossa W.̉K.; Leung, Tse Ngong; Lau, Tze Kin; Chim, Stephen S.C.; Chung, Grace Tin‐Yun; Nicolaides, Kypros H.; Lo, Y.M. Dennis

doi:10.1373/clinchem.2006.074997

Cited by 312 publications

(273 citation statements)

References 14 publications

(12 reference statements)

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“…Apart from placental-specific mRNA transcripts, other types of fetal-specific nucleic acid species in maternal plasma could be used. One example is fetal epigenetic markers (12,21) which have recently been used for the noninvasive prenatal detection of trisomy 18 via the epigenetic allelic ratio (EAR) approach (10). Thus, we predict that digital EAR would be a possible analytical technique.…”

Section: Discussionmentioning

confidence: 99%

Digital PCR for the molecular detection of fetal chromosomal aneuploidy

Lun²,

Chan³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

390

328

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Trisomy 21 is the most common reason that women opt for prenatal diagnosis. Conventional prenatal diagnostic methods involve the sampling of fetal materials by invasive procedures such as amniocentesis. Screening by ultrasonography and biochemical markers have been used to risk-stratify pregnant women before definitive invasive diagnostic procedures. However, these screening methods generally target epiphenomena, such as nuchal translucency, associated with trisomy 21. It would be ideal if noninvasive genetic methods were available for the direct detection of the core pathology of trisomy 21. Here we outline an approach using digital PCR for the noninvasive detection of fetal trisomy 21 by analysis of fetal nucleic acids in maternal plasma. First, we demonstrate the use of digital PCR to determine the allelic imbalance of a SNP on PLAC4 mRNA, a placenta-expressed transcript on chromosome 21, in the maternal plasma of women bearing trisomy 21 fetuses. We named this the digital RNA SNP strategy. Second, we developed a nonpolymorphism-based method for the noninvasive prenatal detection of trisomy 21. We named this the digital relative chromosome dosage (RCD) method. Digital RCD involves the direct assessment of whether the total copy number of chromosome 21 in a sample containing fetal DNA is overrepresented with respect to a reference chromosome. Even without elaborate instrumentation, digital RCD allows the detection of trisomy 21 in samples containing 25% fetal DNA. We applied the sequential probability ratio test to interpret the digital PCR data. Computer simulation and empirical validation confirmed the high accuracy of the disease classification algorithm.circulating fetal nucleic acids ͉ noninvasive prenatal diagnosis ͉ sequential probability ratio test ͉ trisomy 21 ͉ RNA SNP

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Section: Discussionmentioning

confidence: 99%

Digital PCR for the molecular detection of fetal chromosomal aneuploidy

Lun²,

Chan³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

390

328

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“…26 For future studies, RASSF1A, a tumour suppressor gene, may be considered as a potentially useful generic marker for circulating fetal DNA. 27 RASSF1A may represent a universal marker because it is differentially methylated in the placenta compared with the genomic DNA of adults.…”

Section: Discussionmentioning

confidence: 99%

Diagnostic accuracy of noninvasive polymerase chain reaction testing for the determination of fetal rhesus C, c and E status in early pregnancy

Gutensohn¹,

Müller

Thomann

et al. 2010

BJOG

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Objective The aim of the study was to determine the sensitivity, specificity and accuracy of noninvasive tests for the fetal rhesus CcEc (RHCE) alleles C, c and E in early pregnancy.Design A prospective clinical trial was carried out to evaluate diagnostic accuracy.Setting Women were recruited at four centres specialising in prenatal diagnosis. Peripheral blood and amniotic fluid samples were obtained and sent to a single laboratory for analysis.Sample A total of 233 tests (46 for C, 87 for c and 100 for E) were performed on 181 specimens obtained from pregnant women at weeks 12 to 28 (median week 16) of gestation.Methods Following automated extraction of fetal DNA from maternal plasma, two different real-time polymerase chain reaction (PCR) protocols were used for the detection of the C, c and E alleles of RHCE. The results of the PCR were compared with genotyping results for the amniotic fluid.Main outcome measures Failure rate, sensitivity, specificity and accuracy were the main outcome measures.Results Unequivocal results were obtained for all specimens. With the first PCR protocol, the sensitivity was 100% for C, 38% for c and 59% for E. In contrast, with the second protocol, the sensitivity for C, c and E was 100%. The specificity for all assays was found to be between 99% and 100%.Conclusions A highly accurate protocol has been identified for the detection of fetal RHCE alleles in maternal plasma in early pregnancy. This noninvasive approach can be considered as a useful test in the management of pregnancies with anti-c, anti-E or anti-C alloimmunisation.

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“…For the exclusion of the latter case, the detection of paternally inherited single nucleotide polymorphisms [12], the detection of a Y-chromosome speciWc sequence [8], or the detection of an epigenetic marker such as the foetal hypermethylated RASSF1A sequence [3] can be performed. In our current study, we were unable to perform such additional analyses due to very limited amount of sample available.…”

Section: Discussionmentioning

confidence: 99%

High throughput non-invasive determination of foetal Rhesus D status using automated extraction of cell-free foetal DNA in maternal plasma and mass spectrometry

Grill

Banzola

et al. 2008

Arch Gynecol Obstet

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Purpose To examine the potential high throughput capability and eYciency of an automated DNA extraction system in combination with mass spectrometry for the noninvasive determination of the foetal Rhesus D status. Methods A total of 178 maternal plasma samples from RHD-negative pregnant women were examined, from which DNA was extracted using the automated Roche MagNA Pure™ system. Presence of the foetal RHD gene was detected by PCR for RHD exon 7 and subsequent analysis using the Sequenom MassArray™ mass spectrometric system. Results We determined that as little as 15 pg of RHD-positive genomic DNA could be detected in a background of 585 pg of RHD-negative genomic DNA. The analysis of the clinical samples yielded a sensitivity and speciWcity of 96.1 and 96.1%, respectively. Conclusion Our study indicated that automated DNA extraction in combination with mass spectrometry permits the determination of foetal Rhesus D genotype with an accuracy comparable to the current approaches using realtime PCR.

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Hypermethylated RASSF1A in Maternal Plasma: A Universal Fetal DNA Marker that Improves the Reliability of Noninvasive Prenatal Diagnosis

Cited by 312 publications

References 14 publications

Digital PCR for the molecular detection of fetal chromosomal aneuploidy

Digital PCR for the molecular detection of fetal chromosomal aneuploidy

Diagnostic accuracy of noninvasive polymerase chain reaction testing for the determination of fetal rhesus C, c and E status in early pregnancy

High throughput non-invasive determination of foetal Rhesus D status using automated extraction of cell-free foetal DNA in maternal plasma and mass spectrometry

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