Non-invasive prenatal diagnosis of monogenic disorders: an optimized protocol using MEMO qPCR with miniSTR as internal control

Guissart, Claire; Debant, V.; Desgeorges, Marie; Bareil, Corinne; Raynal, Caroline; Toga, C.; Pritchard, Victoria L.; Kœnig, M.; Claustres, Mireille; Vincent, Marie-Claire

doi:10.1515/cclm-2014-0501

Cited by 17 publications

(14 citation statements)

References 10 publications

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“…The previous studies dealing with foetal cfDNA detection and qualitative analysis were mostly based on quantitative PCR 22,[25][26][27] . The hypermethylated RASSF1 sequences were reported as a universal marker for the foetal cfDNA detection and quantification 22 but the authors were not able to detect the foetal fraction in four out of nineteen RhD negative pregnant patients using this methodology.…”

Section: Discussionmentioning

confidence: 99%

Detection of cell-free foetal DNA fraction in female-foetus bearing pregnancies using X-chromosomal insertion/deletion polymorphisms examined by digital droplet PCR

Svobodová

Pazourkova

Laššáková

et al. 2020

Sci Rep

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In families with X-linked recessive diseases, foetal sex is determined prenatally by detection of Y-chromosomal sequences in cell-free foetal DNA (cffDNA) in maternal plasma. The same procedure is used to confirm the cffDNA presence during non-invasive prenatal RhD incompatibility testing but there are no generally accepted markers for the detection of cffDNA fraction in female-foetus bearing pregnancies. We present a methodology allowing the detection of paternal X-chromosomal alleles on maternal background and the confirmation of female sex of the foetus by positive amplification signals. Using digital droplet PCR (ddPCR) we examined X-chromosomal INDEL (insertion/deletion) polymorphisms: rs2307932, rs16397, rs16637, rs3048996, rs16680 in buccal swabs of 50 females to obtain the population data. For all INDELs, we determined the limits of detection for each ddPCR assay. We examined the cffDNA from 63 pregnant women bearing Y-chromosome negative foetuses. The analysis with this set of INDELs led to informative results in 66.67% of examined female-foetus bearing pregnancies. Although the population data predicted higher informativity (74%) we provided the proof of principle of this methodology. We successfully applied this methodology in prenatal diagnostics in a family with Wiscott–Aldrich syndrome and in pregnancies tested for the risk of RhD incompatibility.

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Section: Discussionmentioning

confidence: 99%

Detection of cell-free foetal DNA fraction in female-foetus bearing pregnancies using X-chromosomal insertion/deletion polymorphisms examined by digital droplet PCR

Svobodová

Pazourkova

Laššáková

et al. 2020

Sci Rep

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“…Previous studies were performed on small sample groups and on targeted mutation of the CFTR gene [7,9,10,[16][17][18][19][20]. In contrast, here we analyzed more than 14 samples from high-risk CF pregnancies and over 16 samples from pregnancies with a priori CF risk based on ultrasound findings, the latter secondarily excluded following parental genotyping.…”

Section: Discussionmentioning

confidence: 99%

“…material) in K3 EDTA Vacutainer tubes or cell-stabilizing tubes (Streck blood collection tube [BCT]). Samples were processed up to 24 h (EDTA tubes) or up to 72 h (BCT) after sampling as previously described [9,10].…”

Section: Participants and Processing Of Blood Samplesmentioning

confidence: 99%

A Broad Test Based on Fluorescent-Multiplex PCR for Noninvasive Prenatal Diagnosis of Cystic Fibrosis

et al. 2018

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Background: Analysis of cell-free fetal DNA in maternal plasma is very promising for early diagnosis of monogenic diseases. However, it has been limited by the need to set up patient- or disease-specific custom-made approaches. Here we propose a universal test based on fluorescent multiplex PCR and size fragment analysis for an indirect diagnosis of cystic fibrosis (CF). Methods: The test, based on haplotyping, includes nine intra- and extragenic short tandem repeats of the CFTR locus, the coamplification of p.Phe508del (the most frequent mutation in CF patients worldwide), and a specific SRY sequence. The assay is able to determine the inherited paternal allele. Results: Our simple approach was successfully applied to 30 couples and provided clear results from the maternal plasma. The mean rate of informative markers was sufficient to propose it for use in indirect diagnosis. Conclusions: This noninvasive prenatal diagnosis test, focused on indirect diagnosis of CF, offers many advantages over current methods: it is simple, rapid, and cost-effective. It allows for the testing of a large number of couples with high risk of CF, whatever the familial mutation of the CFTR gene. It provides an alternative method to reduce the number of invasive tests.

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“…Various high-throughput technologies have been explored to detect paternal cystic fibrosis alleles in cffDNA, such as sequencing [ 3 ], microarrays [ 4 ] and conventional fluorescence quantitative PCR [ 5 ]. We propose that ddPCR is a technically unchallenging, flexible and cost-effective addition to the range of tools available for analysing NIPD samples.…”

Section: Discussionmentioning

confidence: 99%

“…The principal challenge of NIPD is that cffDNA makes up only a small proportion of total free circulating DNA (generally less than 10% [ 1 ],[ 2 ]), especially at earlier stages of pregnancy, therefore high-sensitivity technology such as next-generation sequencing [ 3 ], microarrays [ 4 ] or quantitative PCR [ 5 ] is required to accurately detect it. Digital PCR (dPCR) is a highly sensitive and quantitative alternative to conventional qPCR which is based on the nanofluidic partitioning of DNA molecules between many small wells following a high degree of sample dilution.…”

Section: Introductionmentioning

confidence: 99%

A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity

Debrand¹,

Lykoudi

Bradshaw³

et al. 2015

PLoS ONE

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IntroductionNon-invasive prenatal diagnosis (NIPD) makes use of cell-free fetal DNA (cffDNA) in the mother’s bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5–1% risk of fetal loss. We describe a droplet digital PCR (ddPCR) assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in the cffDNA, it is possible to predict whether the fetus will be an unaffected carrier (absence) or whether further invasive testing is indicated (presence).MethodsWe selected a family in which the parents were known to carry different mutated CFTR alleles as our test system. NIPD was performed for three of their pregnancies during the first trimester (at around 11–12 weeks of gestation). Taqman probes were designed against an amplicon in exon 11 of the CFTR gene, to quantify the proportion of mutant (ΔF508-MUT; FAM) and normal (ΔF508-NOR; VIC) alleles at position c.1521_1523 of the CFTR gene.DiscussionThe assay correctly and unambiguously recognized the ΔF508-MUT CFTR allele in the cffDNA of all three proband fetuses and none of the six unaffected control fetuses. In conclusion, the Bio-Rad QX100 was found to be a cost-effective and technically undemanding platform for designing bespoke NIPD assays.

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Non-invasive prenatal diagnosis of monogenic disorders: an optimized protocol using MEMO qPCR with miniSTR as internal control

Cited by 17 publications

References 10 publications

Detection of cell-free foetal DNA fraction in female-foetus bearing pregnancies using X-chromosomal insertion/deletion polymorphisms examined by digital droplet PCR

Detection of cell-free foetal DNA fraction in female-foetus bearing pregnancies using X-chromosomal insertion/deletion polymorphisms examined by digital droplet PCR

A Broad Test Based on Fluorescent-Multiplex PCR for Noninvasive Prenatal Diagnosis of Cystic Fibrosis

A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity

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