Evaluation and validation of the suitable control genes for quantitative PCR studies in plasma DNA for non-invasive prenatal diagnosis

The presence of foetal DNA in the plasma of pregnant women has opened up new possibilities for non-invasive prenatal diagnosis. The use of circulating foetal DNA for the non-invasive prenatal detection of foetal chromosomal aneuploidies is challenging as foetal DNA represents a minor fraction of maternal plasma DNA. In 2007, it was shown that single molecule counting methods would allow the detection of the presence of a trisomic foetus, as long as enough molecules were counted. With the advent of massively parallel sequencing, millions or billions of DNA molecules can be readily counted. Using massively parallel sequencing, foetal trisomies 21, 13 and 18 have been detected from maternal plasma. Recently, large-scale clinical studies have validated the robustness of this approach for the prenatal detection of foetal chromosomal aneuploidies. A proof-of-concept study has also shown that a genome-wide genetic and mutational map of a foetus can be constructed from the maternal plasma DNA sequencing data. These developments suggest that the analysis of foetal DNA in maternal plasma would play an increasingly important role in future obstetrics practice. It is thus a priority that the ethical, social and legal issues regarding this technology be systematically studied.

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Clinical application of non-invasive prenatal diagnosis of phenylketonuria based on haplotypes via paired-end molecular tags and weighting algorithm

BMC Medical Genomics ◽

10.1186/s12920-021-01141-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Dai Peng ◽

Zhao Ganye ◽

Sun Gege ◽

Xia Yanjie ◽

Liu Ning ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Phenylalanine Hydroxylase ◽

Early Stage ◽

Maternal Plasma ◽

Plasma Dna ◽

Target Sequencing ◽

Non Invasive ◽

Hybridization Probes ◽

Irreversible Brain Damage ◽

Flanking Region

Abstract Background Phenylketonuria (PKU) is a metabolic disease that can cause severe and irreversible brain damage without treatment. Methods Here we developed a non-invasive prenatal diagnosis (NIPD) technique based on haplotypes via paired-end molecular tags and weighting algorithm and applied it to the NIPD of PKU to evaluate its accuracy and feasibility in the early pregnancy. A custom-designed hybridization probes containing regions in phenylalanine hydroxylase (PAH) gene and its 1 Mb flanking region were used for target sequencing on genomic and maternal plasma DNA (7–13 weeks of gestation) to construct the parental haplotypes and the proband’s haplotype. Fetal haplotype was then inferred combined with the parental haplotypes and the proband’s haplotype. The presence of haplotypes linked to both the maternal and paternal mutant alleles indicated affected fetuses. The fetal genotypes were further validated by invasive prenatal diagnosis in a blinded fashion. Results This technique has been successfully applied in twenty-one cases. Six fetuses were diagnosed as patients carrying both of the mutated haplotypes inherited from their parents. Eleven fetuses were carriers of one heterozygous PAH variants, six of which were paternal and five of which were maternal. Four fetuses were absence of pathogenic alleles. All results were consistent with the prenatal diagnosis through amniotic fluid. Conclusions The results showed that our new technique applied to the genotyping of fetuses with high risk for PKU achieves an accurate detection at an early stage of pregnancy with low fetal fraction in cell free DNA.

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Faculty Opinions recommendation of Characterization of fetal cells from the maternal circulation by microarray gene expression analysis--could the extravillous trophoblasts be a target for future cell-based non-invasive prenatal diagnosis?

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726854630.793524409 ◽

2016 ◽

Author(s):

Ignatia Van den Veyver

Keyword(s):

Gene Expression ◽

Prenatal Diagnosis ◽

Gene Expression Analysis ◽

Maternal Circulation ◽

Microarray Gene Expression ◽

Non Invasive ◽

Microarray Gene ◽

Microarray Gene Expression Analysis ◽

Extravillous Trophoblasts

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Development of a Specific Monoclonal Antibody to Detect Male Cells Expressing the RPS4Y1 Protein

International Journal of Molecular Sciences ◽

10.3390/ijms22042001 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2001

Author(s):

Silvia Spena ◽

Chiara Cordiglieri ◽

Isabella Garagiola ◽

Flora Peyvandi

Keyword(s):

Monoclonal Antibody ◽

Prenatal Diagnosis ◽

Maternal Blood ◽

Limiting Factors ◽

Inherited Disease ◽

Specific Monoclonal Antibody ◽

Native Form ◽

Fetal Cells ◽

Non Invasive ◽

Reliable Isolation

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.

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Non-invasive approach to prenatal diagnosis from maternal peripheral blood

Prenatal Diagnosis ◽

10.1002/pd.1970120613 ◽

1992 ◽

Vol 12 (6) ◽

pp. 547-548 ◽

Cited By ~ 1

Author(s):

Y.-M. D. Lo ◽

J. S. Wainscoat ◽

K. A. Fleming

Keyword(s):

Prenatal Diagnosis ◽

Peripheral Blood ◽

Invasive Approach ◽

Non Invasive ◽

Maternal Peripheral Blood

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Detection of paternal alleles in maternal plasma for non-invasive prenatal diagnosis of β-thalassemia: A feasibility study in southern Chinese

European Journal of Obstetrics & Gynecology and Reproductive Biology ◽

10.1016/j.ejogrb.2010.02.016 ◽

2010 ◽

Vol 150 (1) ◽

pp. 28-33 ◽

Cited By ~ 19

Author(s):

Kaimin Chan ◽

Irene Yam ◽

K.Y. Leung ◽

Mary Tang ◽

T.K. Chan ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Feasibility Study ◽

Maternal Plasma ◽

Non Invasive ◽

Southern Chinese

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Noninvasive Prenatal Diagnosis of Monogenic Diseases by Targeted Massively Parallel Sequencing of Maternal Plasma: Application to β-Thalassemia

Clinical Chemistry ◽

10.1373/clinchem.2012.189589 ◽

2012 ◽

Vol 58 (10) ◽

pp. 1467-1475 ◽

Cited By ~ 112

Author(s):

Kwan-Wood G Lam ◽

Peiyong Jiang ◽

Gary J W Liao ◽

K C Allen Chan ◽

Tak Y Leung ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Massively Parallel Sequencing ◽

Globin Gene ◽

Maternal Plasma ◽

Massively Parallel ◽

Sequencing Data ◽

Plasma Dna ◽

Parallel Sequencing ◽

Noninvasive Prenatal Diagnosis ◽

Monogenic Diseases

Abstract BACKGROUND A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of maternal plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of maternal plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS Targeted sequencing of maternal plasma DNA for NIPD of monogenic diseases is feasible.

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