Cell free DNA: revolution in molecular diagnostics – the journey so far

2020 ◽  
Vol 41 (1) ◽  
Author(s):  
Kajal Nandi ◽  
Rashmi Verma ◽  
Rajni Dawar ◽  
Binita Goswami
Keyword(s):  
Chorionic Villus ◽  
Prenatal Testing ◽  
Blood Stream ◽  
The Body ◽  
Chorionic Villus Sampling ◽  
Cell Free Dna ◽  
Genetic Profiling ◽  
Non Invasive ◽  
Free Dna

AbstractCell free DNA (cf-DNA) refers to all non -ncapsulated DNA present in the blood stream which may originate from apoptotic cells as a part of the physiological cell turnover, or from cancer cells or fetal cells. Recent studies have highlighted the utility of cfDNA analysis for genetic profiling of cancer, non-invasive prenatal testing besides many other clinical applications. In our review we discuss the sources of cfDNA in the body, the techniques most commonly being used for its isolation and analysis, the applications of cfDNA testing and the associated pros-cons. We conclude that for prenatal testing, cfDNA analysis provides an effective, non-invasive and safer alternative to traditional amniocentesis and chorionic villus sampling tests. Also, in cancer patients, cfDNA analysis is useful for genetic profiling and follow-up during treatment. However, standardization of methods of isolation and analysis has become crucial for the success of widespread use of cfDNA analysis.

Prenatal Screening and Diagnosis

2018 ◽  
Author(s):  
Barbara O’Brien ◽  
Emily Willner
Keyword(s):  
Diagnostic Testing ◽  
Chorionic Villus ◽  
Clinical History ◽  
Maternal Serum ◽  
Specific Gene ◽  
Chorionic Villus Sampling ◽  
Genetic Syndromes ◽  
Cell Free Dna ◽  
Free Dna ◽  
Ultrasound Evaluation

Prenatal genetic testing offers patients and providers the opportunity to screen for aneuploidy, genetic syndromes, and congenital malformations during pregnancy. Screening options include taking a clinical history, evaluation of maternal serum markers or noninvasive cell-free DNA, and ultrasound evaluation during the first and second trimesters. Invasive diagnostic testing such as amniocentesis or chorionic villus sampling allows for further investigation of positive screening results and a directed test to identify aneuploidy as well as specific gene mutations and gain, loss, or rearrangement of genetic information. Laboratory methods for testing fetal samples differ by types of genetic abnormalities that can be detected and turnaround time for results; these methods include karyotype, fluorescence in situ hybridization, and microarray.   This review contains 5 figures, 5 tables and 43 references Key words: amniocentesis, aneuploidy, cell-free DNA, chorionic villus sampling, karyotype, microarray, prenatal genetic screening, ultrasonography  

scholarly journals Implementation of cell-free DNA-based non-invasive prenatal testing in a National Health Service Regional Genetics Laboratory

2019 ◽  
Vol 101 ◽  
Author(s):  
Fiona S. Togneri ◽  
Mark D. Kilby ◽  
Elizabeth Young ◽  
Samantha Court ◽  
Denise Williams ◽  
...  
Keyword(s):  
National Health Service ◽  
High Risk ◽  
Health Service ◽  
National Health ◽  
Prenatal Testing ◽  
Low Risk ◽  
Trisomy 13 ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna

Abstract Background Non-invasive prenatal testing (NIPT) for the detection of foetal aneuploidy through analysis of cell-free DNA (cfDNA) in maternal blood is offered routinely by many healthcare providers across the developed world. This testing has recently been recommended for evaluative implementation in the UK National Health Service (NHS) foetal anomaly screening pathway as a contingent screen following an increased risk of trisomy 21, 18 or 13. In preparation for delivering a national service, we have implemented cfDNA-based NIPT in our Regional Genetics Laboratory. Here, we describe our validation and verification processes and initial experiences of the technology prior to rollout of a national screening service. Methods Data are presented from more than 1000 patients (215 retrospective and 840 prospective) from ‘high- and low-risk pregnancies’ with outcome data following birth or confirmatory invasive prenatal sampling. NIPT was by the Illumina Verifi® test. Results Our data confirm a high-fidelity service with a failure rate of ~0.24% and a high sensitivity and specificity for the detection of foetal trisomy 13, 18 and 21. Secondly, the data show that a significant proportion of patients continue their pregnancies without prenatal invasive testing or intervention after receiving a high-risk cfDNA-based result. A total of 46.5% of patients referred to date were referred for reasons other than high screen risk. Ten percent (76/840 clinical service referrals) of patients were referred with ultrasonographic finding of a foetal structural anomaly, and data analysis indicates high- and low-risk scan indications for NIPT. Conclusions NIPT can be successfully implemented into NHS regional genetics laboratories to provide high-quality services. NHS provision of NIPT in patients with high-risk screen results will allow for a reduction of invasive testing and partially improve equality of access to cfDNA-based NIPT in the pregnant population. Patients at low risk for a classic trisomy or with other clinical indications are likely to continue to access cfDNA-based NIPT as a private test.

P01.05: Preliminary experience of non-invasive prenatal testing with cell-free DNA for aneuploidies in a clinical setting

2014 ◽  
Vol 44 (S1) ◽  
pp. 182-182
Author(s):  
C. Comas ◽  
M. Echevarria ◽  
M. Rodríguez ◽  
P. Prats ◽  
I. Rodríguez ◽  
...  
Keyword(s):  
Clinical Setting ◽  
Prenatal Testing ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna

scholarly journals The fragmentation patterns of maternal plasma cell‐free DNA and its applications in non‐invasive prenatal testing

2020 ◽  
Vol 40 (8) ◽  
pp. 911-917 ◽  
Author(s):  
Min Pan ◽  
Pingsheng Chen ◽  
Jiafeng Lu ◽  
Zhiyu Liu ◽  
Erteng Jia ◽  
...  
Keyword(s):  
Plasma Cell ◽  
Prenatal Testing ◽  
Maternal Plasma ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Fragmentation Patterns

scholarly journals Circulating cell-free DNA levels increase variably following chorionic villus sampling

2010 ◽  
Vol 30 (4) ◽  
pp. 325-328 ◽  
Author(s):  
Neeta L. Vora ◽  
Kirby L. Johnson ◽  
Inga Peter ◽  
Hocine Tighiouart ◽  
Steven J. Ralston ◽  
...  
Keyword(s):  
Chorionic Villus ◽  
Chorionic Villus Sampling ◽  
Cell Free Dna ◽  
Free Dna ◽  
Circulating Cell Free Dna

Prenatal Screening and Diagnosis

2018 ◽  
Author(s):  
Barbara O’Brien ◽  
Emily Willner
Keyword(s):  
Diagnostic Testing ◽  
Chorionic Villus ◽  
Clinical History ◽  
Maternal Serum ◽  
Specific Gene ◽  
Chorionic Villus Sampling ◽  
Genetic Syndromes ◽  
Cell Free Dna ◽  
Free Dna ◽  
Ultrasound Evaluation

Prenatal genetic testing offers patients and providers the opportunity to screen for aneuploidy, genetic syndromes, and congenital malformations during pregnancy. Screening options include taking a clinical history, evaluation of maternal serum markers or noninvasive cell-free DNA, and ultrasound evaluation during the first and second trimesters. Invasive diagnostic testing such as amniocentesis or chorionic villus sampling allows for further investigation of positive screening results and a directed test to identify aneuploidy as well as specific gene mutations and gain, loss, or rearrangement of genetic information. Laboratory methods for testing fetal samples differ by types of genetic abnormalities that can be detected and turnaround time for results; these methods include karyotype, fluorescence in situ hybridization, and microarray.   This review contains 5 figures, 5 tables and 43 references Key words: amniocentesis, aneuploidy, cell-free DNA, chorionic villus sampling, karyotype, microarray, prenatal genetic screening, ultrasonography  

Cell-free DNA and RNA - measurement and applications in clinical diagnostics with focus on metabolic disorders

2020 ◽  
Author(s):  
Markus Hodal Drag ◽  
Tuomas Oskari Kilpeläinen
Keyword(s):  
Metabolic Disorders ◽  
Solid Organ Transplantation ◽  
Prenatal Testing ◽  
Clinical Diagnostics ◽  
Solid Organ ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Alcoholic Fatty Liver ◽  
Non Invasive ◽  
Free Dna

Circulating cell-free DNA (cfDNA) and RNA (cfRNA) hold enormous potential as a new class of biomarkers for the development of non-invasive liquid biopsies in many diseases and conditions. In recent years, cfDNA and cfRNA have been studied intensely as tools for non-invasive prenatal testing, solid organ transplantation, cancer screening, and monitoring of tumors. In obesity, higher cfDNA concentration indicates accelerated cellular turnover of adipocytes during expansion of adipose mass and may be directly involved in the development of adipose tissue insulin resistance by inducing inflammation. Furthermore, cfDNA and cfRNA have promising diagnostic value in a range of obesity-related metabolic disorders, such as non-alcoholic fatty liver disease, type 2 diabetes, and diabetic complications. Here, we review the current and future applications of cfDNA and cfRNA within clinical diagnostics, discuss technical and analytical challenges in the field, and summarise the opportunities of using cfDNA and cfRNA in the diagnostics and prognostics of obesity-related metabolic disorders.

scholarly journals Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Juozas Gordevičius ◽  
Milda Narmontė ◽  
Povilas Gibas ◽  
Kotryna Kvederavičiūtė ◽  
Vita Tomkutė ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Prenatal Testing ◽  
Chromosome 21 ◽  
Whole Genome ◽  
Cell Free Dna ◽  
Chorionic Villi ◽  
Maternal Cell ◽  
Non Invasive ◽  
Fetal Dna ◽  
Free Dna

Abstract Background Massively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test. Methods We employed uTOPseq and hmTOP-seq approaches which combine covalent derivatization of unmodified or hydroxymethylated CG sites, respectively, with next generation sequencing, or quantitative real-time PCR. Results We detected increased 5-hydroxymethylcytosine (5hmC) levels in fetal chorionic villi (CV) tissue samples as compared with peripheral blood. Using our previously developed uTOP-seq and hmTOP-seq approaches we obtained whole-genome uCG and 5hmCG maps of 10 CV tissue and 38 cfDNA samples in total. Our results indicated that, in contrast to conventional whole genome sequencing, such epigenomic analysis highly specifically enriches fetal DNA fragments from maternal cfDNA. While both our approaches yielded 100% accuracy in detecting Down syndrome in fetuses, hmTOP-seq maintained such accuracy at ultra-low sequencing depths using only one million reads. We identified 2164 and 1589 placenta-specific differentially modified and 5-hydroxymethylated regions, respectively, in chromosome 21, as well as 3490 and 2002 Down syndrome-specific differentially modified and 5-hydroxymethylated regions, respectively, that can be used as biomarkers for identification of Down syndrome or other epigenetic diseases of a fetus. Conclusions uTOP-seq and hmTOP-seq approaches provide a cost-efficient and sensitive epigenetic analysis of fetal abnormalities in maternal cfDNA. The results demonstrated that T21 fetuses contain a perturbed epigenome and also indicated that fetal cfDNA might originate from fetal tissues other than placental chorionic villi. Robust covalent derivatization followed by targeted analysis of fetal DNA by sequencing or qPCR presents an attractive strategy that could help achieve superior sensitivity and specificity in prenatal diagnostics.

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