1. Dr Skander Ben Abdelkrim Centre Hospitalier Princesse Grace Monaco
Cell-Free Fetal DNA for Non-Invasive Prenatal Diagnosis and Testing (NIPD , NIPT)
Dr Skander Ben Abdelkrim
Centre Hospitalier Princesse Grace
Monaco

2. CELL-FREE FETAL DNA: Discovery
First described in 1997*
Fetal sex , RHD blood group determination
Advent of Massively Parallel Next- Generation Sequencing (MPS-NGS): widescreen detection of fetal aneuploidy
Fetal genome present in its entirety: whole genome sequencing (WGS)**
CELL-FREE FETAL DNA: Discovery
* Dennis Lo et al, Presence of fetal DNA in maternal plasma and serum, Lancet 350 (1997) 485–487).
** Dennis Lo et al, Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci. Transl. Med. 2 (2010).
Conventionally genetic prenatal diagnosis requires an invasive procedure, namely chorionic villus sampling (CVS) or amniocentesis, which carries a small risk of miscarriage (approximately 0.5–1%).
First description by Pr Dennis Lo in 1997
Prior to this, it was known that fetal cells circulated in the maternal blood stream; however, their clinical application is limited due to the paucity of fetal cells in the maternal circulation.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

3. CLINICAL APPLICATIONS FOR cffDNA: 1- Noninvasive Prenatal Diagnosis
RhD-negative mothers: fetuses at risk of hemolytic disease of the newborn by detecting the presence of fetal RHD in the maternal plasma.
Sex-linked conditions: to triage for invasive testing or treatment by determining fetal sex using markers on the Y chromosome.
Pregnancies at risk of de novo, dominant, or recessive conditions: because of a known family history or ultrasound findings.
CLINICAL APPLICATIONS FOR cffDNA: 1- Noninvasive Prenatal Diagnosis
The purpose of NIPD is to confirm the presence or absence of a specific genomic region in a fetus and is therefore diagnostic, hence the terminology NIPD.
RhD-negative mothers
Sex-linked conditions
Pregnancies at risk of de novo, dominant, or recessive conditions.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

4. Fetal Sex Determination
Can be used to avoid invasive testing in up to 50% of cases at risk of X-linked disorders where a female fetus would not be affected.
Also to guide treatment in pregnancies at risk of congenital adrenal hyperplasia (CAH), where dexamethasone treatment can be administered to mothers bearing female fetuses to reduce virilization of the external genitalia in affected female.
Fetal Sex Determination
X-linked disorders
congenital adrenal hyperplasia
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

5. A useful clinical example: mutations in the HBB gene, which causes sickle cell anemia and β- thalassemia.
Monogenic Diseases
sickle cell anemia and β-thalassemia
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

6. An approach termed relative mutation dosage (RMD) can be used*.
* Lun et al., Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma, Proc. Natl. Acad. Sci. U.S.A. 105 (2008)
Monogenic Diseases
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

7. NIPD for CFTR (CYSTIC FIBROSIS)
Hill et al. (2015) recently described the introduction to clinical practice of a cystic fibrosis assay for noninvasive exclusion of 10 CFTR mutations*.
NIPD for CFTR (CYSTIC FIBROSIS)
* M. Hill, S. Drury et al., NIPD for cystic fibrosis: detection of paternal mutations, exploration of patient preferences and cost analysis. Prenat. Diagn. 35 (2015) 950–958
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

8. CLINICAL APPLICATIONS FOR cffDNA: NIPT for Aneuploidies
Relies on the ability to detect a relatively small overrepresentation of the chromosome in question.
Detection rates for Down syndrome have been reported in excess of 99%, and slightly lower for trisomies 18 and 13, 96% and 92%, respectively* (Nicolaides et al, 2015).
CLINICAL APPLICATIONS FOR cffDNA: NIPT for Aneuploidies
Relies on the ability to detect a relatively small overrepresentation of the chromosome in question.
As in other situations, detection of fetal aneuploidy by analysis of cfDNA is challenging due to the preponderance of cell-free maternal DNA in the plasma.
Thus detection of aneuploidy relies on the ability to detect a relatively small overrepresentation of the chromosome in question
* Nicolaides, Analysis of cell-free DNA in maternal blood in screening for fetal aneuploidies: updated meta-analysis. Ultrasound Obstet. Gynecol. 45 (2015)
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

9. Technical Approaches to NIPT
Bioinformatic analysis is performed to determine whether the chromosome of interest is overrepresented.
If the fetus has trisomy 21, for example, more fragments from chromosome 21 will be present in maternal plasma than expected.
Technical Approaches to NIPT
to determine whether the chromosome of interest is overrepresented.
If the fetus has trisomy 21, for example, more fragments from chromosome 21 will be present in maternal plasma than expected.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

10. Detection of fetal aneuploidy by overrepresentation of fetal DNA at chromosome of interest.
Reproduced from A. Swanson, NIPT: technologies, clinical assays and implementation strategies for women's healthcare practitioners, Curr. Genet. Med. Rep. 1 (2013)
There are three main approaches currently used in practice for the detection of fetal aneuploidy, WGS, targeted sequencing, and SNP analysis.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

11. Overview of the currently available applications of MPS-based NIPT
Overview of the currently available applications of MPS-based NIPT. By MPS of cell-free fetal nucleic acids (DNA and RNA) in maternal plasma which likely originates from apoptotic trophoblasts, the fetal genome (from chromosomal to sub-chromosomal to single-gene levels), methylome and transcriptome may be decoded. Wong , Y.M. Dennis Lo, Prenatal Diagnosis Innovation: Genome Sequencing of Maternal Plasma Annu. Rev. Med :2.1–2.14
MPS
By MPS of cell-free fetal nucleic acids (DNA and RNA) in maternal plasma which likely originates from apoptotic trophoblasts, the fetal genome (from chromosomal to sub-chromosomal to single-gene levels), methylome and transcriptome may be decoded.
For cell-free fetal RNA, target RNA molecules containing SNPs are quantified with the use of a PCR assay.
The abundance of specific chromosome sequences can be compared with normal reference samples.
Other methods have been described, including methods to quantify differentially methylated regions of specific fetal chromosomes with the use of PCR.
These methods await validation.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

12. Fetal Fraction Matters
0-4%
4-8%
8%+
“An aneuploidy sample with a lower FF has a higher probability of resulting in a FN result.”
Musci,Prenatal Perspectives. Volume 1, No
No one gets great results on fetal fractions less than 4%. There is too little fetal DNA to get adequate results and all are no-called
>8% is the fraction at which you are likely to get very good results no matter the method
It is the intermediate 4-8% that causes problems. Counting is the most greatly affected.
FF too low to report
Intermediate FF– decreased sensitivity with counting methodology
FF adequate to achieve best performance
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

13. CELL-FREE FETAL DNA: Fetal Fraction (FF)
10 to 20%.
It is now clear that several factors influence the FF, including gestational age, maternal weight, aneuploidy, maternal disease (e.g. preeclampsia), and the number of fetuses present.
CELL-FREE FETAL DNA: Fetal Fraction (FF)
PE: Although not fully elucidated, it is thought that the mechanism by which elevated cfDNA is generated relates to hypoxia and oxidative stress within the placental intervillous space (between mother and embryo), resulting in increased placental apoptosis and necrosis of trophoblasts, as well as reduced renal clearance of cfDNA in preeclampsia.
In addition to cfDNA in maternal plasma, other non-genomic fetus-specific material can be detected in the circulation, most interestingly mRNA transcripts.
In fact, a majority of mRNA transcripts found in maternal blood are derived from the fetus, and these may represent an attractive substrate for monitoring fetal development and potentially disease during early pregnancy
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

14. CELL-FREE FETAL DNA (cffDNA): Fetal Fraction (FF)
Total cfDNA is directly related to maternal BMI and the relatively lower FF with increasing maternal weight (increased release of maternal cfDNA from adipose cells (Bianchi 2012)).
Trisomy 21: increased FF (Palomaki et al, 2013).
However, trisomies 18 and 13: lower levels of cffDNA (reduction in placental volume)*.
CELL-FREE FETAL DNA (cffDNA): Fetal Fraction (FF)
Several studies have shown that there is an increase in fetal DNA in women with symptomatic preeclampsia and those who will develop preeclampsia
A sharp increase in cffDNA levels have been observed at 32 weeks gestation in normal pregnancies which may indicate impending delivery.
CffDNA is also higher in placenta previa and hyperemesis gravidarum, although the underlying pathology here is unclear.
Important concept:
Trisomy 13 and 18  lower placenta volume
* Rava RP et al. (2014). Clinical Chemistry, 60,
And Wegrzyn et al. (2005). Ultrasound in Obstetrics & Gynecology, 26,
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

15. (Available to all women) Reduced false positives
Reduced risk to fetus
(Available to all women)
Reduced
anxiety
Goals
of NIPT
Reduced false positives
Increased detection
Verinata

16. NIPT Performance for the Common Aneuploidies
More recently, it has become clear that NIPT is highly accurate in the general population* and twin pregnancies.
* Norton et al., Cell-free DNA analysis for noninvasive examination of trisomy. N. Engl. J. Med. 372 (2015) 1589–1597
NIPT Performance for the Common Aneuploidies
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

17. NIPT Performance for the Common Aneuploidies
FP or discordant cases have been reported*.
Predominantly due to confined placental mosaicism (CPM)
CPM is of relevance in T18 and T13 cases, which may have a substantial euploïd cell line in the trophoblasts**.
This can lead to FP (abnormal cells in placenta but normal in fetus); or FN results (abnormal cells in the fetus but not placenta).
NIPT Performance for the Common Aneuploidies
* P. Benn, H. Cuckle, E. Pergament, Non-invasive prenatal testing for aneuploidy: current status and future prospects. Ultrasound Obstet. Gynecol. 42 (2013) 15–33
** Grati et al., Fetoplacental mosaicism: potential implications for false-positive and false-negative noninvasive prenatal screening results. Genet. Med. 16 (2014)
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

18. Confined Placental Mosaicism
Possible explanation for a FP result
Present in 1-2% of 1st trimester placentas

19. NIPT False negatives NIPT demonstrated a small chance for a FN result.
Since the “fetal” DNA in maternal blood originates from the CTB of chorionic villi (CV), some FN results will have a biological origin: absence of chromosome aberration in the CTB*. 
NIPT False negatives
* Van Opstal D et al. (2016) False Negative NIPT Results: Risk Figures for Chromosomes 13, 18 and 21 Based on Chorionic Villi Results in 5967 Cases and Literature Review. PLoS ONE 2016
Firstly, generalised mosaicism confined direct normality (GMDD), characterized by the presence of a chromosome aberration in the fetus and mesenchymal core of the placenta, with the CTB being chromosomally normal.
Secondly, although extremely rare, confined fetal mosaicism (CFM) with a normal karyotype in STC-and LTC-villi and with ultimately abnormal cytogenetic results in the fetus.
Both types of mosaicism will show normal NIPT results due to a normal karyotype in the CTB, the fetus having an abnormal karyotype though.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

20. Early embryonic development from zygote to blastocyst.
The CTB which is studied in short-term cultured villi (STC- villi) and with NIPT is derived from the trophoblast of the blastocyst, whereas the mesenchymal core, investigated in long-term cultured villi (LTC-villi) originates from the extra- embryonic mesoderm (EEM).
Both EEM and fetus are derived from the inner cell mass (ICM) of the blastocyst. 
Early embryonic development from zygote to blastocyst.
The CTB which is studied in short-term cultured villi (STC-villi) and with NIPT is derived from the trophoblast of the blastocyst, whereas the mesenchymal core, investigated in long-term cultured villi (LTC-villi) originates from the extra- embryonic mesoderm (EEM).
Both EEM and fetus are derived from the inner cell mass (ICM) of the blastocyst.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

21. NIPT Performance for the Common Aneuploidies
Another consideration for FP results is maternal malignancy, shedding abnormal cell lines into the maternal circulation*.
Reporting on a case series of 125,000 maternal samples, abnormal results (one or more aneuploidy of chromosome 13, 18, 21, X, or Y) in otherwise asymptomatic pregnant women were found in almost 4000 (3%)*.
From these 4000, 10 cases of maternal cancer were identified*.
NIPT Performance for the Common Aneuploidies
* D.W. Bianchi et al., Noninvasive prenatal testing and incidental detection of occult maternal malignancies. JAMA 314 (2015).
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

22. Other Uses of NIPT: Microdeletions and Microduplication Syndromes
Microdeletions and duplications of clinical significance occur in 1–1.7% of structurally normal pregnancies*
Several of the commercially available NIPT services now also offer testing of common microdeletion syndromes, including di George (22q11-) and Cri-de-Chat (5p-).
Other Uses of NIPT: Microdeletions and Microduplication Syndromes
* R.J. Wapner et al., Chromosomal microarray versus karyotyping for prenatal diagnosis. N. Engl. J. Med. 367 (2012) ** Gross et al., Clinical experience with an SNP-based noninvasive prenatal test for 22q11.2 deletion syndrome, Prenat. Diagn. 35 (Suppl. 1) (2015)
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

23. A minimum of 8% fetal DNA is required to detect common fetal trisomies, which is double the minimum amount required for singleton pregnancies (4%).
Vanishing twins are a clear confounding factor for NIPT and should be carefully monitored to aid in interpreting NIPT results.
NIPT in twins
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

24. What are the odds of being affected given a positive result?
Strongly influenced by the a priori risk.
Even with very high specificity (e.g %), when applied to a low-risk population (e.g. with a prevalence of trisomy 21 of 1 in 700), ‘only’ 3/4 positive results will be confirmed by invasive testing.
In a pre-screened high-risk population, at least 9/10 positive results will be confirmed.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

25. What is the remaining risk for trisomy when NIPT is negative?
Sensitivity for T 18 is nearly as high as that for T 21, while that for T 13 is slightly lower.
In clinical practice, this is overcame by the fact that these diseases are nearly always detectable by a detailed ultrasound examination at around 20 weeks of pregnancy, which is offered routinely to most women in developed countries.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

26. Advanced screening test
Lisa Hui and Diana W Bianchi, Noninvasive Prenatal DNA Testing: The Vanguard of Genomic Medicine, Annu. Rev. Med
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

27. Cell-Free Fetal DNA for Prenatal Diagnosis and Testing
Key Points
Less 95 % invasive procedures
Cost-effective
Not a Karyotype
Not indicated if US or NT abnormality
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

28. Key Points: when to apply for NIPT?
CNGOF and ACLF:
CFTS (Combined first-trimester screening markers) > 1/1000
Maternal Age > 38 years
Known Robertsonian translocation of one parent,
CFTS non feasible (twins, outlimits),
and finally if the future mother had a previous pregnancy with a T21 fetus.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing

29. Cell-Free Fetal DNA for Prenatal Diagnosis and Testing
Conclusions
NIPD can now be performed for definitive diagnosis of some recessive and X-linked conditions, rather than just paternally inherited dominant and de novo conditions.
NIPD/T offers greater choice as these safer methods to avoid the risk of miscarriage associated with invasive testing.
Cell-Free Fetal DNA for Prenatal Diagnosis and Testing