1. Genetic counseling, prenatal diagnosis, PGD, PGS, NIPT; promises and challenges
Dr. Masoud Garshasbi (PhD)
Department of Medical Genetics
Faculty of Medical Sciences
Tarbiat Modares University

2. What is Genetic Counseling?
communication process
address individual concerns relating to development / transmission of hereditary disorder
consultand = individual who seeks genetic counseling
strong communicative and supportive element so that those who seek information are able to reach their own fully informed decisions without undue pressure or stress

3. Steps in Genetic Counseling
Diagnosis - based on history, examination and investigations
Risk assessment
Communication
Discussion of Options
Long-term contact and support

4. Prenatal Diagnosis

5. Techniques
Invasive
Minimally invasive
Non-invasive

6. Invasive Techniques Amniocentesis Chorionic villus sampling (CVS)
FETAL BLOOD SAMPLING (cordocentesis)

7. INVASIVE TESTS AMNIOCENTESIS:
Aspiration of amniotic fluid which contain fetal cells
Fetal cells-cultured – karyotyping (3 weeks).
New technique-QF-PCR, MLPA and FISH-give result in 48 h.
Preferred time of test 16 weeks of pregnancy.

8. Amniocentesis

9. AMNIOCENTESIS---Cont.
Limitations (difficulties)of procedure: if
- Anteriorly placed placenta
- Multiple pregnancy.
- Maternal obesity
- Oligohydramnios
Risks:
- Pregnancy loss 1 % - Bleeding , Infection,
- Rupture of membrane - Preterm labour
- Leaking of Amniotic fluid

10. CHORIONIC VILLUS SAMPLING
Collection of fragments of placental tissue (chorionic villi)- cells
Cytotrophoblastic (rapidly dividing) cells are used for direct karyotyping- result available within h.
Chorionic villi are best source of DNA
CVS can be performed at 11 weeks gestation.
Indications:
- DNA analysis for genetic disorders such as thallasemias, CF. hemophillias and etc…
- Chromosomal abnormalities

12. Minimally Invasive Techniques
Pre-implantation genetic diagnosis (PGD)
Pre-implantation genetic screening (PGS)

13. Pre-implantation Genetic Diagnosis (PGD)
Genetic analysis of a single cell from an eight-cell embryo done in conjunction with in vitro fertilization (IVF) to improve the chances of a “normal” pregnancy.

14. Preimplantation genetic screening (PGS)
The preimplantation genetic screening (PGS) is a procedures that do not look for a specific disease but use PGD techniques to identify embryos at risk.

33. Non-invasive techniques
Cell free fetal DNA (cffDNA)

34. Theoretical Basis of NIPT test
Fundamental Features of Cell-Free Fetal DNA
① The content is 970 times greater than the DNA level of fetal cells in maternal blood.
② Can be detected in maternal plasma from the 5th week of gestation. The concentration increases with the gestation。
③ Disappears in 2 hours after childbirth.
1, That is why we choose it for our analysis
2, The amount of cell-free fetal DNA is increasing with the gestation age, and it can be detect as early as 5 weeks of gestation, but there are individual differences.
3, The cell-free fetal DNA has a short half-life, it’s mean half-life is 16.3 min, which means the fetal DNA will disappear soon after delivery. It allow for independent analysis when a woman have a secondary pregnancy

36. Targeting fetal-specific nucleic acids
• Fetal-specific RNA • Fetal-specific DNA methylation

37. Population coverage: 45%

39. A general approach

40. Principle of NIPT fragments for chrN %chrN = total fragments
Plasma cf-DNA extraction
36 bp
Bioinformatics
Sequencing & alignment
Firstly There are a lot of fetal and maternal DNA fragments circulating in maternal plasma
After the DNA extraction, we need to solve the problem is which chromosome does every fragment comes from? Sequence 50bp of every fragment, it is served as a tag to recognize the specific chromosome, with the help of bioinformatics,fragments can be mapped to chromosome uniquely.
Then, we count the number of every chromosome by counting the uniquely mapped fragments.
After that, we compute the value of chrN% which is a very important value for later statistics analysis.
As we know if a fetus has a third 21 chromosome, the chromosome 21 fragment must be slightly higher than expected. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Chromosomes
counting
%chrN =
total fragments
fragments for chrN
Coverage computation

41. Statistics for t score and L score
Statistic Model
100 cell-equivalent free DNA /ml plasma: 95 from mother, 5 from fetus.
Pregnant woman with health fetus
Pregnant woman with trisomy 21 fetus
Mother Chr 21:
95X2=190
Fetus Chr 21:
5X2=10
Mother Chr 21:
95X2=190
Fetus Chr 21:
5X3=15
In maternal blood Total Chr 21=190+10=200
In maternal blood Total Chr 21=190+15=205
Let us set a simple model to understand this idea. Assume there are…, means volume of free DNA is equal to the volume in a integral cell
In pregnant woman with health fetus, number of chr21 of mother is
Please noted that: we don’t isolate the fetal DNA and count it independently, we count total number. We will make statistics analysis for t score and L score, to detect the discrepancy of total chr 21 between pregnant woman with health fetus and pregnant woman with trisomy 21 fetus
L：likelihood ratio
Statistics for t score and L score
GC correction
Binary hypothesis statistics analysis

42. (Acquired by Illumina) Companies from Germany, UK, Spain, Portugal…
NIPT
NIPS
Cell Free DNA
BGI
Sequenom
(Acquired by Labcorp)
Varinata
(Acquired by Illumina)
Ariosa
(Acquired by Roche)
Natera
Companies from Germany, UK, Spain, Portugal…
2010.9
2012.2
2012.5
After 2013
PreNatest, IONA,
NACE, TOMORROW…

43. Whole Genome MPS based NIPT (NIFTY®, MaterniT21®, Verify®)
Benefits:
Future extensibility
Low assay failure rate
Fast TAT

44. Targeted MPS based NIPT (Harmony®)
Benefits:
lower cost
Drawbacks:
High failure rate
Limited to add new content
Longer TAT

45. Targeted SNPs Sequencing based NIPT (Natera®)
Maternal Blood
cfDNA (Maternal+ Fetal)
Maternal DNA in WBCs
SNP Sequencing
Maternal & Fetal Genotype
Maternal Genotype
Risk Result
Deduce Fetal Genotype
Benefit:
Vanished twin and triploidy
Drawbacks:
High failure rate
Difficult to analyze egg donation, twins and multiple pregnancy
Limited to add new content
Longer TAT

47. Non Reportable Rate NIFTY® 0.7% (79/11184)* MaterniT21® 0.9% Harmony®
Non-Reportable Rate：Cases where no result was obtained on initial and repeat sampling
Non Reportable Rate
NIFTY®
0.7% (79/11184)*
MaterniT21®
0.9%
Harmony®
2% (91/3228)**
Verifi®
2.89% (173/5974)***
Natera®
5.4% ****
*Dan et al., Prenatal Diagnosis, 2012, 32,1–8
**Norton ME, et al., Am J Obstet Gynecol., 2012 Aug;207(2):137
***Futch T, et al., Prenatal Diagnosis, 2013, 33, 569–574
**** Nicolaides KH, et al., Prenatal Diagnosis, 2013, 33, 575–579

49. Selected Chromosome Sequencing Selected SNP Sequencing
BGI NIFTY
Sequenom MaterniT21
Verinata Verifi
Ariosa Harmony
Natera Panorama
Technology
WGS
Selected Chromosome Sequencing
Selected SNP Sequencing
Pubished Paper
>30
>10
>20
Launch Time
2010
2011
2012
2013
Tested Samples (till )
1,000,000
570,000
300,000
600,000
Turnaround Time
4-5 days
Insurance
YES NO
Resampling Rate 4% 5% 6%
10%

50. BGI NIFTY
Sequenom MaterniT21
Verinata Verifi
Ariosa Harmony
Natera Panorama
Failure rate
0.7% (79/11184)
0.90%
2.89% (173/5974)*
2% (91/3228 or [57+91[/3228) **
5.4%***
Sensitivity
T21
99.65%
99.1% (210/212)
100% (90/90)
>99% (231/232)
100% (83/83)
T18
99.66%
100% (59/59)
97.4% (37/38)
>98% (103/105)
>96.4% (27/28)
T13
100 %
91.7% (11/12)
87.5% (14/16)
8/10
100% (13/13)
Specificity
99.99%
99.9% (1484)
99.8% (409/410)
>99.9%
100%
99.98%
99.6% (1929)
99.6% (461/463)
99.7% (1976)
99.9% (485/485)

51. BGI NIFTY
Sequenom MaterniT21
Verinata Verifi
Ariosa Harmony
Natera Panorama
Twin Pregnancy
YES
Gender
Gender Accuracy
97-98%
0.994
97.6%-99.1%
98%
Sex Chromosome Aneuploidies
SCA Accuracy
>95% -
Deletion Syndrome
8 type
7 type NO
Optional
Triploidy
Surrogation

52. BGI NIFTY
Sequenom MaterniT21
Verinata Verifi
Ariosa Harmony
Natera Panorama
Egg Donor
YES NO
Starting Week
10th week
9th week
Mother Blood
10ml
2* 10ml
Father Blood
Fetal Fraction Measurement
Sequencing Data 6M
16.3M
22.8M
1.15M
6.7M
Certification
CAP, ISO15189
CLIA CAP
CLIA