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

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

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

Prenatal Diagnosis

Techniques Invasive Minimally invasive Non-invasive

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

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.

Amniocentesis

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

CHORIONIC VILLUS SAMPLING Collection of fragments of placental tissue (chorionic villi)- cells Cytotrophoblastic (rapidly dividing) cells are used for direct karyotyping- result available within 24-48 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

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

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.

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.

Non-invasive techniques Cell free fetal DNA (cffDNA)

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

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

Population coverage: 45%

A general approach

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

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

(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 2011.10 2012.2 2012.5 2012.12 After 2013 PreNatest, IONA, NACE, TOMORROW…

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

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

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

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

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 2016.3) 1,000,000 570,000 300,000 600,000 Turnaround Time 4-5 days Insurance YES NO Resampling Rate 4% 5% 6% 10%

  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)

  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

  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