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NIPD
Ahmed El-Habashy
A.Lect.OB/GYN.Alex.Univ.
Non-Invasive Prenatal Diagnosis
THE FUTURE ARRIVED
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OUTLINES• TERMINOLOGY
• TICHNIQUE & SAMPLING
• APPLICATION
• ACCURACY
• RECOMMENDATIONS
NIPD Habashy
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What is cffDNA?
• 1st reporting of cffDNA in the Maternal Circulation 1997.
• From Placental Apoptosis.
• cffDNA in Mother is 25 times > Fetus.
• Not contained within cells.• Short life 3h.
Lo YM et al. Lancet 1997.
NIPD Habashy
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cffDNA F/M FRACTIONSNIPD Habashy
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NIPD = NIPT = NIPSDiagnosis Testing Screening.
Although
• cffDNA discovered Since 1997,
• FTS Non-Invasive Prenatal Screening of Fetal Trisomies.
• Its Implementation in Practice was just since 2011.
• NIPD = cffDNA ONLY.
NIPD Habashy
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NIPD GENERATIONS
1st
Quantitative
MPS
SHOTGUN
TARGTED
2nd
SNP
NIPD Habashy
K. Nicolaides Prenat Diagn 2013
Random MPSS
Selective Directed
Single Nucleotide Polymorphism
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Massively Parallel Sequencing MPS
• Millions of DNA fragments From All Chromosomes
• Most Widely Used NIPT
• Digital ANalysis of Selected Regions DANSR
Sparks AB Prenat Diagn 2012K.Nicolaides AJOG 2012Ehrich et al Am J Obstset Gynecol 2011
Shotgun MPSS Targeted / Directed MPS
NIPD Habashy
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MPSS VS DANSR
• ?!?!?WHETHER DANSR or MPSS MORE ACCURATE RESULS.
• Need Further Studies to COMPARE BOTH APPROACHES IN the SAME COHORT of PATIENTS.
DANSR Use 1/10 cfDNA Fragments in MPSSMay ↑ Resampling
This ↑Throughput & ↓ Cost.
RCOG 2014
NIPD Habashy
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STEPS of MPS
PCR Amplification SEQUENCING ANALYTICAL
ALGORITHM
NIPD Habashy
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Chromosome Mapping to a Reference Disomic Genome
NIPD Habashy
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SEQUENCINGSpatial Parallel Segregation
F/M cfDNA PROPORTIONAL RELEATIONSHIP
NIPD Habashy
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2nd Generation NIPD
SNP
• Also a Targeted Sequencing.• Not Need A Disomic Reference Chromosome.
Zimmermann Prenat Diag 2012K.Nicolaides Prenat Diagn2013
NIPD Habashy
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SNPNIPD Habashy
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cffDNA SAMPLING • >10w GA ( SNP can at 9w).• Spontaneous or Assisted Conception.• 10-20 cc Maternal Venous Blood. (?! 2cc)*• Results within 1-2 w. • 5 different Companies (USA,Germany,China).• PRICE : 800-3000 $.
*K.Nicolaides AJOG 2012
NIPD Habashy
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cffDNA APPLICATIONSNot Present in the
Mother
Gender
RH
Paternity
(NIPP)
Single Gene Disorder
In Relation to What is Present in the Mother
Autosomal Trisomy
Sex Chromosomal Aneuploidy
Triploidy
Deletion
NIPD Habashy
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Autosomal Aneuploidy Commonest
•T21•T18•T13
On Request in Some NIPT
•T16•T22
NIPD Habashy
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Sex Chromosomal Aneuploidy (Some NIPT)
• XO (Turner Syndrome)• XXX (Triple X)• XXY (Klienfelter Syndrome)• XYY (Jacob Syndrome)
NIPD Habashy
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SO NIPD Can Detect ~ 85% of Fetal Chromosomal Aneuploidy
NIPD Habashy
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Deletion SyndromesSome NIPD
• 22q11.2 deletion syndrome (DiGeorge)
• Cri-du-chat syndrome (5p minus)
• Prader-Willi/Angelman syndrome (15q)
• 1p36 deletion syndrome
NIPD Habashy
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SensiGene• RhD –ve Mother / RhD +ve Father.• +ve Maternal Indirect Coomb (Sensitized).• ± Hetrogenous Rh +ve Father.• cffDNA♀RhD-ve
Can not Verify the presence of Fetal DNA
= Mother
RCOG 2014
NIPD Habashy
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SensiGene- SNP• Fetal Identifier (FI) control is performed to
Compare M/F genotype.• If ≥ 6 Markers are Observed Verify True
RhD-ve ♀ Fetus:
oNo Antenatal RhIG Prophylaxis .oNo Extra M/F Testing (± Invasive)
RCOG 2014
NIPD Habashy
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Fetal Gender
• If ♀ Fetus 50% ↓INVASIVE TESTING.
• If ♀ Fetus Early Dexamethasone ↓Virilization
• If ♂ Fetus Early cessation of Dexamethasone.
Duchenne Muscle Dystrophy
CAH
RCOG 2014
NIPD Habashy
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Single Gene DisordersPaternally Inherited Allele
Autosomal Dominant
• Huntington Disease.• Achondroplasia.• Thanatophoric Dysplasia.
Autosomal Recessive
• ß Thalassemia.• Cystic Fibrosis.
ABSENT Paternal Allele NO INVASIVE
TESTING.
RCOG 2014
NIPD Habashy
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NIPD Accuracy • SPECIFICITY and SENSETIVITY CLOSE
TO 100% (>98%) FOR T21,18; Less for T13.• FPR < 0.5%.
• 1-10% Chance of No Result (Sample Failure- No Call Rate) Mostly √ in Repeat Sample. (Mostly FREE).
RCOG 2014
NIPD Habashy
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Possible Sources of ERRORSFALSE-ve
• ↓ Fetal cffDNA FractionoEarly GA:<10w
<4-5%.oMaternal Obesity.
FALSE+ve• Confined Placental
Mosaicism (CPM):
Trisomic Placental Cells BUT a NORMAL FETUS.
Maternal Conditions:o Chromosomal Aberrations.o Malignancy.RARELY DISCORDANT RESULTS
TWIN
Still Evolving
RCOG 2014
NIPD Habashy
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Maternal Weight VS cffDNANIPD Habashy
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cffDNA in Twining
MC
• Not only Possible BUT MORE Effective.
DC
• CAN Calculate % Fetal cfDNA Fraction of EACH FETUS.
If 1 Miscarries ?!?Its Fraction Change
Still EvolvingcffDNADx Zygocity *
*Qu JZ.et al. Clin Chem 2013RCOG 2014
NIPD Habashy
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Comparison with Screening Tests
• Combined T21 FTS = NT,ßHCG,PAPPA 90% DR with 3% FPR
• Sensitivity & Specificity of cffDA for T21≈100% .
↓INVASIVE TESTING: o COST o LOSSES
↓
MORE↓
K.Nicolaides Ultrasound Obstet Gynecol 2007
NIPD Habashy
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Prenatal Triosomy Detection Rate DR
FTS NIPD Invasive(Karyotyping)
NIPD Habashy
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ACOG,SMFM,ACMG,ISPD• cffDNA is some FORM
of CONTINGENT Approach offering testing to HIGH RISK Pregnancies:
• Maternal Age ≥35Ys.
• Previous T21.• Parental Balanced
Translocation.• US Aneuploidy
Marker(s).• +ve Biochemical
Screening test.
THE MOST EFFECTIVE ANEUPLOIDY
SCREENING IN HIGH RISK PREG.
ASOG,SMFM 2012ACMG,ISPD 2013
NIPD Habashy
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SOGC• >98% Detection Rate.• <0.5 FPR.• NOT A DIAGNOSTIC TEST.• Can Not Replace US (NT).• +ve NIPS SHOULD do CONFIRMATORY
INVASIVE TEST BEFORE TOP.• NIPT May REPLACE the CURRENT
MATERNAL SCREENING APPROACHES IF:*o↓ COST.o↑ Studies in AVERAGE RISK PREG.
Sholud Included in Pre-Test Pt.
COUNSILING*
*ACOG 2012*SOGC 2013
NIPD Habashy
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NIPT IMPLEMENTATION
• ↑ COST : (COMMERCIALISATION and INTELLECTUAL PROPERTY).
• FTS also Predict PET,FGR.• FTS also Predict Many Non-Chromosomal Structural
Anomalies. • If Contingent (done for +ve FTS) MISS the FALSE –ve of
FTS.• LIMITED STUDIES IN AVERAGE RISK* AND TWINS.• ↓ INVASIVE TESTING ↓ Fetal Medicine Trainee SKILLS.• Limited Geographical Lab. Distribution.
AGAINST
*K.Nicolaides AJOG 2012
NIPD Habashy
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NIPT IMPLEMENTATION
• NO F/M Harm.• ↓ False –ve. (~ 38%*)• ↓ False +ve of FTS • ↓ INVASIVE TESTING COST &• ↓ INVASIVE TESTING Losses. (~ 66%*)• IF REPLACE ßHCG,PAPPA SAVE THERE COST
FOR NIPT.• Multiplexing many patient samples in a SINGLE
SEQUENCING RUN ↓ Individual TEST COST.
WITH
*Garfield S J Manag Care Med. 2012
NIPD Habashy
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NIPD CAN ADJUST FTS RISK
• +ve NIPT x 290 ↑ FTS Risk of T21• -ve NIPT x 110 ↓ FTS Risk of T21
Benn et al.,Ultrasound Obstet Gynecol ;2012
Benn Calculator
NIPD Habashy
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ACMG 2013• No Doubt NIPS Costs WILL ↓.• NIPS is Likely the 1st of major steps toward
the eventual application of WHOLE FETAL GENOME SEQUENCING.
NIPD Habashy
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NIPT Market Competition • Intellectual
Property .• Non-Profit Funding.• ↓ Lab. Geographic
Distribution.• Companies Litigation
• None of them FDA Approved Yet ( in Progress).
• No Insurance Coverage.
• Stick-holders Should Pay Attention to NIPD Commercialization ↑ Patient Access.
Ashwin A .Prenat Diagnos 2013
NIPD Habashy
MARKET MONOPOLY
↑ COST
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Trials • Maternal Urine : cffDNA
(MPS)*.• Maternal Blood :
ocffRNA (more Fetus Specific)oFetal Proteins.oFetal CELLS.
NIPD Habashy
Lo YM , K.Nicolaides Nat Med 2007
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Take Home Message
• NIPD High Accuracy + NO F/M Harm REVOLUTION in FETAL MEDICINE.
• NIPD MAJOR PROBLEM ↑ COST FALLING May REPLACE BIOCHEMICAL MARKERS BUT CAN NOT REPLACE US.
• NIPD are NOT DIAGNOSTIC SHOULD BE CONFERMED BY INVASIVE TEST.
NIPD Habashy
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THANK YOU