Download - NIPD
NIPD
Ahmed El-Habashy
A.Lect.OB/GYN.Alex.Univ.
Non-Invasive Prenatal Diagnosis
THE FUTURE ARRIVED
OUTLINES• TERMINOLOGY
• TICHNIQUE & SAMPLING
• APPLICATION
• ACCURACY
• RECOMMENDATIONS
NIPD Habashy
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.
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cffDNA F/M FRACTIONSNIPD Habashy
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.
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NIPD GENERATIONS
1st
Quantitative
MPS
SHOTGUN
TARGTED
2nd
SNP
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K. Nicolaides Prenat Diagn 2013
Random MPSS
Selective Directed
Single Nucleotide Polymorphism
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
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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
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STEPS of MPS
PCR Amplification SEQUENCING ANALYTICAL
ALGORITHM
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Chromosome Mapping to a Reference Disomic Genome
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SEQUENCINGSpatial Parallel Segregation
F/M cfDNA PROPORTIONAL RELEATIONSHIP
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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
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SNPNIPD Habashy
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
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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
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Autosomal Aneuploidy Commonest
•T21•T18•T13
On Request in Some NIPT
•T16•T22
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Sex Chromosomal Aneuploidy (Some NIPT)
• XO (Turner Syndrome)• XXX (Triple X)• XXY (Klienfelter Syndrome)• XYY (Jacob Syndrome)
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SO NIPD Can Detect ~ 85% of Fetal Chromosomal Aneuploidy
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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
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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
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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
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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
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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
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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
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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
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Maternal Weight VS cffDNANIPD Habashy
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
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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
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Prenatal Triosomy Detection Rate DR
FTS NIPD Invasive(Karyotyping)
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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
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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
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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
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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
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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
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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.
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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
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MARKET MONOPOLY
↑ COST
Trials • Maternal Urine : cffDNA
(MPS)*.• Maternal Blood :
ocffRNA (more Fetus Specific)oFetal Proteins.oFetal CELLS.
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Lo YM , K.Nicolaides Nat Med 2007
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
THANK YOU