NIPD

Ahmed El-Habashy

A.Lect.OB/GYN.Alex.Univ.

Non-Invasive Prenatal Diagnosis

THE FUTURE ARRIVED

OUTLINES• TERMINOLOGY

• TICHNIQUE & SAMPLING

• APPLICATION

• ACCURACY

• RECOMMENDATIONS

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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.

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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