Director Italian Branch Cagliari Regional Director for Europe

Director Italian Branch

Cagliari

Regional Director for Europe

Department of Obstetrics&

Gynaecology

PrenatalPrenatal && PreimplantationPreimplantationGeneticGenetic DiagnosisDiagnosis

Fetal Fetal TherapyTherapy

Ospedale Regionale Microcitemie

CagliariCagliari

WHOWHO

Collaborating Centre for Community Control Collaborating Centre for Community Control of of

Hereditary DiseasesHereditary Diseases

Director Ian Donald School

for Invasive Procedures

12th TURKISH GYNECOLOGY AND OBSTETRICS CONGRESS

Antalya, 15th – 19th Maggio 2014

INVASIVE VS NON-INVASIVE PRENATAL DIAGNOSTIC PROCEDURES

Giovanni Monni

DILEMMAS TO AVOID GENETIC DISORDERS IN THE NEWBORNS

Screening based on maternal age alone? First or second trimester ultrasound and

biochemical screening? Prenatal invasive procedures? Standard karyotype? aCGH analysis? Preimplantation genetic diagnosis? Diagnostic ultrasonography (1st-2nd

trimester)? Fetal cell free-fetal DNA (cff- DNA) in

maternal blood (general or contingent) ?

CHANGES IN THE APPROACH FOR INVASIVE PRENATAL DIAGNOSIS IN 35,127 CASES AT A SINGLE CENTER FROM 1977

TO 2004

• DIAGNOSI SEMPRE Più PRECOCE

Monni, Fetal Diagn Ther 2006Monni, Fetal Diagn Ther 2006

Ekelund, BMJ 2008

Number of amniocenteses and chorionic villus samplings carried out

in Denmark, 2000-2006

Total Number of Diagnostic Procedures in England (2003- 2012)

Morgan,UOG 2013

UK national policy study ofaneuploidy screening after the

implementation of the combined test

1. Reduction of false positive rate from 6% to 3% without significant change of DR of Down Syndrome

2. Progressive reduction in the number of screen-positive cases

3. Significant reduction in the number of invasive prenatal diagnostic procedures

Morgan, Ultrsound Obstet Gynecol 2013

The odds of the fetus being affected after a positive combined test in the

first trimester were much greater than were the odds based on

advanced maternal age alone (1:20 (1:20 vs 1:75).vs 1:75).

So a significantly higher probability of an invasive test would confirm an

abnormal fetal karyotype.

UK NATIONAL POLICY STUDYANEUPLOIDY SCREENING

Morgan, Ultrsound Obstet Gynecol 2013

Redistribution of the proportion of procedures performed by amnio and CVS

- Denmark: in 2006 CVS in 66% of cases

- UK: in 2003 Amnio/CVS 3:1 in 2011 Amnio/CVS 1:1

REDUCTION IN THE FETAL NUMBER OF INVASIVE PROCEDURES PERFORMED

FOR PRENATAL KARYOTYPE

Monni, Zoppi, Ultrasound Obstet Gynecol 2013: Opinion

- Decrease in Fetal Loss due to a reduction in invasive diagnostic

procedures

- Earlier Diagnosis of Chromosomal Aneuploidies

FIRST TRIMESTER EUROPEAN NATIONAL POLICY FOR PRENATAL DOWN

SYNDROME SCREENING Denmark (BMJ 2008) and UK (UOG 2013)

Studies

Monni, Zoppi, Ultrasound Obstet Gynecol 2013: Opinion

FIRST TRIMESTER SCREENING AND INVERSION

OF THE PYRAMID OF PRENATAL CARE

Risk (%)

Age (years)30 35 40 45

100

10

1

0.1

20 250.01

Age riskAge risk

NT

NTNT

Fetal nuchal translucency

•Study in 100, 000 pregnancies•I n 75-80% of trisomy 21 f etuses the NT is increased

Lancet 1998

Nuchal translucency (mm)

Crown-rump length (mm)45 55 65 75 85

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Screening f or Screening f or TrisomyTrisomy 2121

Nicolaides, Prenat Diagn 2012

INVASIVE PRENATAL DIAGNOSIS TECHNIQUE OF 78 CHROMOSOMAL ABNORMALITIESOSPEDALE MICROCITEMICO- CAGLIARI

JANUARY 2011 – DECEMBER 2011

diagnosis by cvs81%

diagnosis by amniocentesis

19%

Distribution of number of fellows for CVS training at the

Ospedale Microcitemico - Cagliari

Period No. %

*1983- 1996 42 28

**1997- 2012 109 72

151 100

* BEFORE NT SCREENING

** AFTER NT SCREENING

FELLOWS TUTORED AT MICROCITEMICO HOSPITAL IN CAGLIARI (No 151)

Other: Argentina, Azerbaijan, Bosnia, Czech Republic, Canada, Japan, France, Germany, India, Lebanon, Mongolia, Morocco,

Netherlands, Portugal, Romania, S. Arabia, Slovenia, Spain, Sudan, Un. Arab Emirates, Venezuela

NEW LABORATORY TECHNIQUES

• Fluorescent in situ hybridization (FISH)

• Amplification of polymorphic chromosome-specific markers by polymerase chain reaction (PCR)

• Most laboratories offer a rapid test (PCR or FISH) to detect trisomy 21, 18, 13 and sex chromosome aneuploidies, as well as tissue culture to provide a full karyotype

• Array comparative genomic hybridization (a-CGH): in cases of multiple congenital abnormalities at ultrasound or for clinical diagnosis?

Advantages of array Comparative Genomic Hybridization (aCGH) or

Chromosomal Microarray Analysis (CMA)

• aCGH allows detection of smaller pathogenic chromosomal variants that are undetectable using standard cytogenetic analyses (G-band karyotyping)

DISADVANTAGES OF ACGH

• aCGH does not allow detection of balanced chromosomal rearrangements triploidy and some instances of mosaicism

• The biggest challenge presented by aCGH is the detection of chromosomal variants of unknown clinical significance (VOUS)

METHODS FOR ANALYSIS OF CELL-FREE (CF) DNA IN MATERNAL

BLOOD• Shotgun massively

parallel

sequencing (s-

MPS)

• Targeted

massively parallel

sequencing (t-

MPS)

• Single nucleotide

polymorphism

(SNP) -based

analysis

CFDNA ANALYSIS FOR T21: A META-ANALYSIS

(18 CITATIONS 2011- 2013)

• Individual studies: – Detection Rate (DR) ranges: 94.4-100%– False Positive Rate (FPR) ranges: 0- 2.05%

• Pooled weighted:– DR: 99.0% (95% CI 98.2- 99.6)– FPR: 0.08% (95% CI 0.03- 0.14)

Gil et Nicolaides, Fetal Diag Ther 2014

CFDNA ANALYSIS FOR T21: A META-ANALYSIS


CFDNA ANALYSIS FOR T18, 13, MONOSOMY X : A META-ANALYSIS


Detection rate False positive rate

Trisomy 18 96.8%

(95% CI 94.5- 98.4)

0.15%

(95% CI 0.08- 0.25)

Trisomy 13 92.1%

(95% CI 85.9- 96.7)

0.20%

(95% CI 0.04- 0.46)

Monosomy X

88.6%

(95% CI 83.0- 93.1)

0.12%

(95% CI 0.05- 0.24)The poor performance of cfDNA analysis in screening for trisomy 13 and monosomy X could be due to the highly variable amplification of chromosome X and 13 because of a lower guanosine- cytosine content

CFDNA ANALYSIS FOR SEX CHROMOSOME ANEUPLOIDIES OTHER

THAN MONOSOMY X

• Pooled weighted:– DR: 93.8% (95% CI 85.9- 98.7)– FPR: 0.12% (95% CI 0.02- 0.28)


CFDNA ANALYSIS FOR TRIPLOIDY

• Diandric (paternal): • Placenta enlarged and partially molar• NT enlarged• Free- beta hCG very high (10 times higher than normal)

• Digynic (maternal): • Placenta very small• Fetus severely growth restricted• Normal NT• Free- beta hCG and PAPP-A very low


The SNP method for cfDNA testing is the only one at present that can detect triploidy because it analyses allele distributions

4 out 8 cases of diandric triploidy have been detected, and suspicion raised for a case of diagynic triploidy

Utility of cfDNA as first-line method of screening because identification of triploidy would be beneficial (diandric triploidy can cause maternal complications including early- onset preeclamsia and

choriocarcinoma)

LIMITATIONS OF CFDNA TESTING

•Failure to provide results

•Receiving results in 1- 2

weeks

•Cost

FAILURE TO PROVIDE RESULTS

In 1- 5% of cases no results is given after first sampling

• Problems with sample collection or with transportation to the laboratory (on repeat sampling result is obtained in about 100%)

• Assay failure (on repeat sampling result is obtained in about 75%)

• Low fetal fraction (on repeat sampling result is obtained in about 50%); if it is a consequence of maternal obesity this problem is difficult to overcome


RECEIVING RESULTS IN 1- 2 WEEKS

• Average interval 10 calendar days

• In 95- 98% of cases a result available within 14 days

• In 2% of cases a result may not be available in less than 3-4 weeks


Such delay may reverse the beneficial shift in screening and diagnosis of aneuploidies from the second to the first

trimester

MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA

TESTING IN MATERNAL BLOOD

•Routine screening for whole population

•Contingent screening based on the result of first trimester combined test

MODELS FOR CLINICAL IMPLEMENTATION OF CFDNA TESTING AS

FIRST-LINE METHOD FOR ALL PREGNANCIES

• 10 weeks, maternal blood to all• 12 weeks first trimester us

Expected:• 99% DR of trisomy 21• 95% DR of trisomy 18 and 13• 1% Invasive testing rate



CONTINGENT SCREENING HIGH RISK GROUP

• Maternal blood in the high risk group (> 1:100)

Expected:• 86% DR of tris. 21• 89% DR of tris.18 /13• 0.4% Invasive test.

rate


cfDNA testing could not detect other aneuploidies


CONTINGENT SCREENING INTERMEDIATE RISK GROUP

• Maternal blood in the Intermediate Risk Group (>1:11<1:2,500)

Expected:• 97.6% DR of tris. 21• 98.1% DR of tris. 18/13• 0.8% Invasive test.

rate


cfDNA testing could not detect other aneuploidies

PRENATAL NONINVASIVE DIAGNOSIS FOR MONOGENIC DISEASE: ACTUALLY

VALIDATED USE

• Fetal sex determination (X-linked diseases in order to avoid invasive procedure in female fetuses) or for congenital adrenal hyperplasia (CAH) for therapeutic options

• RH blood group, D antigen• Paternal inherited autosomal

dominant diseases or de- novo after ultrasound suspicion (chondrodysplasias)

SIGU 2014, Document on the indications of use of performing non-invasive prenatal research

PRENATAL NONINVASIVE DIAGNOSIS FOR MONOGENIC DISEASE: NOT YET VALIDATED USE

• Autosomal recessive diseases

• X linked diseases

• Autosomal dominat diseases of maternal origin

SIGU 2014, Document on the indications of use of performing non-invasive prenatal research

MAIN FEATURES OF FREE DNA IN MATERNAL PLASMA

• Free DNA is always present in peripheral blood with a magnitude of between 145 and 201 bp

• Pregnancy causes an increase in the size of circulating DNA of maternal origin and a progressive increase in the concentration of Fetal DNA that is smaller

• The origin of circulating Fetal DNA in maternal plasma is due to placental apoptotic processes of the syncytium trophoblast

• The Fetal DNA is present since the 7th week of pregnancy and increases during pregnancy. In 10 weeks increases to about 5 or 10% of the total circulating plasma DNA. The fraction of fetal tissue correlates negatively with the maternal weight

• The presence of Fetal DNA in maternal plasma is no longer detected two hours after giving birth. The average half-life of 16.3 minutes (range 4-30 minutes)

FEASIBILITY STUDY OF -THALASSEMIA NIPD IN SARDINIA BY BENCHTOP NEXT GEN

SEQUENCING APPARATUS (PGM LIFE TECHNOLOGIES)

HINC II HIND III HINC II Ava II BamHI

G A

TSPYTSPY

ZFYZFY

SRYSRYChr Y Y Chr XX ZFZFXX

(88 bp) (88 bp)

(139 bp) (139 bp)

(175 bp) (175 bp)

(88 bp) (88 bp)

Chr 11 Cluster Chr 11 Cluster HBB HBB == 48 amplicons (85-197 bp) 48 amplicons (85-197 bp)

LIBRARY PREPARATION (51 AMPLICONS)

TAKE HOME MESSAGES (1)• Maternal age should no longer be the sole

criterium for set the parental choice of invasive prenatal diagnosis

• First trimester combined screening reduces the number of invasive prenatal diagnostic procedures

• After a positive combined test, a significantly high probability of an invasive test would confirm an abnormal fetal karyotype

• First trimester combined test induces reversing the traditional pyramid of prenatal care

• Educational organizations have faced new challenges in providing training for invasive procedures

TAKE HOME MESSAGES (2)

1)aCGH is not a substitute for conventional karyotyping;

2) aCGH should be used for specific diagnostic purposes in selected pregnancies and not for general screening in all pregnancies;

TAKE HOME MESSAGES (3)

1) cff- DNA for NIPT has the role of a screening test2) Evidence from high risk population3) Necessity of implementation of cff- DNA in low risk series4) Genetic counselling is mandatory before and after NIPT

NON-INVASIVE PRENATAL TEST (NIPT)

The expectations regarding cff-DNA for fetal genetic anomalies are very high because it may have the potential to change the landscape of prenatal diagnosis. However, to the disappointment of many, cff-DNA does not have the ability to function as a diagnostic test but is considered at present time as a

“super” screening test.

Monni, Journal of Perinatal Medicine 2014

Documents

Director Italian Branch Cagliari Regional Director for Europe