Transcript Document

Department of Obstetrics
&
Gynaecology
Director
Italian Branch
Cagliari
Regional Director
for Europe
Prenatal & Preimplantation
Genetic Diagnosis
Fetal Therapy
Cagliari
Ospedale Regionale Microcitemie
Director
Ian Donald School
for Invasive
Procedures
WHO
Collaborating Centre for Community Control
of
Hereditary Diseases
INVASIVE VS NON-INVASIVE PRENATAL DIAGNOSTIC
PROCEDURES
Giovanni Monni
12th TURKISH GYNECOLOGY AND OBSTETRICS CONGRESS
Antalya, 15th – 19th Maggio 2014
DILEMMAS TO AVOID GENETIC DISORDERS IN
THE NEWBORNS
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Screening based on maternal age alone?
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Prenatal invasive procedures?
First or second trimester ultrasound and
biochemical screening?
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 2006
Number of amniocenteses and
chorionic villus samplings carried out
in Denmark, 2000-2006
Ekelund, BMJ 2008
Total Number of Diagnostic Procedures
in England (2003- 2012)
Morgan,UOG 2013
UK national policy study of
aneuploidy 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
UK NATIONAL POLICY STUDY
ANEUPLOIDY SCREENING
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 vs 1:75).
So a significantly higher probability of an
invasive test would confirm an abnormal
fetal karyotype.
Morgan, Ultrsound Obstet Gynecol 2013
REDUCTION IN THE FETAL NUMBER OF
INVASIVE PROCEDURES PERFORMED
FOR PRENATAL KARYOTYPE
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
Monni, Zoppi, Ultrasound Obstet Gynecol 2013: Opinion
FIRST TRIMESTER EUROPEAN
NATIONAL POLICY FOR PRENATAL
DOWN SYNDROME SCREENING
Denmark (BMJ 2008) and UK (UOG 2013) Studies
-
Decrease in Fetal Loss due to a
reduction in invasive diagnostic
procedures
Earlier Diagnosis of Chromosomal
Aneuploidies
Monni, Zoppi, Ultrasound Obstet Gynecol 2013: Opinion
FIRST TRIMESTER SCREENING
AND INVERSION OF THE
PYRAMID OF PRENATAL CARE
Screening for Trisomy2
y21
Fetal nuchal translucency
Nuchal translucency (mm)
8.0
7.0
Risk (%)
100
NT
Age risk
10
6.0
NT
5.0
4.0
1
3.0
2.0
0.1
1.0
0.0
45
55
65
75
85 0.0120
Crown-rump length (mm)
25
30
35
40
Age (years)
• Study in 100, 000pregnancies
• In 75-80%of trisomy 21 fetuses the NTis increased
Lancet 1998
45
Nicolaides, Prenat
Diagn 2012
INVASIVE PRENATAL DIAGNOSIS TECHNIQUE
OF 78 CHROMOSOMAL ABNORMALITIES
OSPEDALE MICROCITEMICO- CAGLIARI
JANUARY 2011 – DECEMBER 2011
diagnosis by
amniocentesis
19%
diagnosis by
cvs
81%
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:
•
Pooled weighted:
– DR: 99.0% (95% CI 98.2- 99.6)
– FPR: 0.08% (95% CI 0.03- 0.14)
– Detection Rate (DR) ranges: 94.4-100%
– False Positive Rate (FPR) ranges: 0- 2.05%
Gil et Nicolaides, Fetal Diag Ther 2014
CFDNA ANALYSIS FOR T21: A META-ANALYSIS
Gil et Nicolaides, Fetal Diag Ther 2014
CFDNA ANALYSIS FOR T18, 13,
MONOSOMY X : A META-ANALYSIS
Trisomy 18
Trisomy 13
Monosomy X
Detection rate
False positive rate
96.8%
0.15%
(95% CI 94.5- 98.4)
(95% CI 0.08- 0.25)
92.1%
0.20%
(95% CI 85.9- 96.7)
(95% CI 0.04- 0.46)
88.6%
0.12%
The poor performance of cfDNA analysis in screening for
(95% CI 83.093.1)
(95%
0.05- 0.24)
trisomy 13 and monosomy
X could
be due to
the CI
highly
variable amplification of chromosome X and 13 because of a
lower guanosine- cytosine content
Gil et Nicolaides, Fetal Diag Ther 2014
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)
Gil et Nicolaides, Fetal Diag Ther 2014
CFDNA ANALYSIS FOR TRIPLOIDY
•
•
Diandric (paternal):
• Placenta enlarged and partially molar
• NT enlarged
• Free- beta hCG very high (10 times higher
Digynic (maternal):
• Placenta very small
• Fetus severely growth restricted
• Normal NT
• Free- beta hCG and PAPP-A very
than normal)
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)
Gil et Nicolaides, Fetal Diag Ther 2014
LIMITATIONS OF CFDNA TESTING
• Failure
to provide results
• Receiving
• Cost
results in 1- 2 weeks
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
Gil et Nicolaides, Fetal Diag Ther 2014
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
Gil et Nicolaides, Fetal Diag Ther 2014
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
• 12
•
•
•
weeks, maternal blood to all
weeks first trimester us
Expected:
99% DR of trisomy 21
95% DR of trisomy 18 and 13
1% Invasive testing rate
Gil et Nicolaides, Fetal Diag Ther 2014
MODELS FOR CLINICAL IMPLEMENTATION OF
CFDNA TESTING AS
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
Gil et Nicolaides, Fetal Diag Ther 2014
MODELS FOR CLINICAL IMPLEMENTATION OF
CFDNA TESTING AS
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
Gil et Nicolaides, Fetal Diag Ther 2014
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 noninvasive prenatal research
PRENATAL NONINVASIVE DIAGNOSIS FOR
MONOGENIC DISEASE: NOT YET VALIDATED USE
• Autosomal
•X
recessive diseases
linked diseases
• Autosomal
origin
dominat diseases of maternal
SIGU 2014, Document on the indications of use of performing noninvasive 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
during pregnancy.
circulating plasma
with the maternal
present since the 7th week of pregnancy and increases
In 10 weeks increases to about 5 or 10% of the total
DNA. The fraction of fetal tissue correlates negatively
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)
LIBRARY PREPARATION (51 AMPLICONS)
Chr 11 Cluster HBB = 48 amplicons (85-197 bp)

HINC II
G A
HIND III

HINC II


Ava II
BamHI
TSPY (175 bp)
Chr Y SRY
ZFY
(139 bp)
(88 bp)
Chr X
ZFX
(88 bp)
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 test
2) Evidence from high risk population
3) Necessity of implementation of cffDNA in low risk series
4) 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