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Antoni Borrell BCNatal-Hospital Clínic Barcelona
A comprehensive new paradigm
for Prenatal Diagnosis
Disclosure information: Nothing to declare
Birth Defects Prevalence
conception birth
Chromosomal anomalies 8% 0.6%
Hereditary disorders 1%
Structural anomalies Major Malformations Minor Malformations
15%
3.7% (14%)
TOTAL 5.3%
(Connor & Ferguson-Smith,1993)
(Thomson&Thomson,2016)
Etiology
Birth Defects
FETA
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FETAL STRUCTURAL ANOMALIES Mutltifactorial Origin C
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Prenatal Diagnosis of Birth Defects: - Down Syndrome
- Neural Tube Defects
Prevalences for T21 and NTD: Total and livebirths
(1992-2004)
1/450
1/1000
35+
Prenatal Screening Methods - DS: Advanced Maternal Age
- NTD: MS-AFP and banana & lemon signs
Prenatal Diagnosis:
- DS: Amnio + karyotype
- NTD: AF-AFP and US
FETAL STRUCTURAL ANOMALIES Mutltifactorial Origin C
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FETAL STRUCTURAL ANOMALIES Mutltifactorial Origin C
HR
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Total BD Prevalences (Livebirths + TOP)
(1992-2004)
Total 2.4% Livebirths 1.9% CHD 0.7% TOP 0.5%
Structural anomalies prenatally diagnosed:
(Eurocat)
-
19
20
61
26
18
56
29
22
48
29
21
50
37
15
48
37
18
45
42
24
34
48
18
35
0%
20%
40%
60%
80%
100%
92-9
3
94-9
5
96-9
7
98-9
9
00-0
1
02:03
04:05
:06
no
>24
<24
Evolution of BD prenatally diagnosed (REDCB)
-
0
10
20
30
40
50
60
70
80
90
100
Digest.
Skeletal
Heart
Other
CNS
Facial
Urinary
TOTAL
NN
3T
2T
1T49
30
15
6
Early Prenatal Diagnosis of structural anomalies
(H. Clínic)
Routine US
Prenatal Diagnosis
Incidental
Findings
“Previously undiagnosed medical conditions that are discovered unintentionally and are unrelated to the current medical condition which is being treated or for which tests are being performed.”
• Hepatic metastasis in a MRI for back pain
• Trisomy 21 in a prenatal study for Tuberous Sclerosis
• Charcot-Marie-Tooth in a microarray for fetal hydrops
• ALL FINDINGS in a routine scan
Ultrasound Variants of Uncertain Significance
• Bening Findings Pathogenic Findings
• Uncertain Findings (VOUS):
FETAL STRUCTURAL ANOMALIES Mutltifactorial Origin C
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Chromosomal anomalies prenatally diagnosed
Combined Test: T21 Detection
87% DR for a 5% FPR
Comparing screening and diagnosis methods
Down
Edwards
Patau Turner Other Anom.
Microdel Risk Cost
Invasive Proc. + Microarray
100% 100% 100% 100% 100% 143 1-2‰ 1100
Invasive Proc. + Karyotype
100% 100% 100% 100% 100% 0 1-2‰ 700
Cell free-DNA testing
99% 97% 97% 90% 0 5? 0 600
Genetic Sonogram
95% - - - - - 0 0
Combined Test
90% 90% 90% 90% 50% - 0 0
cfDNA testing: 4 reported Meta-analyses
Efectiveness cfDNA for aneuploidy detection
Detection Rate
DR 1st trim
DR general population
DR consecutive
cases
False Positive
Rate
Trisomy 21 99.3 (n=37)
96.0 (n=7)
95.9 (n=6)
93.2 (n=5)
0.1
Trisomy 18 97.4 (n=32)
92.5 (n=5)
86.5 (n=4)
86.8 (n=4)
0.1
Trisomy 13 97.4 (n=22)
85.0 (n=5)
77.5 (n=4)
85.1 (n=3)
0.1
Failed Results (“No-calls”) (Metaanàlisi Gil, 15)
• 2.2% Inadequate Sample
• 2.4% Laboratory Failure
• ½ due to low fetal fraction (< 4%FF)
• 3-4% aneuploidy risk
N Inadequate sample
Laboratory failure
Low FF (<4%)
Assay failure
Singletons 28606 2.2% - 1.3% -
39730 - 2.4% - 1.1%
Twins 207 - 7.2% 5.3% 1.9%
Adjusted cfDNA efectiveness
Detection Rate
DR in general population
False Positive Rate
FPR including failed results
Trisomy 21 99.3 95.9 0.1 1.9
Trisomy 18 97.4 86.5 0.1 1.7
Trisomy 13 97.4 77.5 0.1 1.9
Efectiveness cfDNA for sex aneuploidies
Detection Rate False Positive Rate
Monosomy X 90.3 0.2
Other sex aneuploidies 93.0 0.1
TOTAL (autosomal trisomies + sex aneuploidies)
0.7
Microdeletion detection by cfDNA
cfDNA:
Which anomaly to detect?
1. TRISOMY 21 The best screening method
2. TRISOMIES 18-13 A good screening method
3. SEX ANEUPLOIDIES Useless in a normal scan
4. MICRODELETIONS Not validated yet
cfDNA:
In which population to be applied?
1. VERY HIGH RISK (Ultrasound Anomaly) -> Not indicated
2. HIGH RISK (≥ 1/250) -> Secondary Screening
3. GENERAL POPULATION -> Primary Screening
4. INTERMEDIATE RISK (1/100- 1/1000) -> Contingent Screening
AMNIOCENTESIS
0.11% (95%CI: -0.04 to 0.026)
CHORIONIC VILLI SAMPLING
0.22% (95%CI: -0.71 to 1.16)
0.1 - 0.2%
Secondary Screening: To avoid iatrogenesis
Primary Screening
3-Branch Contingent Screening (Danemark)
Combined Test
≥1/300
CVS
1/301 - 1/1000
cfDNA
< 1/1000
Combined Test
≥1/10
CVS
1/11 - 1/380
1/381 - 1/1000
cfDNA
< 1/1000
4-Branch Contingent Screning (Switzerland)
Primary
Screening
Secondary
Screening
Contingent
Screening
Microarray
Finland no 1/250 no 1/10
Denmark no 1/300 no all invasive
Sweden no 1/200 (Stockholm) no 1/50
UK no 1/150 (2018) no
Netherlands copay 175€ 1/200 no
Belgium T21 no
Switzerland no si 1/10
1/380-1/1000
1/10
Norway no no no
Germany no no no
Austria no no no
France no no no
Spain no no no
Italy no no no
Ireland no no no
Greece no no no
cfDNA Screening in Europe
FETAL STRUCTURAL ANOMALIES Mutltifactorial Origin C
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T21
T13,T18, MX
Routine Karyotyping in
Pregancy Loss?
• Pathology studies are useful: • To diagnose hydatiform moles • To exclude ectopic pregnancies
• Karyotyping is not the standard practice although: • It may reveal the cause in > 50% • It may establish the recurrence risk • “It is a must” in stillbirths, with few chromosomal anomalies
• It may reduce anxiety and help the grieving process (Nikcevic,99)
CVS vs. POC analysis
CVS + short-term culture + long-term culture provides a greater cytogenetic success than POC analysis:
• 90% cytogenetic success rate • 70% chromosomal anomaly rate • Avoids maternal cell contamination (1.08 sex ratio)
FETAL STRUCTURAL ANOMALIES Mutltifactorial Origin C
HR
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Differential implementation of new prenatal
genetic techniques
cfDNA in maternal plasma:
• To improve the safety of genetic studies
• Described in 2008 and implemented in 2011
Chromosomal Microarray Analysis (CMA):
• To enlarge the spectrum of detectable chromosomal anomalies
• Described in 1997 and implemented in 2005
High risk of microdeletion
(Established Indications)
1. Major or minor birth defect
2. Increased Nuchal Translucency (>p99 or > 3.5 mm)
3. Early Fetal Growth Restriction (<32 wk.) and severe (<p3)
4. Previous or parental microdeletion or microduplication
5. Stillbirth
6. “de novo” balanced rearrangement or “marker” chromosome
Variants of Uncertain Significance (VOUS) (DuplicationCHRNA7 )
(Y. Yaron)
Informed Consent :
Choice in specific pathogenic variants
• Which type: aCGH or SNPa?
• Which resolution: Low or high?
• Parental samples: ideal but impractical
• Triploidies undetected: previous QF-PCR
• Balanced anomalies undetected: irrelevant?
• Reporting VOUS: only in ultrasound anomalies?
• Replacing karyotype in all invasive procedures?
• Universal offer?
CMA: Open Questions
Incremental yield according reason of sampling
3% 6% 1% 1%
FETAL STRUCTURAL ANOMALIES Mutltifactorial Origin C
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Reproductive Carrier Screening (AR, X-l)
1.- Targeted ethnic-based screening (Europe):
• β-talassemia and 6PDHdeficit: Mediterranean, Asian
• Sickle-cell disease: Africans
• Tay-Sachs: Ashkenazis Jews
2.- Targeted panethnic screening (USA):
3.- Expanded screening:
• At least 1/100 carrier frequency and 1/40000 prevalence disease
ACOG ACMG
Cystic Fibrosis
Fragile X
Spinal Muscular Atrophy
Hemoglobinopathies
• 1301 individuals
• NGS technology
• 200 OMIM genes associated to
• 368 monogenic diseases:
• 277 autososmal recessive
• 37 X-linked
Healthy carriers prevalence in Barcelona study
High Carrier Frequency in 10 AR and 3 X-linked
First pregnancy in a 28-y woman:
• Lissencephaly in a female fetus @28 wk.
• Normal QF-PCR and CMA (MDS ruled out: 68% isolated lissencephalies)
• TOP
• LIS1 sequenced: No variants found
Second pregnancy 6 months later:
• Lissencephaly suspected @22 wk. Confirmed @28 wk. TOP
• Lissencephaly NGS panel with 24 genes (20 bening variants) and MLPA for 5 genes (correct)
• WES: 2 likely pathogenic alterations in ASPM gene associated to Microcephaly with simplified gyral pattern (AR)
• Each parent was carrier of one of the 2 heterozygous mutations.
Whole Exome Sequencing (WES)
FETAL STRUCTURAL ANOMALIES Mutltifactorial Origin C
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• GPP: Aneuploidy screening (cfDNA better than Combined) and invasive diagnostic testing
• Karyotyping in pregnancy losses
• GPP: Offer SNP-array • SNP-array in invasive
testing
• GPP: carrier screening (preconcepcional)
• GPP: First and Second Trimester Anomaly Surveys
• NGS panel/WES in malformed fetuses
WES larger series
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• Ofrecer cribado aneuploidia (combinado + DNAfl )
• Cariotipo en perdidas gestacionales
• Ofrecer microarray en malformaciones
• Ofrecer cribado portadores preconcepcional
• Estudio anatómico en primer y segundo trimestres
• NGS panel/exoma en fetos malformados
• La mayoría de pérdidas recurrentes inexplicadas se dan en mujeres sanas, sin patología subyacente, que han sufrido 3 abortos puramente por azar.
• El caso típico es una mujer de edad avanzada (> 40 años) con 3 pérdidas precoces, con un analisis de restos ovulares en el aborto más reciente que muestra una aneuploidía.
• Así pues, se recomienda el cariotipado de los restos ovulares para identificar la pérdida recurrente idiopática que ocurre por azar.
Prevalencia enfermedades genéticas (per 100.000 neixements)
125
101
40
20 20 17 13 10 8 5 4 0
20
40
60
80
100
120
140
T21 22q11 FQ FRAX 1p36 AME T18 PWS Angel 5p- T13
Meta-analysis CMA in cardiac defects
7%
12% Si se incluye la microdeleción 22q11
Meta-analysis CMA in increased NT
5%
Meta-analysis CMA in isolated FGR/SGA
4%
Multicenter study: CMA in early (<32 wk.) FGR (EFW<3p)
Indications N (%) Diagnostic Yield
N (%) 95% CI
Isolated increased NT 63 (17) 2 (3.2%) (0 - 7.5)
Associated increased NT 16 (4.4) 2 (13%) (0 – 28.7)
Isolated FGR 33 (9) 0 (0) -
Associated FGR 18 (4.9) 1 (5.6%) (0 – 16.1)
Isolated structural defects 165 (45) 6 (3.6%) (0.8 – 6.5)
Multiple structural defects 29 (8) 1 (3.4%) (0– 10.1)
Fetal demise 9 (2.5) 1 (11%) (0– 31.6)
Others 31(8.5) 0 (0) -
Total 364 (100) 13 (3.6%) (1.7-5.5)
Experiència BCNatal en microarrays prenatals (N= 364 ; feb 12- ago 15)
SNP-Array
QF-PCR: marcadores de 3-5 cromosomas
Cariotipo y 5 cromosomas diana en QF-PCR
Microarray
NHC: 4903579 arr17p12(14864897-15475024)x3.
16,301 first trimester scans
14,368 complete follow-up
Excluded:
312 chromosomal anomalies
103 single-gene anomalies
230 fetal losses
439 (3.2%) major structural anomalies 49
30
15
6
0
10
20
30
40
50
60
70
80
90
100
Digest.
Skeletal
Heart
Other
CNS
Facial
Urinary
TOTAL
NN
3T
2T
1T
Detection by trimester of gestation (Clinic)
COMMON AUTOSOMAL TRISOMIES
OTHER AUTOSOMAL TRISOMIES
MONOSOMIES
TRIPLOIDIES
STRUCUTRAL ABNORMALITIES
DOUBLE ABNORMALITIES
MOSAICISMS
PRE-EMBRYONIC EMBRYONIC/FETAL
Espectro de anomalías cromosómicas
Pérdidas pre-embrionarias: • 61% tasa anomalías cromosómicas • No hay trisomías 21,18, ni monosomías X
Riesgo de Recurrencia
• Clásicamente: 1% riesgo recurrencia para T21 • Después de anomalía cromosómica, siempre procedimiento • Aplicación 0.4% exceso de riesgo
• NDSCR: exceso riesgo decreciente
– 0.62% @ 20 años – 0.01% @ 46 años
• Post T13,18: RR~4 homotrisomía, también heterotrisomía
• Igarzabal: gestaciones T21, más abortos previos (OR: 1.22)
• Munné,04: más trisomías @ DGP, después trisomía (también no viable)
• Al-Asmar,12: más anom. crom. @ DGP, después trisomía autosómica
Index Trisomy RR Same
trisomy RR Other
viable trisomy
T21 < 30 y.o. 4.7 2.4
T21 ≥ 30 y.o. 1.6 1.7
T13,18,XXY,XXX 2.5 1.6
Non viable trisomy (miscarriage) - 1.8
¿Hay riesgo T21 después de trisomía non-viable?
Variación en número de copias patogénica Microdeleción 22q11 DiGeorge/VCF
(Y. Yaron)
Contenido génico en Microdeleción 22q11
Discapacidad intelectual:
Etiología
Cromosómica Subcromosómica
Monogénica Ambiental
Desconocida
Detection Rate
False Positive Rate
Conventional Screening
82% 4.5%
cffDNA (failed results excluded)
71% 0.7%
cfDNA (failed results assumed positive)
77% 3.7%
• 452,901 screened pregnancies in California (2009-12) • First or first+second screening • 2575 (0.6%) chromosomal anomalies • 601 (23%) undetactable by cfDNA
Cribado primario con DNA libre
-5% -5% -0.8%
Tasa anomalía cromosómica
Sporadic
Miscarriage
Recurrent
Miscarriage
< 35 years
≥ 35 years
56%
44% 44%
56%
78%
22%
69%
31%
• Historia médica personal y familiar • Cariotipo • Cribado genético según etnia del donante: α y β talasemias Mediterráneo, Oriente Medio,
Suroeste Asiático, Pacífico Sur y sur de China.
Anemia falciforme Africanos, afroamericanos y Caribeños.
Enfermedad de Tay-Sachs Judíos (Europa del Este)
Fibrosis Quística Todos los grupos étnicos
Recomendaciones SEF/ASEBIR:
Donantes gametos
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Genetic techniques in the
analysis of pregnancy losses
• Karyotype in STC: avoids MCC
• Karyotype in LTC: less feto-placental discrepancies
• FISH: 5-8 probes
• QF-PCR: 5-8 chromosomes. Detects MCC
• cfDNA: 5 chromosomes
• Array-CGH: no culture needed, detects microdeletions
• SNP-array: detects MCC and UPD
• QF-PCR + SNP-array
Recurrent pregnancy loss: simple work-up
• Recurrent losses (≥3 losses) in 1% couples
• More than 50% remain unexplained after extensive work-up
• 65% Chromosomal anomaly rate
• Similar rates between sporadic and recurrent losses: 70% vs. 60%
• Maternal age was the single predictive factor: 75% vs. 55%
• Differences in the chromosomal anomaly spectrum
common autosomal trisomies
uncommon trisomies
autosomal monosomies
sex trisomies
sex monosomies
polyploidies
unbalanced structural anomalies
double anomalies
mosaicisms
Sporadic miscarriage Recurrent miscarriage
<35years
≥35years
Chromosomal anomaly spectrum
5%
29%
37%
11%
38% 57%
(Thomson&Thomson,1989)
Etiology
Birth Defects
Failed Results (“No-calls”) (Metaanàlisi Gil, 15)
• 4% Inadequate Sample
• 5% Laboratory Failure
• ½ in low fetal fraction (< 4%FF)
• 3-4% aneuploidy risk
N Inadequate sample
Laboratory failure
Low FF (<4%)
Assay failure
Singletons 40847 4.3% 5.1% - -
13225 - - 2.0% 2.0%
Twins 207 - 7.2% 5.3% 1.9%