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

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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

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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

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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%