1. ¿Qué hay más allá de las trisomías más comunes?
Diagnóstico prenatal no invasivo
¿Qué hay más allá de las trisomías más comunes?
Carmina Comas
25 Congreso Nacional SEMEPE. Madrid, octubre 2016
Department of Obstetrics, Gynaecology and Reproduction of Hospital Universitario Quirón Dexeus

2. Background Other genetic inhereted disorders Microdeletions cf-DNA MS
Non invasive prenatal screening (NIPS)
Background
Lo 1997
Devaney 2011, Clausen 2014
Chiu 2011, Ehrich 2011
Palomaki 2012
Canick 2012
Mazloom 2013
Sequenom 2014
Yaron 2015
Lench 2013, You 2014
Lo 2010,
Kitzman 2012
Other genetic inhereted disorders
cf-DNA MS
Microdeletions
Trisomy 21
Twins
X,Y
Rare autosomal trisomies
Whole genome
Fetal sex
Fetal Rh status
Trisomy 18,13

3. Background 2011 Commercialization
Non invasive prenatal screening (NIPS) for fetal aneuploidies
Background
NIPT is offered to > 60 countries in six continents. Primer mercat es USA (65%), despres Europa.
2011 Commercialization
Global availability NIPT for main autosomal trisomies
Allyse M et al. NIPT: a review of international implementation and challenges. International Journal Women’s Health 2015:7;

4. Consensus 2016 Update statment
Non invasive prenatal screening (NIPS) for fetal aneuploidies
Update statment
ACOG/SMFM’s Practice Bulletin 163 May 2016
ACOG/SMFM’s Committee Opinion Sept 2015
ISPD’s Position Satement April 2015
ASHG’s Policy Statement March 2015
“Informing all pregnant women that NIPS is the most sensitive screening option for traditionally screened aneuploidies”
Gregg AR et al. NIPS for fetal aneuploidy, 2016 update: a position statement of the ACMGG. Genet Med 2016
On July 28th, the American College of Medical Genetics and Genomics (ACMG) released a statement supporting the use of noninvasive prenatal tests (NIPT/NIPS) as an optimal, initial option to screen for specific genetic conditions, such as Trisomy 21, during pregnancy for all women, regardless of maternal age.
Growing consensus regarding NIPS for fetal aneuploidies.
Fact: meta-analisis de Gil y Nicolaides 2015: Screening for T21 by cfDNA in singletons is significantly superior to all other traditional strategies
Condition n
DR (%)
FPR (%)
singletons
Trisomy 21
1051
99.2
0.09
Trisomy 18
389
96.3
0.13
Trisomy 13
139
91.0
Gil MM et al. Analysis cfDNA in Maternal Blood in Screening for Aneuploidies: Updated meta-analysis. Ultrasound Obstet Gynecol 2015;45:

5. What else?
¿Are expanded panels currently supported by clinical evidence as screening tests for routine care?

6. Implications of testing for additional conditions
NIPS beyond fetal aneuploidies
Implications of testing for additional conditions
Fetal sex
Fetal Rh status
Paternity (SNPs)
First applications
Mild phenotype
Higher failure rate
Lower DR and higher FPR
50% sex CA are mosaics
High incidence of maternal mosaicisms
Other than 45,XO: indication for testing??
Sex CA
Trisomies 9, 16, 22
Additional FPR 0.1% each
High IU lethality, IUGR
Screening for lethal conditions suspected by US non justified
No currently interventions
Low clinical utility > 10w
Not recommended (ACMG 2016)
Rare autosomal trisomies
Few conditions
Lower prevalence
Lower PPV
High FPR
Few experience
Difficult counselling
Microdeletions
Total and cffDNA higher
Trophoblastic apoptosis
Confliciting results
Future algorithms to estimates risks
Currently no clinical utility
Prediction of APO
Technically faisible
Clinically unpractical
Genome wide sequencing
NIPS beyond fetal aneuploidies: Implications of testing for additional conditions using the currently available technology
First applications
Fetal sex:
The presence of cell-free circulating Y chromosome DNA sequences in the plasma of pregnant women was first described in Since that report, many groups worldwide have validated the initial finding that Y chromosome sequences can be amplified and used to identify male fetuses. The research has been extended using a variety of methodologies, sex-specific markers, and sample types across all gestational ages. In countries such as the Netherlands, the United Kingdom, France, and Spain, this testing has already transitioned to routine clinical care despite the absence of a formal assessment of its performance. More recently, companies have begun offering this technology directly to the consumer over the Internet. The tests are marketed for nonmedical use to curious parents-to-be with promises in some cases of accuracy as high as 95% to 99% at as early as 5 to 7 weeks’ gestation.
Noninvasive prenatal determination of fetal sex using cffDNA provides an alternative to invasive techniques for some heritable disorders. In some countries this testing has transitioned to clinical care, despite the absence of a formal assessment of performance. Sensitivity and specificity for detection of Y chromosome sequences was greatest as GA avanza, but technically faisible from 6-7weeks.
Fetal Rh status: in some parts of Europe, to determine fetal rhesus D status (y identificar los fetos rH+) and prevent rhesus D negative mothers from unnecessary prophilactic treatment
Paternity: to determine
A noninvasive paternity test using informatics-based analysis of single-nucleotide polymorphism array measurements accurately determined paternity early in pregnancy.
Productes directament ofertants al consumidor (sense prescripcio medica)
RARE AUTOSONAL TRiSOMIES (associated to non viable pregnancies)
Trisomies 9, 16, 22 (T16 la mes frecuent AC postconcepcio, T22 non viable except mosaicisms): causa frecuent d’ avortament precoç
Additional FPR 0.1% (casa una)
High IU lethality, IUGR ( sobretot mosaic 22)
No currently interventions to reduce risks or to prevent
Screening for lethal conditions (que generen FP) suspected by US non justified (idem per T13 i 18)
Low clinical utility > 10w
Not recommended by ACMG 2016
PREDICTION OF APO (PE, PP, CIR,…)
Several studies have reported that in women with established PE, the plasma or serum concentrations of both total and fetal cell-free (cf)DNA are higher than
in normotensive controls and the increase is particularly marked in those with severe PE. These findings have been attributed to accelerated apoptosis of trophoblastic cells resulting from placental ischemia and reduced clearance of the cfDNA from the maternal circulation in women with PE. However, data are conflicting as to whether these altered levels precede the onset of the disease, and a recent systematic review was unable to draw definitive conclusions regarding the potential value of fetal cfDNA in the prediction of PE (Martin 2014).
Further studies will be addressed to calculate a parametric statistical algorithm and to estimate a proper posterior risk of the disease by means of fetal DNA alone or combined with other markers
GENOME WIDE SEQUENCING
Actualmente ya se pueden detectar enfermedades genetica AD de origen paterno

7. NIPS for sex CA?

8. Sex CA Background Impact Current situation NIPS
NIPS beyond fetal aneuploidies
Sex CA
Background
Full and mosaic numerical abnormalities interfering with normal sexual development
45,X; XXY; XXX
Prevalence
1/272
MA dependent
Impact
Variable and mild phenotype
Undiagnosed
Social or cognitive deficits
Fertility problems
Current situation
Not detectable by traditional screening
Incidental finding of IT
Difficult counseling and decision making
TOP 36% (≠80-96% T21)
Decreasing trend of TOP (EUROCAT)
NIPS
NIPS makes possible to screen for
Lower accuracy
DR 90%
FPR 1%
PPV 48%
Relevant difference as a screening test
Option for IT with a low PPV (>50% FP)
45,X: High lethality, abnormal US
Ethical questions
Sex discordances 1%
Definition
Full and mosaic numerical abnormalities interfering with normal sexual development
45,X (Turner Sd) and sex-chromosomal trisomies (XXY, XXX) (Klinefelter y triple X sd)
Prevalence
1/272 (Turner 1.5% pregnancies, frequent cause of miscarriage firts trimester)
MA dependent (mes frecuent a major edat)
Not detectable by traditional screening
Impact on general health
Variable and mild phenotype
Frequently remain Undiagnosed
Fertility problems (sols ser la causa del dco)
Normal QI
Current situation
Incidental finding of IT (fins ara)
Difficult counseling and decision making
TOP 36% sex trisomies (a diferencia de: 80-96% T21)
Decreasing trend of TOP (EUROCAT data 2013), associat a un millor assessorament
NIPS
Now NIPS makes possible to screen for
Lower accuracy than for T21,18 (TD menor, mes F+, baix VPP)
Relevant difference as a screening test
Option for IT with a low PPV (>50% del high risk son FP)
45,X: High lethality, si sobreviuen: frequent abnormal US
Ethical questions

9. Fetal sex discordances
NIPS beyond fetal aneuploidies
Sex CA: discordances (≠) ≠
Fetal sex discordances ♀ ♂ 1%
SNPs method
99-100
Counting method
Grati FR. Implications of fetoplacental mosaicism on cfDNA testing.Ultrasound Obstet Gynecol 2016; ♀ ♂

10. Sex CA: Arguments PROS and CONS
NIPS beyond fetal aneuploidies
Sex CA: Arguments PROS and CONS
Pros
NIPS technically available
Individual demand
Already diagnosed by IT
Potential benefits
Early management (fertility preservation)
Prenatal diagnosis of genitalia abnormalities (sex discordance)
Scientific recommendations
AIUM, ACOG 2013: only if medicaly indicated
ISMG 2014: recommended in high-risk
ACMG 2016: inform the availability to all
Cons
Limited data of performance
Lower DR
High cumulative FP
Low PPV
Error 0.7%
Maternal conditions revelead
Mosaics 45,X
Discordance TNI/US/TI
Maternal cancer
Ethical concerns
Psychosocial harm
Loss of equity for access (cost)
Sex selection
ESHG/ASHG 2015: not recommended
ACOG 2016: if requested
Pros
NIPS technically available
Individual demand
Already diagnoses by IT
Potential benefits: Early management (ex: fertility preservation) + prenatal dco anomalias genitales por discrepancia entre sexo por TNI y eco (solo se detectan el 24% de las anomalias de desarrollo sexual prenatalmente (3 casos Lynch Prenat Diagn 2016): 1 caso de Disgenesia gonadal+T21, 2 casos hipospadias severo) + Smith-Lemli-Opitz. Si discrepancia SEXO TNI/ECO: dco por TI
Current US guidelines recommend evaluation of fetals ex: Scientific recommendations:
AIUM, ACOG 2013: in multiple prengancies and when medically indicated
Israelita Society medical Genetic 2014: recommended in high-risk; ACMG American College Medical Genetics 2016: inform the availability to expand NIPT to sex CA all pregnant women
Cons
Limited data of performance
Lower DR
High cumulative FP
Low PPV
Reported error of NIPT in sex CA is 0.7% (45X, 47XXX, 47 XYY)
Mosaicisms (placentaris o fetals) o materns (frecuents Turners en mosaic no descoberts)
Discordancia entre NIPS i Cariotip del 8.5% degut a un cariotip matern anormal no conegut
Es recomana cariotip matern sempre en cas de alt risk per sex CA at NIPS
Maternal conditions revelead
Mosaics 45,X
Maternal cancer
Ethical concerns
Psychosocial harm (estignatitzacio, parental bonding, sobreproteccio, baixa autoestima…)
Equity for access due to the cost
Sex selection (not avoidable in female fetuses, tb amb TI pero encara es facilita mes si el test es de cribratge)
Scientific recommendations: for sex CA
ESHG/ASHG 2015: not recommended
ACOG 2016 if requested

11. NIPS for Microdeletions?

12. Rational for screening
NIPS beyond fetal aneuploidies
Microdeletions
Submicroscopic deletion leading to a monosomy of a chromosomal segment too small to be detected by conventional karyotype (<5Mb)
Microdeletion
Submicroscopic duplication leading to a segmental trisomy too small to be detected by conventional karyotype (<5Mb)
Milder phenotype
Microduplication
Larger deletions and duplications that should be visible by conventional karyotype (>5Mb)
Subchromosomal changes
Depending on size and location
Clinical relevance
Current screening (T21,18,13 and sex CA) detects 82% of CA
(Gap 18% CA missing)
Rational for screening
Microdeletion
Submicroscopic deletion leading to a monosomy of a chromosomal segment too small to be detected by conventional karyotype (<5Mb)
Microduplication
Submicroscopic deletion leading to a segmental trisomy too small to be detected by conventional karyotype (<5Mb)
Milder phenotype
Subchromosomal changes
Larger deletions and duplications that should be visible by conventional karyotype (>5Mb)
Clinical relevance
Depending on size and location
Rational for screening
Current screening (T21,18,13 and sex CA) detects 82% of CA (in AC samples)

13. Justify prenatal screening
NIPS beyond fetal aneuploidies
Microdeletions: Arguments PROS
Prevalence 1.7% (1/60) Wapner 2012
Recommendation: “Microarray should be offered always when IT” ACOG 2013
22q11del >1/1000 (1/992) Grati 2015, Gross 2016
The most common human deletion
2nd cause CHD
Commercial panel 5 microdeletions ≈1/1000
Severity Wapner 2014
Can be more severe than whole CA or T21
Panel of conditions with relevant mortality and morbidity
No risk factors Wapner 2014
Non dependant of MA
Normal US
Benefit from early intervention Handleman 2000, Cheung 2014
Proof-of-principle studies
Counting methods Jensen 2012, Srinivasan 2013, Fan 2012
SNPs methods Wapner 2014
Arguments PRO: why NIPS for general population should be considered:
Prevalence 1,7% structurally normal pregnancies (Wapner 2012, 2014): 1.7% (1/60) pregnancies have clinically relevant subchromosome events
In support of this: Recommendation ACOG 2013: microarray should be offered always when IT (independentment de la EM)
22q11del >1/1000 (Gross 2016)
Prevalence 22q11 del: 1/992 (low risk) Grati 2015
The most common human deletion syndrome
2nd cause CHD (after T21)
This prevalence challenges the concept of “low risk” pregnancies: Suggests offering NIPT microdel to screen general population may be appropiated
Severity
Can be more severe than whole CA/T21
Seleciona un panel de condicions de tamany >3Mb (limit inferior actualment detectable) de morbilitat I mortalitat significativa. Aquest panel de 5 microdel representa el 6-11% del total.
No risk factors
Non dependant of MA (a diferencia de otras aneuploidias asociadas a no-disyuncion)
Normal US (except 22q11del i monosom 1p36)
Benefit from early intervention: Rationale to screen: early detection improves prognosis
Justify prenatal screening
Proof-of-principle studies : para detectar microdel y microdup
- en metodo de contaje: requiere high sequence reads (deletion sd affects <1% genoma), deep of sequencing very high, high incidence of VOUS
- Targeted SNPs (Wapner 2014): permite distinguir si la delecion procede del cromosoma materno o paterno , facilita el asesoramiento cuando se trata de genes imprintados (identical chromosome 15 deletion, Prader Willy or Angelman sd) (depends on the informative SNPs in each critical region, not appropiate for egg donors)

14. Microdeletions: Arguments PROS
NIPS beyond fetal aneuploidies
Microdeletions: Arguments PROS
Incidence out of Live Births
Incidence 5 microdel panel /1000
5 microdeletions Panel
Down Syndrome
Prevalence
Maternal Age
<29 years
prevalence 5 microdel >> T21
Figure 1: Incidence of conditions out of Live births
22q11del: mes frequent que T18 y 13 ( I a mes son letals, menor importancia de cara al cribratge prenatal, tampoc cumpleixen criteris de cribratge classics)
Incidencia global 5 microdel testadas: 1/1000 (superior a la incidencia de SD en gestantes jovenes)
Figure 2: Risk of T21 increases with maternal age.
Risk of Microdeletions is consistent across all ages.
Younger women (<29 años) have a higher risk of a microdel than T21

15. >93% >99% Microdeletions: Proof-of-principle Sensitivity
NIPS beyond fetal aneuploidies
Microdeletions: Proof-of-principle
469 samples tested
SNPs
PlasmArt
PPV 5%
Sensitivity
Specificity
22q11.2 Deletion
45/47
95.7%
(CI: %)
419/422
99.3%
(CI: %)
Prader-Willi
15/16
93.8%
(CI: %)
453/453
100%
(CI: %)
Angelman
21/22
95.5%
(CI: %)
447/447
1p36
1/1
( %)
468/468
Cri-du-Chat
24/24
(CI: %)
444/445
99.8%
(CI: %)
>93%
>99%
Proof-of principle with targeted SNPs methodology: Wapner 2014
469 samples tested
PlasmArt (artificial plasma mixtures) due to low prevalence
(Include 70% of the causal mutations in this panel) + Aquest panel de 5 microdel representa el 6-11% del total
PPV 5%
Proof-of principle requieres clinical validation studies.
Wapner et al. Expanding the scope of NIPT: Detection of fetal mycrodeletion syndromes. AJOG 2014

16. Microdeletions: Clinical validation
NIPS beyond fetal aneuploidies
Microdeletions: Clinical validation
VPP 22q11.del
Deep of read
Regular
18%
High
42% US
Normal 5%
Abnormal
89%
VPP 22q11.del
60%
100%
Upgrade August 2016
22q11del:
DR 95.7% + PPV 44%
Adjusted risk score from 1/19 to 1/5
Less FPR (/6)
PPV 20% (x4)
A diferencia de los estudios de validacion clinica del NIPS para aneuploidias, hay pocos estudios en microdeleciones. 3 estudios actuales en microdel in clinical practice.
Diferencias.
Gross:
SNPs
retrospective
High risk 0,5%
N TP:11
PPV 18% regular DOPR (4,9% si US normal, 89% si US abnormal-high risk referrals))
PPV 42% resequencyng at a higher deep of read HDOR in high-risk results, keeping the same DR but lowering F+ to 0,12% (similar to PPV of addition T18,13 in conventional screening, or PPV of 45,X0)
Helgeson 2015 (incluye tambien 3 microdel mas: 4p-, 8q-, 11q-)
MPS (contaje masivo, no selectivo)
High risk 0,03%
N TP:14
PPV %
Newest data from Panorama: Upgrade August 2016 NIPT microdel panel (refining the test strategy): all + high risk samples are reanalyzed at a higher DOR + incresing the algorithm quality control confidence threshold. Large clinical cohort presented at ESHG 2016 and ISPD 2016
Upgrade August 2016
No impact on DR (22q11del DR 95,7% 3Mb)
22q11del PPV 44%
22q11del Adjusted risk score from 1/19 to 1/5
Overall Less FPR (x/6)
Overall PPV 20% (x4)
From Natera, presented at ESHG and ISPD Congress 2016

17. 1/20 2/3 1/5 Microdeletions: PPV PPV: TP/T + FP DR: TP /T PPV cfDNA
NIPS beyond fetal aneuploidies
Microdeletions: PPV
PPV: TP/T + FP
DR:
“What percentage of fetuses affected
are detected”
PPV:
“What percentage of fetuses is truly affected
when the test is positive?”
DR: TP /T
PPV
 cfDNA
Combined*
T21
T18
T13
45X n
222
154 29 21 18 TP
184
140 27 8 9 FP 38 14 2 13
83%
91%
93%
38%
50%
Gross SJ et al.
Ultrasound Obstet Gynecol 2016;47:177-83
Gross 2016: 6 primers mesos d’ experiencia clinica (primer article). n=21948 samples. 95 high risk for 22q11del (0.5%): Dco confirmacion x TI prenatal en 57% (follow-up available 88%). VPP 18% (1 de cada 5). Prevalencia microdeletion 22q11: 1/1000. Test SNPs cubreix el 87% de totes les 22q11del. VPP 18% (range 11-47%); VPP 89% si te anomalies ecografiques pre-test, i 5% si no te anomalies ecografiques. Prevalencia 22q11 del 1/946 (higher than estimated in RN).
Opcio per incrementar VPP de 22q11del: resequencing high risk sampler at a higher deepth. Llavors el VPP puja a 42%.
1/20
2/3
1/5
Dar P, et al.
Am J Obstet Gynecol 2014;211:527.e1-17.

18. Overall DR <<< 100% in all methods
NIPS beyond fetal aneuploidies
Microdeletions: Considerations
Size imbalance (3Mb) FF
DNA base composition (GC)
Factors affecting accuracy
Adjustement for GC content
Increasing DOR
Deep of sequencing
Additional normalizing of data
Selection of the most informative sequences
Use of probabilistic statistical model for computing LR of imbalance
Algorithm refinements
False negatives:
Variant partial deletions
Alternative genetic mechanisms
False positives:
Fetoplacental discrepancies
Maternal CA: mall partial imbalances, rare mosaicism (T8)
Non-pathogenic CNV close to critical region
Aneuploid vanishing twin
Maternal malignancy
Limitations
Microarray (no FISH)
High risk 22q11del at NIPS
22q11del
Factors affecting accuracy
Size imbalance (alrededor de 3Mb limite inferior con la tecnologia actual) (22q11del tienen este tamaño, resto de PW, Angelman, Cri du chat, 1p36 del tienen deleciones mayores, mayor TD) FF
DNA base composition (contenido GC aumenta la sensibilidad)
Ej 22q11del: TD según FF
Algorithm refinements
Adjustement for GC content
Increasing DOR
Deep of sequencing
Additional normalizing of data
Selection of the most informative sequences
Use of probabilistic statistical model for computing LR of imbalance
Limitations
False negatives:
Genetic variants (Variant partial deletions que no incluyen la zona critica)
Alternative genetic mechanisms (point mutations, inversions, DUP,…)
False positives:
Fetoplacental discrepancies: menos frecuentes que en aneuploidias, pero desconocemos frecuencia (ej: marcadores, rearrangements aparentmente balanceados pero que tienen un 8% de riesgo de desequilibrios relevantes), no hay datos actualmente
Small partial maternal imbalances
Rare maternal mosaicism (T8), non-pathogenic CNV in proximity to the 22q11del region (2 casos FP, Mather Prenat Diagn 2016)
Aneuploid vanishing twin
Maternal malignancy
Overall DR <<< 100% applicable to all methods
Overall DR <<< 100% in all methods
Dugoff et al. Prenat Diagn 2016;36:1-8

19. Microdeletions: Arguments CONS
NIPS beyond fetal aneuploidies
Microdeletions: Arguments CONS
Yaron Y et al. Obstet Gynecol 2015;126:1095-9
Microdel included in regular panel (algunos incluyen tambien Sd Horschhorn por del 4p)
Low prevalences
Detection if >3Mb (current limit for the detection): 15% of Di George are smaller (sols detecta el 85% de casos), aixo explica el low VPP (5%) y alta TFP 0.8% (sol 22qlldel)
Other causal etiopatogenesis of the conditions (DUP, mutations, inversions,…)
Regular panel > 3Mb 5 microdel: only detects 70% of the causal conditions
Aspecto practico a recordar:
Si alto riesgo 22q11del at TNI (o dco de CC en eco prenatal): Array es la prueba de eleccion (no FISH, utilizada historicamente para este dco, porque deleciones mas pequeñas o de otras regiones podrian no ser detectadas). (FISH solo detecta deleciones en la region LCR22A a LCR22B). 7% de CC con cariotipo y FISH normales tienen otras CNV patogenicas (en array).
DiGeorge syndrome: Only detects 85% of the causal conditions
Regular panel: Only detects 70% of the causal conditions
Only 6-11% of overall conditions

20. Firm conclusions not possible
NIPS beyond fetal aneuploidies
Microdeletions: Lessons to be learned
> pregnancies
Incomplete data (prevalence, clinical referral details, US findings, cytogenetic confirmation, pregnancy outcome)
Still lack of basic performance (S and NPV)
Firm conclusions not possible
Lessons from Newborn Screening Application
New proposal 2011, USA
Advisory Committee on Heritable Disorders in Newborns and Children
PCR 22q11del as universal screening in newborns
Rejection
Performance screening test not assessed
Unclear impact of testing
Benefit of early treatment not demonstrated
No evidence-based
All applicable in prenatal screening
+ Potential harms
Miscarriage after confirmatory IT
Postnatal phenotype difficult to predict
TOP unaffected cases may occur
22q11del
only partially fullfills these criteria
Have a severe phenotype ?
Have a consistent natural history ?
Excellent performance screening test ?
Be common
Criteria for screening for a health condition
Overall > pregnancies,
But we still lack basic performance, studies cannot evaluate S and NPV in clinical practice (principla limitacion: falta de seguimiento, no podemos detectar F- porque fenotipicamente los RN son normales a diferencia de las tris. Autosomicas)
LESSONS FROM NEWBORN SCREENING APPLICATION
2011, USA: solicitud per incorporar 22q11del en el screening universal del RN, basat en una PCR afegida al panel de condicions cribades (Committee on Heritable Disorders in Newborns and Childs)
Advisory Committee on Heritable Disorders in Newborns and Children
PCR 22q11del as universal screening in newborns
Rejection: Reasions
Performance screening test not assessed
Unclerar implact of testing
Benefit of early treatment not demonstrated
No evidence.based
All these arguments are still applicable in prenatal screening
Potential harms
Miscarriage after confirmatory IT
Postnatal phenotype difficult to predict
TOP in unaffected cases may occur
CLASSICAL CRITERIA FOR SCREENING FOR A HEALTH CONDITION
Be common ok
Have a severe phenotype ? Variable
Have a consistent natural history ? Variable
Excellent performance screening test:
Validations studies more difficult than for T21, accuracy not well defined, genetic variants 22q11del
>3Mb 87%
additional genetic mechanisms
Ethical Implications in cases inherited
CONCLUSION: 22q11del: only partially fulfills the criteria

21. Microdeletions: Arguments PROS and CONS
NIPS beyond fetal aneuploidies
Microdeletions: Arguments PROS and CONS
Pros
Significant high prevalence
Severity
No risk factors
Benefit from early intervention
Hypocalcemia
Critical CHD
Need for delivery at 3 center
Preventing “postnatal diagnostic odyssey”
Opportunity for TOP
Proof-of – principle
PPV similar to FTS
No previous screening test
Personal utility
Familiar utility
Cons
Prevalence
Limited conditions tested (6-11%)
Only 70% of causal conditions
Similar risk pre and post test (1.7% and 1.6%)
Technical accuracy
More challenging
Clinical validity: not demonstrated
Variable DR 60-99%
High FPR (cumulative) (0.8-3%)
Clinical utility: not demonstrated
Low PPV
Low penetrance/expressivity
Disadvantages from early diagnosis
Benefit not demonstrated prospectivelly
Excess of medicalization ?
Negative postnatal effects
Complex and unreallistic prenatal counseling
Doesn’t feet criteria for screening
PROS
Benefits of early diagnoses and intervention (neonatal hypocalcemia as a cause of mental retardation, critical CHD –DA dependent- preventing postnatal “diagnostic odyssey”, nneds for delivery at a terciary hospital, option for TOP …
PPV: Low PPV (depend de la performance i prevalencia), 5% , similar a FTS tradicional
No previous screening test (detected only as incidental findins)
Opportunity for TOP
Personal utility (in high risk patients that decline IT despite US abnormalities: “knowledge as an end in itself”) (aunque estaria indicado un array no desean riesgo de aborto)
Familiar utility: 10% (up to 28%) 22q11del are inhereted from a mildly affected parent (ventajas; estudio familiar, 50% recurrencia,…)
CONS
Prevalence
Limited conditions tested (Aquest panel de 5 microdel representa el 6-11% del total)
Only 70% of causal conditions
Similar pre and post-risk (residual risk); panels NIPTS 6-11% of all microdel/dup sd.; a priory risk 1,7%; post-screening o residual risk 1,6%
High FPR (cumulative): cumulative effect on FPR (due to low specificity):
0,8% only 22q11del
Up to 1% (5 microdel panel)
and 3% (all conditions in extended panel)
Variable DR: 60-99%
Performance depends on deletion size.
3Mb lower limit for detection using current technology
15% 22q11del <3Mb
Other causes
Technical accuracy (Validez analitica (technical accuracy) (capacidad tecnica)
More challenging
Technically more challenging: Non pathogenic CNV near critical regions may cause F+ for clinical syndromes (ex: 22q11 o 15q11 regions), present in <0.1% normal population ; Benigh CNVs amongst controls is a major cofounder and needs to be bioinformatically corrected/inter-individual variation in genomic CNVs in controls DNA samples is a major confounder
Limitations technology: Counting (<1% genoma, exceptionally high DOS, VOUS in non targeted) and SNPs (non informative, eggs donnors)
Clinical validity Validez Clinica (TD,Esp): not demonstrated: Solo hay proof of principle, not clinical validation
Variable DR 60-99%
High FPR (cumulative) (0.8-3%)
Clinical utility: not demonstrated
Utilidad clinica (VPP, VPN)
Low penetrance/expressivity (difficult decision for TOP, ethically more complex than T21) (difficult to counsel phenotypic consequences of a particular genotype)
Disadvantages from early diagnoses
Excess of medicalization ?
Negative postnatal effects: negative effects on bonding and parental behaviour
Complex prenatal counseling
Benefits of intervention: pendiente de demostracion prospectiva
Complex prenatal counseling (is realistic??) (high confusion for clinitians and patients)
We are not currently ready
Not feet classical criteria for screening

22. Current consensus at 2016 Public health perspective
NIPS beyond fetal aneuploidies: Routine prenatal screening for microdeletions
Current consensus at 2016
Scientific societies
Current status
Near future
None endorses routine NIPS for microdeletions
Still in its infancy
Refined strategies
Evolving nature of the technology
Cost reduction
Some advice explicity against it
Cost/benefit analysis
Not ready for wide-scale implementation
Some inconsistences
ACOG, ISPD, ESHG, ASHG, HGSA,AACG,BSGM,CMGS
Insufficent evidence
Public health perspective
Limited utility
Clinical perspective
Appropiate pretest counseling
Appropiate counseling resources
Sufficient justification?
Personal utility
Routine NIPS for microdel/microdupl sd.
is currently premature
and should be only offered only
under a research protocol
Recommendations scientific societies
None endorses routine NIPS for microdeletions
Some advice explicity against it
Some inconsistences (ex: ACOG recomienda SSD, con un VPP del 2% pero no recomienda expanded NIPT por VPP superior)
Current status
Still in its infancy
Evolving nature of the technology
Not ready for wide-scale implementation
Near future
Refined strategies (results at a lower FF, for smaller deletions, selection of more informative sequences or probes)
Cost reduction
Cost/benefit analysis
Public health perspective: Insufficent evidence
Clinical perspective: Limited utility
Personal utility:
Appropiate pretest counseling
Appropiate counseling resources
Sufficient justification?
Routine NIPS for microdel/microdupl sd. is currently premature
and should be only offered under a research protocol until we understand its true S and Sf, technical and biological variables that impact the accuracy, relevance to phenotypic penetrance

23. NIPT for
the entire fetal genome?

24. NIPT for the entire fetal genome
NIPS beyond fetal aneuploidies
NIPT for the entire fetal genome
From August 2015
The most advanced information available from a NIPT
Genome-wide events >7Mb + other selected microdeletions <7Mb*
Level of information previously offered only from karyotype analysis
* Performance dependent on sequencing depth, FF and size of event
August 215 (Sequenom): The MaterniT™ GENOME offers the most scientifically-advanced information available from a noninvasive prenatal test (NIPT). The MaterniT GENOME test offers a novel capability of noninvasive testing, identifying > 95% of genome-wide deletions or duplications ≥ 7 Mb. This enables the most comprehensive fetal chromosomal test currently available noninvasively.
Detection of Genome-wide events >7Mb (del o dupli, similar than karyotype)(S 95% Sp >99.9%) + other selected microdeletions<7Mb*
* Performance dependent on sequencing depth, FF and size of the event
Similar technology but deeper sequencing, simulated sensitivity metrics, in silico modeling using ISCA database (International Standards for cytogenetic Arrays), n clinical samples 448

25. Arguments PRO and CONS CONS PROS More difficult/unrealistic counseling
NIPS beyond fetal aneuploidies
Arguments PRO and CONS
“Just because you can, does not mean you should”
Catherine Russell
More difficult/unrealistic counseling
More difficult reproductive choices
Implications for close relatives
Ethical principles (beneficence, autonomy, justice)
Violation of child’s right to open future
Protection interests and autonomy rights future child
“Just for information” justified???
Recommendations from scientific societies
NOT NOW
CONS
“Why not?”
Hilary Duff
More difficult/unrealistic counseling
Just because you can, does not mean you should”
Catherine Russell (Jazz singer)
More difficult reproductive choices
Implications for close relatives
Ethical principles (beneficence, autonomy, justice)
Violation of child’s right to open future
Protection interests and autonomy rights future child
“Just for information” justified???
Recommendations from scientific societies
NOT NOW
Why not?”
Hilary Duff
Looking for everything
Why only disorders included in conventional prenatal screening?
Autonomy paradigm
Inconsistences of recommendations from scientific societies
PROS
Looking for everything
Why only disorders included in conventional prenatal screening?
Autonomy paradigm
Inconsistences of recommendations from scientific societies

26. Recommendations and guidelines
NIPS beyond fetal aneuploidies
Recommendations and guidelines
Scientific society
Sex CA
Microdeletions
Israel SMG 2014
Recommended in high-risk for CA -
ESHG/ASHG 2015
Not recommended
ISPD 2015
As an option
ACOG 2016
Only if requested
(level B)
SMFM 2016
ACMG 2016
Only information
HGA, AACG, BSGM, CzMGS,
PHG Foundation
Level B of evidence: limited or inconsistent evidence
Sex CA: Inconsistences
Microdeletions: Not recommended
Genome-wide CNV: Not recommended currently (if this level of information is desired: TI+array)
Majority
Inconsistences
Not supported
Sex CA: Inconsistences
Microdeletions: Not recommended
Genome-wide CNV: Not recommended

27. NIPS beyond fetal aneuploidies
Conclusions
Only letal or serious congenital childhood onset disorders
Sex CA and microdeletions NOT INCLUDED
Contemporary approach recognizes individual values and choices
In locations where expanded NIPT is available, women need to be informed
Current scope of prenatal screening for fetal abnormalities
Looking for everything: Why NOT?
Not all we technically can: WE CAN, BUT WE SHOULDN’T
What the scope of prenatal screening should be?
For fetal abnormalities (autonomy-aimed screening)
For conditions relevant to a healthy outcome of pregnancy for mother and child (prevention-aimed screening)
Prenatal screening for different purposes
All chromosome abnormalities
Mendelian disorders
Fetal genome scan
Genetic risk profiles for multifactorial disorders
Fetal personalized medicine
Future scenario (“omics era”): broad-scope NIPT
Current scope of prenatal screening for fetal abnormalities
Only letal or serious congenital childhood onset disorders
EX: Sex CA and microdeletions NOT INCLUDED/Justified currently
Contemporary approach recognizes individual values and choices
In locations where expanded NIPT is available, women need to be informed
What the scope of prenatal screening should be? 2 enfoques:
Looking for everything Why NOT?
Not all we technically can WHY NOT ALL?
Prenatal screening for different purposes 2 objetivos de cribado:
For fetal abnormalities (autonomy-aimed screening)
For conditions relevant to a healthy outcome of pregnancy for mother and child (prevention-aimed screening) EX: Rh, Preeclampsia, pretermino, CIR
Future scenario (“omics era”): broad-scope NIPT
All chromosome abnormalities
Fetal genome scan
Mendelian disorders
Genetic risk profiles for multifactorial disorders
Fetal personalized medicine