Oltre la trisomia 21: un approccio alternativo al counseling genetico per gli screening e la diagnosi prenatale - Dovremmo continuare a concentrarsi sulle aneuploidie ricorrenti? / Beyond Trisomy 21 - an alternative approach to prenatal fetal screening and diagnosis counseling - subtitle: Should we keep focusing on the common aneuploidies? (J. Ferreira)

1. Beyond Trisomy 21:
an alternative approach to prenatal fetal
screening and diagnosis counseling
Should we keep focusing on the common aneuploidies?

2. Not so long ago….
• Tr 21 (+18 + 13) are the most common anomalies, among the
anomalies detectable by invasive based testing
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~1%?)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 - ~1%
for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

3. Not so long ago….
• Tr 21 (+18 + 13) are the most common anomalies, among the
anomalies detectable by invasive based testing - karyotype
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~1%?)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

4. Not so long ago….
• Tr 21 (+18 + 13) are the most common anomalies, among the
anomalies detectable by invasive based testing - karyotype
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~1%?)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

5. Not so long ago….
• Tr 21 (+18 + 13) are the most common anomalies, among the
anomalies detectable by invasive based testing - karyotype
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~1%?)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

6. The Fetal Medicine
Foundation 1960-20131960-2013
100,000 pregnancies
Trisomy 21Trisomy 21 N=200N=200
METHOD OF SCREENINGMETHOD OF SCREENING DetectedDetectedDRDR
Serum biochemistry at 16 wksSerum biochemistry at 16 wks 14014070%70%
Combined testCombined test at 12 wksat 12 wks 18018090%90%
Combined test PLUS at 12 wksCombined test PLUS at 12 wks 19419497%97%
9999,8,800 normal00 normal
49904990
49904990
29942994
False positiveFalse positive
5%5%
5%5%
3%3%
Maternal ageMaternal age 606030%30% 499049905%5%
ScreeningScreening
forfor Trisomy 21Trisomy 21

7. Not so long ago….
• Tr 21 (+18 + 13) are the most common anomalies, among the
anomalies detectable by invasive based testing - karyotype
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~1%?)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

8. Amniocentese
CVS
2014

9. Not so long ago….
• Tr 21 (+18 + 13) are the most common anomalies, among the
anomalies detectable by invasive based testing - karyotype
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~0.1 or 0.2%)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

10. Not so long ago….
• Tr 21 (+18 + 13) are the most common anomalies, among the
anomalies detectable by invasive based testing - karyotype
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~0.1%)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

11. 11-15 weeks 15-20 weeks
Hecht and Hook, 1994 – T21, Grati et al., non-published data on 90,000 amnioc and 40,000 CVS,
Frequency of karyotype anomalies per maternal age at 11-15 weeks and 15-20 weeks

12. Eurostat - http://appsso.eurostat.ec.europa.eu/nui/mainPage.do?lang=en
Genomic Defect 2003 2012 2003 2012
Mos>30% 612 660 16.7 15.0
DIC 21 21 0.6 0.5
Struct_unb 362 355 9.8 8.0
SMC_abn 105 103 2.9 2.3
Complex 34 33 0.9 0.7
TZ 19 21 0.5 0.5
69XXX 25 24 0.7 0.6
47,XXY 327 381 8.9 8.6
MX 152 149 4.1 3.4
T13 132 149 3.6 3.4
T18 263 367 7.1 8.3
T21 1626 2153 44.2 48.8
Estimated proportions of karyotype defects in Italian population,
>15 wks, in 2003 and 2012
Hecht and Hook, 1994 – T21
Grati et al., non-published data
Excluded
balanced
translocations,
mosaics less than
30%, trisomy X
and 47,XYY

13. Eurostat - http://appsso.eurostat.ec.europa.eu/nui/mainPage.do?lang=en
Genomic Defect 2003 2012 2003 2012
Mos>30% 282 359 17.4 17.7
DIC 14 15 0.8 0.7
Struct_unb 234 257 14.4 12.7
SMC_abn 68 75 4.2 3.7
Complex 22 24 1.3 1.2
TZ 8 10 0.5 0.5
69XXX 16 18 1.0 0.9
47,XXY 125 171 7.7 8.4
MX 98 108 6.0 5.3
T13 54 72 3.3 3.5
T18 81 115 5.0 5.7
T21 625 806 38.4 39.7
Estimated proportions of karyotype defects in Poland population,
>15 wks, in 2003 and 2012
Hecht and Hook, 1994 – T21
Grati et al., non-published data

14. Not so long ago….
• Tr 21 (+18 + 13) are 60% of the anomalies detectable by invasive
based testing – karyotype – in Italy, 2012!
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~0.1%)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

15. Year 2000
• Draft sequence of the HUMAN GENOME

16. RAPID TECHNOLOGY ADVANCES
• Multiplexing
• MicroArrays
• Sequencing

17. RAPID TECHNOLOGY ADVANCES
• Multiplexing
• MicroArrays
• Sequencing
FETAL DIAGNOSIS
~2010

18. RAPID TECHNOLOGY ADVANCES
• PNBoBsTM
• ArrayCGH
• cfDNA NGS
FETAL DIAGNOSIS
~2010

19. The Fetal Medicine
Foundation
• cfDNA testing is far superior to other methods for T21 and T18
• Can be offered to all women irrespective of risk
• Can provide results in the 1st
trimester of pregnancy
• Has the potential for detection of all aneuploidies
1960-20131960-2013
100,000 pregnancies
Trisomy 21Trisomy 21 N=200N=200
METHOD OF SCREENINGMETHOD OF SCREENING DetectedDetectedDRDR
Serum biochemistry at 16 wksSerum biochemistry at 16 wks 14014070%70%
Combined testCombined test at 12 wksat 12 wks 18018090%90%
Combined test PLUS at 12 wksCombined test PLUS at 12 wks 19419497%97%
9999,8,800 normal00 normal
49904990
49904990
29942994
Cell-free DNACell-free DNA >199>199>99%>99% <100<100
False positiveFalse positive
5%5%
5%5%
3%3%
<0.1%<0.1%
Maternal ageMaternal age 606030%30% 499049905%5%
ScreeningScreening
forfor Trisomy 21Trisomy 21

20. RAPID TECHNOLOGY ADVANCES
• PNBoBsTM
• ArrayCGH
• cfDNA NGS
FETAL DIAGNOSIS
~2012

21. RAPID TECHNOLOGY ADVANCES
• PNBoBsTM
• ArrayCGH
• cfDNA NGS
FETAL DIAGNOSIS
~2012
NICHD study – Wapner et al., NEJM, 2012
CNVs in prenatal samples with normal karyotype
3822 samples, of which 3067
•>35
•Positive mat serum screening
•Mat anxiety
•Family history of T21

22. Frequency of arrayCGH detectable anomalies per maternal age at 15-20 weeks
Hecht and Hook, 1994 – T21, Grati et al., non-published data on 90,000 amnioc, Wapner et al., 2012 - arrayCGH

23. Eurostat - http://appsso.eurostat.ec.europa.eu/nui/mainPage.do?lang=en
Genomic Defect 2003 20122003 (%) 2012 (%)
Expert re-classified CNVs 3193 3133 25.1 23.6
Incomplete / low penetrance variable expressivity CNVs 3370 3308 26.5 24.9
Other rare CNVs 1951 1915 15.3 14.4
Common recurring and well defined CNVs 532 522 4.2 3.9
Mos>30% 612 660 4.8 5.0
DIC 21 21 0.2 0.2
Struct_unb 362 355 2.8 2.7
SMC_abn 105 103 0.8 0.8
Complex 34 33 0.3 0.2
TZ 19 21 0.1 0.2
69XXX 25 24 0.2 0.2
47,XXY 327 381 2.6 2.9
MX 152 149 1.2 1.1
T13 132 149 1.0 1.1
T18 263 367 2.1 2.8
T21 1626 2153 12.8 16.2
Estimated proportions of genomic defects in Italian population,
>15 wks, in 2003 and 2012
Hecht and Hook, 1994 – T21, Grati et al., non-published data, Wapner et al., 2012 - arrayCGH
Excluded mosaics less than
30%, trismoy X and 47,XYY

24. T(21+18+13)
Karyotype
(Amnioc. or Chor. villi)
T(21+18+13)
arrayCGH
(Amnioc. or Chor. villi)

25. Not so long ago….
• Tr 21 (+18 + 13) are NOT the most common anomalies, among the
anomalies detectable by invasive based testing - arrayCGH
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~0.1%)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

26. Not so long ago….
• Tr 21 (+18 + 13) are NOT the most common anomalies, among the
anomalies detectable by invasive based testing - arrayCGH
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~0.1%)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Risk assessment (Tr21 and 18 screening):
• Maternal age
• Biochemical serum markers (1st and/or 2nd trim)
• Ultasound markers (1st and/or 2nd trim)

27. 0.01 = 1/100
0.02 = 1/50
0.03 = 1/33
0.001 = 1/1000
Hecht and Hook, 1994 – T21, Grati et al., non-published data, Wapner et al., 2012 - arrayCGH
25 years old

28. 0.01 = 1/100
0.02 = 1/50
0.03 = 1/33
0.001 = 1/1000
(Targeted defects – e.g. 22q11 del)
= 0.001 = 1/1022
= 0.004 = 1/279
= 0.006 = 1/161
= 0.006 = 1/170
Hecht and Hook, 1994 – T21, Grati et al., non-published data, Wapner et al., 2012 - arrayCGH
25 years old

29. 0.01 = 1/100
0.02 = 1/50
0.03 = 1/33
0.001 = 1/1000
+
= 0.018 = 1/57
}
Hecht and Hook, 1994 – T21, Grati et al., non-published data, Wapner et al., 2012 - arrayCGH
(Targeted defects – e.g. 22q11 del)
25 years old

30. 0.01 = 1/100
0.02 = 1/50
0.03 = 1/33
0.001 = 1/1000
+
}
Hecht and Hook, 1994 – T21, Grati et al., non-published data, Wapner et al., 2012 - arrayCGH
(Targeted defects – e.g. 22q11 del)
= 0.006 = 1/178
25 years old

31. Selected disease associations with rare CNVs and common CNPs
MR, idiopathic mental retardation
Assuming a 1% prevalence of autism, 1% prev of schizophrenia, 1.5% prev of IMR
Manolio TA, Collins FS, Cox NJ, et al. Finding the missing heritability of
complex diseases. Nature 2009;461(7265):747-753.
doi:10.1038/nature08494.
Disease Locus Type of CNV Size (kb) Population
frequency
Case frequency Effect size (OR) Est PPV*
Rare CNVs
Autism/IMR59
16p11.2 De novo deletion 600 1 × 10−4
1% 100 50%
Autism59
16p11.2 Rare duplication 600 3 × 10−4
0.50% 16 14%
Schizophrenia60
,78
1q21.1 Rare deletion 1,400 2 × 10−4
0.30% 15 13%
IMR79
1q21.1 Rare deletion 1,400 2 × 10−4
0.47% Not observed in
4,737controls
(?) >19% schiz,
>26% IMR
Schizophrenia60
,78
15q13.3 Rare deletion 1,600 2 × 10−4
0.20% 12 9% schiz, 13% IMR
Epilepsy80
15q13.3 Rare deletion 1,600 2 × 10−4
1.0% Not observed in
3,699 controls
(?) >33% schiz
IMR79
,81
15q13.3 Rare deletion 1,600 2 × 10−4
0.30% Not observed in
960 controls
(?) >18% IMR
Schizophrenia82
22q11.2 Rare deletion 3,000 2.5 × 10−4
1% 40 29% schiz
Common CNPs
Crohn’s
disease83
IRGM Deletion
polymorphism
20 7% 11% 1.5
Body mass
index61
NEGR1 Deletion
polymorphism
45 65% Quantitative trait <1 kg
Psoriasis84
LCE3C Deletion
polymorphism
30 55% 65% 1.3

32. Not so long ago….
• Tr 21 (+18 + 13) are NOT the most common anomalies, among the
anomalies detectable by invasive based testing - arrayCGH
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~0.1%)
• Expensive
• Invasive testing only acceptable at high risk - ≥ 35 yo (±1/280 for T21 -
~1% for T21+18+13)
• Minimum risk is 1/60 to 1/200 for any age!!!!

33. Not so long ago….
• Tr 21 (+18 + 13) are NOT the most common anomalies, among the
anomalies detectable by invasive based testing - arrayCGH
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~0.1%)
• Expensive
• Invasive testing for everyone who wants it!!
• Minimum risk is 1/60 to 1/200 for any age!!!!

34. Not so long ago….
• Tr 21 (+18 + 13) are NOT the most common anomalies, among the
anomalies detectable by invasive based testing - arrayCGH
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~0.1%)
• Expensive!!!
• Invasive testing for everyone who wants it!!
• Minimum risk is 1/60 to 1/200 for any age!!!!

35. Not so long ago….
• Tr 21 (+18 + 13) are NOT the most common anomalies, among the
anomalies detectable by invasive based testing - arrayCGH
• Diagnostic – invasive based testing
• Disadvantages:
• Risk of miscarriage (~0.1%)
• Expensive and higher counseling complexity!!!
• Invasive testing for everyone who wants it!!
• Minimum risk is 1/60 to 1/200 for any age!!!!

36. Prices (Euros) in Italy
Government Private
Serum screening NA 260
Karyotype+inv proc
574 (CVS)
294 (AF)
720 (CVS)
620 (AF)
arrayCGH+inv proc 1087
1000-1200 (CVS)
900-1100 (AF)
cfDNA test 500-1200

37. Alternative model

38. Proposed classification of genomic
defects• 1-Lethal diseases: defects resulting in a lethal phenotype
• 2-Highest severity diseases: Defects in moderate to severe
phenotype, non lethal, with long term effects, with mental and other
health problems, some of them, sometimes, treatable
• 3-Intermediate severity diseases: Defects resulting in phenotypes
mostly mild to moderate
• 4-Lowest severity diseases: Defects resulting in phenotypes
mostly mild
• 5-Incomplete penetrance and variable expressivity defects
• 6-Possibly, but not surely, diseases of undetermined
severity
• 7-Risk of fetal loss at reproductive age

39. Eurostat - http://appsso.eurostat.ec.europa.eu/nui/mainPage.do?lang=en
Genomic Defect 2003 20122003 (%) 2012 (%)
Expert re-classified CNVs – Possible, undetermined severity 3193 3133 25.1 23.6
Incomplete / low penetrance variable expressivity CNVs 3370 3308 26.5 24.9
Other rare CNVs – Highest 1951 1915 15.3 14.4
Common recurring and well defined CNVs – Highest 532 522 4.2 3.9
Mos>30% - Intermediate 612 660 4.8 5.0
DIC – Highest 21 21 0.2 0.2
Struct_unb – Highest 362 355 2.8 2.7
SMC_abn – Highest 105 103 0.8 0.8
Complex – Highest 34 33 0.3 0.2
TZ – Lethal 19 21 0.1 0.2
69XXX – Lethal 25 24 0.2 0.2
47,XXY – Intermediate 327 381 2.6 2.9
MX – Intermediate 152 149 1.2 1.1
T13 – Lethal 132 149 1.0 1.1
T18 – Lethal 263 367 2.1 2.8
T21 – Highest 1626 2153 12.8 16.2
Estimated proportions of genomic defects in Italian population,
>15 wks, in 2003 and 2012

40. Alternative model

41. 38 y.o., interested in:
Lethal, severe, moderate and low severity, incomplete penetrance and possibly causing disease
At the time of counseling (16 weeks) and at birth
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive

42. 38 y.o., interested in:
Lethal, severe, moderate and low severity, incomplete penetrance and possibly causing disease
At the time of counseling (16 weeks) and at birth
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive

43. 38 y.o., interested in:
Lethal, severe, moderate and low severity, incomplete penetrance and possibly causing disease
At the time of counseling (16 weeks) and at birth
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive

44. 38 y.o., interested in:
Lethal, severe, moderate and low severity, incomplete penetrance and possibly causing disease
At the time of counseling (16 weeks) and at birth
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive

45. 24 y.o., interested in:
Lethal, severe, moderate and low severity, incomplete penetrance and possibly causing disease
At the time of counseling (16 weeks) and at birth
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive

46. 38 y.o., interested in:
Lethal, severe, moderate
At the time of counseling (16 weeks) and at birth
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive

47. 24 y.o., interested in:
Lethal, severe, moderate
At the time of counseling (16 weeks) and at birth
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive
Classic
screenings
Primary
cfDNA
Classic +
cfDNA
Contingent +
cfDNA
Classic +
arrayCGH
Invasive