1. Down Syndrome : screening evolution and natural history
Professor Joan Morris
20th Annual CARIS meeting
Princess of Wales Hospital
Bridgend
Tuesday 13th November 2018
Population Health Research Institute
St George’s University of London
Cranmer Terrace SW17 ORE

2. Down Syndrome : screening evolution and natural history
History of screening for Down Syndrome
Principles of free fetal DNA testing
Proposed method of screening using free fetal DNA
But first news of the NDSCR…..

3. Update on National Down Syndrome Cytogenetic Register
From all cytogenetic laboratories in England and Wales notified the NDSCR of any diagnosis of trisomy 13,18 or 21 or related karyotype
Pre- and postnatal diagnoses
Follow-up information from clinicians and midwives
> 94% Complete
Crude measures of national trends can be derived directly from the NDSCR.
More sophisticated measures derived by linking NDSCR with other data.
35,429 Down syndrome cases
1,714 Patau syndrome cases
4,297 Edwards syndrome cases

4. Maternal age and risk of trisomy birth
Down syndrome
Edwards syndrome
Patau syndrome
Prenatal Diagnosis, 2009 doi: /pd.2403.

5. Survival of trisomy 21 pregnancies diagnosed at 10 weeks (95% CI)
Upper 95% CI
Lower 95% CI
American Journal of Medical Genetics Part A 146A:827–832 (2008)

6. Recurrence risk of trisomy 21

7. Recurrence risk by age at 1st pregnancy
Age related risk
Recurrence risk
Maternal age (years) 25 30 35 40 45
Number of Down syndrome pregnancies per 100 pregnancies .5 1 2 3 4
15277 free trisomy 21 pregnancies
95 women had 2+ trisomy 21 pregnancies

8. How accurate are the risk estimates ?

9. Current Situation
National Down Syndrome Cytogenetic Register is now part of NCARDRS (National Congenital Anomaly and Rare Diseases Registration Service)
Data collected from all of England (not including Wales)
Annual report published July 2017

10. Future research
Health and Education of children with Down syndrome across Europe
EUROlinkCAT – includes Welsh data on children with Down Syndrome
Educational achievements of children with Down syndrome
Linking NDSCR (English Data) to Hospital Episode Statistics and National Pupil Database

11. History of antenatal screening for Down syndrome

12. Screening using maternal age (1970’s)
Penrose LS. J Genet 1933;27:
Amniocentesis offered to women 35 years of age and over

13. Screening using maternal age and second trimester AFP
1984 : Cuckle HS, Wald NJ & Lindenbaum RH. Lancet 1984;i:

14. Measurement (eg Human chorionic gonadotrophin )
THEORY OF SCREENING
Detection Rate = the proportion of unaffected individuals with a positive result
False-positive rate = the proportion of unaffected individuals with a positive result
Screen Negative
Cut-off
Screen Positive
Unaffected
Down’s syndrome
Detection Rate
False-positive Rate
Low Values
High Values
Measurement (eg Human chorionic gonadotrophin )

15. Screening using maternal age and second trimester AFP
Detection Rate = 44%
False-positive Rate = 5%

16. Screening using maternal age and the second trimester triple test
Alphafetoprotein (MoM)
Unconjugated estriol (MoM)
+ Maternal age
Human chorionic gonadotrophin (MoM)
Wald NJ et al. BMJ 1988;16:

17. Risk of Down's syndrome (early mid-trimester)
screening-Iran v06.pptx
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Antenatal screening for Down’s syndrome using the second trimester Triple test
1:108
1:106
1:104
1:102
1:1
102:1
104:1
106:1
Risk of Down's syndrome (early mid-trimester)
Unaffected
Down’s syndrome
Lower risk
Higher risk
Risk cut-off
1:150
Detection Rate = 78%
False-positive Rate = 5%
SURUSS 11w
DS: ,
Unaffected: ,
Cutoff:
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18. Detection rate for a 5% false-positive rate
100
Integrated test* 96
NT, PAPP-A, AFP, uE3, hCG, Inhibin-A
1999 T1 T2
Combined test* 88
NT, free β-hCG, PAPP-A
1996 T1
Quad test* 86
1996
AFP, uE3, hCG, Inhibin-A T2
Triple test* 78
AFP, uE3, hCG
1988 T2 80
Detection
Rate
(%) 60
AFP test* 44
1987 40 33
AFP 20 T2
Maternal Age
Year
1970s
Wald NJ, Bestwick JP and Huttly WJ. J Med Screen 2013;20:7-14
*Include maternal age

19. Should interim risks be revealed with the Integrated test?
screening-Iran v06.pptx
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Should interim risks be revealed with the Integrated test?
Example: 43 year old woman
1st trimester test results:
NT MoM
Free β-hCG 1.25 MoM
PAPP-A 0.76 MoM
Should this be reported?
Risk = 1 in 40
2nd trimester test results:
Risk = 1 in 5000
AFP MoM
uE MoM
free β-hCG MoM
Inhibin-A MoM No
One pregnancy, one risk
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20. Principles of free fetal DNA test in screening for Down syndrome

21. DNA fragments (100-200 DNA bases) in maternal plasma
85%
15%
Fetal
DNA
1ml of maternal plasma contains about 100 million DNA fragments

22. DNA from chromosome 21 in maternal plasma
ReflexDNA-Prague v06.pptx
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DNA from chromosome 21 in maternal plasma
Relative amounts of chromosome 21 DNA fragments in maternal plasma
From mother
From fetus
Total
Unaffected 85 15
100
x 1.5
Down's syndrome 85
22.5
107.5
Down's syndrome / Unaffected = 107.5/100 = 1.075
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23. % of DNA on all chromosomes
ReflexDNA-Prague v06.pptx
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Human Chromosomes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Chromosome
% of DNA on all chromosomes
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24. % of DNA on all chromosomes
ReflexDNA-Prague v06.pptx
13/01/2019
Human Chromosomes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Chromosome
% of DNA on all chromosomes
1.3%
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25. DNA from chromosome 21 in maternal plasma
ReflexDNA-Prague v06.pptx
13/01/2019
DNA from chromosome 21 in maternal plasma
Unaffected pregnancy
1.3%
Down's syndrome pregnancy
1.3% x = 1.4%
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26. DNA fragments from chromosome 21
Distributions of DNA fragments from chromosome 21 in affected and unaffected pregnancies
Unaffected
Down’s syndrome
1.2
1.25
1.3
1.35
1.4
1.45
1.5
DNA fragments from chromosome 21
(%)

27. DNA fragments from chromosome 21
Distributions of DNA fragments from chromosome 21 in affected and unaffected pregnancies
Unaffected
Down’s syndrome
1.2
1.25
1.3
1.35
1.4
1.45
1.5
DNA fragments from chromosome 21
(%)

28. DNA fragments from chromosome 21 Unaffected
ReflexDNA-Prague v06.pptx
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Distributions of DNA fragments from chromosome 21 in affected and unaffected pregnancies
Canick et al. Prenat Diagn 2013;33:667-74
DNA fragments from chromosome 21
Unaffected
1.4
1.5
1.2
1.25
1.35
1.45
1.3
(%)
Down’s syndrome
Need to count about 10 million DNA fragments in maternal plasma
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29. Down’s syndrome screening performance: Universal DNA screening
screening-Iran v06.pptx
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Down’s syndrome screening performance: Universal DNA screening
Palomaki et al. Genet Med 2011;13:
Wald & Bestwick, J Med Screen 2015;22:168-74
98.7
Detection rate
False-positive rate*
2.70
Percent
100 20 40 60 80
*DNA test failures (2.5%) classed screen positive
clear
input ct ct10 ct20 dnapos dnaquad
end
set scheme lean2
graph bar (mean) ct (mean) ct10 (mean) ct20 (mean) dnapos (mean) dnaquad , legend(off) /*
*/ ylabel(0 "0.0" 10(10)100, nogrid) plotregion(margin(zero))
*/ ylabel(0(0.5)3, nogrid) plotregion(margin(zero))
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30. Down’s syndrome screening performance: Universal DNA screening
screening-Iran v06.pptx
13/01/2019
Down’s syndrome screening performance: Universal DNA screening
Palomaki et al. Genet Med 2011;13:
Wald & Bestwick, J Med Screen 2015;22:168-74
98.7
Detection rate
False-positive rate*
2.70
Percent
100 20 40 60 80
*DNA test failures (2.5%) classed screen positive
Move and change scale
clear
input ct ct10 ct20 dnapos dnaquad
end
set scheme lean2
graph bar (mean) ct (mean) ct10 (mean) ct20 (mean) dnapos (mean) dnaquad , legend(off) /*
*/ ylabel(0 "0.0" 10(10)100, nogrid) plotregion(margin(zero))
*/ ylabel(0(0.5)3, nogrid) plotregion(margin(zero))
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31. Down’s syndrome screening performance: Universal DNA screening
screening-Iran v06.pptx
13/01/2019
Down’s syndrome screening performance: Universal DNA screening
100 20 40 60 80
Detection rate (%)
98.7
Universal DNA screening
0.5
1.0
1.5
2.0
2.5
3.0
False-positive rate (%)
2.70*
Palomaki et al. Genet Med 2011;13:
Wald & Bestwick, J Med Screen 2015;22:168-74
*DNA test failures (2.5%) classed screen positive
clear
input ct ct10 ct20 dnapos dnaquad
end
set scheme lean2
graph bar (mean) ct (mean) ct10 (mean) ct20 (mean) dnapos (mean) dnaquad , legend(off) /*
*/ ylabel(0 "0.0" 10(10)100, nogrid) plotregion(margin(zero))
*/ ylabel(0(0.5)3, nogrid) plotregion(margin(zero))
N:\graphics\jpbestwick\WaldNJ\ x Nick Iran screening

32. False-positive rate (%)
screening-Iran v06.pptx
13/01/2019
Down’s syndrome screening performance: Universal DNA screening compared with screening using the Integrated test
100 20 40 60 80
Detection rate (%)
98.7
Universal DNA screening
Integrated test
93.0
0.5
1.0
1.5
2.0
2.5
3.0
False-positive rate (%)
2.70*
2.70
Palomaki et al. Genet Med 2011;13:
Wald & Bestwick, J Med Screen 2015;22:168-74
*DNA test failures (2.5%) classed screen positive
clear
input ct ct10 ct20 dnapos dnaquad
end
set scheme lean2
graph bar (mean) ct (mean) ct10 (mean) ct20 (mean) dnapos (mean) dnaquad , legend(off) /*
*/ ylabel(0 "0.0" 10(10)100, nogrid) plotregion(margin(zero))
*/ ylabel(0(0.5)3, nogrid) plotregion(margin(zero))
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33. Main issues with antenatal DNA screening
screening-Iran v06.pptx
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Main issues with antenatal DNA screening
Test failures – about 2-9%
Could be reduced by more than half by using a 2nd aliquot of the plasma sample collected
High cost
Time taken to complete test – 7 to 14 days
From out PlosOne paper. Ref 1 = Sequenom = 11.5%, ref 2=Sequenom = 3.4%; Ref 3 = Verinata = 7%, Ref 4 = Ariosa = 0% and 5%, Ref 5 = Ariosa = 4.9%, Ref 6 = BGI = 1.6%, Ref 7 = Natera = 13%
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34. Solution: Reflex DNA screening
screening-Iran v06.pptx
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Solution: Reflex DNA screening
All women have Combined test, extra blood sample collected
Those with highest Combined test risk (eg top 20%) have a DNA test using the extra blood sample. Remainder (80%) are screen negative
If DNA test fails on two separate samples women return for Integrated test
From out PlosOne paper. Ref 1 = Sequenom = 11.5%, ref 2=Sequenom = 3.4%; Ref 3 = Verinata = 7%, Ref 4 = Ariosa = 0% and 5%, Ref 5 = Ariosa = 4.9%, Ref 6 = BGI = 1.6%, Ref 7 = Natera = 13%
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35. Solution: Reflex DNA screening
Reflex DNA test using other sample, repeated if test fails
Higher risk
Lower risk
Positive (offer amniocentesis)
Higher risk
Lower risk
Combined test using one sample
Negative
Test fails on two separate samples
Two blood samples taken (11-13 weeks)
Higher risk
Lower risk
Positive (offer amniocentesis)
Negative
Integrated test
Negative

36. False-positive rate (%)
screening-Iran v06.pptx
13/01/2019
Down’s syndrome screening performance: Universal DNA screening compared with screening using the Integrated test
100 20 40 60 80
Detection rate (%)
98.7
Universal DNA screening
Integrated test
93.0
0.5
1.0
1.5
2.0
2.5
3.0
False-positive rate (%)
2.70*
2.70
Palomaki et al. Genet Med 2011;13:
Wald & Bestwick, J Med Screen 2015;22:168-74
*DNA test failures (2.5%) classed screen positive
clear
input ct ct10 ct20 dnapos dnaquad
end
set scheme lean2
graph bar (mean) ct (mean) ct10 (mean) ct20 (mean) dnapos (mean) dnaquad , legend(off) /*
*/ ylabel(0 "0.0" 10(10)100, nogrid) plotregion(margin(zero))
*/ ylabel(0(0.5)3, nogrid) plotregion(margin(zero))
N:\graphics\jpbestwick\WaldNJ\ x Nick Iran screening

37. *DNA test failures (2.5%) classed screen positive
screening-Iran v06.pptx
13/01/2019
Down’s syndrome screening performance: Universal DNA screening compared with screening using the Integrated test and Reflex DNA screening
100 20 40 60 80
Detection rate (%)
98.7
Universal DNA screening
94.2
Reflex DNA screening**
Integrated test
93.0
0.5
1.0
1.5
2.0
2.5
3.0
False-positive rate (%)
2.70*
2.70
Palomaki et al. Genet Med 2011;13:
Wald & Bestwick, J Med Screen 2015;22:168-74
*DNA test failures (2.5%) classed screen positive
**20% reflexed, Combined test risk cut-off 1 in 2000
clear
input ct ct10 ct20 dnapos dnaquad
end
set scheme lean2
graph bar (mean) ct (mean) ct10 (mean) ct20 (mean) dnapos (mean) dnaquad , legend(off) /*
*/ ylabel(0 "0.0" 10(10)100, nogrid) plotregion(margin(zero))
*/ ylabel(0(0.5)3, nogrid) plotregion(margin(zero))
N:\graphics\jpbestwick\WaldNJ\ x Nick Iran screening

38. *DNA test failures (2.5%) classed screen positive
screening-Iran v06.pptx
13/01/2019
Down’s syndrome screening performance: Universal DNA screening compared with screening using the Integrated test and Reflex DNA screening
100 20 40 60 80
Detection rate (%)
98.7
Universal DNA screening
94.2
Reflex DNA screening**
Integrated test
93.0
0.5
1.0
1.5
2.0
2.5
3.0
False-positive rate (%)
2.70*
2.70
Palomaki et al. Genet Med 2011;13:
Wald & Bestwick, J Med Screen 2015;22:168-74
*DNA test failures (2.5%) classed screen positive
**20% reflexed, Combined test risk cut-off 1 in 2000
clear
input ct ct10 ct20 dnapos dnaquad
end
set scheme lean2
graph bar (mean) ct (mean) ct10 (mean) ct20 (mean) dnapos (mean) dnaquad , legend(off) /*
*/ ylabel(0 "0.0" 10(10)100, nogrid) plotregion(margin(zero))
*/ ylabel(0(0.5)3, nogrid) plotregion(margin(zero))
0.046
Reflex DNA screening**
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40. Reflex DNA screening implementation project
Five hospitals involved
Royal London
Newham
Whipps Cross
Kingston
Liverpool Women’s
22,812 pregnancies screened
73 trisomy 21
25 trisomy 18
08 trisomy 13
Wald et al. Genet Med 2017; doi: /gim

41. Reflex DNA Screening: Predicted vs. observed screening performance
ReflexDNA-Prague v06.pptx
13/01/2019
Reflex DNA Screening: Predicted vs. observed screening performance
Observed
Predicted
Reflexed to DNA
11%
11%
90% 0%
20%
40%
60%
80%
100%
Detection
Rate
(trisomy 21,
18 or 13)
95%
0.02%
False-Positive Rate
0.06%
Wald et al. Genet Med 2017; doi: /gim
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42. Reflex DNA Screening: Predicted vs. observed screening performance
ReflexDNA-Prague v06.pptx
13/01/2019
Reflex DNA Screening: Predicted vs. observed screening performance
Predicted
Observed
Reflexed to DNA
11%
11%
95%
T21 0%
20%
40%
60%
80%
100%
90%
96%
T18
89%
T13
100%
78%
Detection
Rate
False-Positive Rate
0.06%
0.02%
Observed, Wald et al. Genet Med 2017; doi: /gim ; Predicted Bestwick & Wald J Med Screen 2016;23:171-4
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43. Half a Century of Progress in Antenatal Screening for Down’s Syndrome
screening-Iran v06.pptx
Half a Century of Progress in Antenatal Screening for Down’s Syndrome 20 40 60 80
100 33
1.0
2.0
3.0
4.0
5.0
Detection rate (%)
False-positive rate (%)
1970’s
Age Screening
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44. Half a Century of Progress in Antenatal Screening for Down’s Syndrome
screening-Iran v06.pptx
Half a Century of Progress in Antenatal Screening for Down’s Syndrome 20 40 60 80
100 78
1.0
2.0
3.0
4.0
5.0
Detection rate (%)
False-positive rate (%)
1988
Triple Test
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45. Half a Century of Progress in Antenatal Screening for Down’s Syndrome
screening-Iran v06.pptx
Half a Century of Progress in Antenatal Screening for Down’s Syndrome 20 40 60 80
100 88
1.0
2.0
3.0
4.0
5.0
Detection rate (%)
False-positive rate (%)
1996
Combined Test
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46. Half a Century of Progress in Antenatal Screening for Down’s Syndrome
screening-Iran v06.pptx
Half a Century of Progress in Antenatal Screening for Down’s Syndrome 20 40 60 80
100 93
1.0
2.0
3.0
4.0
5.0
2.7
Detection rate (%)
False-positive rate (%)
1999
Integrated Test
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47. Half a Century of Progress in Antenatal Screening for Down’s Syndrome
screening-Iran v06.pptx
Half a Century of Progress in Antenatal Screening for Down’s Syndrome 20 40 60 80
100 94
1.0
2.0
3.0
4.0
5.0
0.05
Detection rate (%)
False-positive rate (%)
2014
Reflex DNA screening*
*20% reflexed, Combined test risk cut-off 1 in 2000
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48. Half a Century of Progress in Antenatal Screening for Down’s Syndrome
screening-Iran v06.pptx
Half a Century of Progress in Antenatal Screening for Down’s Syndrome
True Positives
False Positives : 1
100 4
Age Screening
Reflex DNA screening
A 400-fold improvement
and an increase in detection from 33% to 94%
The odds of being affected given a positive result
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49. Thank you