10/1/2014
CLINICAL EXPERIENCE OF
MICRODELETION AND EXPANDED
TRISOMY DETECTION BY
NONINVASIVE PRENATAL TESTING
(NIPT)
2014 NSGC
Jenna Wardrop, MS, CGC
Sequenom Laboratories
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Disclosure
 The speaker is a full-time employee of Sequenom
Laboratories
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Introduction
 Noninvasive prenatal testing (NIPT) for fetal aneuploidies has
become a component of the standard of care for high risk
pregnancies
 A whole genome sequencing approach readily allows for
identifying subchromosomal deletion/duplication events
 A novel algorithm to identify fetal microdeletion and
microduplication events has been developed by Sequenom
Laboratories
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10/1/2014
Delivering on the promise of massively parallel
sequencing (MPS)
MPS allows for genome-wide
information that can expand
as clinical need dictates
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TCCGCCCAGGCCATGAGGGACCTGGAAATGGCTGAT
GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT
GACACGGTGGAGCTCGGCCACACCAGGCCCAGCTGG
GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT
ACAGTGGTGGGGCCCATCCCTGGGTGAGGCTCAGTT
GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT
GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT
GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT
TCCGCCCAGGCCATGAGGGACCTGGAAATGGCTGAT
GACACGGTGGAGCTCGGCCACACCAGGCCCAGCTGG
GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT
ACAGTGGTGGGGCCCATCCCTGGGTGAGGCTCAGTT
GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT
GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT
GACACGGTGGAGCTCGGCCACACCAGGCCCAGCTGG
GGCCCTGGGGACAGTCTCCAATCCACTGAGTCATCT
11 12 13 14 15
16 17 18 19 20
chr21
chr10
chr14
chr10
chr21
chr10
chr10
chr10
chr21
chr14
chr10
chr21
chr10
chr10
chr14
chr10
21 22
X
Y
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Four key elements to evaluate microdeletions noninvasively
The detection of subchromosomal microdeletions/duplications is limited by
four main factors
– Size of the variation
(biology dependent)
– Number of sequencing counts
(can be influenced)
– Fetal fraction
(sample dependent)
– Sequencing noise in that
area (method and biology dependent)
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The MaterniT21™ PLUS test
Enhanced Sequencing Series Validation
Method validation
Sensitivity
94.4%
95% CI (71-99%)
17 of 18
Specificity
99.4%
95% CI (95-99%)
156 of 157
 Blinded plasma samples and genomic DNA with karyotypic anomalies were
used for clinical validation
 The validation was not syndrome-specific due to low prevalence rates, but
rather size specific for events ranging from 3Mb-40Mb
 Validation was genome wide
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10/1/2014
The MaterniT21™ PLUS test
Enhanced Sequencing Series performance
Analytical performance based on size of abnormality**
>99%
>99%
>91% >99%
>99% >99%
85-90%
60-85%
3-6 Mb deletion 7-11Mb deletion >12Mb deletion Trisomy 16/22
 Analytical Sensitivity  Analytical Specificity n = 48
** Absence of an Additional Finding does not indicate a negative result
** Analytical performance modeled on genomic DNA with plasma mixtures
** Performance dependent on size of deletion, number of reads, fetal fraction, etc.
** Although deletions as small as 1.5Mb have been detected, sensitivity in this range is
variable due to size of the deletion
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The Enhanced Sequencing Series
Frequency of
condition
(# of births)
Name
Location
Deletion size
DiGeorge/22q
22q
1.5-3Mb
1/4,000
Cri-du-chat
5p
5-40Mb
1/50,000
Angelman
15q
5-6Mb
1/20,000
Prader-Willi
15q
5-6Mb
1/20,000
1p36
1p
1.5-10Mb
1/10,000
Trisomy 16
Chromosome 16
NA
1/50,000
Trisomy 22
Chromosome 22
NA
1/40,000
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Clinical laboratory review process
 Over- or under-representation of the chromosome
region is flagged by bioinformatics pipeline
 The pipeline flag is pulled up in the classification report,
and data around the flag is reviewed:
– Size
– Z-score
– Log Odds Ratio
– FF determined from the subchromosomal event
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10/1/2014
ESS laboratory metrics
Total samples screened: 120,726 (October 2013 – July 2014)
Result
Number
Average fetal
fraction (%)
Size Range
Average
z-score
22q11
24
13.35
0.65 – 5.0 Mb
Maternal= -26.36
Fetal= -9.89
1p36
4
14.95
4.3 - 19.9 Mb
-13.77
5p
6
11.25
16.2 - 29.5 Mb
-15.24
15q
4
14.85
1.25 – 5.45 Mb
-14.28
T16
38
10.21
NA
16.94
T22
25
11.56
NA
20.34
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Algorithm flag examples: 22q11
You may want to insert a normal Chromosome 22 cadet view and
then compare with the abnormal, i.e. 22qel
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Algorithm flag examples: 22q11 deletions
Heart defect, short long bones, brain anomaly, confirmed by microarray
2.25 Mb
0.85 Mb
Tetralogy
of Fallot
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5.0 Mb
Tetralogy of
Fallot noted on
ultrasound.
2.30 Mb
Tetralogy
of Fallot,
confirmed
by FISH
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Rates of positive calls compared to expected
Total samples screened: 120,726 (October 2013 – July 2014)
Condition
Incidence
Trisomy 16
32/100,0001
Trisomy 22
9-20/100,0001
22q11 deletion
1/40002
Cri-du-chat (5p-)
15q11 deletion
(PW/AS)
1p36 deletion
Expected
Actual
39
38
11-24
25
30
24
2-8
4
6-12
6
12
4
1/15,0001/50,0003,4
1/10,0001/20,0005,6
1/10,0007
1. Wolstenholme J. Prenat Diagn. 1996;16(6):511-24. 2. Wilson DI, et al. Am J Hum Genet. 1994; 55:A169. 3. Higurashi M, et al. Brain Dev. 1990; 12: 770-773. 4. Niebuhr
E. Hum Genet. 1978; 44: 227-275. doi: 10.1007/BF00394291.5. Driscoll DJ, et al. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1330/. 6. Clayton-Smith J, et al. J
Med Genet. 1992;29:412-5. 7. Slavotinek A, et al. Monosomy 1p36. J Med Genet. 1999; 36:657-63.
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Obtaining outcomes
 A laboratory genetic counselor contacts the provider when the
report is released to review the result, gather relevant known
clinical information, and discuss the need for follow up confirmatory
testing
 Genetic counselor recontacts the provider in 1-2 weeks to inquire
about follow-up testing
 If no diagnostic testing is performed, the case is flagged for
postnatal follow-up at the estimated date of delivery
 Multiple attempts are made to gather follow-up information
 All ESS positive cases are tracked in a database and updated as
more information becomes available
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ESS Outcome Data
Total samples screened: 120,726 (October 2013 – July 2014)
Condition
Total
Confirmed
Strongly
Suspected
No
Information
False
Positive
T16
38
6
18
12
2
T22
25
3
8
13
1
22q11
24
15
6
3
0
5p
4
1
0
1
2
15q
6
2
2
2
0
1p36
3
2
0
0
1
Totals
100
29
34
31
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Summary
 Automated bioinformatics flags are critically reviewed by the
laboratory directors
 Rates of positive calls align with expected numbers
 Minimal incremental increase in false positive rate with
addition of content to test
 Positive predictive value is overall high
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Related posters
Thank You!
 R&D team
 Clinical laboratory team
– Juan-Sebastian Saldivar
– Nilesh Dharajiya
– Tom Monroe
– Theresa Boomer
– Julie Jesiolowski
 Providers who contributed
feedback
 Poster 152: Detection of maternal
22q deletions by noninvasive
prenatal testing (NIPT)
 Poster 146: Clinical experience
reporting trisomy 16 and 22 on
noninvasive prenatal testing
(NIPT): Test performance and
implications for genetic
counseling.
Breakfast Symposia
Noninvasive Prenatal Testing for
Microdeletions: One Year Later
 Saturday 7:00 am- 7:45 am
These laboratory-developed tests were developed and their performance characteristics determined by Sequenom Laboratories. They have
not been cleared or approved by the U.S. Food and Drug Administration (FDA). Although laboratory-developed tests to date have not been
subject to U.S. FDA regulation, certification of the laboratory is required under CLIA to ensure the quality and validity of the tests. This
laboratory is accredited and certified to perform high complexity clinical laboratory testing.
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