Download The ABCs of Genetic Testing

THE ABC’S OF GENETIC
TESTING
Garrett K. Lam, MD
Professor and Chair, Dept of Ob/Gyn
University of Tennessee COM Chattanooga
Regional Obstetrical Consultants
LEARNING OBJECTIVES
1. Compare the specificity and sensitivity of
serum aneuploidy screening against NIPS
2. Explain the difference between NIPS vs true
diagnostic testing
3. Assess the utility of cfDNA testing for
detecting other chromosomal and genetic
issues
4. Describe the utility of microarray in the
workup of fetal demise
Credits
1. Thanks to Dr. Owen Phillips (UT Memphis)
and Janet Ulm (genetic counselor, ROC) for
their direct contributions to this talk
2. Some of their slides have been incorporated
into this talk with their express permission
3. Some of the slides are adapted from the
Sequenom/LabCorp speaker’s slide deck.
They are used for teaching purposes only, and
do not mention their product and names are
redacted.
Survey says:
What percentage of early pregnancy loss is
attributable to aneuploidy?
A. <5%
B. 10%
C. 25%
D.50%
Survey says:
What is the proportion of pediatric inpatients
with some type of genetic disorder?
A. <5%
B. 10%
C. 25%
D.50%
Epidemiology of genetic disease
Reproductive
 Approximately half of early pregnancy loss
attributable to aneuploidy
 Average number of recessive traits each adult
carries is 2-3
 Incidence of microdeletion or duplication in
structurally normal fetuses is about 1%;
incidence is about 6% in structurally
abnormal fetuses
Epidemiology of genetic disease
Childhood
 3/1000 newborns have aneuploidy, and 9.2/1000
newborns have some type of structural
rearrangement of chromosomes
 7/1000 have autosomal dominant traits, 2.5/1000
have autosomal recessive traits, and 0.5/1000
have X-linked trait
 Among pediatric inpatients, more than 25% have
a genetic disorder
 Over 1/3 pediatric deaths attributed to
congenital anomalies – 17% of these due to
chromosome abnormalities, and 12% due to
some other recognized malformation syndrome
Prenatal screening in US 10
years ago
 Define a woman’s risk for having a child
with a chromosomal abnormality (age,
previously affected) via risk factors
 With this information, offer serum
screening or invasive testing (CVS or
amniocentesis)
Prenatal Screening in US NOW
 First Trimester (Combination)




Screening
Second Trimester Maternal
Serum Screening
NIPT
CMA
????
Prenatal Trisomy 21 Screening
Detection
Rate
& Testing Performance
Screening Tests1
2nd Tri Triple Test 69 %
2nd Tri Quad Test 81%
Screen
Positive
Rate
NonDetection
Rate
5%
5%
31%
19%
1st Tri Combined
(+NT)
85%
5%
15%
1st & 2nd Full
Integrated (+NT)
95%
5%
5%
Diagnostic Confirmatory Tests2,3
Chorionic Villus
Sampling (CVS)
> 99.2%
<0.8%
Amniocentesis
>99.7%
<0.3%
1. Malone FD, Canick JA, Ball RH, Nyberg DA, et al. First-trimester or second-trimester screening, or both, for Down syndrome. First- and Second-Trimester Evaluation of Risk (FASTER) Research
Consortium. N Engl J Med. 2005 Nov 10;353(19):2001-11.
2. Hsu LY, Perlis TE.United States survey on chromosome mosaicism and pseudomosaicism in prenatal diagnosis. Prenat Diagn. 1984 Spring;4 Spec No:97-130.
3. Ledbetter DH, Martin AO, Verlinsky Y, et al. Cytogenetic results of chorionic villus sampling: High success rate and diagnostic accuracy in the U.S. Collaborative Study. Am J Obstet Gynecol 1990;162:495.
Newest Screening Test:
NIPS (Non-Invasive Prenatal
Screen)
 Done at 9-10 weeks or greater
 Screening test; not diagnostic
 Utilizes circulating cell free DNA
(ccfDNA) in the mother’s blood
 “Fetal” DNA is actually of placental
origin
Screens for:






Trisomy 21
Trisomy 18
Trisomy 13
X and Y chromosome abnormalities
Microdeletions optional in some labs
Detects sex of baby
Detection rate 85-99%. False positive rate <1%
Black- euploid fetus
Red- Fetus with Trisomy 21
NIPS Tests Available







MaterniT21Plus (Sequenom)
Verifi (Progenity)
Harmony (Ariosa)
Panorama (Natera)
InformaSeq (LabCorp)
Qnatal Advanced (Quest)
Informed Pregnancy Screen (Counsyl/PathGroup)
Survey says:
 NIPS is such a highly sensitive test, that I feel
comfortable ordering it to diagnose or rule
out an aneuploidy with a positive serum
screening test result
1. I agree
2. I disagree
3. I don’t know
Procedure
 Blood sent to processing lab
 Determine fetal fraction (at least 4% to run
specimen)-thus GA at time of testing is important
 Sequence all DNA in specimen (shot gun
sequencing) or use of site specific probes
 Analyze for 13, 18, 21, presence of Y
 Report ‘positive’ if excess 13, 18 or 21 is present
DATA ON DETECTION RATE OF TRISOMY
21, 18 AND 13 BY CCFF DNA
Trisomy 21
Number of cases
Detected
Performance
210/212
Sensitivity 99.1%
Specificity 99.9%
Trisomy 18
59/59
Sensitivity 99.9%
Specificity 99.6%
Trisomy 13
11/12
Sensitivity 91.7%
Specificity 99.7%
Palomaki GE, et al.. DNA sequencing of maternal plasma
reliably identifies trisomy 18 and trisomy 13, as well as Down
syndrome: An international collaborative study. Genet Med.
2012 Jan 26. doi: 10.1038/gim.2011.73.
NIPS: Accuracy= Detection
rates and FPR: Not 100%
NIPT result
N (%)
True positive for T21
119/126 (94.4%)
False positive for T21
7/126 (5.6%)
True positive for T18
25/42 (59.5%)
False positive for T18
17/42 (40.5%)
True positive for T13
12/27 (44.4%)
False positive for T13
15/27 (55.6%)
True positive for SCA
11/29 (37.9%)
False positive for SCA
18/29 (62.1%)
Wang et al, Genet Med 2015
Why NIPS can be wrong
 False positives
 Confined placnetal mosaicism
 vanishing twins
 Microdeletion/duplication
 Maternal conditions
 False negatives




Obesity
‘no calls’ from low fetal fraction
trisomies other than T21
chromosome rearrangements (translocations,
microdeletions/duplications)
ACOG Opinion
“Given the performance of conventional
screening methods, the limitations of cell free
DNA screening performance, and the limited
data on cost-effectiveness in the low-risk
obstetric population, conventional screening
methods remain the most appropriate choice
for first-line screening for most women in the
general obstetric population.”
-Committee Opinion
Number 640, September 2015
Microdeletions: The “Seek” tests
 Microdeletion syndromes-covers
five clinically relevant
microdeletions.
References
3. Data on file, Illumina, Inc.
Microdeletion detection by NIPT
The “Seek tests”)
 ACMG and ACOG recommend against
screening
 Played on fears “Test for everything”
 Serious conditions, but exceedingly rare (PPV
issues)
 Most common (1/2000): del 22q11.2
 Rare disorders: PPV an issue 5.3%
 Kids may have heart defect
 Kids may have just a psychiatric disorder
 How do you counsel for that?
ACOG Opinion
 Routine cell free DNA screening for
microdeletion syndromes should not be
performed.
 The PPV for microdeletions is much lower than
for the Trisomies because the prevalence is
lower.
 Cell free DNA screening is not recommended for
women with multiple gestations.
-Committee Opinion
Number 640, September 2015
Professional Recommendations for
NIPS
•
•
•
•
•
•
Advanced Maternal Age
Previous Pregnancy with a Trisomy
Family History (i.e. balanced translocation)
Positive Multiple Marker Screening
Abnormal Ultrasound Findings
Soft Markers for Aneuploidy
Why just high risk groups?
Why not all patients?
1. Understanding PPV
2. Issue of rarer trisomies and other
chromosome abnormalities in younger
patients
3. Large studies yet to be published
Detection rate/Sensitivity vs
PPV
 The # true positives/total number of cases x 100
 How many affected cases did the test pick up
 EX: 212 cases in a population. Test detected 209
and missed 3. DR= 209/212 (X 100)= 98.6%
 PPV=What are the odds that a positive result
means the fetus is truly affected.
 Since there are false positives, the PPV depends on the
prevalence of the disease in the population.
 If there is low prevalence, the positive result is most
likely a false positive as the disease is so rare.
For trisomies, PPV differs
based on age
Sensitivity Specificity Age 25
PPV (%)
Age 40
PPV (%)
Trisomy 21 99.3
99.8
33
87
Trisomy 18 97.4
99.8
13
68
Trisomy 13
99.9
9
57
91.6
-In younger women, a positive result is more likely to be a false
positive than in older women because fewer cases are seen.
-Since not 100% accurate-should report out an adjusted
risk
-Regardless, positive predictive value means all positive tests need
diagnostic confirmation
AJMG: 2010; Grati et al
 In women > 35 y/o
 NIPT detected 65.1% of all clinically
significant chromosome abnormalities
 Why?– AMA=More trisomies, specifically 21,
18 and 13
 In women < 35 y/o
 NIPT detected 45.8% of all clinically
significant chromosome abnormalities.
 Fewer abnormalities present were
trisomies
Traditional serum screening in low risk
population
 Norton et al, Obstet Gynecol, 2014
 Traditional screening (first trimester and quad
screening) in low risk population followed by
amniocentesis
 Found 17% of clinically significant
chromosomal abnormalities that would not
have been by NIPT
 ccfDNA does outperform traditional
screening for T21, but that is about all we
can say, so ACOG recommends in low risk
population: “Traditional” serum screening
should be performed
“Invasive” (Diagnostic)
Testing: is it dangerous?
Danish Fetal Medicine Study Group
147,987 women who underwent
FTS for Down syndrome
5072 CVS (transabdominal, 18/20 g
double needle technique)
1809 amniocentesis (20 g needle)
141,106 women had no procedure
CVS loss difference ranged from
-0.08% at 3 days to -0.21% at
21 days after screening
Amniocentesis loss difference
ranged from 0.56% at 28 days
to 0.52% at 42 days after
screening
Conclusion: No statistically significant differences
between those women having a procedure and
those not having one
Wulff CB et al; Ultrasound Obstet Gynecol 2016;47:38-44.
‘Prenatal Diagnosis’
Advantages
 Chromosomal abnormalities will be missed by ccfDNA,
but detected with testing.
 Some women who receive a screen positive result
from MSS may ask about cell free technology rather
than definitive PD
 May delay definitive diagnosis and management plan as well
risk missing some pregnancies with aneuploidy
 Risks of invasive nature are negligible compared to
benefit in high risk patients
Obstet Gynecol: 2015; Benachi
et al
 Compared effectiveness of NIPT in 2 groups:
 Group 1: indications like AMA, screen positive on serum
screening
 Group 2: Ultrasound abnormalities
 NIPT DR (Confirmed by amnio): both groups
 T21 100%
 T18 88%
 T13 100% BUT:
 Group 1 (n=510):
 2.5% cases were NIPT negative but had other significant
findings on Invasive testing
 Group 2 (n= 376)
 7.7% cases were NIPT negative but had other significant
findings on Invasive testing
Reasonable approach
 Offer low risk patients: MSS or PD (amnio or CVS)
 Offer high risk patients (AMA, +MSS, previously affected child)
ccfDNA or PD (amnio or CVS)
 (ACOG) cfDNA really for 21, 18
 Evidence: PD Safe and detects 99.7% of all chromosome abnormalities
 2nd screen delays PD by 7-10 days
 Offer patients with fetal ultrasound anomalies: PD (amnio or
CVS)
 ACOG: cfDNA really for 21, 18. Go to amnio
 Evidence: PD Safe and detects 99.7% of all chromosome abnormalities and
can follow up with microarray
Positive ccfDNA
Recommendations:
Do not use ultrasound
for risk reduction. Go to
CVS or amnio
Evidence: US has lower
sensitivity than ccfDNA.
PD Safe and detects 99.7% of all
chromosome abnormalities.
 All women should be offered the option of aneuploidy screening or
diagnostic testing….choice of screening test is affected by many factors
 NIPS not recommended for women with multiple gestations.
 All positive NIPS results should have a diagnostic test before considering
pregnancy termination
 NIPS should not be used as a substitute for diagnostic testing
 Cell-free DNA for microdeletions has not been validated clinically and
should not be offered at this time
 Women whose NIPS results are inconclusive or not resulted because of low
fetal DNA content should be offered testing
Survey Says:
 38 y/o (AMA) G3P1011 presents with
IUFD at 20.5 wk found at anatomy US.
 CVS = 46,XX
 MSAFP only elevated at 3.5MoM
 US with abnormal fetal body positioning
(hyperextension) and clefting of face
 Your next step is:
1. Fetal autopsy
2. Send fascia lata/achilles for FISH &
karyotype
3. Send placenta for chromosomal
microarray
Diagnostic Technology:
Microarray
 Aka Comparative Genomic Hybridization (CGH),
Chromosomal microarray (CMA), SNP array
 Helps further characterize abnormalities seen by
karyotyping (e.g. unbalanced translocations, marker
chroms) and detects chromosome imbalances such as
deletions or duplications that are too small to be seen
by the microscope and may cause multiple congenital
abnormalities and/or known genetic syndromes.
 Gains or losses are called copy number variants (CNVs)
 Will not detect balanced translocations.
Google Scavenger Hunt
What tissue sample needs to be sent for
a CMA?
-use your smartphone to find the
answer
• Chromosomal microarray analysis have been found to detect a pathogenic
(or likely) copy number variant in approximately 1.7% of patients with a
normal ultrasound exam and a normal karyotype
• CMA detected all aneuploidies that routine karyotyping detected
• It is recommended that microarray be made available to any patient
choosing diagnostic testing, and may eventually replace conventional
karyotype for patients undergoing prenatal diagnosis
ACOG Opinion
“In patients with a fetus with one or more major
structural abnormalities identified on
ultrasonographic examination and who are
undergoing invasive prenatal diagnosis,
chromosomal microarray analysis is recommended.”
-Committee Opinion , Number 581, December 2013
Microarray Statistics
 In samples from fetuses with growth or structural
anomalies, 6% had clinically relevant findings on
microarray that were not found on karyotyping.
 With indication of AMA or abnormal screening for Tri
21, 1.7% had clinically relevant finding on microarray
not detected on routine karyotyping.
Wapner, et al. NEJM, December 6, 2012
Issues with Microarray
 Variants of unclear significance
 Coincidental findings
 Cost
Conclusions
 ACOG Committee Opinion:
no. 640; September 2015
 NIPT(S) does not replace CVS or amniocentesis - should remain
options (the gold standard for diagnosis)
 NIPS, though powerful, is a screening, not diagnostic tool
 Sensitivity does not equal positive predictive value
 the performance of the test is affected by disease prevalence
 Similarly, microdeletion testing by ccfDNA is of limited use
 CMA is valuable at detecting specific genetic mutations
that are below the karyotypic (subchromosomal) level, at
a scope beyond what microdeletion testing can offer
 Good for IUFD and searching for answers when karyotype does
not offer any help
Extra slides for discussion
Terminology: Basics of
traits
 Germline (versus somatic) mutation
 Heterozygous/Homozygous/X-linked
 Microsatellite (as in microsatellite
instability) – normally occurring repetitive
sequence of DNA within the genome that
can be used as a genetic “marker”, but
also may predispose to mutation and
gene disruption
Terminology: Diagnostic
studies
 Genome-wide association studies –
examination of genetic “markers” among
many individuals to find common genetic
variations associated with disease states
 Hybridization (as in comparative genomic
hybridization) – annealing of a single-strand
of nucleic acid (usually DNA that carries some
type of chemical identification, such as
fluorescence) to its complementary strand
Terminology: Diagnostics
 Next-gen sequencing – new technologies that
allow for more rapid and less costly DNA
sequencing
 Whole genome sequencing
 Whole exon sequencing
 Oncogene (and proto-oncogene)
 Tumor suppressor gene
Microdeletion syndromes
(examples)
 Cri-du-Chat…5p minus (46,XX,del(5p) )
 Wolf-Hirschhorn…4p minus (46,XY,del(4p)
)
 DiGeorge…22q11.2 deletion,
aka Velo-Cardio-Facial syndrome,
Sphrintzen syndrome
46,XX,ish del(22)(q11.2q11.2) (D22S75-)
OR arr cgh 22q11.2 (D22S75)X1
Case Example
 Abnormality noted on U/S: micrognathia
 Amnio FISH: Normal male
 Microarray: “Normal result with the
exception of a recessive disease risk”
 A 56.10 kb heterozygous deletion …was
detected. Individuals with this gene
deletion are generally asymptomatic
carriers for glycogen storage disease IXb.
Newer Diagnostic Technology:
Whole Exome Sequencing
 Sequencing of the protein coding regions
of the genome (exons) (approximately
1% of the genome)
 Useful in prenatal when chromosomal
analysis and microarray have been
normal and particularly when there has
been a recurrence of the same
abnormalities
Modes of Inheritance
 Mitochondrial inheritance
e.g. Leber hereditary optic neuropathy, MERRF
(Myoclonic Epilepsy with Ragged-Red Fibers)
 Mitochondrial DNA only transmitted by the
mother.
 NO transmission by the male. Thus affected
males do not have affected offspring.
 Affected females MAY have a very high
chance of having affected offspring
Mitochondrial Inheritance
Uniparental Disomy (UPD)
 Both chromosomes or portions of
chromosomes inherited from the SAME
parent.
 Examples: Prader-Willi syndrome
-mat UPD15
Angelman syndrome
-pat UPD 15
Beckwith-Wiedemann syndrome
-pat UPD 11
Web resources
 ACOG.org
 Genome.gov
 OMIM.org
 Genereviews.org
(www.ncbi.nlm.nih.gov/books/NBK1116/)
 ACMG.net
 NSGC.org
 Genetests.org
What about multiples?
 An issue with serum screening
 Fetal DNA into maternal circulation detected
 Canick et al
 25 twins: 17 were euploid
 5 discordant for trisomy 21 (100% DR)
 2 concordant for trisomy 21 (100% DR)
 1 discordant for trisomy 13 (100% DR)
 No cases of trisomy 18 in this set
Results of prenatal testing
 Traditionally
 Cell Culture (10 days to 3 weeks)
 Karyotype (computer-assisted)
 +/- Fluorescent In situ
hybridization (rapid 1-2 days)
Karyotype
FISH
 Interphase cells
 No culture needed
 Cells are 3 dimensional View can distort count of
signals
First Trimester Screening
 Biochemistry
 Free beta hCG
 PAPP-A
 Ultrasound
 Nuchal Translucency
 Nasal Bone
First Trimester Screening
 Biochemistry:
Can be drawn as early as 9 weeks gestation until
13+6.
 Ultrasound measurements:
CRL must be 45-84 mm
Advantages of First Trimester
Screening
 Higher detection rate (85-95% for
Trisomy 21 and 18)
 Earlier detection
 Separate risk estimates for twins
 Will take into account previous
pregnancy with Trisomy 21 or 18.
Disadvantages to First
Trimester Screening
 Sonographer must be specially certified
for NT and NB measurements
 Some patients do not present for care
early enough for screening.
 Does not detect open neural tube
defects (can draw AFP ONLY in 2nd
trimester)
Maternal Serum Screening in
Second Trimester
 Quad screen
 Penta screen
AFP
hCG
Estriol
Inhibin
Hyperglycosylated hCG (Penta only)
Maternal Serum Screening
 Done at 15-20 weeks gestation
 Screens for:
Open NTDs (AFP only) 85% detection rate
Trisomy 21 80% detection rate
Trisomy 18 60% detection rate
False positive rate 5%
Advantages of Prenatal
Diagnostic Testing
 If you go from MSS (5% positive rate)
to PD with a karyotype will detect
additional 17% chromosomal
abnormalities (not T21, 13, 18)
 If microarray is used, an additional
2% (over karyotype) chromosomal
abnormalities (deletion or
duplication syndromes) will be
detected