THE NEW GENETICS:

PARADIGM SHIFTS IN
PRENATAL DIAGNOSIS

Jennifer Hoskovec, MS, CGC

Assistant Professor
Director, Prenatal Genetic Counseling Services
Department of Ob/Gyn and Reproductive Sciences
UT Health
 OUTLINE

Overview of standard screening and testing options for

aneuploidy
Non-Invasive Prenatal Testing for aneuploidy (NIPT)
Chromosomal Microarray Analysis in the prenatal setting
 Standard Screening and Testing Options for
Invasive Prenatal
Screening Fetal Aneuploidy
Diagnosis

Screening Options: Testing Options:

First Trimester Screening
Quadruple Marker Screen
Integrated, Sequential, or
Contingency Screens
Anatomy Scan
Benefit(s)
Non-invasive = no risk
Identifies women from low
risk pool who are at
increased risk
Disadvantage(s)
Risk calculation only
False positive/negative
Limited to trisomy 18, 13,
21
Timing, insurance coverage
Patient anxiety
Chorionic Villus
Sampling (CVS)
Amniocentesis
Benefit(s)
Diagnostic information
on all aneuploidies
Additional testing
available such as
microarray, PCR
Disadvantage(s)
Invasive, risk of
pregnancy loss (1/300-
1/500)
NON-INVASIVE
PRENATAL NIPT
TESTING
 NIPT

Available clinically since November 2011 in the United States

Analyzes cell-free fetal DNA circulating in maternal blood:
(cf fDNA)
Placental and fetal-derived cells
Possibly through the breakdown of fetal cells in circulation
~10-15% of cell-free DNA circulating in maternal blood is from
the fetus
Quantitative dif ferences in chromosome fragments can
identify fetuses with Down syndrome, trisomy 18, trisomy 13,
and sex chromosome abnormalies
Two dif ferent techniques
MPSS
DANSR + FORTE
 MASSIVELY PARALLEL SHOTGUN
SEQUENCING

Simultaneously sequence millions of short segments from

amplified DNA
Hundreds of sequences generated in a single run
Amplifies maternal and fetal DNA together
Increases number of samples run
Decreases cost
Dif ferent platforms used

Technique currently used by Sequenom and Verinata

 NIPT WITH MPSS
Extra Chromosome
Fragments = Affected
Each diagrammatic fragment
Unaffected represents many thousands of
sequenced fragments from
chromosome 21.

The quantitative over-

abundance of Trisomy 21
fragments in an affected
pregnancy is significant and can
be measured with high
precision.

Unaffected Fetus Fetus with Trisomy 21

Slide adapted from Sequenom

 DANSR + FORTE

DANSR- Digital Analysis of Selected Regions

Chromosome selective approach: selectively evaluates specific
genomic fragments from cfDNA
Determines fraction of fetal cfDNA in maternal plasma as well as the
chromosome proportion by assaying polymorphic and
nonpolymorphic loci
FORTE- Fetal-fraction Optimized Risk of Trisomy Evaluation
Algorithm that takes into account additional data: prior risk (based
on maternal age and gestational age) as well as fetal fraction
Enables determination of chromosome proportion and fetal fraction
at same time

Requires less DNA sequencing and can analyze ~750 patient

samples per run
Technique currently used by Ariosa
 NIPT VALIDATION STUDIES

NIPT has been validated by multiple groups:

In high risk pregnancies
AMA
Abnormal serum screen
Family or personal hx of child with aneuploidy
Abnormal ultrasound suggestive of aneuploidy
Between 10-22 weeks gestation
 25 twin pregnancies
17 normal pairs
5 with Down syndrome in one fetus of twin pair
2 with Down syndrome in both fetuses of twin pair
1 with trisomy 13 in one fetus of twin pair
All pregnancies were correctly classified
25/25 confidence interval [59-100]
Two triplet pregnancies studied
Unaffected; correctly classified
Authors note twin pregnancies have higher placental mass
and therefore might have higher fetal fraction and thus better
separation of af fected and unaf fected fetuses despite the
presence of multiples.
 2049 pregnant women undergoing routine screening at 11 -13
weeks gestation
86 pregnancies (4.3%) had karyotype via CVS or amniocentesis
1963 pregnancies were phenotypically normal at birth (assumed euploid)
Harmony risk scores available for 1949 (95.1%) pregnancies
46 (2.2%) had low fetal fraction
54 (2.6%) had assay failure
Trisomy 21
Detected 8/8 cases, all having risk scores >99%
Trisomy 18
Detected 2/3 cases, both having risk scores >99%, the third was an
assay failure
1939 euploid pregnancies
1937 has risk scores of <1% (cutoff for low risk)
2 = risk scores for trisomy 18 were 9.8% and 11.7%
 RAPID CLINICAL EVOLUTION

Verinata
Reporting sex chromosomes (Normal = XX, XY) and identification of
sex chromosome aneuploidies (XXX, XXY, XYY, monosomy X)
Sequenom
Reporting absence or presence of Y chromosome material on all
patients (99.4% accuracy quoted)
 Three
separate
groups
have now
shown
high
sensitivity
NIPT IN CLINICAL CARE and
specificity
with low
false
positive
rate
 NIPT

Ver y high specificity and sensitivity

Detection Rates
Down syndrome: >99% (0.2% FPR)
Trisomy 18: 97-100% (0.2% FPR)
Trisomy 13: 79-92% (1.0% FPR)
* Detecti on rates and FPR var y slightly between labs
Results
Typically reported as positive or negative
Some labs distinguish between results close to or distant from
the cut-off
Results close to the cut-off would have less confidence
Some labs classify those results as unclassifiable, some
would place results on a continuum scale
Confirmatory testing via CVS or amniocentesis is
recommended for positive results
 INCORPORATION INTO CLINICAL
CARE

Assume 100,000 women at high risk

1:32 of affected:unaffected
Diagnostic testing cost of $1000/patient
Procedure loss rate of 1/200

Complete uptake of Complete uptake of MPSS

diagnostic testing: followed by diagnostic testing
Detects 3000 cases
for positive results:
Cost of $100 million
500 procedure-related Detect 2958 cases (miss 42)
losses Cost of $3.9 million
20 procedure-related losses

Palomaki GE et al. Genet Med 2011

 NIPT: LIMITATIONS

Current limitations
Validation
Limited validation studies in low risk women
Validation study in twins had only 25 sets
Not validated in triplet or higher order multiples
Not validated in pregnancies conceived with egg donors
Not validated past 22 weeks gestation
Cost and insurance coverage
Does not include screening for ONTD
 NIPT Specifics
Laboratory Technology Conditions Sensitivity Specificity Reporting
(Test name) Tested For

Sequenom MPSS Trisomy 21 T21 = 99.1% T21 = 99.9% Positive

(MaterniT21Plus) Trisomy 18 T18 = >99.9% T18 = 99.6% Negative
Trisomy 13 T13 = 91.7% T13 = 99.7% Failure

Verinata MPSS Trisomy 21 T21 = 100% T21 = 100% Positive

(Verify) Trisomy 18 T18 = 97.2% T18 = 100% Negative
Trisomy 13 T13 = 78.6% T13 = 100% Aneuploidy
Sex 45X = 95% 45X = 100% suspected
Chromosomes XXX, XXY, XYY = Failure
Limited data
Ariosa DANSR Trisomy 21 T21 = 100% T21 = 99.9% Risk Ratio via
(Harmony) (assay) Trisomy 18 T18 = 97.4% T18 = 99.9% algorithm
Partnered with
+ Trisomy 13 1/10,000
Integrated
Genetics FORTE 99/100
(LabCorp) (algorithm) (0.5% results fell
between the two
extreme values)
NONINVASIVE PRENATAL TESTING/NONINVASIVE PRENATAL DIAGNOSIS
(NIPT/NIPD): The National Society of Genetic Counselors currently supports
Noninvasive Prenatal Testing/Noninvasive Prenatal Diagnosis (NIPT/NIPD) as
an option for patients whose pregnancies are considered to be at an
increased risk for certain chromosome abnormalities. NSGC urges that
NIPT/NIPD only be offered in the context of informed consent, education, and
counseling by a qualified provider, such as a certified genetic counselor.
Patients whose NIPT/NIPD results are abnormal, or who have other factors
suggestive of a chromosome abnormality, should receive genetic counseling
and be given the option of standard confirmatory diagnostic testing. (Adopted
February 18, 2012)
 ACOG/SMFM COMMITTEE OPINION
N UM BE R 5 4 5 D E C E M B E R 2 01 2

Noninvasive Prenatal Testing for Fetal Aneuploidy

ABSTRACT: Noninvasive prenatal testing that uses cell free

fetal DNA from the plasma of pregnant women of fers
tremendous potential as a screening tool for fetal aneuploidy.
Cell free fetal DNA testing should be an informed patient
choice after pretest counseling and should not be part of
routine prenatal laboratory assessment. Cell free fetal DNA
testing should not be of fered to low -risk women or women
with multiple gestations because it has not been suf ficiently
evaluated in these groups. A negative cell free fetal DNA test
result does not ensure an unaf fected pregnancy. A patient
with a positive test result should be referred for genetic
counseling and should be of fered invasive prenatal diagnosis
for confirmation of test results.
 NIPT FUTURE DIRECTIONS

Additional validation studies on use in low -risk

population and multiple gestations
Other chromosomal disorders and
microdeletions/duplications
Use for Mendelian disorders
New technology may increase accuracy
MeDiP: enriches for fetal-specific hypermethylated DNA
regions
Whole genome sequencing
Within the next 10 years, the complete fetal genome
will be successfully sequenced from maternal plasma
Lo (Prenat Diagn 2010;30:702-3)
 SUMMARY

So many options!
Accurate and balanced discussion of options with patient is ver y
impor tant
Benefits
Limitations
Risks
Assist the patient in making informed, autonomous decision
Be sensitive to per sonal nature of decision
Family values
Religious beliefs
Family and life situations
Concerns about having a child with an abnormality
Concerns about risk of miscarriage
 PRENATAL DIAGNOSTIC TESTING

CVS and amniocentesis

Routine karyotype
FISH
Aneuploidy FISH (13, 18, 21, X, Y)
Site specific FISH for deletion syndromes (22q11.2)
Chromosomal Microarray Analysis
CHROMOSOMAL MICROARRAY ANALYSIS

CMA platforms use thousands of DNA probes spread across the

genome to detect gains and losses of genetic material.
Extracted DNA from the patient (fetus) is compared with a
reference (normal) genome.
Allows identification of abnormal copy number changes (gains
and losses).
Aneuploidy
Duplications and deletions too small to be seen by conventional
cytogenetics
Limitations:
Cannot detect balanced chromosome rearrangements (identifies dosage
differences, not positional differences)
Cannot identify triploidy
Possible Pitfalls:
Identification of a copy variant of unknown significance (~1.5%)
Requires parental bloods for comparison
Possible out of pocket expense to the patient
Overview of CMA Process
Patient Control
1
Mix
Hybridization
to Array CMA
Methodology

Laser Scanner

2 Data analysis 3 FISH confirmation

del 22:q11.21
EXAMPLE- NORMAL RESULTS
EXAMPLE- TRISOMY 21
 CHROMOSOMAL MICROARRAY ANALYSIS
IN PRENATAL CLINICAL PRACTICE

Each individual syndrome incidence low

Ex: 22q11.2 deletion syndrome, AKA DiGeorge syndrome (22q11.2 del;
>95% detection; 1 in 4000-6000 incidence)
Ex: Williams Syndrome (7q11 del; 95% detection; 1 in 10,000 incidence)
Ex: Prader Willi Syndrome (15q11 del; 70% detection; 1 in 25,000
incidence)
Detailed detection potential for CMA version 6.3 oligo at BCM:
http://www.bcm.edu/geneticlabs/index.cfm?pmid=16202

Likelihood of finding a clinically relevant information not

identified on routine karyotype ( N E N G L J M E D 3 6 7 ; 2 3 D e c 6 , 2 01 2 )
1.7% of women referred for routine indications (AMA, pos screen, etc)
with normal ultrasound and karyotype had a clinically significant finding
on CMA
6% of women with abnormal ultrasound findings and normal karyotype
had a clinically significant finding on CMA
QUESTIONS
 REFERENCES

Lo et al (1997) Presence of fetal DNA in maternal plasma and serum.

Lancet
Finning et al (2002) Prediction of fetal D status from maternal plasma:
introduction of a new noninvasive fetal RHD genotyping ser vice.
Transfusion
Bianchi DW (2004) Circulating fetal DNA: its origin and diagnostic
potential- a review.
Ding et al (2004) MS analysis of single -nucleotide dif ferences in
circulating nucleic acids: application to non -invasive prenatal
diagnosis. PNAS
Gautier et al (2005) Fetal RhD genotyping by maternal serum analysis:
a two-year experience. AJOG
Scheffer et al (2011) Noninvasive fetal blood group genotyping if rhesus D,
c, E, and of K in alloimmunised pregnant women: evaluation of a 7-year
clinical experience. BJOG
Chiu et al (2011), Non-invasive prenatal assessment of trisomy 21 by
multiplexed maternal plasma DNA sequencing: large scale validity study.
BMJ
 REFERENCES

Palomaki et al (2011), DNA sequencing of maternal plasma to detect Down

syndrome: an international clinical validation study. Genetics in Medicine
Palomaki et al (2012), DNA sequencing of maternal plasma reliably identifies
trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative
study. Genetics in Medicine
Bianchi et al (201 2) Genome -wide fetal aneuploidy detection by maternal
plasma DNA sequencing. Obstetric s and Gynecology
Sparks et al (201 2) Non -invasive prenatal detection and selective analysis of
cell-free DNA obtained from maternal blood: evaluati on for trisomy 21 and
trisomy 1 8. AJOG
Ashoor et al (201 2) Chromosome -selective sequencing of maternal plasma cell -
free DNA for fir st -trimester detection of trisomy 21 and trisomy 1 8. AJOG
Colah et al (2011) Invasi ve and non -invasive approac hes for prenatal diagnosis
of haemoglobinopathi es : experiences from India. Indian J Med Res
Nor ton et al (201 2) Non Invasive Chromosomal Evaluati on (NICE) Study: results
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trisomy 1 8. AJOG.
Canick et al (201 2) DNA sequencing of maternal plasma to identify Down
syndrome and other trisomies in multiple gestations . Prenat Diagnosis
 REFERENCES

Benn et al (2011) Prenatal detection of Down syndrome using massively parallel

shotgun sequencing : a rapid response position statement from a committee on
behalf of the board of the international society for prenatal diagnosis.
NSGC (201 2) Position statement on noninvasive prenatal testing/noninvasive
prenatal diagnosis.
Sehner t et al (2011) Optimal detection of fetal chromosomal abnormalities by
massively parallel DNA sequencing of cell -free fetal DNA from maternal blood.
Clinic al Chemistr y
Ladha, S (201 2) A new era of non -invasive prenatal genetic diagnosis: exploiting
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 REFERENCES

Nicolaides et al (201 2) Noninvasive prenatal testing for fetal trisomies in a

routinely screened fir st -trimester population. AJOG
Wapner et al (21 2) Chromosomal Microarray ver sus Kar yotyping for Prenatal
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