The New Genetics: Paradigm Shifts in Prenatal Diagnosis

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:
• 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
• Testing Options:
• 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:
(cffDNA)
• 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 differences in chromosome fragments can
identify fetuses with Down syndrome, trisomy 18, trisomy 13,
and sex chromosome abnormalies
• Two different 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
• Different platforms used
• Technique currently used by Sequenom and Verinata

NIPT WITH MPSS
Unaffected
Each diagrammatic fragment
represents many thousands of
sequenced fragments from
chromosome 21.
The quantitative overabundance
of Trisomy 21
fragments in an affected
pregnancy is significant and can
be measured with high
precision.
Extra Chromosome
Fragments = Affected
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 affected and unaffected 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

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)

NIPT IN CLINICAL CARE
Three
separate
groups
have now
shown
high
sensitivity
and
specificity
with low
false
positive
rate

NIPT
• Very 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)
* Detection rates and FPR vary 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
diagnostic testing:
Detects 3000 cases
Cost of $100 million
500 procedure-related
losses
Complete uptake of MPSS
followed by diagnostic testing
for positive results:
» Detect 2958 cases (miss 42)
» 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
(Test name)
Technology
Conditions
Tested For
Sensitivity Specificity Reporting
Sequenom
(MaterniT21Plus)
MPSS Trisomy 21
Trisomy 18
Trisomy 13
T21 = 99.1%
T18 = >99.9%
T13 = 91.7%
T21 = 99.9%
T18 = 99.6%
T13 = 99.7%
Positive
Negative
Failure
Verinata
(Verify)
MPSS Trisomy 21
Trisomy 18
Trisomy 13
Sex
Chromosomes
T21 = 100%
T18 = 97.2%
T13 = 78.6%
45X = 95%
XXX, XXY, XYY =
Limited data
T21 = 100%
T18 = 100%
T13 = 100%
45X = 100%
Positive
Negative
Aneuploidy
suspected
Failure
Ariosa
(Harmony)
Partnered with
Integrated
Genetics
(LabCorp)
DANSR
(assay)
+
FORTE
(algorithm)
Trisomy 21
Trisomy 18
Trisomy 13
T21 = 100%
T18 = 97.4%
T21 = 99.9%
T18 = 99.9%
Risk Ratio via
algorithm
1/10,000 –
99/100
(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
NUMBER 545 DECEMBER 2012
• Noninvasive Prenatal Testing for Fetal Aneuploidy
• ABSTRACT: Noninvasive prenatal testing that uses cell free
fetal DNA from the plasma of pregnant women offers
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 offered to low -risk women or women
with multiple gestations because it has not been sufficiently
evaluated in these groups. A negative cell free fetal DNA test
result does not ensure an unaffected pregnancy. A patient
with a positive test result should be referred for genetic
counseling and should be offered 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 very
important
• Benefits
• Limitations
• Risks
• Assist the patient in making informed, autonomous decision
• Be sensitive to personal 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
1
Patient
Control
Hybridization
to Array
Mix
CMA
Methodology
Laser Scanner
2 3
Data analysis
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.cfmpmid=16202
• Likelihood of finding a clinically relevant information not
identified on routine karyotype ( N ENGL J MED 367;23 Dec 6, 2012)
• 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 service.
Transfusion
Bianchi DW (2004) Circulating fetal DNA: its origin and diagnostic
potential- a review.
Ding et al (2004) MS analysis of single-nucleotide differences 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 (2012) Genome-wide fetal aneuploidy detection by maternal
plasma DNA sequencing. Obstetrics and Gynecology
Sparks et al (2012) Non-invasive prenatal detection and selective analysis of
cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and
trisomy 18. AJOG
Ashoor et al (2012) Chromosome-selective sequencing of maternal plasma cell -
free DNA for first-trimester detection of trisomy 21 and trisomy 18. AJOG
Colah et al (2011) Invasive and non-invasive approaches for prenatal diagnosis
of haemoglobinopathies: experiences from India. Indian J Med Res
Norton et al (2012) Non Invasive Chromosomal Evaluation (NICE) Study: results
of a multicenter prospective cohort study for detection of fetal trisomy 21 and
trisomy 18. AJOG.
Canick et al (2012) 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 (2012) Position statement on noninvasive prenatal testing/noninvasive
prenatal diagnosis.
• Sehnert et al (2011) Optimal detection of fetal chromosomal abnormalities by
massively parallel DNA sequencing of cell -free fetal DNA from maternal blood.
Clinical Chemistry
• Ladha, S (2012) A new era of non-invasive prenatal genetic diagnosis: exploiting
fetal epigenetic differences. Clin Genet
Devaney et al (2011) Noninvasive fetal sex determination using cell -free fetal
DNA: a systematic review and meta -analysis. JAMA
Hill et al (2011) Non-invasive prenatal determination of fetal sex: translating
research into clinical practice. Clin Genet
Geifman-Holtzman et al (2006) Diagnostic accuracy of noninvasive fetal Rh
genotyping from maternal blood- a meta-analysis. AJOG
Lo, Y (1994) Non-invasive prenatal diagnosis using fetal cells in maternal blood. J
Clin Pathol
Benn et al (2012) Non-invasive prenatal diagnosis for Down syndrome: the
paradigm will shift, but slowly. Ultrasound Obstet Gynecol

REFERENCES
• Nicolaides et al (2012) Noninvasive prenatal testing for fetal trisomies in a
routinely screened first-trimester population. AJOG
• Wapner et al (212) Chromosomal Microarray versus Karyotyping for Prenatal
Diagnosis. NEJM