Jennifer Hoskovec, MS, CGC

Assistant Professor

Director, Prenatal Genetic Counseling Services

Department of Ob/Gyn and Reproductive Sciences

UT Health

THE NEW GENETICS:

PARADIGM SHIFTS IN

PRENATAL DIAGNOSIS

Overview of standard screening and testing options for

aneuploidy

Non-Invasive Prenatal Testing for aneuploidy (NIPT)

Chromosomal Microarray Analysis in the prenatal setting

OUTLINE

Screening

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

Invasive Prenatal Diagnosis

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)

Standard Screening and Testing Options for

Fetal Aneuploidy

NIPT NON-INVASIVE

PRENATAL

TESTING

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

NIPT

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

MASSIVELY PARALLEL SHOTGUN

SEQUENCING

NIPT WITH MPSS

Unaffected

Unaffected Fetus Fetus with Trisomy 21

Each diagrammatic fragment

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.

Extra Chromosome

Fragments = Affected

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 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

NIPT VALIDATION STUDIES

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 <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

and

specificity

with low

false

positive

rate

NIPT IN CLINICAL CARE

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 sl ightly 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

NIPT

Complete uptake of

diagnostic testing:

Detects 3000 cases

Cost of $100 million

500 procedure-related

losses

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 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

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: LIMITATIONS

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)

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 of fered 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 of fered invasive prenatal diagnosis for confirmation of test results.

ACOG/SMFM COMMITTEE OPINION NUMBER 545 DECEMBER 2012

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)

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

SUMMARY

CVS and amniocentesis

Routine karyotype

FISH

Aneuploidy FISH (13, 18, 21, X, Y)

Site specific FISH for deletion syndromes (22q11.2)

Chromosomal Microarray Analysis

PRENATAL DIAGNOSTIC TESTING

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

Mix

Patient

Hybridization to Array

Control

Laser Scanner

Data analysis

del 22:q11.21

FISH confirmation

CMA Methodology

1

2 3

Overview of CMA Process

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 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 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 (2012) Genome-wide fetal aneuploidy detection by maternal

plasma DNA sequencing. Obstetr ics and Gynecology

Sparks et al (2012) Non-invasive prenatal detection and selective analysis of

cel l - free DNA obtained from maternal blood: evaluation for tr isomy 21 and

trisomy 18. AJOG

Ashoor et al (2012) Chromosome-selective sequencing of maternal plasma cel l -

free DNA for first -tr imester detection of tr isomy 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 tr isomy 21 and

trisomy 18. AJOG .

Canick et al (2012) DNA sequencing of maternal plasma to identify Down

syndrome and other tr isomies in multiple gestations . Prenat Diagnosis

REFERENCES

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

shotgun sequencing : a rapid response posit ion statement from a committee on

behalf of the board of the international society for prenatal diagnosis.

NSGC (2012) Posit ion statement on noninvasive prenatal testing/noninvasive

prenatal diagnosis.

Sehnert et al (2011) Optimal detection of fetal chromosomal abnormalit ies by

massively paral lel DNA sequencing of cel l - free fetal DNA from maternal blood.

Clinical Chemistry

Ladha, S (2012) A new era of non -invasive prenatal genetic diagnosis: exploit ing

fetal epigenetic dif ferences. Clin Genet

Devaney et al (2011) Noninvasive fetal sex determination using cel l - free fetal

DNA: a systematic review and meta -analysis. JAMA

Hil l et al (2011) Non-invasive prenatal determination of fetal sex: translating

research into cl inical 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 cel ls in maternal blood. J

Clin Pathol

Benn et al (2012) Non-invasive prenatal diagnosis for Down syndrome: the

paradigm wil l shif t , but slowly. Ultrasound Obstet Gynecol

REFERENCES

Nicolaides et al (2012) Noninvasive prenatal testing for fetal tr isomies in a

routinely screened first -tr imester population. AJOG

Wapner et al (212) Chromosomal Microarray versus Karyotyping for Prenatal

Diagnosis. NEJM