![Page 1: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/1.jpg)
New Genetic Approaches to Prenatal Diagnosis:
THE TRANSITION TO GENOMICS
Michael Urban Medical Geneticist
Division of Molecular Biology and Human Genetics
Tygerberg Hospital and University of Stellenbosch
![Page 2: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/2.jpg)
Prenatal screening and diagnosis for chromosome disorders
SCREENING
Primarily for Down syndrome
• 1970’s: Maternal age
• 1980’s: Triple test
• 1990’s: 1st tri biochemistry
• 2000: Combined screen – 1st tri nuchal & biochemistry
• 2012: NIPT
DIAGNOSIS
For chromosome disorders
• 1970’s: Amniocentesis &
karyotype
• 2000’s: CVS &
aneuploidy qPCR
• 2010’s: array CGH
![Page 3: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/3.jpg)
Genetic technology - diagnostics
• Chromosome analysis • Detects >5-10Mb
• Sanger sequencing & PCR • Few hundred base pairs • Single genes / exons
![Page 4: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/4.jpg)
Roles of traditional genetic testing
• Diagnostic / confirmatory testing
• Prenatal testing (and PGD)
• Predictive testing
• Carrier testing
• Population screening – usually reserved for populations at high risk on basis of ethnicity
![Page 5: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/5.jpg)
The clinical genetic approach
Approach: Unique aspects:
Case history Family history
Clinical examination Dysmorphology
Investigations Genetic tests --> Genomic tests
Medical care Where relevant…
Genetic counselling Information: • On clinical and lab findings • On personal and familial implications
Support: • To adjust to the condition • To make decisions (e.g. regarding TOP)
Family follow-up
![Page 6: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/6.jpg)
Genomics
A branch of biotechnology…
…applying new techniques of genetic testing…
…to sets of genes…
…or even the complete DNA sequence
… and analysis using ‘bioinformatic’ databases and computational methods
![Page 7: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/7.jpg)
Genomic diagnostic testing
• Array CGH:
– ‘Comparative genomic hybridization
– ‘Molecular karyotyping’
• 2nd generation sequencing:
– Non-invasive prenatal testing
– Whole genome sequencing
– Whole exome sequencing
(all protein coding regions)
![Page 8: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/8.jpg)
Pro’s and cons of genomic approach
• Pro’s:
– Lots more information:
• New understanding of genome structure and function
• New tests and treatments
• Cons:
– Too much information:
• What do detected genetic variants mean? – Particularly important in prenatal diagnosis
• How much information should be known/disclosed?
![Page 9: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/9.jpg)
Array CGH
• ‘High resolution karyotype’:
– Detects very small chromosome deletions / duplications
e.g. 10,000 - 500,000 base pairs in size
• Limitations:
– As resolution increases so does detection of normal or unknown variants (higher false positive rate)
– Less good than karyotype for a few indications: • Mosaicism
• Triploidy
• ‘Balanced’ chromosome abnormalities
![Page 10: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/10.jpg)
Application of array CGH in paediatrics
• Now a first-line test in paediatrics for:
– Intellectual disability
– Autism
– Multiple congenital abnormalities (MCA)
• Detection rate is 15-20% (vs ~3% for karyotype)
(E.g. Lu et al, 2009)
![Page 11: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/11.jpg)
Tygerberg experience with aCGH
In SA, availability of aCGH is still limited even in paediatrics
OUR EXPERIENCE:
• Carefully selected patients with: – MCA and/or intellectual disability
– Normal karyotype
• 24% disease-causing variants
• 12% variants of unknown significance (VOUS)
![Page 12: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/12.jpg)
Karyotype: 46,XY
Photos of dysmorphic baby
![Page 13: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/13.jpg)
7Mb duplication
7Mb deletion
aCGH result
![Page 14: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/14.jpg)
How did this test result help?
• Did not alter medical care
(in some cases may affect management)
• Gave the parents an answer
• Reduced further investigations
• Informed us of possible recurrence risk
• Can offer parents testing for carrier status
• Option of prenatal diagnosis in future pregnancies
![Page 15: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/15.jpg)
Prenatal use of aCGH … a tale
• Fetal anomaly scan at 18 weeks:
– Isolated CL&P
• Karyotype requested, but failed
• Lab arranged aCGH --> result:
– Arr 9p22.3(14,210,804-15,317,498) x3
– 1.1 Mb duplication of material from short arm of chromosome 9
![Page 16: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/16.jpg)
• Paediatrician informed parents that aCGH showed duplication of 9p
• Referred to an excellent website - information:
– Severe condition
– Limited ability to communicate
http://www.rarechromo.org/html/home.asp
![Page 17: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/17.jpg)
Closer assessment…
VOUS (variant of unknown significance)
![Page 18: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/18.jpg)
Genetic counselling
• Parents informed of uncertain significance
• For them:
– Better than expected
– Continued pregnancy
– Ongoing anxiety
– ‘An emotional rollercoaster thanks to conflicting opinions they received’
At 3 ½ months age
Photo of normal looking baby with cleft lip
![Page 19: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/19.jpg)
Current aCGH in prenatal diagnosis
• aCGH is increasingly recommended as primary genetic diagnostic test for investigation of ultrasound abnormalities (ACOG, Dec 2013)
• Still debate about:
– Array resolution
– Reporting of results
– Clinical criteria for testing:
• E.g. for single or multiple sonar abnormalities?
• Still very limited access in South Africa
![Page 20: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/20.jpg)
One approach: Guy’s Hospital
Array CGH:
• Limit resolution to predefined level (currently 3Mb)
• Except for known microdeletn regions (e.g. DiGeorge)
Dx test Indication
OLD APPROACH
Amnio & karyotype
• High risk Down syndrome • Fetal anomalies
NEW APPROACH
CVS and Aneuploidy qPCR (Since 2007)
• High risk Down syndrome
Array CGH (Since 2012)
• Fetal anomalies • NT>3,0mm • IUGR…
Courtesy of Dr Christine Patch
Detection rate: ~7%
![Page 21: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/21.jpg)
Non-invasive prenatal testing (NIPT) for fetal chromosome abnormalities
![Page 22: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/22.jpg)
Cell-free DNA in Maternal Blood
• Short DNA fragments in plasma
• In pregnancy, cell-free fetal DNA (cffDNA): – Comprises ~10% of total cell-free DNA (after ~10 weeks gestation)
– Probably mainly placental in origin
– Rapidly cleared after birth
• The entire genome of a fetus has been sequenced from cffDNA
![Page 23: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/23.jpg)
Clinical availability of cffDNA tests
Available for 5-10 years (internationally): • Fetal sexing • Fetal Rh status in Rh- women (Caucasian) Available for 2 years: • Aneuploidy
In the future? • Other chromosome abnormalities • Other genetic disorders
![Page 24: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/24.jpg)
NIPT methods for aneuploidy
Test* (Company) Method
Harmony (Ariosa) Comparison of counts of targeted chromosomes
Verifi (Verinata/Illumina) Comparison of counts of all chromosomes
Panorama (Natera) Analysis of SNPs (single nucleotide polymorphisms)
*These tests are mentioned because they are available through local ‘agents’ Note that different tests have different methods, and different pros and cons
![Page 25: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/25.jpg)
In practice NIPT for aneuploidy is currently a: • highly accurate… • screening test… • for Down syndrome
![Page 26: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/26.jpg)
NIPT can detect…
Trisomy 13
Trisomy 18
Down syndrome
Turner syndrome
22q11 del/DiGeorge
![Page 27: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/27.jpg)
Accuracy of NIPT
Detection rate False pos rate
Trisomy 21* 99.3% 0.16%
Trisomy 18* 97.3% 0.15%
Trisomy 13* 79% 0.41%
Sex chromosome abnormalities
~80% ~0.2%
Other chromosome abnormalities
Limited data ?
Benn et al, UOG 2013 *Data from ~6000 pregnancies
![Page 28: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/28.jpg)
Advantages of NIPT
COMPARED TO INVASIVE TESTS:
• No risk of procedure-related miscarriage
COMPARED TO OTHER SCREENING TESTS:
• Highly accurate for Down syndrome:
– Low false negative rate (but some cases are missed)
– Low false positive rate = far fewer invasive tests
• Anytime from 10 weeks onward
![Page 29: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/29.jpg)
Comparison with combined screen
0.01
0.1
1
10
100
5 15 25 35
Tris
om
y R
isk
(%)
Fetal Fraction (%)
0.01
0.1
1
10
100
5 15 25 35
Tris
om
y ri
sk (
%)
Fetal Fraction (%)
Nicolaides et al., Am J Obstet Gynecol 2012
Trisomy
False positive
Non-trisomy
• Both figures have the same number of patients • 10 trisomy cases • 1,939 non-trisomy cases
FPR: 4.5% FPR: 0.1%
![Page 30: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/30.jpg)
NIPT is not a diagnostic test
Test accuracy:
99% detection
0.1% false positive
T21 prevalence:
1 in 500
1,000 women
2 T21 998 non-T21
2 0
Test + Test -
1 997
Test + Test -
Example:
Positive test result is correct only 2/3 of the time
![Page 31: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/31.jpg)
Clinical guidelines for NIPT
• ACOG guideline (Dec 2012):
– Indications for NIPT (high risk of Down syndrome)
• ACMG guideline (Genet Med, 2013):
– Pro’s and cons of NIPT
– Pretest and post-test counselling
• Concerns about very rapid implementation esp. in low risk population:
– But evidence is accumulating that NIPT accurately detects Down syndrome in this situation
![Page 32: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/32.jpg)
Pretest counselling for NIPT • Assess if NIPT is appropriate:
– Check family history – Check why couple want NIPT – Check what they would do with NIPT result
• Discuss each condition (implications & test accuracy): – T21 – T13/18 – Turner and Klinefelter – 47,XXX and 47,XYY – Gender
• Discuss / compare alternative approaches: – Other screening tests – Invasive diagnostic testing
• Discuss need for confirmatory invasive testing – if ‘high risk’ NIPT result
• Discuss failure rate and logistics • Couple decides whether to test, and what to test
![Page 33: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/33.jpg)
Practical implementation of NIPT
Several options (not mutually exclusive)
• 10 weeks: – Primary screening procedure
– Allows for: • Modified first trimester scan
• Early invasive testing
• 12 weeks: – Contingent on NT/combined screen
• Later gestation: – Instead of triple screen
![Page 34: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/34.jpg)
Fetal Assessment Center NIPT implementation (Harmony)
Numbers Offered NIPT 171
Declined NIPT after counselling 17 (10%)
Accepted NIPT: 10-11 weeks 12-13 weeks 14-19 weeks
20+ weeks
154 (90%) including 5 twins 41 (27% of tests) 60 (39%) 42 12 (35%)
Results: ‘High risk’ ‘Low risk’
Failed
1 => confirmed positive 150 3
![Page 35: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/35.jpg)
Emerging workflow for prenatal diagnosis
SCREENING TEST(S)
RISK or SONAR FINDINGS
INVASIVE TESTING
NIPT
Risk Down syndrome
qPCR for aneuploidy
Combined screen
Sonar
Fetal anomalies Increased nuchal
Array CGH (Karyotype)
P R E T E S T
C O U N S S E L I N G
P O S T E S T
C O U N S S E L I N G Adapted from: Lichtenbelt et al, 2011
![Page 36: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/36.jpg)
New directions for genomics
• Extended NIPT – microdeletions
• Extended carrier screening
![Page 37: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/37.jpg)
Summary
• Genomics is superceding old genetic approaches:
– Array CGH for prenatal diagnosis
– NIPT for prenatal screening of Down syndrome
• Will continue to evolve rapidly
• Complementary to ultrasound
• Need for genetic counselling
![Page 38: New Genetic Approaches to Prenatal Diagnosis: THE ... Indications for NIPT (high risk of Down syndrome) •ACMG guideline (Genet Med, 2013): –Pro’s and cons of NIPT –Pretest](https://reader031.vdocuments.site/reader031/viewer/2022030511/5abbd42b7f8b9a297f8d5863/html5/thumbnails/38.jpg)
Acknowledgements
• For permission to use photographs:
– Dr H Ramdhani
– Parents