An Introduction to Prenatal Chromosomal Microarray

Developed by Dr. June Carroll, Ms. Shawna Morrison and Dr. Judith Allanson

Last updated April 2015

Disclaimer

• This presentation is for educational purposes only and should not be used as a substitute for clinical judgement. GEC-KO aims to aid the practicing clinician by providing informed opinions regarding genetic services that have been developed in a rigorous and evidence-based manner. Physicians must use their own clinical judgement in addition to published articles and the information presented herein. GEC-KO assumes no responsibility or liability resulting from the use of information contained herein.

Objectives• Following this session the learner will be able to:– Refer to their local genetics centre and/or order genetic

testing appropriately for prenatal chromosomal microarray– Discuss and address patient concerns regarding prenatal

chromosomal microarray– Find high quality genomics educational resources

appropriate for primary care

Case 1• 29-year-old G1P0 woman, in good health• No significant family history or history of prenatal exposure • Integrated Prenatal Screening (IPS) was negative

– 1 in 2,000 versus her age related risk to have a baby with Down syndrome of about 1 in 1,095

• 19 week fetal morphology ultrasound showed ventricular septal defect (VSD), polyhydramnios and suspected cleft lip and palate

• Patient is seen in Genetics and offered amniocentesis with QF-PCR* to rule out common aneuploidies (Down syndrome, trisomy 18, trisomy 13 and sex chromosome differences)

QF-PCR is a PCR-based technique that consists of amplifying markers located on the chromosomes of interest to determine the number of copies of those chromosomes present per cell. This method only detects chromosome number of the select chromosomes (13, 18, 21, X and Y), not structural arrangement.

Case 1

• No common aneuploidy is detected (normal male on QF-PCR)

• Patient is then offered chromosomal microarray for further, more detailed analysis (testing will be performed on the same amniotic sample)

• Results take about 4 weeks

Case 2• 42-year-old G3P2 woman• No significant family history or history of

prenatal exposure • Integrated Prenatal Screening (IPS) was

positive – 1 in 100 versus her age related risk to have a baby

with Down syndrome of about 1 in 61

Case 2• The patient is offered the options of: no further

testing, non-invasive prenatal testing, or amniocentesis

• She chooses the diagnostic certainty of amniocentesis

• This genetics centre has implemented a new algorithm for all prenatal invasive testing so that all normal QF-PCR samples are sent for chromosomal microarray testing

• Results take about 2-3 weeks

Typical Prenatal Testing Algorithm

Offer PN screening to all pregnant womenOffer PN screening to all pregnant women

18-20 week fetal morphology scan18-20 week fetal morphology scan

FTS/IPS/SIPSFTS/IPS/SIPS NIPT for AMA+ and for women willing to payNIPT for AMA+ and for women willing to pay Family historyFamily history Ethnicity-based

screening*Ethnicity-based

screening*

If positive

*for ethnicity-based screening, if both members of the couple are carriers of the same condition

If negative or decline

Refer to GeneticsRefer to Genetics

If indicated (e.g. fetal anomalies )

+generally maternal age of 40 years or older at estimated date of birth

Additional Testinge.g. Chromosomal

microarray

Updated Prenatal Testing Algorithm for Women at Increased Risk

IndicationAdvanced maternal age, multiple soft markers on ultrasound, ultrasound

anomaly, positive prenatal screen, etc.

IndicationAdvanced maternal age, multiple soft markers on ultrasound, ultrasound

anomaly, positive prenatal screen, etc.

Genetic counselling with testing optionsGenetic counselling with testing options

No further testingNo further testing Screening Teste.g. NIPT

QF-PCRDetects common aneuploidies: Down syndrome, Trisomy 18, Trisomy 13 and sex chromosome aneuploidies

QF-PCRDetects common aneuploidies: Down syndrome, Trisomy 18, Trisomy 13 and sex chromosome aneuploidies

KaryotypeKaryotype

No further testingNo further testing

If positiveIf negative

Depending on indication:•No further testing•Consider additional testing

If negativeIf positive

Invasive Testing(diagnostic)

Invasive Testing(diagnostic)

What is Chromosomal Microarray (CMA)?• CMA is a technology used to determine if there are small extra (micro-duplication)

or missing (micro-deletion) pieces of genetic information. These gains and losses are called copy number variants (CNVs). A CNV can be: of no medical consequence; pathogenic, resulting in physical and/or intellectual consequences; or protective against disease (e.g. HIV infection).

Reference DNA from control labeled Red

Test DNA from patient labeled Green

Glass microarray slide

Denature the DNA (separate the strands) and Hybridize to slide

Computer scans and analyzes signal outputs

Areas of loss (deletion)

Area of gain (duplication)

Use of Prenatal Chromosomal Microarray

• Canadian College of Medical Geneticists (CCMG) and Society of Obstetricians and Gynaecologists of Canada (SOGC) state:

• Chromosomal microarray (CMA) may be an appropriate investigative measure in cases with fetal structural abnormalities detected on ultrasound or fetal MRI

• CMA is generally not recommended in pregnancies at increased risk for a numerical chromosomal abnormality (aneuploidy) e.g. advanced maternal age, positive maternal serum screen

Duncan et al 2011 J Obstet Gynaecol Can

What do microarray results mean? Normal

No copy number variant (microdeletion/microduplication)

detected Does not exclude a syndrome caused by

a mutation within a single gene or detect a balanced translocation

Pathogenic

Copy Number Variant has been previously described and associated with a known phenotype

Variant of uncertain significance (VOUS/VUS)

Not yet described in the literature, is challenging to interpret and benefits from knowledge of parental status

Incidental finding

Results that are not apparently relevant to indication for which test

was ordered

VUS identified in fetus Test parents

Neither parent has the VUS identified in the pregnancy (both have a normal result)

One parent has same CMA result as child

Barring non-paternity, the finding in fetus is new, de novo, and likely pathogenic

Finding in the fetus is a normal familial variant and not pathogenic

Finding in the fetus is pathogenic, and the parent displays reduced penetrance (not everyone with the CNV will have symptoms), variable expressivity (individuals with this CNV have varied presentation)

Library analogy for explaining genetic testing

• Clinical examination =• Observing the outside

of building– Number of windows– Doors – Roof– Height of the windows

Wikimedia

Library analogy for explaining genetic testing

• Karyotype = • Standing in one spot in the

library and looking at the number of rows (46 rows, 2 row 1s, 2 row 2, etc… the location of the rows, large extra or missing pieces

Wikimedia

Library analogy for explaining genetic testing

• Microarray = • Walking through the library

and seeing if there are extra or missing shelves

• A shelf may be thought of as a collection of books or genes, that are closely located and extra or missing shelves would be called microduplication or microdeletions

Flikr.com

Library analogy for explaining genetic testing

• Sequencing– Next-gen sequencing, Sanger

sequencing

= • Reading through the books

word by word, letter by letter to detect small changes: substitutions, extra or missing words

Wikimedia.orgwww.2dayfm.com.au

What are the benefits of prenatal chromosomal microarray?

• A systematic review of the literature was conducted to calculate the utility of prenatal microarrays in the presence of a normal conventional karyotype. 12,362 cases from all prenatal ascertainment groups1

i.e. abnormal ultrasound, advanced maternal age, prenatal screening, parental anxiety

2.4% had a clinically significant copy number variant

(CNV) (295/12,362) 3,090 abnormal ultrasound 1 6.5% had clinically

significant CNV (201/3,090)

4,164 other indications1

5,108 AMA1 1.0% had clinically significant CNV (50/51,08)

[1] Callaway et al 2013 Prenat Diagn[2] Shaffer et al 2012 Prenat Diagn[3] Wapner et al 2012 NEJM

6.5% had clinically significant CNV (201/3,090)

1.1% had clinically significant CNV (44/4,164)

Variant of Unknown

significance (VUS) are found in about 1% of

cases2,3

What are the limitations of prenatal chromosomal

microarray?

• Normal result does not rule out the possibility of a genetic change causing health and/or developmental concerns – CMA is unable to detect single gene changes, balanced genomic

rearrangements (e.g. inversions, reciprocal translocations), low level mosaicism

• Interpretation of copy number variants (CNVs)– Some CNVs have highly variable clinical expressivity associated with

incomplete penetrance, which can cause significant diagnostic, counselling, and ethical dilemmas

• Incidental findings– CNVs may be identified that are unrelated to the indication for testing,

but could possibly predict other health problems in the future– Insurance discrimination

• Genetic testing may affect an individual’s ability to obtain life, disability, critical illness, long-term care and/or extended health insurance if the test reveals a predisposition to other medical issues, e.g. increased cancer risk

• Non-paternity could be disclosed following parental sample analysis

Crolla Prenat Diagn 2014

Case 1• 29-year-old G1P0 woman, in good health• 19 week fetal morphology ultrasound showed ventricular

septal defect (VSD), polyhydramnios and suspected cleft lip and palate

• Patient was seen in Genetics and offered amniocentesis with QF-PCR to rule out common aneuploidies (Down syndrome, trisomy 18, trisomy 13 and sex chromosome differences)

• QF-PCR showed normal male• Chromosomal microarray was offered and the results showed

a 2.54-Mb deletion within 22q11.2• The patient is now about 23weeks gestation

22q11.2 deletion syndrome

• Caused by a sub-microscopic deletion on chromosome 22 – 85% of individuals will have the typical deletion

size and about 15% will have smaller atypical deletions within the critical region

• About 93% of affected individuals have a de novo deletion of 22q11.2 and about 7% have inherited the deletion from a parent

McDonald-McGinn, 2015

• Multi-system disorder with variable expressivity – Clinical presentation will vary between affected individuals even within the

same family (variable expressivity)

• Features include:

22q11.2 deletion syndrome

McDonald-McGinn, 2015

Case 2• 42-year-old G3P2 woman• No significant family history or history of

prenatal exposure• Integrated Prenatal Screening (IPS) was

positive – 1 in 100 versus her age-related risk to have a baby

with Down syndrome of about 1 in 61

Case 2

• Patient chose amniocentesis• QF-PCR showed normal female • This genetics centre has implemented a new

algorithm for all prenatal invasive testing so that all normal QF-PCR samples are then sent for chromosomal microarray testing

• Chromosomal microarray results showed a pathogenic deletion that includes the BRCA1 gene

Case 2

• This incidental finding has diagnosed the fetus with an adult-onset hereditary cancer predisposition syndrome

• Consider:– Was disclosure of incidental results, including adult onset

conditions, part of the pre-test counselling and consent?– Implications for autonomy and insurance discrimination

for the fetus– Implications if either parent carries this deletion and is at

increased risk for cancer

Prenatal Chromosomal Microarray Pearls

• Chromosomal microarray (CMA) has a greater yield (~6%) than traditional karyotype, particularly in high risk pregnancies

• There is variability in practice with regards to who will be offered prenatal CMA

• Consent process, pre- and post- test counselling are complicated

Bernhardt 2014 J Genet Counsel 23:938

Prenatal Screening Summary

• Offer all pregnant women, regardless of age:– PN screening for fetal aneuploidy (trisomy 13, 18, 21)

through FTS, IPS, SIPS or Quad screening– Second trimester ultrasound for dating, assessment of

fetal anatomy and detection of multiples

• Prenatal testing menu continues to evolve and expand with new screening and diagnostic tests– Consider NIPT in your counselling

• More, complex options may add to patient’s decisional conflict

Don’t forget• Take a family history to identify familial and/or

ethnicity-specific disorders and screen accordingly

• Consider consanguinity and screen and test accordingly

• Refer or consult genetics when in doubt

References• Bernhardt BA, Kellom K, Barbarese A, et al. An exploration of genetic counselors' needs

and experiences with prenatal chromosomal microarray testing. J Genet Couns 2014; 23(6):938-47

• Callaway JL, Shaffer LG, Chitty LS, et al The clinical utility of microarray technologies applied to prenatal cytogenetics in the presence of a normal conventional karyotype: a review of the literature. Prenat Diagn 2013; 33(12):1119-23

• Crolla JA, Wapner R, Van Lith JM. Controversies in prenatal diagnosis 3: should everyone undergoing invasive testing have a microarray? Prenat Diagn 2014; 34(1):18-22

• Donnelly JC, Platt LD, Rebarber A et al. Association of copy number variants with specific ultrasonographically detected fetal anomalies. Obstet Gynecol 2014;124(1):83-90

• Duncan A, Langlois S; SOGC Genetics Committee; CCMG Prenatal Diagnosis Committee. Use of array genomic hybridization technology in prenatal diagnosis in Canada. J Obstet Gynaecol Can 2011;33(12):1256-9

• McDonald-McGinn DM, Emanuel BS, Zackai EH. 22q11.2 Deletion Syndrome. 1999 Sep 23 [Updated 2013 Feb 28]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2015.

• Shaffer LG, Dabell MP, Fisher AJ, et al. Experience with microarray-based comparative genomic hybridization for prenatal diagnosis in over 5000 pregnancies. Prenat Diagn 2012; 32(10):976-85

• Wapner RJ, Martin CL, Levy B, et al. Chromosomal microarray versus karyotyping for prenatal diagnosis. N Engl J Med 2012; 367(23):2175-84