1. An Introduction to Prenatal Chromosomal Microarray Developed by Dr. June Carroll, Ms. Shawna Morrison and Dr. Judith Allanson Last updated April 2015

2. 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.

3. 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

4. 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. 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.

5. 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

6. 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

7. Case 2 The patient is offered the options of: no further testing, non-invasive prenatal testing, or amniocentesisnon-invasive prenatal testing 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

8. Typical Prenatal Testing Algorithm Offer PN screening to all pregnant women 18-20 week fetal morphology scan FTS/IPS/SIPS NIPT for AMA + and for women willing to pay Family history 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 Genetics If indicated (e.g. fetal anomalies ) + generally maternal age of 40 years or older at estimated date of birth

9. Additional Testing e.g. Chromosomal microarray Additional Testing e.g. Chromosomal microarray Updated Prenatal Testing Algorithm for Women at Increased Risk Indication Advanced maternal age, multiple soft markers on ultrasound, ultrasound anomaly, positive prenatal screen, etc. Indication Advanced maternal age, multiple soft markers on ultrasound, ultrasound anomaly, positive prenatal screen, etc. Genetic counselling with testing options No further testing Screening Test e.g. NIPT Screening Test e.g. NIPT QF-PCR Detects common aneuploidies: Down syndrome, Trisomy 18, Trisomy 13 and sex chromosome aneuploidies QF-PCR Detects common aneuploidies: Down syndrome, Trisomy 18, Trisomy 13 and sex chromosome aneuploidies Karyotype No further testing If positive If negative Depending on indication: No further testing Consider additional testing Depending on indication: No further testing Consider additional testing If negative If positive Invasive Testing (diagnostic) Invasive Testing (diagnostic)

10. 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)

11. 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

12. 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 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 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 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 Incidental finding Results that are not apparently relevant to indication for which test was ordered

13. 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)

14. Library analogy for explaining genetic testing Clinical examination = Observing the outside of building – Number of windows – Doors – Roof – Height of the windows Wikimedia

15. 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

16. 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

17. 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.org www.2dayfm.com.au

18. What are the benefits of prenatal chromosomal microarray?

19. 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 groups 1 i.e. abnormal ultrasound, advanced maternal age, prenatal screening, parental anxiety 12,362 cases from all prenatal ascertainment groups 1 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 indications 1 5,108 AMA 1 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 cases 2,3

20. What are the limitations of prenatal chromosomal microarray?

21. 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

22. 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

23. 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

24. 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 —Characteristic facial appearance —Congenital heart disease (>70%) —Palatal anomalies (~70%) —Learning difficulties (70-90%) —Immune deficiency (>70%) —Hypocalcemia (50%), most serious in neonatal period —Developmental delay —Autism (~20%) —Psychiatric illness in adults (~25%), particularly schizophrenia McDonald-McGinn, 2015

25. 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

26. 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

27. 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

28. 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

29. 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

30. Don’t forget Take a family history to identify familial and/or ethnicity-specific disorders and screen accordinglyfamily history ethnicity-specific disorders Consider consanguinity and screen and test accordinglyconsanguinity Refer or consult genetics when in doubtconsult genetics

31. 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