1. Prenatal Chromosomal Microarray Developed by Dr. Judith Allanson, Ms. Shawna Morrison and Dr. June Carroll Last updated November 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: – Appropriately refer to their local genetics centre and/or order 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 2-3 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 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 ) AMA – Advanced Maternal Age, ≥40y@EDB

9. Additional Testing e.g. Chromosomal microarray Additional Testing e.g. Chromosomal microarray 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)? A cytogenetic test used to determine if there are chromosomal imbalances, either large (e.g. whole extra or missing chromosomes, also detected by standard karyotype) or smaller extra (micro-duplication) or missing (micro-deletion) pieces of genetic information, also called copy number variants (CNV)

11. Single Nucleotide Polymorphism Array There are various chromosomal microarray (CMA) platforms, increasingly a single nucleotide polymorphism (SNP) based approached is being used SNPs, pronounced ‘snips’, are the most common type of genetic variation Each SNP represents a difference in a single DNA building block, a nucleotide (guanine, cytosine, adenine, thymine) An individual inherits one SNP from their mother and one SNP from their father, these can be the same (homozygous) or different (heterozygous) An individual can be one of three possibilities at each SNP – AA e.g. A/T and A/T – BB e.g. C/G and C/G – AB e.g. A/T and C/G TAC AGA CCA ACT TAC AGA CCA ACC Maternal allele Paternal allele

12. Single Nucleotide Polymorphism Array Licence for use of figures: https://creativecommons.org/licenses/by/3.0/ https://creativecommons.org/licenses/by/3.0/

13. Prenatal Chromosomal Microarray Canadian College of Medical Geneticists (CCMG) states (2009): 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

14. What do the genetic test results mean? 1.Normal 2.Pathogenic 3.Variant of Uncertain Significance (VUS) 4.Incidental Finding

15. What do the genetic test results mean? Normal: No clinically significant copy number changes were identified in the DNA of this specimen in the areas tested Excludes a micro-deletion/micro-duplication (CNV) within the limits of resolution of the test (typically very high) Limitations: CMA is not able to detect balanced genomic rearrangements, low levels of mosaicism, and mutations within single genes Next Steps: Referral for genetic consultation should be considered, depending on the initial reason for the invasive prenatal testing – additional testing may be indicated

16. What do the genetic test results mean? Pathogenic: A copy number variant known to be associated with an abnormal phenotype Provides insight to the genomic etiology of ultrasound findings and may assist in counselling about prenatal and postnatal outcomes and management options Limitations: Not all pathogenic findings are associated with a severe clinical presentation, and the clinical presentation can be extremely variable. Uncertainty often remains and may cause anxiety for a pregnant couple. Next Steps: Genetic counselling is recommended to review significance, provide information, resources and support and if indicated discuss further testing/change in medical management (e.g. fetal echocardiogram) and parental testing

17. 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)

18. What do the genetic test results mean? Variant of Uncertain Significance (VUS): This is a genomic variant that has not yet been categorized as benign or pathogenic, either because too few cases have been reported in the literature or the affected gene’s content and/or function are not yet understood Next Steps: Parental testing: Parental status can help determine whether or not the CNV is familial, and less likely to be pathogenic, or de novo (new in the affected individual) and more likely pathogenic Refer for genetic counselling Next Steps: Parental testing: Parental status can help determine whether or not the CNV is familial, and less likely to be pathogenic, or de novo (new in the affected individual) and more likely pathogenic Refer for genetic counselling Occurs in about 1% of pregnancies

19. What do the genetic test results mean? Incidental Finding (IF): Genetic variant(s) – either benign or pathogenic - identified by a genetic test that is unrelated to the primary indication for testing An IF may signify: Presence of late-onset disorder with result having clinical utility e.g. hereditary cancer syndrome Presence of late-onset disease without therapeutic possibilities e.g. Alzheimer disease risk Carrier status for autosomal recessive or X-linked diseases e.g. cystic fibrosis (CF), Duchenne Muscular Dystrophy (DMD) Parental consanguinity An IF may signify: Presence of late-onset disorder with result having clinical utility e.g. hereditary cancer syndrome Presence of late-onset disease without therapeutic possibilities e.g. Alzheimer disease risk Carrier status for autosomal recessive or X-linked diseases e.g. cystic fibrosis (CF), Duchenne Muscular Dystrophy (DMD) Parental consanguinity Next Steps: Genetic counselling is recommended Rare (1/1,500). Depends on a patient’s motivation for testing

20. Normal result can provide reassurance Increased diagnostic yield over traditional karyotype – A pathogenic copy number variant (CNV) will be identified in more than 6% of pregnancies following a normal karyotype Potentially valuable information for parents making reproductive decisions, and may have significant value in the future management of the child Benefits of Chromosomal Microarray

21. 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 PN ascertainment groups 1 i.e. abnormal u/s, AMA, prenatal screening, parental anxiety 12,362 cases from all PN ascertainment groups 1 i.e. abnormal u/s, AMA, prenatal screening, parental anxiety 2.4% had a clinically significant 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

22. Back to 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. Back to 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. Back to 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. Back to 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 Microarray Chromosomal microarray (CMA) has a greater yield than traditional karyotype, particularly in high risk pregnancies There is variability in practice with regards to who will be offered prenatal CMA Consent, pre- and post- test counselling is complicated

29. Resources Visit www.geneticseducation.ca to connect to your local genetics centre and for the GECKO on the run resourcewww.geneticseducation.cayour local genetics centreGECKO on the run You may also wish to consult your local maternal-fetal medicine (MFM) specialist or high risk obstetrician/gynaecologist depending on the reason CMA has being considered If there are terms that require further elaboration please visit the GECKO Glossary in Educational ResourcesGECKO Glossary Unique – Disorder Guides Disorder Guides – Unique has been collecting information about specific chromosome disorders in their offline database for nearly 30 years and produces family-friendly, medically-verified, disorder-specific information guides. Orphanet – A reference portal for information on rare diseases and orphan drugs, for all audiences

30. How to explain genetic testing to patients

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

32. 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

33. 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

34. 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