Prenatal Chromosomal Microarray Developed by Dr. Judith Allanson, Ms. Shawna Morrison and Dr. June Carroll Last updated November 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: 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

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 2-3 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 women FTS/IPS/SIPS NIPT for AMA and for women willing to pay Family history Ethnicity-based screening AMA – Advanced Maternal Age, ≥40y@EDB If negative or decline If positive* *for ethnicity-based screening, if both members of the couple are carriers of the same condition SOGC Nov 2011 age 40 = AMA Family Hx Q: hx of genetic conditions, ID/DD, congenital anomalies, infertility, consanguinity 18-20 week fetal morphology scan Refer to Genetics If indicated (e.g. fetal anomalies )

Prenatal Testing Algorithm for Women at Increased Risk Indication Advanced maternal age, multiple soft markers on ultrasound, ultrasound anomaly, positive prenatal screen, etc. Genetic counselling with testing options No further testing Invasive Testing (diagnostic) Screening Test e.g. NIPT If positive If negative QF-PCR Detects common aneuploidies: Down syndrome, Trisomy 18, Trisomy 13 and sex chromosome aneuploidies Depending on indication: No further testing Consider additional testing Recommend that all women be offered prenatal screening CCMG/SOGC Guidelines Jan 2011 Use of a DNA Method, QF-PCR, in the Prenatal Diagnosis of Fetal Aneuploidies – http://sogc.org/wp-content/uploads/2013/01/gui265CPG1109E.pdf CMA 2.4% CMA will pick up abnormality for any indication 6.4% if u/s abnormality ~1% VUS If positive Karyotype If negative No further testing Additional Testing e.g. Chromosomal microarray

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)

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 There are about 10 million SNPs in the human genome, occurring about once in every 300 nucleotides TAC AGA CCA ACT TAC AGA CCA ACC Maternal allele Paternal allele

Single Nucleotide Polymorphism Array DNA is hybridized to a slide which contains DNA with known sequences and use computer scanning to analyse the signal hybridization DNA from the patient is hybridized to the slide This technology distinguishes between the two possible SNP alleles (A or B) for a specific position in the genome allowing distinction between copy number changes, since AA or AB or BB at a specific position would be indicative of two copies of that genome site versus any other combination would signal a gain (AAA, AAB, ABB, BBB) or loss (A-, B-, -- ) at that site. Karampetsou E, Morrogh D, Chitty L. Microarray Technology for the Diagnosis of Fetal Chromosomal Aberrations: Which Platform Should We Use? Journal of Clinical Medicine. 2014; 3(2):663-678.Liscence for use of figures: https://creativecommons.org/licenses/by/3.0/ Licence for use of figures: https://creativecommons.org/licenses/by/3.0/

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 RE soft markers: Since the vast majority of fetuses in these situations are clinically unaffected, the positive predictive value of a detected CNV would likely be low.

What do the genetic test results mean? Normal Pathogenic Variant of Uncertain Significance (VUS) Incidental Finding

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 Limitations: so a normal CMA result does not completely rule out a genomic abnormality Next steps: So that additional genetic testing may be offered where appropriate. For example, if the ultrasound finding that triggered invasive testing was a cystic hygroma, your patient may be offered single gene testing for Noonan syndrome. Next Steps: Referral for genetic consultation should be considered, depending on the initial reason for the invasive prenatal testing – additional testing may be indicated

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. Pathogenic: There are over 200 known pathogenic CNVs and this number is increasing with CMA use. Example Dx: 22q11 deletion, also known as DiGeorge syndrome, characterized by congenital heart disease (74%), palatal abnormalities (69%); learning difficulties (70%-90%); immune deficiency (77%); hypocalcemia (50%); renal anomalies (31%); autism or autistic spectrum disorder (20% of children); psychiatric illness (specifically schizophrenia, 25% of adults). 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

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 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) Finding in the fetus is a normal familial variant and not pathogenic Barring non-paternity, the finding in fetus is new, de novo, and likely pathogenic

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 Occurs in about 1% of pregnancies

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 In the prenatal setting, IF more commonly refers to findings which do not have a direct consequence for the fetus, but may have implications postnatally for the individual or for his/her relatives. Next Steps: Genetic counselling is recommended Rare (1/1,500). Depends on a patient’s motivation for testing

Benefits of Chromosomal Microarray 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

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 groups1 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) 6.5% had clinically significant CNV (201/3,090) Variant of Unknown significance (VUS) are found in about 1% of cases2,3 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 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 Shaffer 2012 3,506 total non-demise cases 4.2% VUS (n=163) (reported as higher because a result wasn’t determined to be benign just because a parent had the same) 1.1% if VUS was defined as 0.39% (n = 15) had apparently de novo and 0.75% (n = 29) unknown inheritance. 5.3% clinically significant CNV (n=207) 0.3% AMA 6.6% ultrasound anomaly 2.6% soft signs Wapner 2012 3822 Total cases (51.4% were AMA) with normal karyotype VUS was decreased from 3.4% to 1.5% VUS after reclassification from 2007-2012 1.7% clinically significant in AMA 6.0% clinically significant in ultrasound anomaly 1.1% had clinically significant CNV (44/4,164) 4,164 other indications1 1.0% had clinically significant CNV (50/51,08) 5,108 AMA1 [1] Callaway et al 2013 Prenat Diagn [2] Shaffer et al 2012 Prenat Diagn [3] Wapner et al 2012 NEJM

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

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 22q11.2 del includes: DiGeorge syndrome Velocardiofacial syndrome Conotruncal anomaly face syndrome Autosomal dominant Opitz G/BBB syndrome 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. McDonald-McGinn, 2015

22q11.2 deletion syndrome Multi-system disorder with variable expressivity Clinical presentation will vary between affected individuals even within the same family (variable expressivity) Features include: Characteristic facial appearance Hypocalcemia (50%), most serious in neonatal period Congenital heart disease (>70%) Developmental delay Autism (~20%) Palatal anomalies (~70%) Psychiatric illness in adults (~25%), particularly schizophrenia Learning difficulties (70-90%) Immune deficiency (>70%) 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. McDonald-McGinn, 2015

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

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

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

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

Resources Visit www.geneticseducation.ca to connect to your local genetics centre and for the GECKO on the run resource 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 Resources Unique – 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 

How to explain genetic testing to patients

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.org www.2dayfm.com.au