When and how to use chromosomal microarray (CMA) in prenatal diagnosis Brian L Shaffer MD 12/14/2018 Associate Professor Maternal Fetal Medicine Doernbecher Fetal Care Clinic

Objectives – Chromosomal Microarray (CMA) in Prenatal Diagnosis Review counseling for prenatal diagnosis in Obstetrics Understand indications for CMA In the context of neonatal disease Understand benefits and limitations of chromosomal microarray (CMA) in prenatal diagnosis With structural malformations (US/MRI) Aneuploidy – traditional karyotype Chromosomal Deletion/Duplication – array CGH/CMA Understand the benefits of SNP array Cases 40 min

Disclosures I have no relevant financial relationships

Relative contribution of fetal/neonatal disease

Case: 32 yo G1 – abnormal sequential screen neg cf DNA, “normal” 18 week anatomic survey Dating – 7 weeks US “Second opinion” - ?soft markers Dates by 1st TM US; risk of T21 1 in 7 ICU RN Ob scan in office

Case: 32 yo G1 – abnormal sequential screen neg cf DNA, “normal” 18 week anatomic survey

Case: 32 yo G1 - abnormal sequential screen neg cf DNA, “normal” 18 week anatomic survey

Case: 32 yo G1 – abnormal sequential screen neg cf DNA, “normal” 18 week anatomic survey

Case: 32 yo G1 – referred for abnormal sequential neg cf DNA, “normal” 18 week anatomic survey

Case: 32 yo G1 – referred for abnormal sequential neg cf DNA, “normal” 18 week anatomic survey

Case: 32 yo G1 – referred for abnormal sequential neg cf DNA, “normal” 18 week anatomic survey

Case: 32 yo G1 – abnormal sequential screen neg cf DNA, “normal” 18 week anatomic survey Structural malformations / abnormalities Cardiac – Interrupted arch, VSD Rocker bottom feet, clenched hands Renal abnormalities – small echogenic cystic kidneys Severe IUGR Cell free fetal DNA (cf DNA) – negative (from referring) Abnl sequential screen – 1 in 8 for trisomy 21 Opts for diagnostic testing

Case: 32 yo G1 – abnormal sequential screen neg cf DNA, “normal” 18 week anatomic survey Structural malformations / abnormalities Cardiac – Interrupted arch, VSD Rocker bottom feet, clenched hands Renal abnormalities – small echogenic cystic kidneys Severe IUGR Cell free fetal DNA (cf DNA) – negative (from referring) Abnl sequential screen – 1 in 8 for trisomy 21 Opts for diagnostic testing -- 46, XY (425 bands)

Relative contribution of fetal/neonatal disease

Case: 32 yo G1 – referred for abnormal sequential neg cf DNA, “normal” 18 week anatomic survey Structural malformations/dysmorphism – cardiac, rocker bottom feet, clenched hands, renal abnormalities Severe IUGR Diagnostic testing - Amniocentesis: 46, XY (425 bands) Additional testing – Chromosomal Microarray Arr [hg19] 15q26.1q26.3 – 9.3MB deletion (93,107,153_102,383,479)x1

Development of Cytogenetics Establishment of chromosome banding (1970s) Structural alterations 5-10 megabases Prometaphase – prophase banding Chromosome 15 has 102 MB

Comparative Genome Hybridization: Evolution to Arrays Subject and control DNA compete for hybridization CGH applied to chromosomes 5 Mb resolution An array consists of DNA fragments of known sequence printed on a platform (a glass slide) genomic imbalances produce differential fluorescent signals as determined by a laser scanner “virtual or molecular karyotype”

Comparative Genomic Hybridization

Single nucleotide polymorphism (SNP) array

Case: 32 yo G1 – referred for abnormal sequential neg cf DNA, “normal” 18 week anatomic survey Structural malformations/dysmorphism – cardiac, rocker bottom feet, clenched hands, renal abnormalities Severe IUGR Amniocentesis: Arr [hg19] 15q26.1q26.3 – 9.3MB deletion (93,107,153_102,383,479)x1

Chromosomal Microarray - ADVANTAGES Better resolution – 50-100kb vs. 50-10Mb Higher diagnostic yield: when traditional karyotype normal ~1.0-1.5% - AMA ~ 6.5% - abnormal US (major abnormality) Faster turnaround time Direct preparation (uncultured cells) Improved yield in IUFD (87 vs. 70%) and miscarriage Different platforms – CGH vs. SNP Comparative Genomic Hybridization (CGH) Detects Deletions/Duplications – compares sample with reference Targeted -- Associated with specific structural abnormalities or phenotypic features Whole genome - more coverage in gene rich areas, with “backbone” coverage Single nucleotide polymorphism (SNP) Assess for continuous regions of homozygosity – Consanguinity/Triploidy/UPD

Chromosomal Microarray - DISADVANTAGES Inability to detect balanced rearrangements Majority of balanced rearrangements result in normal outcomes Trisomy 21/13 or Robertsonian translocation – cannot provide information on recurrence Poor performance in certain circumstances (potentially) Triploidy (e.g. 69, XXX) (SNP) Low level mosaicism (<20%, SNP) Variants of uncertain significance (1-3%) Lower in targeted CMA Susceptibility deletion/duplication / Variants of Uncertain Significance Variable expressivity – Increased autism risk  Parental anxiety May be unable to accurately predict phenotype Does not detect point mutations (single gene changes) Cost

Who should be offered CMA? Fetal structural abnormalities - recommend Fetal demise - recommend Apparently balanced rearrangement Find a deletion/duplication at breakpoint Identify a marker or ring chromosome on karyotype Identify origin of material Suspect a common aneuploidy? -- Karyotype US consistent with trisomies 13, 18, 21 or 45,X All of those undergoing diagnostic testing? ACOG & SMFM – either karyotype or CMA with normal US

Case: 28 yo G1, referral for CDH

Case: 28 yo G1

Case: 28 yo G1

Case: 28 yo G1

Case: 28 yo G1

Case: 28 yo G1

Case: 28 yo G1 Left congenital diaphragmatic hernia LHR 1.3-1.4, Liver Down Mild cerebral ventriculomegaly – 11-12 mm Echogenic renal parenchyma and mild hydronephrosis Enlarged nuchal fold 6-7 mm Counseling: Amniocentesis Traditional Karyotype Chromosomal Microarray (CMA) / array CGH Single gene(s) / Exome

Case: 28yo G1, referral for CDH aCGH 1.4MB deletion 17q12 CNS/Brain Malformations Developmental delay – variable learning Autism, Anxiety Seizures Renal anomalies – large, echogenic Mullerian abnormalities Predisposition to Diabetes Less often, CDH Variability in phenotype Parental results – without deletion, de novo

Case – CVS for Abnormal sequential screen 23 yo G2P1 – 1 in 108 for trisomy 21 CVS: Mosaic trisomy 15 [46, XY (8)/47, XY +15(11)] Amniocentesis: Normal male karyotype 46, XY (425 bands) Fetal anatomic survey - normal Any additional testing which could elucidate any issues/etiology? Opts for karyotype

Uniparental Disomy (UPD) Usually inherit one chromosome from each parent haploid UPD - both members of a chromosome pair are inherited from the one parent May be without adverse consequence Can be quite significant: Two copies of a deleterious single gene for a recessive disease CFTR – AR disease without paternal input (isodisomy) Difference in phenotype may also be due to parent of origin differences We will get to some of these phenotype in a moment But first, lets review how we get UPD

Uniparental Disomy (UPD) - Mechanism Nondisjunction during meiosis (EVENT #1) Gamete contains 2 copies of a chromosome (disomic)..or Gamete has 0 copies (nullisomic) Resulting conception trisomy or monosomy Rescue -- EVENT #2 Loss of extra chromosome or…. Duplication of the single chromosome

Uniparental Disomy (UPD) - Mechanism EVENT#1 Most UPD “events” occur during maternal meiosis I EVENT#2 Thus – it is more likely for a trisomy to consist of two maternal chromosomes – and mat UPD will occur if the paternal component is lost So, in trisomy rescue, the most likely outcome is HETERODISOMY Shaffer LG, ACMG Statement on Diagnostic Testing for UPD. 2001 Genetics in Med

Uniparental Disomy (UPD) - Mechanism EVENT #1 EVENT #2 Duplication during mitosis Monosmy rescue is less common and results most often in pat ISODISOMY 15 – TWO identical homologues

Case – CVS for Abnormal sequential screen 23 yo G2P1 – 1 in 108 for trisomy 21 CVS: Mosaic trisomy 15 [46, XY (8)/47, XY +15(11)] Amniocentesis: Normal male karyotype 46, XY (425 bands) Fetal anatomic survey - normal Any additional testing which could elucidate any issues/etiology? Risk of UPD from CVS T15 mosaicism – 10-25% SNP aCGH Opts for karyotype

Why is UPD potentially important? Some genes are active based on the parent of origin Which copy is active depends on the parent of origin Some genes are normally active only when they are paternally inherited Others only when inherited from mother “Stamped “– methylation 11p15 15q11-15q13 Prader Willi – hypotonia, short stature, feeding difficulties MISSING activity which is paternally derived Two maternal copies Angelman – cognitive disability, seizures, abnormal movement, “happy” Missing activity which is maternally derived No maternal copy, abnormal mom copy, two paternal copies

CGH SNP array can detect UPD 38 fetuses with US abnormalities DNA banked until after delivery, termination, IUFD 16% of those with a normal karyotype (n=5) - pathologic CNV 6% with CNV of uncertain significance (n=2) 6% with UPD One child with maternal UPD 16 (SUA, IUGR, hypospadius, single kidney) One child with paternal UPD 4 (renal anomalies, dysmorphic features, normal development) Can detect areas of consanguinity, triploidy Cannot detect imprinting Should detect ~50% UPD Isodisomy/Heterodisomy

Imprinting Disorders (IDs) Differential expression of a gene(s) depending on the sex of the transmitting parent Some genes are expressed only From a paternally or maternally inherited chromosome/gene cluster Differentially Methylated regions (DMR) – CpG islands Cytosine residues are methylated according to parent of origin Phenotype may be attributable to: Over expression (Beckwith-Wiedemann) Lack of expression (Russell-Silver) Maternal 7,14,15 and Paternal 6,11,14,15 Have a definite phenotypic effect due to uniparental inheritance of imprinted regions

Uniparental Disomy/Imprinting – Definite Phenotype Solid black – UPD not described Solid dark Pink/ dark Blue – mat/pat with abnl PHENOTYPE Light pink/Light blue – UPD occurs, no clear phenotype Hatched – no abnl pheno could be defined due to imprinting

Summary Recommend CMA in context of prenatal diagnosis with fetal structural malformations or IUFD Review option of aCGH vs. traditional karyotype in context of diagnostic testing without malformation SNP array can identify – UPD, triploidy Review potential benefits: better resolution, higher yield, faster Review potential limitations: VUS, May identify abnormalities with variable phenotype SNP array may identify consanguinity or non-paternity May identify adult onset conditions which are inherited