1. 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

2. 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

3. Disclosures
I have no relevant financial relationships

4. Relative contribution of fetal/neonatal disease

5. 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

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

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

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

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

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

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

12. 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

13. 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)

14. Relative contribution of fetal/neonatal disease

15. 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

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

17. 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”

18. Comparative Genomic Hybridization

19. Single nucleotide polymorphism (SNP) array

20. 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

21. Chromosomal Microarray - ADVANTAGES
Better resolution – kb vs Mb
Higher diagnostic yield: when traditional karyotype normal
~ % - 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

22. 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

23. 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

24. Case: 28 yo G1, referral for CDH

25. Case: 28 yo G1

26. Case: 28 yo G1

27. Case: 28 yo G1

28. Case: 28 yo G1

29. Case: 28 yo G1

30. Case: 28 yo G1 Left congenital diaphragmatic hernia
LHR , Liver Down
Mild cerebral ventriculomegaly – 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

31. 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

33. 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

34. 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

35. 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

36. 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 Genetics in Med

37. 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

38. 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

39. 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

40. 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

41. 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

42. 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

43. 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