Eva Pompilii, MD Genetic Counselor , TOMA Advanced Biomedical Assays, S.p.A., Gynepro Medical Bologna, Policlinico S.Orsola Malpighi Bologna • OBJECTIVES: At present, a precise guideline establishing chromosome microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The actual controversial question is whether CMA technologies can or should shortly replace the standard karyotype in prenatal diagnosis practice • CONCLUSIONS: Presently CMA analysis can be considered a second-tier diagnostic test to be used after a standard karyotype in selected group of pregnancies, such as those with single (apparently isolated) or multiple US fetal abnormalities, with de novo chromosomal rearrangements, even if apparently balanced, and those with supernumerary markers chromosomes

1. Microarray in diagnosi prenatale: la complessità dellaMicroarray in diagnosi prenatale: la complessità della
consulenza geneticaconsulenza genetica
Prenatal chromosomal microarray analysis: the complexityPrenatal chromosomal microarray analysis: the complexity
of genetic counselingof genetic counseling
LA DIAGNOSI PRENATALE E GLI SCREENING PRENATALI: cosa sta cambiando?LA DIAGNOSI PRENATALE E GLI SCREENING PRENATALI: cosa sta cambiando?
TOMA lab, Busto Arsizio –TOMA lab, Busto Arsizio – 18 September 2015
Eva Pompilii, MDEva Pompilii, MD
Genetic Counselor , TOMA Advanced Biomedical Assays, S.p.A.,Genetic Counselor , TOMA Advanced Biomedical Assays, S.p.A.,
Gynepro Medical Bologna, Policlinico S.Orsola MalpighiGynepro Medical Bologna, Policlinico S.Orsola Malpighi
BolognaBologna
eva_pompilii@yahoo.iteva_pompilii@yahoo.it

2. CMA in Prenatal testingCMA in Prenatal testing
• OBJECTIVES:
At present, a precise guideline establishing chromosome microarray analysis (CMA) applications and
platforms in the prenatal setting does not exist. The actual controversial
question is whether CMA technologies can or should shortly replace the standard karyotype in prenatal
diagnosis practice
• CONCLUSIONS:
Presently CMA analysis can be considered a second-tier diagnostic test to be used after a standard
karyotype in selected group of pregnancies, such as those with single
(apparently isolated) or multiple US fetal abnormalities, with de novo chromosomal rearrangements,
even if apparently balanced, and those with supernumerary markers chromosomes

3. Chromosomal microarray as second-tier test afterChromosomal microarray as second-tier test after
karyotypekaryotype
CMA in Prenatal testingCMA in Prenatal testing
400 bande 550 800

4. CMA in known abnormal fetal karyotypesCMA in known abnormal fetal karyotypes
In cases with a known fetal balanced rearrangement the detection rate for clinicallyIn cases with a known fetal balanced rearrangement the detection rate for clinically
significant cryptic abnormalities is 10%significant cryptic abnormalities is 10% Shaffer et al, 2012Shaffer et al, 2012

5. CMA in Definition of sSMCs and RingsCMA in Definition of sSMCs and Rings
In cases with a homogeneous or mosaic sSMC or ring chr the detection rate byIn cases with a homogeneous or mosaic sSMC or ring chr the detection rate by
CMA for clinically significant abnormalities is ~50%CMA for clinically significant abnormalities is ~50% Shaffer et al, 2012Shaffer et al, 2012

6. Challenges in counsellingChallenges in counselling
 Increasing and earlier clinical, genetic and genomic fetal informationIncreasing and earlier clinical, genetic and genomic fetal information
 Counseling more complex for elementsCounseling more complex for elements
often not readableoften not readable
 Increasing analytical complexityIncreasing analytical complexity
 Increased difficulty makingIncreased difficulty making
 CostCost
 Ethical and legal issuesEthical and legal issues
 TIME

7.  May uncover unwanted information (incidental findings)May uncover unwanted information (incidental findings)
 Adult on-set condition in a prenatal settingAdult on-set condition in a prenatal setting
 Known pathological conditions unrelated to the reasonKnown pathological conditions unrelated to the reason
for the test (with/out current management options)for the test (with/out current management options)
 Consanguinity (SNP arrays)Consanguinity (SNP arrays)
 Non paternity (SNP arrays)Non paternity (SNP arrays)
 May identify regions of unclear clinical significanceMay identify regions of unclear clinical significance (VOUS)(VOUS)
 Counseling dilemmasCounseling dilemmas
 Uncertain outcomeUncertain outcome
 Unknown outcomeUnknown outcome
 NICHD clinical trial (WapnerNICHD clinical trial (Wapner
et al, 2012) identified anet al, 2012) identified an
alteration in:alteration in:
 6% of abnormal fetuses and6% of abnormal fetuses and
normal karyotypenormal karyotype
 1.7% of normal fetuses and1.7% of normal fetuses and
normal karyotypenormal karyotype
 NICHD clinical trial (Wapner et al,NICHD clinical trial (Wapner et al,
2012) identified2012) identified VOUS in 3.4% ofVOUS in 3.4% of
normal karyotype fetusesnormal karyotype fetuses::
 1.8% likely benign1.8% likely benign
 1.6% potential for clinical1.6% potential for clinical
significancesignificance
Microarray and counsellingMicroarray and counselling

8. European Common FrameEuropean Common Frame

9. Copy Number Alterations: how many “ClinicallyCopy Number Alterations: how many “Clinically
Significant “Significant “
Anomaly Detection Rate
Single Anomaly 99/1772 (5.6%)
Anomalies in 2 or more organ systems 78/808 (9.6%)
Isolated abnormalities of growth 2/76 (2.6%)
One or more soft ultrasound markers* 2/78 (2.6%)
* Increased nuchal translucency excluded
Increased NT Isolated Other findings Total
< 4mm 1/113 (0.9%) 1/7 (14.3%) 2/120 (1.7%)
>4mm 6/96 (6.3%) 2/12 (16.7%) 8/108 (7.4%)
Total 10/303 (3.3%) 6/49 (12.2%) 16/352 (4.5%)

10. Normal karyotypeNormal karyotype in increased NT (>3.5 or 4mm) and fetalin increased NT (>3.5 or 4mm) and fetal
hydrops:hydrops: clinically significant findings by CMAclinically significant findings by CMA
I ncreased NT
Shaffer et al,
2012
Donnelly et
al, oral
communica
tion
I SPD2013
Isolated 6,3% 3,3%
In association with str. Abn. 16,7% //
Isolated or in association (all) 7,40% 5,1%
I solated
hydrops/ ascites/ oedema
Shaffer et al,
2012
Donnelly et
al, oral
communica
tion
I SPD2013
Isolated 7,0% NR
In association with str. Abn. 9,0% NR
Isolated or in association (all) 8,0% NR

11. Anomalies Single or ComplexAnomalies Single or Complex
Organ System or
Single Anomaly
Detection
Rate
CNS 25/381
(6.6%)
Heart 6/237
(2.5%)
Facies (dysmorphism) 6/88 (6.8%)
Diaphragmatic hernia 4/48 (8.3%)
Omphalocele 4/49 (8.2%)
Musculoskeletal 18/203
(8.9%)
Genitourinary 7/115
(6.1%)
Nuchal or other body
fluid accumulation
27/628
(4.3%)
Anomaly Detection
Rate
Holoprosencephaly 9/85
(10.6%)
Posterior fossa
defects
21/144
(14.6%)
Skeletal anomalies 15/140
(10.7%)
Ventricular septal
defect
14/132
(10.6%)
Hypoplastic left
heart
11/68
(16.2%)
Cleft lip/palate 14/136
(10.3%)

12. Case Example 1Case Example 1
• 42-year-old healthy pregnant woman
• PARA 0020
• Family history: no congenital anomalies or genetic-ereditary
disease. No consanguinity
• CVS karyotype analysis (AMA): normal male karyotype (46,XY)
• Second trimester ultrasound examination (20w): isolated
bilateral club feet
• CGH-array (CVS): del5p15.2 (breaking CTNND2 gene). Maternal
origin of del5p15.2.

14. • 34-year-old healthy pregnant woman
• PARA 1001
• Family history: no congenital anomalies or genetic-ereditary disease.
No consanguinity
• ICSI - pregnancy
• First trimester ultrasound examination (11w): increased nuchal
translucency (6mm)
• CVS karyotype analysis: normal female karyotype (46,XX)
Case Example 2Case Example 2

15. •Fetal Echocardiography (15w): right heart
hypoplasia, hyperechogenic bowel, SUA
•Second trimester ultrasound examination
(20w): previous echographic findings and
corpus callosum agenesis
•VMTP
•CGH-array (CVS): del1p36.21p36.33
(13Mb)
•Maternalkaryotype: 46,XX,inv(1)
(p36.21q44)

16. • 35-year-old healthy pregnant woman
• PARA 0000
• Dichorionic diamniotic twins pregnancy
• Family history: no congenital anomalies or
genetic-ereditary disease. No consanguinity
• Second trimester ultrasound examination
(20w): reduced fetal movement and fetal
growth restriction (fetus 1); normal anatomy
(fetus 2)
• LA karyotype analysis: 46,XX,del(13)(q14q31)
in fetus 1; 46,XY in fetus 2
• CGH-array (LA): 38 OMIM genes (PCDH9,
DACH1, KLF5, KLF12, CLN5, EDNRB e GPC5…)
Case Example 3Case Example 3

17. A chromosomal breakpoint that separates the esterase
D and retinoblastoma predisposition loci in a patient
with del(13)(q14q31)
John K. Cowell John Hungerford Paul Rutland Marcelle Jay
Received: March 5, 1986; Accepted: August 25, 1986;
A patient with severe mental retardation
and other congenital abnormalities who
developed retinoblastoma was shown to
have a deletion on the long arm of
chromosome 13 with breakpoints in
regions q14 and q31. Quantitation of
enzyme activity of the esterase-D gene
which, together with the retinoblastoma
locus, is located in region 13q14 showed
levels that were equal to those of normal
controls. The 13q14 breakpoint, therefore,
appears to have occurred between the two
loci, which places the esterase D gene in a
more proximal position in this band than
the retinoblastoma locus.

18. Case Example 4Case Example 4
• 36-year-old healthy pregnant woman
• PARA 2002
• Family history: no congenital anomalies or genetic-ereditary disease. No
consanguinity
• CVS karyotype analysis (AMA): normal male karyotype (46,XY)
• Second trimester ultrasound examination (20w): marked enlargement of
the cisterna magna and corpus callosum hypoplasia
• CGH-array (CVS): dupXq22.1mat including BEX5 gene
• Gene expressed in brain tissue but not in international medical
literature related to pathology

19. Case Example 5Case Example 5
• 40-year-old healthy pregnant woman
• PARA 0000
• Family history: epiplepsy/cerebral
malformation? (brother); intrauterine
fetal death (mother). No consanguinity
• CVS karyotype analysis (AMA): normal
male karyotype (46,XY)
• Second trimester ultrasound examination
(20w): Dandy-Walker malformation, CLP,
complex cardiac anomaly and esophageal
atresia
• VMTP
• CGH-array (LA): dupXq11.1 and
del16p13.11 (NDE1 gene)
• Parental karyotype/CGH-array: not
available
-Cytogenetic alteration associated with neuro-cognitive disorders
-NDE1 is reported to be involved in the development of the brain and
in the organization of the cerebral cortex as well as in the
proliferation and neuronal migration.
-Deletion also described as risk factor for multiplecongenital
anomalies

20. GRAZIE! THANK YOU FOR YOUR ATTENTION!