Chromosomal microarray will replace conventional karyotype in near future..

1. Neharika Malhotra Bora
Asst Prof BVP medical college,Pune
Rainbow hospitals,Agra
Narendra Malhotra,MD,FICOG
FOGSI rep to FIGO
Vaidehi Jobanputra, Ph.D., M.S., FACMG
President ,
Advanced Genomics Institute & Laboratory Medicine (AGILE), New Delhi
Keshav Malhotra
Rainbow Hospital, Agra

3. STUDY METHOD
 CMA(Chromosomal micro-array) is a
new technology which provides a
whole genome scan to detect copy
number changes (CNCs).
 This study is to compare CMA
versus conventional fetal genetic
testing in collaboration with AGILE
lab ,New Delhi and RAINBOW
hospitals, Agra.
 The study started from June 2013
and until now 50 cases have been
done.
 Still ongoing .
 Long term follow-up of babies born
is being kept for five years.and will
be analyzed at the five year cutoff.

4. Inclusion criterias for prenatal
testing
 Advanced maternal age
 Previous child with de novo chromosomal
aneuploidy
 Woman 30 yrs, child with T21: Increased Recurrence risk for
any chromosomal abnormality (1/100) versus age-related risk
(1/390)
 Parental structural chromosome abnormality
 Family history of a genetic disorder
 Elevated risk based on maternal serum screening
 Fetuses with abnormal ultrasound examination

5. Its all in your “genes”
and
the genes are on your
chromosomes

6. Gold standard in prenatal
analysis
invasive procedures and
conventional karyotyping

7. Karyotyping versus microarray
analysis
Detects
– large aberrations only (10Mb)
– balanced translocations
– triploidy
• Does not detect
– smaller aberrations
Detects
– large and small aberration,
variants
of unknown significance
– unbalanced translocations

8. OBSERVATIONS
OUR EXPERIENCE WITH SOME CASE REPORTS
Example of a Normal Result

9. Interpretation of
TRISOMY 21

10. Amniotic fluid specimen
28 yrs old with absent nasal bone, increased NT
Normal FISH for aneuplodies and Karyotype
Case
1

11. Amnio
28 yrs old with absent nasal bone, increased NT
Normal FISH and Karyotype

12. 52 Protein Coding Genes
14 Disease associated genes
Common clinical features included feeding problems, behavioral
problems, abnormally shaped teeth, scoliosis, developmental delay,
MR.
Deletion of chromosome 3 – 4.9Mb

13. CVS
31yrs old, fetus with Holoprosencephaly at 13 wksCase
2

14. 13 Disease associated genes including TRPS1 and EXT1, Langer-
Giedion syndrome
Common clinical features included in Langer-Giedion syndrome
include multiple dysmorphic facial features, multiple cartilaginous
exostoses, craniofacial and skeletal abnormalities
Deletion on chromosome 8 – 20.9MbCVS
31yrs old with fetus with
Holoprosencephaly

15. Amniotic Fluid Sample
25 yrs old with Abnormal Quad Screen
Case
3

16. 35 Protein Coding Genes
6 Disease associated genes
Common clinical features included ASD, VSD,
developmental delay, MR, micrognathia, deafness
Amniotic Fluid Sample
25 yrs old with Abnormal Quad
Screen
Duplication on chromosome 9: 9.1
Mb

17. 45 Protein Coding Genes
15 Disease associated genes including TCF4
Common clinical features included hypotonia, microcephaly, short
stature, speech delay, cardiomyopathy, developmental delay, MR.
Amniotic Fluid Sample
25 yrs old with Abnormal Quad
Screen
Deletion on chromosome 18 –
26.2Mb

18. 40 yrs, AMA Case
4

19. 4 genes
1 Disease associated gene: STS, Ichthyosis X-linked
Ichthyosis X-linked is a keratinization disorder manifesting with mild
erythroderma and generalized exfoliation of the skin within a few weeks after
birth
Affected boys later develop large, polygonal, dark brown scales, especially on
the neck, extremities and trunk
Deletion on chromosome X– 1.15 MbAmnio
40 yrs, AMA

20. Results and Discussion
Chromosomal microarrays already in mainstream use for
postnatal genetic diagnosis are increasingly used for prenatal
diagnosis, mainly in pregnancies with sonographic anomalies
but also for routine screening after any invasive procedure.
Arrays have demonstrated the ability to detect submicro-
scopic copy number variations, providing an approximately 2.1
% increase in the detection rate of pathogenic copy number
variations regardless of the referral indication, and rising to an
approximately 5.3 % increase above conventional
karyotyping in the presence of sonographic anomalies.

22. CAS
ES
AGE NT NB Biochemi
cal
FISH
Karryot
ype
CMA Life
threate
ning
Not
life
threat
ening
1 29 N ABS + N ABN NO YES
2 34 ABN ABS - ABN ABN YES -
3 42 ABN P + ABN ABN YES -
4 40 ABN P + N ABN NO YES
5 27 ABN P + N N - -
6 28 ABN ABS + N ABN NO YES
7 28 N ABS + N N - -
8 27 ABN P + N N - -
9 39 ABN P + N ABN YES YES
10 25 N P + N ABN YES -
11 38 ABN P + N N N N
12 33 N ABS + N N - -
13 35 ABN P - N N - -
14 36 ABN ABS - ABN YES NO YES-

23. CASES AGE NT NB Bioche
mical
FISH
Karyot
ype
CMA Lifethr
eatenin
g
Not life
threate
ning
15 40 ABN P + N N - -
16 31 ABN ABS + ABN ABN YES -
17 33 ABN P + N NOT
DONE
- -
18 32 N ABS + N NOT
DONE
- -
19 37 ABN P + N ABN NO YES
20 34 N ABS + N N - -

24. CASE
S
AGE NT NB Biochemic
al
FISH
Karyoty
pe
CMA Lifethr
eatenin
g
Not life
threate
ning
21 34 N ABS + N ABN NO YES
22 34 ABN ABS - ABN ABN YES -
23 40 ABN P + ABN ABN YES -
24 27 ABN P + N ABN NO YES
25 31 ABN P + N N - -
26 33 ABN ABS + N ABN NO YES
27 37 N ABS + N N - -
28 27 ABN P + N N - -
29 39 ABN P + N ABN YES YES
30 34 N P + N ABN YES -
31 38 ABN P + N ABN NO YES
32 29 N ABS + N N - -
33 31 ABN P - N N - -
34 36 ABN ABS - ABN YES NO YES-

25. CASE
S
AGE NT NB Biochemic
al
FISH
KAryoty
pe
CMA Lifethr
eatenin
g
Not life
threate
ning
35 34 N ABS + N ABN NO YES
36 34 ABN ABS - ABN ABN YES -
37 40 ABN P + ABN ABN YES -
38 27 ABN P + N ABN NO YES
39 31 ABN P + N N - -
40 37 ABN ABS + N ABN NO YES
41 37 N ABS + N N - -
42 27 ABN P + N N - -
43 39 ABN P + N ABN YES YES
44 36 N P + N ABN YES -
45 38 ABN P + N ABN NO YES
46 29 N ABS + N N - -
47 31 ABN P - N N - -
48 36 ABN ABS - ABN YES NO YES-

26. CASES AGE NT NB Bioche
mical
FISH
Karyot
ype
CMA Lifethr
eatenin
g
Not life
threate
ning
49 40 ABN P + N N - -
50 31 ABN ABS + ABN ABN YES -

27. -42% HAD Normal NT showed But had a Abnormal CMAin
whch 70% HAD POSITIVE BIOCHEMICAL TEST
POSITIVE and 51% HAD LIFE THREATENING
ANOMALIES IN THE RESULT
NT
NB
BIOCH
LIFE
THREAT

28. IN OUR STUDY
 Microarray analysis yielded results more often than
did karyotype analysis (87.4% vs. 70.5%, P<0.001)
and provided better detection of genetic
abnormalities (aneuploidy or pathogenic copy-
number variants, 8.3% vs. 5.8%; P=0.007).
 Microarray analysis also identified more genetic
abnormalities among antepartum stillbirths (8.8% vs.
6.5%, P=0.02) and stillbirths with congenital
anomalies (29.9% vs. 19.4%, P=0.008). As compared
with karyotype analysis, microarray analysis provided
a relative increase in the diagnosis of genetic
abnormalities of 41.9% in all stillbirths, 34.5% in
antepartum stillbirths, and 53.8% in stillbirths with
anomalies.

30. Challenges in CMA
 Detection of balanced chromosomal
rearrangements
 Interpretation of CMA in the prenatal setting is
more challenging than in the postnatal
population
 Whole Genome Scan provides data that
requires careful clinical interpretation
 Bioinformatic information vs Clinical
correlations
 Accurate counseling required when results of
unknown clinical significance are discovered

31. Ethical dilemmas
• Prenatal diagnosis and abortion are major issues of moral
sensitivity
• Molecular techniques allow narrowing or broadening of the scope of
diagnostic testing
• Range of conditions tested and why?
• Testing marked driven / within public health care
• Basic principles of biomedical ethics
– respect for person, beneficience, non-maleficience, justice
– proportionality, equity of access, informed consent
- Ensure abortion decision remains personal, not turned into
instruments of social goals

32. Current Recommendations
 American College of Obstetricians and Gynecologists
(ACOG) and European Best Practice Guidelines do not yet
include offering CMA to all patients undergoing invasive
prenatal testing
 ACOG has endorsed CMA an appropriate test in
prenatal cases with abnormal ultrasound findings and a
normal karyotype

34. Summary
 Advances in invasive prenatal diagnosis: Chromosomal
microarray (array-CGH, molecular karyotyping) will replace
conventional karyotyping
 Non-invasive prenatal analysis in maternal plasma will
replace other screening tests and further decrease the
need for invasive testing
 Diagnosis of monogenic disorders in prenatal analysis
will become feasible by panel testing
 Importance of pre- and post-test counseling
discussion of ethical issues

35.  More choices, more uncertainty
 The availability of these two new technologies presents
expectant parents with complex choices. Where NIPT presents
no risk of pregnancy loss, it only tests for a limited number of
conditions, so it may miss a potentially serious genetic disorder.
On the other hand, CMA can detect a much a wider range of
genetic disorders, but is invasive and carries a very small risk of
miscarriage.
 In addition, CMA occasionally produces findings where it is not
clear how seriously the child we be affected, which can create
further challenges for expectant parents. These findings, called
variants of unknown significance, were found to occur in up
to1.5% of cases.
 These variants put patients in a difficult situation, where they
must make decisions on how to proceed without definitive
information.

36.  The general consensus is growing that arrays can be
implemented in the prenatal setting. Those arrays provide a
higher diagnostic yield, detecting submicroscopic CNVs
as well as larger imbalances, and within a faster
timeframe. The ethical concerns have long been hampering
their introduction. There was (and still is) concern about how
to deal with unsought findings, late onset disorders, CNVs
with variable expressivity and penetrance and variants with
mild phenotypic anomalies.
 Current experience reveals that the proportion of cases
where difficulties in counselling arise is limited to 1–2 %.
Nevertheless, more research and consensus guidelines in
this area would help both clinicians and families.
 Because an invasive procedure is required, associated with
a procedure-related risk, it seems likely that high-resolution
chromosomal microarrays will mainly be offered for the
analysis to those fetuses at significant increased risk of a
chromosome abnormality.