genetic prenatal diagnosis

1. NARENDRA MALHOTRA
M.D., F.I.C.O.G., F.I.C.M.C.H
• Prof. Dubrovick International university,croatia
• Indian FOGSI representative to FIGO
• President FOGSI (2008)
• Dean of I.C.M.U. (2008)
• Director Ian Donald School of Ultrasound
• National Tech. Advisor for FOGSI-G.O.I.—Mc Arthur Foundation EOC Course
• Hon Prof Ob Gyn at DMIMS,Sawangi,Advisor ART unit at MAMC & SMS Jaipur
• Editor od SAFOG journal
• Chairman publication committee of AOFOG
• Practicing Obstetrician Gynecologist at Agra. Special Interest in High Risk Obs., Ultrasound, Laparoscopy
and Infertility, ART & Genetics
• Member and Fellow of many Indian and international organisations
• FOGSI Imaging Science Chairman (1996-2000)
• Awarded best paper and best poster at FOGSI : 5 times, Ethicon fellowship, AOFOG young gyn. award,
Corion award, Man of the year award, Best Citizens of India award
• Over 30 published and 100 presented papers
• Over 50 guest lectures given in India & Abroad.Presented 10 orations.
• Organised many workshops, training programmes, travel seminars and conferences
• Editor 8 books, many chapters, on editorial board of many journals
• Editor of series of STEP by STEP books
• Revising editor for Jeatcoate’s Textbook of Gynaecology (2007) and DONALD OBS MANNUAL(2012)
• Very active Sports man, Rotarian and Social worker
MALHOTRA NURSING & MATERNITY HOME
84, M.G. Road, Agra-282 010
Phone : (O) 0562-2260275/2260276/2260277, (R) 0562-2260279, (M) 98370-33335; Fax : 0562-2265194
E-mail : mnmhagra3@gmail.com
RAINBOW HOSPITALS
GLOBAL RAINBOW HEALTH CARE
Consultant for IVF at jalandhar,ludhiana,ambala,bhiwani,gwalior,allahabad,gorakhpur,udaipur,bariely,jaipur,delhi
Neapal & Bangladesh

2. Beyond ultrasound abnormalities
what next ? and what more ?…
time for FISH & CHIPS
Narendra Malhotra,MD,FICOG
FOGSI rep to FIGO
Vaidehi Jobanputra, Ph.D., M.S., FACMG
President ,
Advanced Genomics Institute & Laboratory Medicine (AGILE), New Delhi
Jaideep Malhotra
President elect ASPIRE
Neharika Malhotra Bora
Asst Prof BVP medical college,Pune

3. Nature, editorial Nov.2011

4. Prenatal Diagnosis for fetal anomalies
• 20% of stillborn babies have a major malformation
• 3% of live births have congenital anomalies
• Diagnosis of a malformation syndrome requires
• a number of “broad spectrum” analyses
• followed by more sophisticated and targeted tests
• Detailed ultrasound and invasive fetal testing
• Etiology
• Prognosis
• Recurrence risk
• Prevention / options in future pregnancies

5. Indications for Prenatal Diagnosis
• 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 ultrasound examination

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

7. • Cytogenetics, the beginnings…
• • 1956 Tjio and Levan
• – human species has 46 chromosomes
• • 1959 Léjeune
• – Detection of the first chromosomal anomaly, trisomy 21
• • „Etude des chromosomes somatiques de neuf enfants
mongoliens.“
• • 1963 Léjeune
• – Diagnosis of first structural aberration, crit du chat
syndrome, 5p-
• • 1966 Steele and Berg
• – fetal karyotyping on amniotic fluid
• • 1968 Nadler et al.
• – diagnosis of trisomy 21 in amniotic fluid
• • 1984 Ward
• – CVS in first trimester

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

9. Chromosome Analysis
Most widely used whole genome analysis
method for clinical cytogenetics

10. Chromosomal analysis
classical cytogenetics molecular cytogenetics
1960 1980 1990 2000
FISH Array- CGH
Chromosome banding

11. Advances in invasive prenatal diagnosis:
• 1.Chromosomal microarray (array-CGH,
molecular karyotyping) versus conventional
karyotyping to detect chromosomal
aneuploidies
• 2. Non-invasive analysis in maternal plasma
• 3. Beyond chromosomal aberrations,
diagnosis of monogenic disorders in prenatal
analysis

12. 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
• Does not detect
– balanced translocations, triploidy

13. Chromosomal Microarray Analysis
(CMA)
AKA aCGH, SNP array, genomic
microarray
Compares differences in DNA
copy number (dosage)
between two genomes
Single step genome scan
Much higher resolution than
chromosome analysis

14. A microarray is a multiplex lab-on-a-chip. It is a 2D array on
a solid substrate (usually a glass slide or silicon thin-film cell)
that assays large amounts of biological material using high-
throughput screening methods. The concept and
methodology of microarrays was first introduced and
illustrated in antibody microarrays (also referred to
as antibody matrix) in 1983 in a scientific publication and a
series of patents.As the "gene chip" industry started to grow
in the 1990's, with the establishment of companies, such
as Affymetrix, Illumina, and others, the technology of DNA
microarrays has become the most sophisticated and the
most widely used

15. Types of microarrays include:
DNA microarrays, such as cDNA microarrays, oligonucleotide
microarrays and SNP microarrays
MMChips, for surveillance of microRNA populations
Protein microarrays
Peptide microarrays, for detailed analyses or optimization of
protein-protein interactions
Tissue microarrays
Cellular microarrays (also called transfection microarrays)
Chemical compound microarrays
Antibody microarrays
Carbohydrate arrays (glycoarrays)
Phenotype microarrays
interferometric reflectance imaging sensor (IRIS)

16. A DNA microarray (also commonly known as DNA chip
or biochip) is a collection of microscopic DNA spots attached to a
solid surface. Scientists use DNA microarrays to measure
theexpression levels of large numbers of genes simultaneously
or to genotype multiple regions of a genome. Each DNA spot
contains picomoles (10−12 moles) of a specific DNA sequence,
known as probes (or reporters or oligos). These can be a short
section of a gene or other DNA element that are used
to hybridize a cDNA or cRNA (also called anti-sense RNA) sample
(called target) under high-stringency conditions. Probe-target
hybridization is usually detected and quantified by detection
of fluorophore-, silver-, or chemiluminescence-labeled targets to
determine relative abundance of nucleic acid sequences in the
target.

17. Four Types of Testing
Programs
• Newborn screening
• Carrier testing
• Prenatal testing
• Presymptomatic (predictive) testing

18. Its now a story of FISH & CHIPS

21. Copy Number Changes
• AKA Copy Number Variants (CNV)
• Common cause of abnormal phenotypes
– Congenital anomalies
– Autism
– Syndromic and nonsyndromic intellectual disability
– Speech delay
– Sezuires
– Growth retardation with or without anomalies
• Copy number changes are identified in ~20% of cases
American College of Medical Genetics:
CMA as first-line diagnostic test for postnatal cases

24. Prevalence of genomic changes in Fetuses with Selected
Isolated and Multiple Sonographically Detected Abnormalities
Abnormality Abnormal CMA (%)
Isolated Multiple findings
Cystic hygroma 52 71
Nuchal edema 19 45
Heart Defects 16 66
Duodenal atresia 38 64
Ventriculomegaly 2 17
Choroid Plexus cysts 1 48
Diaphragmatic hernia 2 49
Examphalos 8 46
Renal abnormalities 3 24

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

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

27. 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

28. CVS
31yrs old, fetus with Holoprocencephaly at 13 wks
Case 2

29. 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 Holoprocencephaly

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

31. 35 Protein Coding Genes
6 Disease associated genes
Common clinical features included ASD, VSD, developmental delay,
MR, micrognatia, deafness
Amniotic Fluid Sample
25 yrs old with Abnormal Quad Screen
Duplication on chromosome 9: 9.1 Mb

32. 45 Protein Coding Genes
15 Disease associated genes including TCF4
Common clinical features included hypotonia, microcephaly, short strature, speech delay,
cardiomyopathy, developmental delay, MR.
Amniotic Fluid Sample
25 yrs old with Abnormal Quad Screen
Deletion on chromosome 18 – 26.2Mb

33. • Patient elected to terminate the pregnancy
• CMA suggests possible balanced translocation in
the parents
• Follow up parental karyotypes:
– Mom 46,XX
– Dad 46,XY,t(9;18)(p24;q21.2)
• Increased risk for miscarriages and offspring with
congenital abnormalities and/or MR
Counseling

34. 40 yrs, AMA Case 4

35. 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

36. Challenges in CMA
• Detection of balanced chromosomal rearrangements
• Interpretation of CNV 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

37. 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

38. Indications for CMA in Prenatal Diagnosis
• Fetal structural anomaly: cardiac, central nervous system,
skeletal, urogenital, renal, increased nuchal translucency
and intrauterine growth restriction (IUGR)
• Delineating marker chromosomes: origin and genomic
content
• Precisely determine the breakpoints of genomic
alterations detected with conventional cytogenetic
methods
• Apparently balanced reciprocal balanced translocations

39. Indications for CMA in Still Births
• About 5% of structurally normal stillborn fetuses
have an abnormal karyotype
• 35–40% of stillbirths that are structurally
abnormal or macerated
 Underestimates: poor quality of the karyotype
 Tissue culture failure in 50% of cases

40. Summary
• CMA is a new technology which provides a whole
genome scan to detect copy number changes (CNCs)
• Defines genomic aberrations with extraordinary
precision and at “ultra-high” resolution
• Prenatal CMA can detect clinically significant
genomic aberrations in fetuses with U/S abnormality
• Provide accurate diagnosis and prognosis both
prenatally and postnatally to parents

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

42. 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

43. Acknowledgements
our partners in
GENETIC ANALYSIS & DIAGNOSIS
Contact Info::
Email: vaidehi@labassure.com
Website: www.labassure.com
info@rainbowhospitals.org
rainbowgenetics@rainbowhospitals.org
yourgenes@rainbowhospitals.org