Download Preimplantation Genetic Testing An Overview

DEVELOPMENTS AND LANDMARKS
OF GENETIC TESTING IN WEST
AFRICA
PROF. OLADAPO ASHIRU
M.B.B.S, MS, PHD. HCLD/CC(ABB-USA), FASN, FNSEM, OFR
48th Annual General Meeting and
Scientific Conference
SOGON ASABA 2014.
ASSISTED REPRODUCTIVE
TECHNOLOGY (ART)

Conception requiring
the complex handling
or manipulation of
male and female
gametes in-vitro to
facilitate pregnancy
LANDMARKS IN ART

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
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
1969 Sperm Capacitation
Chang & Yamagimachi
1976 IVF in London
Steptoe & Edwards
1978 IVF baby London
Steptoe & Edwards
(First IVF Baby – Louise Brown)
1977 LHRH
Schally & Guileman
1978 FSH +ve feedback
Ashiru & Blake
1980 IVF in Melbourne, Australia
Carl Wood
1982 IVF in Virginia
Jones
1984 IVF in Lagos, Nigeria
Ashiru & Giwa-Osagie
– 1986/1989
1992 ICSI
Jacques Cohen
LANDMARKS IN PGD


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
Jacobson CB & Barter RH 1960 - 1st prenatal
diagnosis
Saiki et al 1985 – PCR
Alan Handyside and Robert Winston 1989 –
Embryo Biopsy
Verlinsky Y et al 1999 – Polar body testing
LANDMARKS IN PGD

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1990: Sexing Human Preimplantation Embryo,
& Polar Body Analysis for Mendelian Disease;
- First PGD Baby Born
1993: FISH Analysis for Sexing and for PGD of
Aneuploidies
1996: PGD for Chromosomal Translocations
1999: PGD for Late Onset Common Disorders
with Genetic Predisposition
LANDMARKS IN PGD

2000: Pre-implantation HLA Typing
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2002: 1st Thousand PGD Babies Born

2002 onwards: Many technological
developments CGH, aCGH, NGS, SNP,qPCR
TYPES OF PREIMPLANTATION
GENETIC TESTING

Preimplantation Genetic Diagnosis
(PGD)
– Targeted genetic testing of known
genetic abnormality in the couple:
Single gene disorder
Structural chromosomal
abnormality
TYPES OF PREIMPLANTATION
GENETIC TESTING

Preimplantation Genetic Screening for
Aneuploidy (PGS)
–

Screening for numerical abnormality of select
chromosomes: 13, 18, 21, X, Y, etc
Preimplantation Gender Determination or
Gender Selection
INDICATIONS FOR PGD
Autosomal dominant disorders
– One of the couple is a carrier of the
genetic defect (e.g., Huntington
Disease or dwarfism)
 Autosomal recessive disorders:
– Both couple are carriers of the genetic
defect (e.g., Sickle Cell Disease)

INDICATIONS FOR PGD

X-linked disorder
–


One of the couple is a carrier of a x-linked genetic
defect (e.g., Hemophilia)
Human leukocyte antigen (HLA) matching
Structural chromosomal abnormality
–
One of the couple is a carrier of a chromosome
abnormality (translocation, inversion, deletion,
insertion)
INDICATIONS FOR PGS
(ANEUPLOIDY SCREENING)



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Advanced maternal age
Recurrent pregnancy loss
Repeated IVF failure
Severe male factor infertility
INDICATIONS FOR GENDER
SELECTION

One of the couple is a carrier of x-link
disorder

Family balancing
BIOPSY TECHNIQUES
•
•
•
May be largely diagnostic
No paternal/meiosis II/post-zygotic information
May require later follow-up testing (d3/d5)
• Accounts for meiosis II errors
• No paternal/post-zygotic information
• Most expensive option diagnostically
• Biopsy most detrimental
• Chromosomal mosaicism common
• Associated with Low implantation
•
•
•
•
•
Highly recommended
3-10 (?) cells (high accuracy/reliability)
Significance of mosaicism?
Least expensive per patient (diagnosis)
May require vitrification
BIOPSY OBJECTIVE
Methods of Genetic Analysis:
PGD single gene disorder
–
–
–
–
Whole genome amplification
Polymerase chain reaction (PCR)
Direct detection of specific mutation
Genotyping of linked polymorphic markers to
ensure:


Bi-parental inheritance
Infer the inheritance of normal copy of the gene (Allele)
Methods of Genetic Analysis:
Structural chromosomal abnormality

Fluorescent in situ hybridization (FISH)

PCR-based testing
–
–
Whole genome amplification
Genotyping of linked polymorphic markers
METHODS
FISH Gender Determination
Orange: X chromosome
Green: Y chromosome
FISH Aneuploidy Screening
Normal Male
Trisomy 21 Male
Normal Female
Red: chromosome 13
Cyan: chromosome 18
Green: chromosome 21
Purple: chromosome X
Yellow: chromosome Y
ARRAY COMPARATIVE GENOMIC
HYBRIDIZATION (aCGH)
• A molecular cytogenetic technique
• Detects 'copy number changes' of chromosomes on a
genome
• Embryo DNA is compared with a known normal DNA
specimen utilising thousands of specific genetic markers
• More accurate results (fewer false normal or false abnormal
results)
• Error rate of 2% compared to 5 - 10% of FISH
aCGH versus Conventional CGH
• Better resolution (as low as 5 – 10 kilobytes of DNA
sequences)
• Cloned DNA fragments with exact chromosomal location
replaces reference metaphase spread
• Better detection of aberrations
• Better identification of micro deletions and duplications
• Increases the possibility of mapping changes directly onto the
genomic sequence
ARRAY COMPARATIVE GENOMIC
HYBRIDIZATION (aCGH) - METHOD
 Same principle as conventional CGH
 Labelling of DNAs (control and test) with 2 different
'flurophores' (green and red)
 Green flurophore (cyanine 3) for test / patient and red
flurophore (cyanine 5) for control / reference are used as
'probes'
 Competitive cohybridisation of probes onto nuclei acid targets
(cloned genomic fragments (BACs / plasmids), cDNAs or
oligonucleotides) with thousands of genetic markers.
 Digital imaging / specialised microscopes to capture and
quantify fluorescence intensities of hybridised flurophores.
ARRAY COMPARATIVE GENOMIC
HYBRIDIZATION (aCGH) – RESULT
Interpretation of results.
 Ratio of the fluorescence intensities is proportional to the ratio
of 'copy number of DNA sequences' in the test and reference
genomes.
 Altered Cy 3 : Cy 5 ratio indicates a loss or gain of the patient
DNA at that specific genomic region
 If Cy 3 : Cy 5 ratio is equal on one probe (equal intensities of
the flurophores), patient's genome is interpreted as having
equal quantity of DNA as in the reference sample.
ARRAY COMPARATIVE GENOMIC
HYBRIDIZATION (aCGH)

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Array CGH remains the ‘gold standard’ for
PGS with the greatest clinical experience to
date
RCTs show great promise for PGS
Numerous RCTs are ongoing
Relatively easy to set up
Use of a reference laboratory recommended
as a first step
DETECTION OF WHOLE CHROMOSOME
ANEUPLOIDIES IN SINGLE CELLS
Trisomy 13 male
(47,XY,+13)
Sequencing
Results
24Sure Results
PGD/PGS SINCE 2009
(MART DATA)

Chromosomes Aneuploidy (CA)
Single gene disorders (Sickle cell – SC)
Family balancing (sex selection – FB)

So far over 75 cases have been done


SUMMARY OF REASONS FOR
PGD/PGS (N=75)
45%
43%
40%
35%
32%
30%
23%
25%
20%
15%
10%
5%
0%
CA
FB
SC
SUMMARY FOR PGS (N= 32)
SUMMARY OF EMBRYO BIOPSED FOR
CHROMOSOMES ANEUPLIODY (N=300)
59%
60%
50%

40%
30%

59% of embryos
biopsied were
abnormal
15% No result rate
26%
20%
15%
10%
0%
Normal
Abnormal
No Result
? Number and quality
of cells tested
SICKLE CELL DIAGNOSIS (PGD) N = 17
SUMMARY OF EMBRO BIOPSED
FOR SICKLE CELL DIAGNOSIS
(N= 133)
50%

41%
34%
40%
25%
30%

20%
10%
RESULT…
0%
Normal
Abnormal
No Result
41% of biopsied
embryos were
homozygous and
heterozygous normal
34% of embryos
undiagnosed
FAMILY BALANCING – SEX SELECTION
(N=26)
SUMMARY OF GENDER ANALYSIS
IN FAMILY BALANCING
100%
100%
90%
80%
70%
60%
47%
47%
50%
40%
30%
20%
10%
0%
Male
Total
Female

No difference in the
proportion of male
and female embryos
SUMMARY OF PREGNANCY RATE
70%
59%
60%
50%
40%
32%
30%
20%
9%
10%
0%
CA
FB
SC
SUMMARY OF CLINICAL PREGANCY
RATE
35
35
30
30
25
20
15
9
10
5
0
CA (%)
FB (%)
Clinical Rate
SS (%)
SETTING UP A PGD LAB
Two ways


IVF centre and PGD centre in the same
institute – preferred
Transport PGD
ORGANIZATION OF THE
PGS/PGD CENTRE
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Highly successful IVF unit
Patients need genetic and specific PGD counselling
Biopsy performed by trained embryologist
Diagnosis performed by molecular biologist/cytogeneticist
Patient information leaflets and consents
Excellent communication between IVF centre and
diagnosis lab
Join the PGD Consortium
WHAT MAKES A GOOD PGD
CENTRE

COMMUNICATION

Excellent IVF Platform

Excellent Diagnostics Laboratory

Integration of Services

Commitment to Follow-up

TRANSPORT PGD
FUTURE OF PGD/PGS
• PGD
– New technology to allow diagnosis
of more disorders
– Whole genome amplification
– SNP microarrays, array-CGH,
quantitative real time PCR (qPCR)
– Blastocyst biopsy and vitrification
FUTURE OF PGD/PGS
• PGS
– Randomised controlled trials to see if valid
procedure with clinical significance
– NOT cleavage stage biopsy, NOT FISH
– Try polar body or trophectoderm biopsy
– SNP, array-CGH, or sequencing
BEETHOVEN
“I RECOGNIZE NO
SUPERIORITY IN
MANKIND OTHER
THAN GOODNESS.”
THE TEAM
THANK YOU
FOR LISTENING!