Preimplantation Genetic Testing Flashcards
name genetic diseases - healthy legacy
- Physical disability
- Mental disability
- Neonatal and childhood
death - Chronic disease and
early demise - Recurrent pregnancy
loss - Late onset disease
describe Avoiding transmission of genetic disease - reproductive options for those with serious recurrent genetic risk
if find out have disease/carrier = can do adoption, no children, donor gametes (art) or become pregnant and undergo prenatal diagnosis - terminate or not if affected
name 2 methods of prenatal diagnosis
testing on established pregnancy= chorionic villi sampling or amniocentesis
describe chorionic villus sampling
transcervical, more common
arounf 10-12 weeks
more invasive = slightly higher risk
could also test mom blood around 10 weeks
describe amniocentesis
more direct = tests on fetus
always little bit of risk
15-18 weeks, much later
5% fetus loss
what is preimplantation genetic testing
If none of options of prenatal testing available
A test performed to analyze the DNA from oocytes (polar bodies) or embryos (cleavage stage or blastocyst) for HLA (Human Leukocyte Antigen) -typing or for determining genetic abnormalities (test before going into mom, not 100% accurate tho)
what is pgd and pgs
Preimplantation Genetic Diagnosis (PGD) and screening (PGS)
These terms have now been replaced by “preimplantation genetic testing” (PGT)
consensus def of pgt
Preimplantation genetic testing (PGT)- A test performed to analyze the DNA from oocytes (polar bodies) or embryos (cleavage stage or blastocyst) for HLA-typing or for determining genetic abnormalitie
name types of pgt
pgt-a = for aneuploidies
pgt-m = for monogenic/single gene defects
pgt-sr = for chromosomal structural rearrangements
name types of genetic risk
Recurrent: As a result of inherited disorders
Sporadic: Random, often age related
what is required to preform pgt
ivf, need excess = multiple eggs and embryos
also diagnosis done in vitro
name and describe the 3 stages of pgt
Patient evaluation and management = done by fertility doctor and nurse in clinic
Gamete or embryo manipulation= embryologist/scientist in lab
Genetic analysis (diagnosis)= Single gene defects, Chromosome analysis- done by geneticist in specialized lab
Sometimes in one place or 3 diff places = need doctor + ivf lab and send samples to geneticist
name types of ways of doing pgt
polar body biopsy
cleavage stage biopsy
blastocyst biopsy
describe Early Human Embryo Development - gen
polar bodies, day 1 = one cell
day 2 = 2-4 cell, cleavage stage
then day 3 = around 8 cell, cleavage biopsy
day 4 = morula
day 5-6= blastocyst biopsy
describe polar body biopsy
aspirate polar bodies and then do diagnosis = see if chromosome related or single gene related
describe polar body biopsy = for normal segregation in m1
pb 1 and 2 have one chrom in it
describe polar body biopsy = for nondisjunction in m1
pb can have no chroms = disomic gamete
pb can have 2 chroms = nullsomic gamete
describe polar body biopsy = for prevision of one univalent in m1
pb has 1 chromatid, gamete = 23+1
or pb has 1 chromatid + 1 chrom = 22+1/2 gamete
describe polar body biopsy = for prevision of both univalent in m1
22 + 1/2 + 1/2
pb has 2 chromatids and sod does egg
principle of PB-based PGT for single-gene disorders- ex = cftr mutation
ctfr=autosomal recessive, gene defect
if first pb normal homozygous = egg retained mutation
if first pb homozygous mutant = egg retained normal
but if crossing over = depends on how far gene of interest is, farther from chrom = more crossing over, higher % MUST GET RESULTS FROM 2ND pb
if first pb normal + cf then if 2nd pb normal = know oocyte retained cf, if 2nd pb cf = oocyte retained normal
MUST SEEEEE
ADVANTAGES - polar body biopsy
*Patients with religious or moral objections to embryo biopsy = not comfy with embryo biopsy, so use pb therefore embryo wont be affected
*Time for Fresh embryo transfer = 5-6 days to do all tests and can still transfer as blastocyst
DISADVANTAGES - polar body biopsy
*High diagnostic failure rate = especially in 1st polar body bc in arrested state for long
*Excludes paternal genome = polar bodies are only mat genome
*Most expensive= 2 blastocysts but 10 octets = expensive and time consuming
*time consuming approach
describe embryo biopsy for pgt - day 3
make small hole on zona and aspirate out one blastomere
ADVANTAGES - day 3 cleavage stage biopsy
*High worldwide experience = first biopsy procedure applied, many embryologists know it, first successful baby born
*Time for fresh embryo transfer= still have 2-3 days to diagnose
*Cells are not differentiated= bc at cleavage pluripotent = should not affect bc has not decided to be te or icm
DISADVANTAGES - day 3 cleavage stage biopsy
*Reduction in embryo viability= remove 1/8th of blastomere = reduction of vol
*All day-3 embryos will not reach blastocyst stage –extra work= if do 10, only a few could actually reach Blastomere stage, so waste of time (like if fails to amplify)
*Higher diagnostic failure compared to blastocyst stage (if miss = done)
describe blastocyst biopsy
do not touch icm cells
only te cells (extra embryonic tissue)
sex election for x linked chrom abnormality or pcr for single gene defects
ADVANTAGES - blastocyst biopsy
*Larger number of cells can be removed providing more robust analysis (5-10 cells = more dna, more robust)
*Smaller proportion of embryonic mass removed (only 5-10cells from 60-100)
*Not removing cells that are destined to contribute to fetus (only remove te cells)
DISADVANTAGES - blastocyst biopsy
*Shorter time for diagnosis (bc need to freeze on day 6, so not as much time)
*Not all embryos will reach the blastocyst stage
*In most cases embryos will be required to freeze (another invasive process, could reduce viability, also more time)
*Labour intensive as not all embryos will reach the blastocyst stage on the same day (day 5-6) (could be any day of week = must come into lab and see = rough)
principle of pgt = overview
egg retrieval
on day 3 after ivf
day 3 = biopsy of blastomere
or day 5/6 = te cells
single blastomere undergoes genetic analysis and diagnosis of embryos
fresh or frozen cycle = transfer of unaffected embryo
few weeks later = pregnancy tests
Genetic risk: Disease dependent - single gene defects
Autosomal Recessive: 1in 4 Autosomal dominant: 1in 2
X-linked recessive (female obligate carrier): 1 in 2 male, females are either normal or carriers
describe pgt-m
blastocyst, cleavage or pb biopsy
pcr with reg or f primers and thne do f labeled dna fragment analysis or dna analysis on gel
describe single cell nest pcr - pgt-m
amplify with pcr since small amount of dna
1 = primers far away from target
then reamplify with diff set primers = smaller sequence
get rid of unspecific bands = get cleaner gene product
then run on polyacrylamide gel or agarose gel or florescent based pcr and abi system for fragment analysis
describe karyotyping pcr - pgt-m
A universal method for genome wide analysis of genetic disease
SNP genotyping performed with 300k bead chip (Human CytoSNP-12, Illumina: Data analysed by dedicated karyomapping software using Bluefuse Multi, version 4, Illumina
HAPLOTYPING = run and amplify and use diff algorithms, mat and pat dna, dna library
name Two types of mutation detection for PGT-M
Diseases caused by Specific deletion/insertion/point mutation etc
Triplet repeat diseases e.g. DM1, HD, fragile X etc
explain ex of trinucleotide repeat diseases
expansion in diff regions of genes of tri nts
could be anywhere= in 3’ utr, to 5’, intron, exon, anywhere
describe pgt for triplet repeat disease
Fragment analysis
what is myotonic dystrophy type 1 - steinert disease generally
-most common adult onset muscular dystrophy
Varying severity of phenotype and age of onset - different based on expansion of repeat
affects many organs
what is myotonic dystrophy type 1 - steinert disease saguenay
in saguenay = 1 in 500
but everywhere else = 1 in 10 000-12 000
= founders mutation = small community and not much movement
what is myotonic dystrophy type 1 - steinert disease genetic mutation
Genetic mutation: dynamic expansion of CTG repeat in 3’UTR of DMPK gene (19q13.3) <– mapped to
what is myotonic dystrophy type 1 - steinert disease phenotypes kinda
Unaffected 5-37 CTG
mildly affected 50-150
adult onset 100-1000
congenital up to 6kb
we all have these repeats
how to diagnose mutation - steinert disease
when do diagnosis from few cells = not easy to amplify these cg areas especially with single cells = must do special technique
describe dm-str markers
can see which affected vs which normal
run on gel
describe family pedigrees
how to diagnose mutation
haplotyping
use of linked markers in pgt-m
disease mapped to specific chroms = if inherited = have disease
Preimplantation genetic Testing of CF (deltaF508) -gen
Cystic fibrosis is one of the most common autosomal recessive disorder among Caucasian population
1 in 2,000 live births and carrier frequency 1 in 20-25
The disorder is caused by mutation in the CFTR gene on chromosome 7
The most common mutation is deltaF508 deletion, a 3 bp deletion at AA position 508 causing loss of phenyl alanine
Number of organs are affected including lung airways, pancreas and sweat glands
Preimplantation genetic Testing of CF (deltaF508) - technique
amplification of deltaF508 = 193/190bp
do pcr amplification from single blastomere and run on gel = can see
use fluorochrome based pedigree and fragment sizes and link markers - different for each person and fam so time consuming
most ppl use universal approach = less time consuimg = karyotyping
describe Development of PGT-M test requires DNA from 2 generations
must test at multiple loci
want to reduce misdiagnosis rate and make sure info correct
what is good about pgt-m with linked markers (and explain) - 4
Detects Allele drop out (failure of amplification of one of the alleles during pcr can lead to misdiagnosis)
Detects contamination (multiple link markers and attaches to specific fam dna profile, see extra dna profile = dna contamination, so misdiagnosis so will not use)
Detects problems with RE digestion
May detect chromosome abnormality e.g. Haploidy, monosomy
describe how to do Preimplantation Genetic Testing for X-linked recessive disorder by sex selection
Using FISH
explain describe how to do Preimplantation Genetic Testing for X-linked recessive disorder by sex selection BY FISH
faster and easier
can transfer only female embryos = sex selection
X-linked recessive: 1 in 2 male, females are either normal or carriers
what is chromosome translocation
Chromosome translocation occurs when there is a transfer/exchange of genetic materials between chromosomes (2): breakage of both chromosomes and re-arrangement of chromosomes involved
what is balanced translocation
if the exchange results in no loss or gain of DNA (ie genes), the individual is clinically normal and is said to have a “balanced translocation.”
what can chromosome translocation - imbalance result in
A balanced translocation carrier is at risk of producing chromosomally unbalanced gametes.
This chromosome imbalance may result in abortion, stillbirth, malformation and/or mental retardation.
name types of chromium translocations
Robertsonian and reciprocal
explain robertsonian translocations
between chroms 13,14,15 and 21,22
only
acrocentric chromosomes
short arms end up lost
what is most common robertsonian translocation
Fusion between one chromosome 13 and one chromosome 14
Robertsonian
(13;14) translocation
Male Karyotype: 45,XY,der(13;14)(q10;q10)
explain robertsonian translocations - balanced?
Alternate segregation = 33% normal balanced
adjacent segregation and 3:0 segregation = unbalanced 67%
describe reciprocal translocation- gen
rest of chromosomes
describe reciprocal translocation- outcomes
16 possible outcomes of segregation of translocation chroms, only 2 produce balanced or normal gametes
describe a reciprocal translocation disease - how to diagnose
46XY,t(2;4 )(q33.1;q35)
use fish to diagnose
Commercial subtelomere and centromere probes
and look under microscope
Balanced = see it but cannot distinguish if normal or balanced like parents, kids might be carriers or have issue, could go for amniocentesis and confirm, then can know if child carrier or not
if unbalanced = do not use for transfer
describe age and fertility
fertility decreases and spontaneous abortion increased with increased maternal age
describe aneuploidy and maternal age
increase in aneuploidy and miscarriage and decrease in live births with increased mat age
describe cytogenic analysis of miscarriages
about 50-80% of abortuses carry chromosome abnormalities = one of 2 main causes for pregnancy wastage
trisomy, polyploidy, monosomy x, structural abnormality
when and what type pgt is used for chrom abnormalities
Women of Advanced maternal age , Repeated implantation failure, Recurrent miscarriage = Normal karyotype
Carriers of chromosome translocations= Parental karyotype with known rearrangement
Gonadal mosaics= Parental karyotype normal
describe how to know if gonadal moasics
young woman = 1st preg lost to trisomy 21, 2nd = same, 3rd= same
then think maybe something wrong in gonad = test woman peripheral blood but nothing wrong = must be gonadal mosaic
what are New strategies and Techniques for PGT-A and PGT-SR
ngs
high res ngs
describe pgt - blastocyst biopsy
or even cleavage stage = diagnose then freeze immediately
if normal = embryo transfer
if abnormal = discard or donate to research
what is ngs
next generation sequencing
biopsy embryo = amplify genome dna, dna fragmentation and put adapter
describe pgt with ngs
like barcode = add during amplification = fragments with adapters
computer will analyze and then sort embryos
describe Illumina Sequencing Workflow
Sequencing by Synthesis
1 = library preparation
2 = cluster growth
3 - in situ amplification
4 = sequencing
5 = image acquisition
6= base calling, computer read out
Describe High Resolution Next Generation sequencing
sequences are compared - aligned to known human genome (reference dna)
thousands of dna fragments mapped to each chrom
then see if normal, trisomies, mosaics, check how many copies of chrom
Enables reliable detection of mosaicism
describe normal ngs profile
46, xy and 46, xx
everything at 2 (copy number, except y chrom at 1 ish)
describe abnormal ngs profile
47,xx+16 = trisomy 16
46,xx;+14, -16 = trisomy and monosomy
43,xx;+21,-9,13,-16,-18 = complex
46,xy mosaic del (7)(p.11.1-pter):45%= partial deletion chrom 7
describe PGT-SR by NGS on balanced translocation carrier
46,XY,t(3:7)(q26.32; q11.23)
3 copies chrom 3, partial monosomy
detect translocation
compare ngs to fish
ngs = easier to detec mosaic since can detect abnormalities for many chroms = other chrom abnormalities too
fish targets only translated chrom
describe Identifying embryos in PGT - false results
Technical limitations may give False results (false pos/neg, nothing 100%)
Biological problems may also give false results (no control what happens in nature = mosaicism could cause misdiagnosis)
describe clinical implications of chromosome mosaicism - gen
normal = normal preg/no preg/abnormal preg
Abnormal = loss of euploid embryo-possible viable pregancy
describe clinical implications of chromosome mosaicism - for specific types of mosaic embryos
pick one of blastomere and randomly could get false pos or neg
based on where you biopsy =
could get all euploid, all aneuploid or some % mosaic
describe blastocyst biopsy mosaicism and clinical implications
if blastocyst mosaic then misdiagnosis may occur
do not where mosaic cells are
describe mosaic individuals - diff phenotypes
could be low level mosaicism, 50% mosaicism, high level mosaicism
Constitutional mosaicism = all over place, can detect with blood sample
germline mosaicism = gonadal, difficult to diagnose = until reach reproductive age
describe Choosing embryos for social reason using pgt
Social sex selection= Family balancing –OK?, Some countries have more men than women- skew sex pops in society, not allowed, but is allowed in states
Selecting other traits= Height/IQ/etc? = ethics issues, multifactorial
Deafness, dwarfism: Traits Vs disabilities
Slippery slope? = Misunderstanding about genetics, Parents expectations, Rights of unborn child? = what happens when become adults
MANY ETHICAL ISSUEs, =sometimes have to go through ethics board and approve of what we doing
describe Choosing embryos for social reason using pgt - NOT PERFORMED AT MCGILL - 5
- Sex selection for family balancing = no but yes for x linked diseases
- Specific genetic profile: multifactorial (do not do multifactorial)
- Risk of passing on less severe disorders: How do you define? (should we allow requests?)
- Risk of late onset disease -Case by case basis (only when embryo becomes 70-80 y/o, ethics board)
- Cancer –Case by case basis= just bc have mutation does not mean will get cancer (ex =brca, for breast cancer)
The Society of Obstetricians and Gynaecologists of Canada: conclusions
Preimplantation genetic Testing is an alternative to prenatal diagnosis for the detection of genetic disorders in couples at risk of transmitting a genetic condition to their offspring. The recommendations were made according to guidelines developed by the Canadian Task Force on Preventive Health Care
The Society of Obstetricians and Gynaecologists of Canada: step 1
Before Preimplantation Genetic Testing is performed, genetic counselling must be provided to ensure that patients fully understand the risk of having an affected child, the impact of the disease on an affected child, and the benefits and limitations of all available options for preimplantation and prenatal diagnosis
The Society of Obstetricians and Gynaecologists of Canada: step 2
Couples should be informed that Preimplantation Genetic Testing can reduce the risk of conceiving a child with a genetic abnormality carried by one or both parents if that abnormality can be identified with tests performed on a single cell.
The Society of Obstetricians and Gynaecologists of Canada: step 3
Invasive prenatal testing to confirm the results of Preimplantation Genetic Testing is encouraged because the methods used for PGT have technical limitations that include the possibility of a false negative result
