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ANAT 381 > Preimplantation Genetic Testing > Flashcards

Preimplantation Genetic Testing Flashcards

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1
Q

name genetic diseases - healthy legacy

A
  • Physical disability
  • Mental disability
  • Neonatal and childhood
    death
  • Chronic disease and
    early demise
  • Recurrent pregnancy
    loss
  • Late onset disease
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2
Q

describe Avoiding transmission of genetic disease - reproductive options for those with serious recurrent genetic risk

A

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

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3
Q

name 2 methods of prenatal diagnosis

A

testing on established pregnancy= chorionic villi sampling or amniocentesis

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4
Q

describe chorionic villus sampling

A

transcervical, more common
arounf 10-12 weeks
more invasive = slightly higher risk
could also test mom blood around 10 weeks

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5
Q

describe amniocentesis

A

more direct = tests on fetus
always little bit of risk
15-18 weeks, much later
5% fetus loss

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6
Q

what is preimplantation genetic testing

A

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)

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7
Q

what is pgd and pgs

A

Preimplantation Genetic Diagnosis (PGD) and screening (PGS)
These terms have now been replaced by “preimplantation genetic testing” (PGT)

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8
Q

consensus def of pgt

A

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

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9
Q

name types of pgt

A

pgt-a = for aneuploidies
pgt-m = for monogenic/single gene defects
pgt-sr = for chromosomal structural rearrangements

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10
Q

name types of genetic risk

A

Recurrent: As a result of inherited disorders
Sporadic: Random, often age related

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11
Q

what is required to preform pgt

A

ivf, need excess = multiple eggs and embryos
also diagnosis done in vitro

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12
Q

name and describe the 3 stages of pgt

A

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

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13
Q

name types of ways of doing pgt

A

polar body biopsy
cleavage stage biopsy
blastocyst biopsy

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14
Q

describe Early Human Embryo Development - gen

A

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

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15
Q

describe polar body biopsy

A

aspirate polar bodies and then do diagnosis = see if chromosome related or single gene related

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16
Q

describe polar body biopsy = for normal segregation in m1

A

pb 1 and 2 have one chrom in it

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17
Q

describe polar body biopsy = for nondisjunction in m1

A

pb can have no chroms = disomic gamete
pb can have 2 chroms = nullsomic gamete

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18
Q

describe polar body biopsy = for prevision of one univalent in m1

A

pb has 1 chromatid, gamete = 23+1
or pb has 1 chromatid + 1 chrom = 22+1/2 gamete

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19
Q

describe polar body biopsy = for prevision of both univalent in m1

A

22 + 1/2 + 1/2
pb has 2 chromatids and sod does egg

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20
Q

principle of PB-based PGT for single-gene disorders- ex = cftr mutation

A

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

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21
Q

ADVANTAGES - polar body biopsy

A

*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

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22
Q

DISADVANTAGES - polar body biopsy

A

*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

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23
Q

describe embryo biopsy for pgt - day 3

A

make small hole on zona and aspirate out one blastomere

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24
Q

ADVANTAGES - day 3 cleavage stage biopsy

A

*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

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25
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)
26
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
27
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)
28
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)
29
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
30
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
31
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
32
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
33
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
34
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
35
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
36
describe pgt for triplet repeat disease
Fragment analysis
37
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
38
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
39
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
40
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
41
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
42
describe dm-str markers
can see which affected vs which normal run on gel
43
describe family pedigrees
how to diagnose mutation haplotyping use of linked markers in pgt-m disease mapped to specific chroms = if inherited = have disease
44
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
45
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
46
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
47
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
48
describe how to do Preimplantation Genetic Testing for X-linked recessive disorder by sex selection
Using FISH
49
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
50
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
51
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.”
52
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.
53
name types of chromium translocations
Robertsonian and reciprocal
54
explain robertsonian translocations
between chroms 13,14,15 and 21,22 only acrocentric chromosomes short arms end up lost
55
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)
56
explain robertsonian translocations - balanced?
Alternate segregation = 33% normal balanced adjacent segregation and 3:0 segregation = unbalanced 67%
57
describe reciprocal translocation- gen
rest of chromosomes
58
describe reciprocal translocation- outcomes
16 possible outcomes of segregation of translocation chroms, only 2 produce balanced or normal gametes
59
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
60
describe age and fertility
fertility decreases and spontaneous abortion increased with increased maternal age
61
describe aneuploidy and maternal age
increase in aneuploidy and miscarriage and decrease in live births with increased mat age
62
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
63
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
64
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
65
what are New strategies and Techniques for PGT-A and PGT-SR
ngs high res ngs
66
describe pgt - blastocyst biopsy
or even cleavage stage = diagnose then freeze immediately if normal = embryo transfer if abnormal = discard or donate to research
67
what is ngs
next generation sequencing biopsy embryo = amplify genome dna, dna fragmentation and put adapter
68
describe pgt with ngs
like barcode = add during amplification = fragments with adapters computer will analyze and then sort embryos
69
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
70
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
71
describe normal ngs profile
46, xy and 46, xx everything at 2 (copy number, except y chrom at 1 ish)
72
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
73
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
74
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
75
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)
76
describe clinical implications of chromosome mosaicism - gen
normal = normal preg/no preg/abnormal preg Abnormal = loss of euploid embryo-possible viable pregancy
77
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
78
describe blastocyst biopsy mosaicism and clinical implications
if blastocyst mosaic then misdiagnosis may occur do not where mosaic cells are
79
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
80
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
81
describe Choosing embryos for social reason using pgt - NOT PERFORMED AT MCGILL - 5
1. Sex selection for family balancing = no but yes for x linked diseases 2. Specific genetic profile: multifactorial (do not do multifactorial) 3. Risk of passing on less severe disorders: How do you define? (should we allow requests?) 4. Risk of late onset disease -Case by case basis (only when embryo becomes 70-80 y/o, ethics board) 5. Cancer –Case by case basis= just bc have mutation does not mean will get cancer (ex =brca, for breast cancer)
82
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
83
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
84
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.
85
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

ANAT 381 (24 decks)

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