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FRC Path 2023 > Session 6: Prenatal Genetics > Flashcards

Session 6: Prenatal Genetics Flashcards

1
Q

cffDNA

A

Discovered by Lo et al (1997)
Orignates from placental cells
Fetal fraction 10-20% of total cfDNA
Detectable from 4-5 weeks (reliably tested 7-9 wks)
Level increases with gestation
Rapidly cleared from circulation- within 1h after delivery
Av size 143bp (mat frag 166bp)

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2
Q

cffDNA technical challenges

A

Low abundance
Mat nucleated cells must be stabilised - STRECK tubes
cffDNA is outnumbered by mat cfDNA
Nearly indestinguishable from mat cfDNA

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3
Q

Sources of fetal genetic material in mat blood

A

cffDNA
Intact fetal cells - nucleated cells: erythroblasts, cytotrophoblasts and leukocytes
cffmRNA

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4
Q

What stages of oogenesis occur in the in the fetal ovary?

A
  • in the female embryo the primordial female germ cells migrate to the primitive ovary and rapidly proliferate to form the oogonia
  • after month 7 most oogonia die- those that remain enter the 1st meiotic division to produce a primary oocyte
  • the primary oocyte is arrested in prophase 1 of meiosis until puberty
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5
Q

What stages of oogenesis occur in the adult ovary ?

A

at puberty groups of oocytes periodically resume meiosis
- oocytes are in metaphase II at ovulation and do not complete meiosis II until fertilisation

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6
Q

What is the difference between cell division in oogenesis and spermatogenesis?

A

In oogenesis there is unequal cytokinesis in meiosis-most of the cytoplasm is retained by the oocyte and the polar body contains little more that the haploid nucleus

In spermatogenesis there is equal division

In meiosis I the primary spermatocyte divides to produce 2 secondary spermatocytes
in meiosis II the secondary seprmatocytes divide to produce 2 haploid spermatid (4 total)

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7
Q

what is thought to be responsible for the different mutation rates between the sexes?

A

there are 2 more mitotic divisions in the production of sperm than of an ovum

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8
Q

what is the structure of the oocyte?

A

oocyte cytoplasm contains: mitochondria, ribosomes, DNA/RNA polymerases and protective chemicals
- plasma membrane is surrounded by a thick extracellular matrix called the zona pellucida
- the oocyte is surrounded by a layer of follicle cells called umulus cells which nurture the occyte just before and after fertilisation.

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9
Q

what are the stages of spermatogenesis?

A

in male embryo development primordial germ cells migrate to the male testes and undergo proliferation to generate spermatogonia

some spermtogonia stop dividng and differentiate into spermatocytes- in meisosi 1 there is symmetrical division to produce 2 spermatocytes and in meiosis II 4 equally sized haploid spermatids are produced

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10
Q

Where the sperm mature?

A

haploid spermatids undergo morphological differentiation into sperm and escape in the lumen of the seminiferous tubules and then into the epididymus

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11
Q

when does spermatogenesis occur?

A

starts in puberty and continues until death but the quality of sperm deteriorates
- get new sperm each month

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12
Q

what is the structure of sperm?

A

sperm are small with reduced cytoplasm
- head piece contains acrosomal vesical with digestive enzymes required to fertilise the oocyte
- mid piece with mitochondria and
- flagellum in tail provides propulsion

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13
Q

Define fertilisation, where does it occur?

A

Process by which a sperm and egg fuse to create ans new individual and occur in the ampula of the fallopian tube

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14
Q

What are the stages of fertilisation?

A
  1. sperm attaches to outer cumulus cells of the oocyte
  2. sperm reaches the zona pellucida (ZP)
  3. acrosome bursts releasing enzymes which digest the ZP so the sperm can come into contact with the oocyte membrane- acrosomal reaction
  4. cortical reaction occurs to prevent fertilisation by more than 1 sperm
  5. oocyte undergoes 2nd meiotic division to produce a secondary oocyte and a polar body
  6. sperm tail and mitochondria degrade- explaining maternal inheritance of mitochondria
  7. haploid sperm and oocyte nucelai form the pronuclei
  8. first mitotic division occurs

fertilised oocyte = totipotent zygote

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15
Q

what occurs in the cortical reaction?

A

prevents fertilisation of an oocyte with more than 1 sperm

cortical granules within the oocyte fuse with the plasma membrane of the cell- enzymes in these granules are then expelled by exocytosis to the ZP. this causes crosslinking of gycoproteins in the ZP making the matrix impermeable to sperm

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16
Q

Define embryogenesis?

A

process of cell division and differentiation of the human embryo

Embryo = fetus at 8 weeks gestation

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17
Q

What happens to the oocyte post fertilisation?

A

fetilised egg is propelled along the oviduct and is prevented from adhering to the oviduct wall by the ZP (if this does not happen you can get an ectopic pregnancy)

in the uterus degrades and there is implantation of the blastocyst

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18
Q

what are the stages of embryogenesis?

A

cleavage
compaction
blastocyst
implantation
gastrulation

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19
Q

what happens in the cleavage stage of embryogenesis?

A

zygote divides rapidly to form a number of smaller cells- the blastomere
- controlled but zygotic genome

cells do not always divide at the same time so result in embryos with odd numbers of cells

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20
Q

what happens in compaction?

A

at the 8 cell stage the cells latten against each other to from the tightly packed morula

compaction introduces some level of polarity

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21
Q

what is the 16 cell embryo?

A

blastocyst with external polarised cells, internal non-polar cells

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22
Q

what is the blastocyst

A

16 cells embryo with fluid filled cavity called the blastocoele
- outer layer of trophoblast cells with give rise to the fetal portion of the placenta (the chorion)
- inner cell mass (ICM) which congregate at one end- the embryonic pole

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23
Q

What cells does the ICM give rise to?

A

all the cells of the organism plus the yolk sac, amnion and allantosis

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24
Q

when can monozygotic twins arise?

A

at any time until the late blastocyst stage

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25
Q

what happens in implantation?

A

at day 5 an enzyme is released which makes a hole in the ZP releasing the blastocyst- the blastocyst then attaches the the uterine membrane

  • trophoblast cells rapidly divide to form the cytotrophoblast ans syncytiotrophoblast and invade the connective tissue of the uterus
  • a fluid filled cavity forms within the ICM generating the amniotic cavity enclosed by the amnion
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26
Q

what happens in gastrulation?

A

Gastrulation is the process in which the orientation of the body is established and the 3 germ layers are formed:
ectoderm
endoderm
mesoderm

The characteristic gastrulation is the formation of the primitive streak

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27
Q

what is the primitive streak?

A

establishes the antero-posterior body axis

forms during gastrulation
starts as a primitive streak and then develops into a groove then a pit.

cells migrate through the primitive streak to the space between the epiblast and hypoblast to generate the 3 germ layers- 1st endoderm, 2nd mesoderm and 3rd ectoderm

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28
Q

what structures develop from the endoderm?

A

lungs, liver, thymus, endocrine glands

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29
Q

what structures develop from the mesoderm?

A

blood vessels, muscle tissue, connective tissue

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30
Q

what structures develop from the ectoderm?

A

skin and the CNS

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31
Q

What happens to the epigenetic imprint in the fetal germline?

A

All DNA methylation patterns are erased and the pattern specific to the sex of the fetus is set- when fertilised the imprint is maintained stably in all somatic cells of the fetus and throughout life

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32
Q

what is IUI?

A

interuterine insemination- injection of sperm directly into the womb following stimulated ovulation of the woman

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33
Q

what is IVF?

A

adding sperm samples to eggs (havested from a women following stimulated ovulation) in a petri dish. They are incubated together and monitored for successful fertilisation. Successfully developing embryo is transferred back to womans womb

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34
Q

what is ICSI?

A

microscopic injections of a sperm directly into an egg nuclei in a petri dish. Similar to IVF but maximises the chance of fertilisation

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35
Q

what is MESA?

A

Microsurgical Epididymal Sperm Aspiration- surgical sperm extraction method and can be used with IUI, IVF or ICSI for men with azoospermia (no sperm in semen)

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36
Q

what is triploidy?

A

additional full set of chromosome
69,XX/XY

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37
Q

what is the incidence of triploidy?

A

1-3% of pregnancies
99.9% abort in 1st or second trimester- those that make it to term die in the neonatal period <1 month
advanced maternal age is not a risk factor

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38
Q

what is the proportion of diandry and digyny triploidy?

A

60-80% are diandry- most common

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39
Q

what is diandry triploidy?

A

2 paternal and 1 maternal set of chromosomes

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40
Q

what is the cause of diandry triploidy?

A

fertilisation of a normal egg by 2 sperm- failure of cortical reaction- 69,XXX or 69,XXY or 69,XYY
69,YYY not viable so not seen

minority are due to fertilastion of a normal egg by diploid sperm by a failure of NDJ in spermatogenesis for a complete set of chromosomes

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41
Q

what is the characteristic feature of a diandric triploid?

A

partial hydatidiform mole which is a trophoblastic hyperplasia of the cystic villi

increased hCG levels

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42
Q

what are the clinical features of triploidy?

A

IUGR
head to chest fusion
limb growth abnormalities
macrocephaly- digyny
neural tube defect
hydrocephalus- digyny
syndactyly
heart/renal defects
cleft lip/palate

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43
Q

what is digyny triploidy?

A

2 maternal and 1 paternal set of chromosomes
non hydropic villi
NO MOLE and placenta is generally small

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44
Q

what causes digyny triploidy?

A

fertilisation of a diploid egg

  • due to NDJ of a complete set of chromosome during meiosis
  • retention of a polar body in the fertilised egg- failure of meiosis II
  • fusion of 2 eggs and fertilisation by a haploid sperm
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45
Q

what causes the difference in presentation between digyny and diandry triploids?

A

due to the influence of differing imprinted states

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46
Q

what is the recurrence risk of triploidy?

A

usually sporadic
- diandry triploidy - recurrence risk is 1-1.5%
- digyny triploidy has been reported as recurrent in several families

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47
Q

what is gestational trophoblastic disease (GTD)?

A

a group of rare diseases in which abnormal trophoblast cells grow inside the uterus after conception

includings
- non invasive: mole and partial mole
- malignant- invasive mole, choriocarcinoma and placental site trophoblastic tumours (PSRR; very rare), Epithelioid trophoblastic tumors (ETT; even more rare).

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48
Q

what is a complete mole?

A

46,XX/XY but only paternal chromosomes present

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49
Q

what are the characteristics of a complete mole?

A

high levels of hCG
bunch of grapes appearance
NO formation of a fetus

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50
Q

how do complete moles arise?

A

fertilisation of a enucleated egg with a haploid sperm and duplication of the haploid genome (46,XX)

remaining are fertilisation of an enucelated egg with 2 different sperm 46,XY or 46,XX

46,YY are not seen as unviable

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51
Q

How are molar pregnancies diagnosed?

A

high hCG
uterus may be enlarged
more vomiting
vaginal bleeding

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52
Q

what is the recurrence risk of a complete mole?

A

1 in 100 after 1st
1 in 5 after 2nd

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53
Q

what is familial recurrent hydatiform mole?

A

maternal affect
AR
results in recurrent molar pregnancies
the moles that are present are usually diploid and genetically bi parental which is different to sporadic moles which are diandric

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54
Q

what is a malignant gestational trophoblastic disease (GTD)?

A
  • malignant transformation of a mole
  • also known as a persistent mole
  • usually arise form a complete mole
  • patients present with elevated hCG after a previous molar pregnancy

also presents with abormal bleeding abdominal pain or swelling

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55
Q

What other malignancies may arise following molar pregnancy?

A

malignant choriocarcinoma- highly invasive and can cause symptoms locally e.g. uterine bleeding and from distant metastasis in the lungs, CNs and liver (most common)

diagnosed by raised hCG and reproductive history

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56
Q

what is the treatment for gestational trophoblastic disease (GTD)?

A

suction evacuation recommended to remove complete and partial molar pregnancies.
- after molar pregnancy hCG levels should be monitored until they return to normal. usually ~6 weeks and not adviced to get pregnant during this period

if hCG does not return to normal, choriocarcinoma or metastasis is present chemotherapy with methotrexate is recommend

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57
Q

what is a dizygotic twin?

A

fraternal
2 different eggs fertilised by 2 different sperm
more common than identical twins

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58
Q

what are the risk factors for dizygotic twin pregnancy?

A
  • increased maternal age
  • mutations in genes in the TGF9 pathway a.e.g BMP
  • can be familial
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59
Q

what is a monozygotic twin?

A

formed from complete cleavage of single embryo

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60
Q

What are the different types of MZ twins and when are they formed?

A

dichorionic diamniotic (DCDA) twins -division at ~ day 4, at morula stage

monochorionic diamniotic (MCDA) twins - division at day 4-7 at the blastocyst stage

monochorionic monoamniotic (MCMA) twins - embryos that divide after the amnion is formed at day 9

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61
Q

are MZ twins identical?

A

have identical genes but rarely identical due to epigenetic and prenatal environmental post-zygotic mechanisms

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62
Q

what pregnancy complications are associated with twinning and what is the highest risk twin type?

A
  • 6 fold increase in perinatal mortality
  • preterm delivery
  • IUGR

MZ twins have poorer outcomes than DZ
MCMA have highest perinatal mortality rate

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63
Q

what is an anastomoses?

A

vascular connections between the feto-placental circulation in each twin

  • risk on MCDA - monochorionic diamniotic (MCDA) twins
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64
Q

what is TRAP (Twin reversed arterial perfusion)?

A

Twin reversed arterial perfusion (TRAP sequence) is a rare condition of monochorionic twin pregnancies.

It arises when the cardiac system of one twin does the work of supplying blood for both twins.

The twin supplying the blood is known as the “pump twin” and develops normally in the womb. However, the increased pumping of the heart puts this twin at risk for cardiac failure. The other twin — known as the “acardiac twin” — lacks a heart or has one that is not fully formed. It usually has a poorly developed body and may also be missing a head, limbs and torso

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65
Q

what causes TRAP (Twin reversed arterial perfusion)?

A

MC twins
artery to artery and vein-vein anastomoses

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66
Q

What are the features of TRAP?

A

one twin is acardiac and perfused in a reverse direction by the pump twin
- acardiac twin has abn development of head, thorax and or limb

  • ‘normal’ pump twin has poor prognosis and 50% chance of survival due to cardiac overload

some are chromosomally abn

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67
Q

What is TTTS?

A

twin twin transfusion syndrome

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68
Q

what causes TTTS (twin twin transfusion syndrome)?

A

Affects monochorionic twins

there is an imbalance in the blood exchange between the twins and the placenta. One twin — the donor twin — gives away more blood than it receives in return and runs the risk of malnourishment and organ failure. The recipient twin receives too much blood and is susceptible to overwork of the heart and other cardiac complications.

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69
Q

What are the features of TTTS?

A

dihydramnios and IUGR in donor twin

polyhydramnios and enlarged bladder and larger size in recipient twin

can result in preterm labour due to severe polyhydramnios

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70
Q

what is the testing and treatment for TTTS?

A
  • amnio reduction by serial amniocentesis from recipient twin to reduce pressure
  • laser ablation of connecting anastomoses (vessels)

amnio may be sent for testing but array is not indicated

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71
Q

What is a vanishing twin?

A

when 1 twin suffers early fetal death (1st trimester) and is not aborted

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72
Q

How may a vanishing twin be identified?

A
  • areas of degenerate villi or amorphous debris- but often difficult to detect
  • may be identified by the presence of an empty sac on USS
  • can explain otherwise inexplicable anomalous maternal serum and alpha fetoprotein and CVS chromosome result
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73
Q

what is a fetus papyraceaus?

A

when a twin suffers death and isn’t aborted but unlike in a vanishing twin the dead fetus persist (fetal death occurs beyond the first trimester)

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74
Q

What is zygosity testing?

A

determine the degree of identity in the genome of twins

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75
Q

when is zygosity testing performed?

A

generally requested when one twin has developed a clinical phenotype that is thought to be genetic in origin

also used to in transplantation where a twin is a HLA match- if they are identical there is less likely to be GVHD in the recipient

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76
Q

How is zygosity calculated?

A

Use QF-PCR to detect microsatellie markers
the likelihood of zygosity is then calculated using bayes.

the prio risk of monozygosity is 1/3 and dizygosity is 2/3
-if sex is know this can be added (1/2)
- the probability of being dizygotic but with the same alleles by chance is calculated from the possible combination of parental alleles.

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77
Q

What is the method for QF-PCR

A
  • amplifies microsatellite markers on target chromosomes for semi-quantitative detemination of ploidy
  • multiplexed and uses conditional to allow multiple PCR reactions to be run together
  • products are differentiated by using different colour dyes and product sizes using capilliary electrophoresis
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78
Q

how is QF-PCR quantitative?

A

reaction is stopped in the exponential phase- PCR reagents are not depleted

In the exponential phase the level of product is proportional to the amount of starting material and can be used to determine the relative CN of the chromosomes tested

BPG recommend 24-26 cycles

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79
Q

how many markers are required for reporting QF-PCR?
what are the requirements for choosing markers?

A

Need 2 informative markers- remaining can be inconclusive but not contradictory to the result being reported.

recommended to have at least 4 markers per chromosome to increase the chance of an informative result

when choosing markers need?
- high heterozygosity in the general population
- SNP checked
- majority are 4bp repeats as these have fewer stutter peaks than dinucleotide repeats

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80
Q

What sex chromosome markers are used for QF-PCR?

A

sexing may or may not be included depending on local policy- risk of sex selection of fetus

AMEL- present on X and Y and gives peaks 4bp apart. cannot detect sex chr no. but can detect their ration e.g. 1:1 XY or 2:1 XXY

SRY- Y chromosome specific non-polymorphic marker to confirm presence or Y

TAF9- present on chr3p and Xq used to determine X chromosome copy number based on the assumption that 3p will have a CN of 2
- can be used to distinguish XO from XX with all homozygous markers

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81
Q

how is peak ratio determined for QF-PCR?

A

peak height/area of smaller peak divided by the peak height/area of the larger peak

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82
Q

what is normal PCR ratio?

A

1:1 alleles with a range from 0.8-1.4

for alleles >24bp apart a ratio of 1.5 is acceptable because of preferential amplification of the smaller allele

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83
Q

Examples of trisomy PCR result?

A

1:1:1 - triallelic and confirms meiosis 1 NDJ

2:1 ratio 1.8-2.4
1:2 ratio 0.46-0.65

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84
Q

what is the significance of a biallelic PCR result

A

a biallelic pattern could indicate meiosis II NDJ or mitotic NDJ and CPM should be considered for CVS - report cautiously

  • meiotic NDJ not confirmed
  • follow-up karyotype on cultured cells which contain the mesenchyme and are more representative of the fetus
  • suggest detailed USS and AF sampling
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85
Q

what are the inconclusive ratio in PCR and their implication?

A

0.6-0.8 and 1.4-1.8

  • can be resolved by running extra single chromosome markers
  • cannot report a sample as trisomic if there is a single inconclusive marker or single normal marker for that chromosome
  • may be due to preferential amplification of the smaller allele and can result in inconclusive ratios the greater the distance between the 2 markers.
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86
Q

does QFPCR result need to be confirmed?

A

2018 BPG
trisomic results do not need confirmation by another method but sample ID must be confirmed before reporting- usually by repeating the original sample

normal result do not need to be repeated

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87
Q

can single allele result be reported

A

occasionally if normal but this should be stressed on the report- ideally another method e.g. karyotype of FISH should then be used as follow-up to confirm the result for the chromosome

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88
Q

how may MCC present on PCR results?

A
  • skewed/inconclusive results for all chromsomes
  • normal female results
  • need to request maternal blood

if there is a mixed genotype there will be 3 alleles:
- 1 fetal (pat) specific,
- 1 mat and fetal shared and
- 1 mat specific.

In a normal sample: the sum of the peak of the maternal and fetal specif alleles should add up to the fetal+mat shared allele and give a normal ratio

AMEL can be used to quantify the fetal genotype in a male- if a normal result with non skewed XY is obtained a normal result can be issued

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89
Q

what types of MCC result can and can’t be reported?

A

BPG
- low level MCC - majority fetal genotype with no inconclusive markers- report
- single fetal genotype with no inconclusive alleles- report
- inconclusive alleles with mixed genotype- do not report

QF-PCR is sensitive to MCC to ~10%

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90
Q

how can the risk of MCC be reduced for CVS and AF?

A

AF-MCC may be obvious due to bloodstaining wither before or after centrifuging
- if normal female result obtained need to request mat sample before reporting
- could have a tissue plug resulting in MCC with no obvious bloodstaining
- culturing will remove MCC from blood as the culture will favour growth of the amniocytes, but contamination from mat tissue may grow

CVS- carfeul dissection to remove maternal decidua required to prevent MCC risk
- analysis of cultured cells may help but need to be cautious as maternal tissue may still be present and could predominate

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91
Q

How is mosaicism detected on PCR?

A

presence of extra peaks, skewerd markers for just one chromsome

markers may be subtle with alleles still in the normal or inconclusive range

diploid/triploid mosaicism can be difficult to distinguish from MCC

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92
Q

what are the causes of mosaicism?

A
  • mitotic rescue of a trisomic cell line
  • mitotic NDJ and generation of a trisomic cell line in a normal conceptus

Need to be wary of CPM in CVS samples

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93
Q

what may cause a single abnormal marker?

A

SMM
PSP
SMD
partial chromosome imbalance
CNV

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94
Q

can markers 2bp apart be used for reporting?

A

No - stutter peaks may be incorporated into the peak for the shorter allele resulting in misinterpretation of allele ratios

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95
Q

what is an somatic microsatellite mutation (SMM)?

A

somatic microsatellite mutation
- single marker with 1:1:1 ratio and unequl peak heights (2 will add up to the larger one)

  • due to mutation of an allele and mosaicism for the de novo and orginal allele
  • caused by defective proofreading during DNA rep in early embryogenesis

cant use as an informative marker for reporting, lower annealing temp to distinguish from a PSP

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96
Q

what is a (polymorphism to primer binding site) PSP?

A

polymorphsim to primer binding site resulting in efficient amplifcation of an allele and a single inconclusive result 0.6-0.8 or 1.4-1.8

repeating with a lowered anealing temp should normalise the ratio

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97
Q

what is an submicroscopic microsatellite dup (SMD)?

A

submicroscopic micrsatellite dup
- trisomic result for a single marker- all other consistent with normal
- some are inherited and benign- list on known inherited SMDs available
- if flanked by 2 normal markers assumed to represent an SMD and not reported
- parents may be requested to check if SMD is inherited and not associate with abn phenotype

distinguish from PSP be repeating at lower annealing temp

detail findings on report if not considered benign and extra testing with flanking markers may be required

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98
Q

How may a partial chromosome imbalance appear on PCR?

A

normal and abnormal results for the same chromosome- 2 or more consecutive markers showing the same result

follow up with karyotype, array, parental bloods

99
Q

How may a partial chromosome imbalance appear on PCR?

A

normal and abnormal results for the same chromosome- 2 or more consecutive markers showing the same result

follow up with karyotype, array, parental bloods

100
Q

what are the considerations for PCR in twin pregnancies?

A

twins can be identified as dizygotic or probably monozygotic which may be clinically useful
- likelihood of monozygosity is determined by the number of informative markers and the maternal/paternal relatedness

sampling the same twin twice cant be excluded

which twin the report is for should be clearly identified e.g. twin 1 upper left

101
Q

what are the advantages of PCR over FISH?

A

cheaper
needs less sample
less labour intensive
can detect MCC in all pregnancies not just male
can detect UPD
may detect some unbalanced rearrangements

102
Q

what should be included on QF PCR reports?

A
  • assumption that fetal material was tested
  • result is consistent with trisomy as only specifc markers tested
  • include interpretative sentence e.g. consistent with a diagnosis of down syndrome
    2 alleles results- risk that it may represent CPM- follow up karyotype, AF and USS
  • should perform karyotype follow-up of abnormal results to determine the structure and recurrence risk
103
Q

what are the limitations of FISH for aneuploidy?

A
  • does not give structure
  • higher resolution that G-banding
  • cant detect MCC in female pregnancies but may be suspected if there is mosaicism
  • more expensive ans time consuming that PCR
  • higher failure rate that PCR
  • need to scan lots of cells are there can be a range of artefactural staining e.g. monomsomy from co-localisation of 2 signals or trisomy from 2 cells being on top of each other.

need to scan even more if referral indicates mosaicism

104
Q

what is the problem with using centromere probes in FISH?

A

repetitive sequence probes are targeted to the alpha satellite sequences found at the centromere - composed on array on tandomly repeated monomers

the are sufficiently different to allow distinction of all chromosome except
chr13 and 21 and chr14 and chr22
so need to look for 5 signals for a trisomy and follow-up testing required to confirm the chromosome involved.

target specific probes targeted to specific sequences on the q arms of chromosomes are therefore preferred

105
Q

what percentage of pregnancies have major congenital abnormalities and what percentage of these are chromosomally abnormal?

A

1-1.5% of pregnancies have major congenital abnormalities
20-25% of these are chromosomal

106
Q

what are the benefits of prenatal microarray?

A

higher resolution than karyotype increases diagnostic yeild
- BPG recommend minimum resolution of 400Kb
- 2.4% increase in diagnostic rate for all and 7% for abscan referrals
- carriers of familial rearrangements not detected
- culturing generally not required (except small samples or MCC cases

107
Q

what are the limitations of prenatal array?

A
  • increased resolution can increase the pick up of VUS which can be difficult to interpret- may need MDT.
  • should only report variants that are class 4 or 5 in prenatals, may also need to request parents to aid interpretation
  • may miss low level mosaisicism
  • may not detect triploidy
  • may detect IFs- need reporting policy (report IFs that are clinically actionable to the family)
  • culturing may be required for follow-up as array does not give structure
108
Q

what was the EACH study?

A

Evaluation of ArrayCGH in prenatal diagnosis of fetal anomalies

  • multi centre study comparing aCGH to karyotype in fetus with 1+ USS abn + NT>3.5mm
  • 500 recrutied
  • QF-PCR followed by aCGH for those with a -ve result
  • ISCA arrays
  • variants categorised as benign, VUS or pathogenic (arrays are now using a 5 clas system as with SNV interpretation)
  • only reported variants associated with a known clinical syndrome of significant chromosomal imbalance
109
Q

what were the aims of the EACH study?

A
  • to optimise prenatal DNA extraction techniques
  • incoporate aCGH into standard lab service
  • all CNVs recorded in a standard way and in a central database
110
Q

what were the findings of the EACH study?

A

CMA is a robust, cost effective, acceptable diagnostic technique for prenatals with a NT >3.5mm and US abn and should replace karyotype

1-3% increased diagnosis compared to karyotype

111
Q

what are the recommendations for use of microarrays in pregnancy?

A

2015 working group on behalf of the joint committee on genomics in medicine
- gudiance on reporting in prenatal samples e.g. whether come low penetrances susceptibility loci should be reported

recommends that:
NIPA1 dels and dups are not reported
15q13 dups not reported
STS dups not reported
16q13 dups not reported
het del of gene that cant be linked to phenotype i.e. carrier status should not be reported

112
Q

what percentage of pregnancies are chromosomally abnormal?

A

1%

113
Q

what is a very early conceptus and what percentage end in miscarriage?

A

at the morula to blastocyst stage many abnormal embryos arrest

~ 30% of pregnancies fail to implant and another 30% implant transiently - this results in little disruption to the menstrual cycles

cause is commonly polyploidy or monosomy and frequency increases with advancing maternal age

114
Q

what gestation distinguishes miscarriage from still birth?

A

fetus is incapable of surviving independently < 24 weeks, therefore below this is classed as miscarriage and above this is a still birth

115
Q

what are the common causes of 1st trimester pregnancy loss?

A

50% are chromosomally abnormal
majority are trisomy, triploidy and monosomy X

60% single trisomy
12-15% monosomy X
12% triploidy
3% tetraploidy
~5% structural rearrangement
~5% multiple aneuploidy

116
Q

what percentage of 1st trimester pregnancies end in miscarriage?

A

~10-15% usually towards the end where the placenta takes over the nourishment of the fetus so the body recognises abnormality

117
Q

what are the most common trisomies in pregnancy loss?

A

T16
T22
T21
T15
T13/18

only T21,18 and 13 are seen in liveborns non-mosaically

the frequency of trisomy in pregnancy is greater than in live birth as many are lethal and will result in miscarriage

118
Q

what is the prevalence of T16 in miscarriage

A

T16 is seen in 1% of pregnancies and accounts for 10% of pregnancy loss

those that survive are mosaic or CPM and only in placenta- this can result in placental insufficiency and IUGR

119
Q

When are most trisomies derived from?

A

Majority are from an error (NDJ) in maternal meiosis I

  • T18 associated with NDJ in meiosis II

significantly associated with maternal age

120
Q

what is the miscarriage rate in the second trimester?

A

2-5% with 20% chromosome abn rate

121
Q

what are the common chr abn in 2 trimester miscarriage and what percentage are chromosomally abnormal?

A

20% chromosomally abnormal
- more likely to be viable trisomies (21, 18, 13) as others lost in 1st trimester)
- increased proportion of structural rearrangments

122
Q

what percentage of 3rd trimester pregnancies result in miscarriage?

A

0.5%

123
Q

what are the genetic findings in 3rd trimester miscarriage?

A

reduced proportion of aneuploidy (T21, T18, T13 and 45,X)

increased proportion if sex chr abn and structural rearrangements and there is less genetic imbalance than in a trisomy

124
Q

what percentage of the following chr abn are lost during pregnancy?

T16
T21
T18 + T13
45,X
tertraploid
triploid
balanced rearrangement
unbalanced rearrangement
normal karyotype

A

T16- 100% unless mosaic
T21 - 80%
T18 + T13 - 95%
45,X- 95-99%
tertraploid- almost 100%
triploid- almost 100%
balanced rearrangement- 15%
unbalanced rearrangement- 95% (depending on size of imbalance)
normal karyotype- 8%

125
Q

what is the role of tetraploidy on miscarriage?

A

4 copies- usually due to failed early mitotic division resulting in duplicated genome and 92,XXX or 92,XXYY

2-3% of spontaneous miscarriage

126
Q

what is the role of triploidy on miscarriage?

A

10% of miscarriages

2/3 are diandric due to dispermy
- partial hyadatiform mole and IUGR
- most miscarry in 1st trimester a few make it to second trimester

1/3 digyny due to diploid egg from NDJ of complete chromosome set in oogenesis or failure to expel polar body
- misacarry in 1st trimester
- severe IUGR, macrocephaly and small placenta (non molar)

127
Q

what is the role of 45,X on miscarriage?

A

95-99% die in utero
hypothesised that complete 45,X is lethal and that all TS patients have some level of mosaicism

128
Q

what is the role of monosomy on miscarriage?

A

Only seen for chr 21 ~1 in 1000 karyotyped abortions

129
Q

what is the role of uniparental diploidy on miscarriage?

A

diandric diploidy- complete mole due to 2 sperm fertilising and empty egg
- increased risk of choriocarcinoma

digyny diploidy- ovarian teratoma. fatal. may contain fully differentiated tissue e.g. hair and nails

130
Q

what is the role of structural rearrangements on miscarriage?

A

likelihood of miscarriage of abnormal offspring is dependent on the size and genetic content of the imbalanced region (if unbalanced);

balanced rearrangements seen in the same frequency in liveborns

131
Q

what is the role of chromosome mosaicism and CPM on miscarriage?

A

CPM with the abnormality only in the placenta can still result in pregnancy loss due to abnormal development of the placenta which can have a physiological effect on the fetus

132
Q

What referrals are eligible for testing following a miscarriage?

A

3rd consecutive recurrent miscarriage (<24weeks)-n RCOG = test the POC and follow- up in parents if an abnormality is detected. Only test parents by karyotype in exceptional circumstances

pregnancy loss with significant fetal abnormalities or termination (irrespective of gestation)

still birth (pregnancy loss >24 weeks)

133
Q

how many women experience recurrent miscarriage?

A

1%

134
Q

What causes are there for miscarriage other than genetics?

A

epidemiological factors
parental/ embryonic factors
infections
acquired thrombophilic defects
anatomical abnormalities (uterus, cervix)
maternal endocrine and immune anomalies (thyroid dysfunction)
UNEXPLAINED

135
Q

what is the genetic testing methodology for POCs

A

QF-PCR for common aneuploidies and sex
- some centres also include other trisomies common in miscarriage 16, 22, 14, 15

ArrayCGH

Karyotype no longer performed as tissue can be hard to culture and prone to infection or failure plus array provides a higher resolution and expecting to find imbalance.

Donaghue et al 2017- PCR and array = lower failure rate and higher yeild that karyotype or MLPA strategies

136
Q

when is the 1st trimester USS offered and what are the aims

A

11-14 weeks
- pregnancy dating
- ID multiple pregnancies
- placental location
- ID fetal anomalies (50%) can be detected at 1st trimester scan

137
Q

what is the nuchal translucency?

A

subcutaneous fluid under the skin at the nape of the neck
- most powerful measure for detecting aneuploidy
- included in the combined score (with mat age, PAPP-A and B-hCG)
- requires a highly trained sonographer to be measured and variation in measurement has been reported

138
Q

what is the significance of the NT measurement?

A

increasing size is correlated with increased risk of chromosomal abn
>3.5mm is significant and may be due to chromosomal abnormality (T21, 18, 13, XO), cardiac defect/failure or delayed development of the lymphatic system

can either regress or evolve into cystic hygroma, oedema, fetal hydrops in second trimester

139
Q

what are the USS soft markers ‘normal variants’

A

USS markers that may be associated with anuploidy but are also seen in normal pregnancies

  • absent nasal bone (seen in 2% of normal fetus and 9% in normal afro-Caribbean pregnancies)
  • dilated cisterna magna
  • echogenic foci in the heart
  • 2 vessel cord
  • echogenic bowel with density lower than bone
140
Q

what is the significance of normal variants?

A

DS screening programme has increased the detection of aneuploidy at first trimester therefore if normal variants are detected at the second trimester and the mother had a normal combined screen score they are not referred for genetic testing

141
Q

what second USS abn are offered genetic testing?

A

ventriculomegaly >10mm
echogenic bowel with density greater than bone
nuchal fold >6mm
renal pelvic dilation

142
Q

what major congenital abnormalties can be detected in 1st trimester and what conditions do they indicate?

A

holoprocencephaly- failure of brain to divide into 2 lobes (T13, 18, SHH gene)

Exomphalos- herniation of fetal abdomen 9t18, 13 or BWS)

megacystis - enlarged fetal bladder (T13 and 18)

skeletal abn e.f. T21, 18 and X) associated with shortened long bones

143
Q

What are the USS abn associated with iso(12p)?

A

diaphragmatic hernia
short limbs
abnormal hands and feet

144
Q

What are the USS abn associated with wolf hirschorn?

A

IUGR
microcephaly
cleft palate
heart defect

145
Q

What are the USS abn associated with cri-du-chat?

A

heart defect
cleft lip/palate

146
Q

What are the USS abn associated with Miller Dieker?

A

lissencephaly (LISS1)

147
Q

What are the USS abn associated with 22q11.21?

A

cardiac abn (TOF)
renal abn
polyhydramnios
cleft lip and palate

148
Q

What are the USS abn associated with Williams?

A

IUGR, cardiac defects

149
Q

What are the USS abn associated with BWS?

A

overgrowth,
exomphalos

150
Q

What are the USS abn associated with AS?

A

asymmetrical IUGR

151
Q

What are the USS abn associated with achondroplasia?

A

short long bones
frontal bosing

152
Q

What are the USS abn associated with T21?

A

cardiac defects-VSD
duodenal atresia
IUGR
hydrops
echogenic bowel
renal anomalies
syndactyly

153
Q

What are the USS abn associated with T18?

A

rocker bottom feet
talipes
clenched hands
exomphalos
VSD
oesopageal atresia
IUGR
renal abn
neural tube defect

154
Q

What are the USS abn associated with T13?

A

holoprosencephaly
diaphragmatic hernia
cleft lip/palate
IUGR
renal abn

155
Q

What are the USS abn associated with 45,X?

A

cystic hygroma
hydrops
increased NT and nuchal fold
fetal oedema
IUGR
renal abn
2 vessel cord

156
Q

What are the USS abn associated with triploidy?

A

IUGR
holoprosencephaly
2 vessel cord

partial mole in diandric

IUGR, macrocephaly and small placental in digyny

157
Q

what is PIGD?

A

PIGD aims to select embryos that are unaffected for transfer to the uterus to prevent the birth of a baby with a disease causing genetic mutation.

158
Q

at what stages can cells be sampled for PIGD

A
  1. biopsy of polar body just prior to conception (1st polar body) or just after fertilisation (2nd polar body)
  2. biopsy of day 3 cleavage stage embryo (5-cells)
  3. biopsy of trophoectoderm from bastocyst (day 5-6 embryos)
159
Q

what techniques are used fro PIGD?

A

used to be FISH but now being replaced by array (structural) and PCR (molecular diagnosis by linkage) can also do direct mutations analysis by sanger

160
Q

what diseases has PGD been added to?

A

CF, HD, NF1, DMD/BMD, chromosomal rearrangments

161
Q

what is PGS

A

Preimplantation genetic screening-screens embryos for aneuploidies with the aim of selecting euploid embryos for transfer

the use for PGS is controversial, at least 11 randomised control trials have found no clinical benefit and 1 study has shown that PGS significantly lowered the birth rate from IVF when applied to women of advanced maternal age

Not currently supported by or offered by the NHS

162
Q

Which group is in charge of determining the clinical criteria for offering PIGD in the NHS?

A

NHS reference group for medical genetics published a clinical commissioning policy for PGD which outlines the conditions under which PGD can be commissioned

163
Q

what are the aims of the PGD NHSE clinical commissioning group?

A
  • provide guidance, consistency and clarity in the commissioning of PGD services
  • improve equality of access to PGD
  • ensure PGD is offered for diseases where there is sufficient evidence of clinical benefit and cost effectiveness
  • reduce variation in clinical practice and conditions referred for PGD
164
Q

what are the requirements for a couple to receive PGD?

A

NHS will commission up to 3 courses of PGD for a couples

  • at risk of having a child with a sever genetic disease >10% risk
  • fertile- different to IVF treatment for infertility
  • HEFA licence for the disease
  • genetic counselling
  • female partner <40yrs and BMI between 19 and 30
  • both partners should be non-smokers
165
Q

what are the exclusions for PGD?

A

not offered for sex selection for family balancing in the UK
- PGS is not commissioned in the NHS as there is no evidence that it provides clinical benefit
- not to be used to address fertility problems or recurrent miscarriage with unknown aeitiology

166
Q

what is the HFEA and what do they do?

A

TheHuman Fertilisation and Embryology Authority(HEFA) oversees the use of gametes and embryos in fertility treatment and research

human fertility embryology authority
- licence and monitor UK fertility clinics and all research involving embryos on the UK
- licence that diseases for which PGD can be offered

167
Q

what is the method for PGD?

A

IVF–> biopsy cells –> genetic test –> transfer unaffected embryos

168
Q

what are the stages in IVF?

A

in IVF a women undergoes ovarian stimulation then oocyte removal. The oocyte can then be fertilised by a sperm in a petri dish. In IVF the sperm and the egg and incubated in the same petri dish and the sperm are left to fertilise the egg. Improved fertilisation can be achieved by ICSI where the sperm is injected into the egg.

sperm for ICSI are morphologically selected- sperm with a large vacuole are more likely to result in aneuploidy

169
Q

what are the different stages that cells can be biopsied for PGD

A
  • 1st and 2nd polar body
  • cleavage stage embryo (day 3)
  • blastocyst (day 5-7) trophoectoderm
  • balstocyst- blastocoele fluid
170
Q

what are the advantages and disadvantages of the sampling 1st and 2nd polar body?

A

longer time to complete genetic testing without requiring crypreservation,

can only detect maternal mutations

generally only used in countries where testing cleavage stage embryos is illegal - Austria, Switzerland and Germany

171
Q

what are the advantages and disadvantages of the sampling cleavage stage embryo (day 3)?

A

well assessed methodology
may hamper embryo viability
60% show mosaicism for anueploidy but rectified by the blastocyst stage

172
Q

what are the advantages and disadvantages of the sampling blastocyst (day 5-7) trophoectoderm?

A

more cells to test = more DNA for analysis
may not be representative of the inner cell mass which goes on to develop into the embryo

173
Q

what are the advantages and disadvantages of the sampling balstocyst- blastocoele fluid?

A

less invasive and safe and contains DNA from ICM and trophoectoderm

only ~ 50% of samples contain DNA suitable for analysis and there can be low concordance with cleavage stage embryos

174
Q

what is the non-invasive preimplantation genetic screening approach?

A

embryoonic DNA is isolcated from the blastocyst culture medium combined with blastocoele fluid can be used for genetic testing.

  • aspiration of blastocoele fluid is less technically challenging and invasive than TE cell biopsy

origin of DNA in the blastocoele fluid is still under investigation and is routinely discarded during hr vitrification process to prevent formation of crystals

175
Q

what is PGD used for?

A

Monogenic disorders
- 1st PGD cases where directed at the Y chromosome for X-linked disease
- HD is the most commonly tested for AD disease - direct and indirect testing is available
- CF- due to the high number of mutations a muliplex linkage based approach has been used so that the same design can be used for all mutations.

176
Q

how can PGD be applied to mitochondrial disease?

A

PGD can be used to select embryos with a low level of the mutatn mtDNA

less prone to allele drop our as there is a high copy number of mtDNA

when there is a high level of mut mtDNA or homoplasmy PGD is not suitable

main issue is even if an embryo is selected with a level of heterpolasmy below that associated with disease they may still be affected due to the mitochondrial bottleneck which could result in higher levels in the fetus

177
Q

what is PGH?

A

PG haplpotyping

used for diagnosis of monogenic disorders by using linkage to determine if the high or low risk allele has been inherited
- same test can be used for all families rather than needing to design mutation specific tests for each mutation
- can be used for disease where the causative mutation has not been determined
- ideally need DNA from parents and an affected proband to ID the high risk allele but an unaffected family member can be used for exclusion testing
- useful if mutation is not amenable to PCR detection
- need markers either side of gene and should determine that there are enough informative markers before offering test
- need to determine the risk of a double recombination resulting in low risk haplotype carrying the causative mutations
- where there is an informative marker either side of the gene the risk of a double recombination is much lower than if the informative markers are only on one side.

178
Q

what is MDA- mutiple displacement amplification?

A

used bacteriophage for whole genome amp meaning lots of DNA can be generated from small samples for genotyping multiple polymorphic markers

179
Q

what are the limitations of PGD that can lead to misdiagnosis?

A

confusion of embryo and cell line number
transfer of the wrong embryo
mat/pat contamination
allele drop-out
probe/primer failure
chromosomal mosaicism

180
Q

how can PGD impact epigenetics?

A

children born from ART are at a greater risk of suffering from an imprinting disorder -> 10% increased risk of BWS

this is because the epigenetic genome undergoes 2 waves of reporgramming
- in primordial germ cells
- during preimplantation genetic development

ART is though to interfere with this

however there is not a large cohort to study and this also makes analysis of trangenerational effects on imprinting difficult to study
- most parents will still chose to have PGD despite the associated risks

181
Q

what are some of the ethical concerns of PGD?

A
  • morally acceptable? many people put higher value on the fetus than embryo making PGD preferable to PND
  • the risk of designer babies if exaggerated and improbable providing there is adequate legislation
  • need to consider the severity of disease, age of onset (number of disease free years), treatments available
  • variable penetrance diseases?
182
Q

what is non-disclosure testing?

A

not permitted in most centers
client may not want to know if they have the disease but this is unethical to uphold if you find they have zero risk as they will be undergoing unnecessary and risky procedures for no reason

183
Q

Is HLA typing?

A

used to conceive a child that is a HLA to a sibling so they may be a stem cell donor for a critically ill sibling if no other donor is available

preferential procedure may be to harvest embryonic stem cells from matched embryos

184
Q

what is the reported rate of mosaicism in CVS and AF?

A

1-2% in CVS
0.1-0.3% in AF

lower level in AF likely represents pregnancy loss in the time between CVS and AF sampling and CPM

185
Q

give a brief description of embryology?

A

fertilisation –> zygote–> early blastocyst
- trophpectoderm goes on to form the placenat and extra-embryonic material
- ICM form the amnion and embryo

the amniotic cavity and embryo are formed from a different cell lineage from the chorion. The chorion differentiates early and is rapidly dividing do more likely to contain mitotic errors.

186
Q

what cells are in the AF?

A

AF contains cells shed from the embryo- skin, fetal urine, cells from the fetal respiratory tract

cell are derived directly from the phenotype and are more likely to be a true representative of the fetal genotype

187
Q

How can mosaicism develop?

A

Mitotic NDJ
Trisomy rescue

188
Q

what is level I , II and III mosaicism?

A

level 1- 1 cell in 1 culture - pseudomosaic

level 2- > 1 cell in 1 culture - may be pseudomosaic or true mosaicism

level 3 - > 1 cell in > 1 culture- likely to represent true fetal mosaicism and reported

type 1 and 2 may be a cultural artefact

189
Q

what are the BPG required work up for exclusion of mosaiscism in AF and CVS? Hsu and Benn 1999

A

basic- 20 cells from 2 cults, 1 of which may contain an abnormal metaphase

moderate- 20 cells from an additional separate culture

extensive - 20 cells from 2 additional separate cultures

190
Q

how can mosaicism be excluded in FBS?

A

single cell abn can be clinically significant and 30 additional metaphases should be screened

If FISH is used 50 cells should be screened for exclusion of mosaicism

191
Q

what are the BPG for reporting mosaicism?

A

pseudomosaicism is considered an artefact by application of Hsu and Benn

if level II mosaicism is sen on CVS it may be appropriate to mention on report and recommend additional follow-up with USS and AF

for level III mosaicism the number of cells in each cell line should be included in the report with the caveat that the levle may be different in other tissues

if a chr involved in UPD is involved UPD studies should be activated

192
Q

describe pallister killian

A

iso(12p)
- normal karyotype in peripheral blood due to the high turnover rate of cells and instability of the iso(12p) meaning it is lost from these cells
- can be detected in skin, AF, CVS
- only ever seen as mosaic

193
Q

what is a marker chromosome?

A

small piece of extra chromosomal material - smaller than chr 22 and unable to be clearly identified by G-banding alone

  • usually present in additional to the normal chromosome complement and termed supernumary marker chromosome or extra structurally abnormal chromosome
194
Q

what structure can a marker have?

A

ring
inv dup
centric minute

195
Q

what determines the phenotypic consequence of a marker chromosome?

A

size
genetic content
level of mosaicism
euchromatic/heterochromatic
result in copy number gain of the region in the marker

196
Q

what is the most frequent marker chromosome?

A

markers derived from chromosome 15 are the most common

197
Q

what causes cat eye syndrome?

A

+ psu idic (22)(q11.2)

198
Q

what causes Emanuel syndrome?

A

+ der(22)t(11;22)(q23;q11)

199
Q

When may marker chromosome be identified?

A

prenatal or postnatal array- detect presence of duplication and investigated by follow-up karyotype to confirm as a marker

karyotype investigation of an infertile male/ POF female

200
Q

what should be the management if a marker is detected?

A
  • determine genetic content
  • check to see if it is mosaic
  • test parents to inform on pathogenicity and recurrence risk
  • if marker involves a chromosome involved in UPD it is relevant to check for UPD for the sister chromosome for UPD
201
Q

How can the genetic content of a marker be determined?

A

arrayCGH best first option

If the marker only contains heterochromatin may require FISH to determine origin

chr 15 most common

202
Q

what is the consideration of inheritance of a marker?

A

if inherited more likely to be benign
- phenotype expected to be similar to parent

however may be different if mosaic as level and tissue distribution can affect penetrance

203
Q

how can cyto banding help ID a marker?

A

provide structural info
main limitation is resolution

can be used to determine level of mosaicism and is more sensitive than array

204
Q

what is the use of C-banding to investigate a marker chromosome

A

C-banding can be used to determine the amount of heterochromatic and determine if a marker is acrocentric or not.

can also determine the number of centromeres to ID is mono or dicentric

markers that are all het are expected to have low risk as they contain little active genetic material

205
Q

what is the use of silver staining to investigate a marker?

A

stains ribosomal RNA in the nucelolar organiation region (NOR)
- found on short arms of acrocentric chromosomes

206
Q

what is the use of FISH when investigating a marker?

A

can use sequential FISH
- if acrocentric start with 15
- limited benefit if inhertied from normal parent so may just want to start with chromosomes involved in UPD (6,7,11,14,15,)
- costly and inefficient

  • if abn pheno may also want to FISH for chr 12, 18 and 22 and iso chro associated with severe pheno
207
Q

Markers derived from sex chromosomes?

A
  • if karyotype is correlated with TS the marker is likely to be derived from a sex chr e.g. 45,X + mar
  • important to determine if Y chr derived due to the risk of gonadoblastoma
  • if X chr derived can test for Xist- if present marker will be inactive and more likely benign

XIST = X Inactive Specific Transcript

208
Q

what the use of array to investigate a marker?

A
  • can ID full extend of marker and hence gene content and clinical significance
  • may miss low level mosaic and het markers
  • can ID markers consisting or more than 1 chromosome
  • cannot ID the structure
209
Q

what should be considered if a chr15 marker is identified?

A

need to distinguish between benign small marker idic(15) and larger pathogenic marker idic(15) which contains the PWS/AS region and is associated with LD, epilepsy and ASD

210
Q

what should be considered if a ring chr marker is detected?

A

may see dynamic mosaicism and loss, gain, doubling up in different cells resulting in generalised ring syndrome and short stature

211
Q

what is cffDNA derived from?

A

cells apoptosed from the outer layer of the placenta - cytotrophoblasts and syncytiotrophoblast - therefore it is at risk of CPM

212
Q

from what gestation can cffDNA be tested?

A

can be observed from ~ 5 weeks but generally need to wait till 8-10 weeks before there is enough for reliable testing (high enough fetal fraction)

benefit is testing in early pregnancy

213
Q

what size cffDNA, how does this differ from maternal?

A

cffDNA is ~200-300bp long
shorter than mat cffDNA and this difference can be used to enrich for cffDNA for genetic testing ang

214
Q

what are the components of CVS?

A

inner mesenchyme, diofferentiates later and more closely related to fetus

outer cytotrophoblast

215
Q

when can CVS be sampled?

A

early CVS is associated with fetal limb defects

taken in 1st trimester 10-13+6 weeks gestation

216
Q

what is the miscarriage risk associated with CVS?

A

1-2% according to RCOG, risk influenced by experience of operator and due to infection, amniotic leakage or damage to the placenta

217
Q

How is CVS sampled?

A

10-25mg of CVS is taken from the placenta via a needle and syringe under USS guidance

218
Q

what is the advantage and disadvantage of CVS compared to AF?

A

can be taken in earlier gestation giving more time for pregnancy decision making and follow-up testing

Risk of CPM
slightly increased miscarriage rate

219
Q

what proportion of CVS show CPM?

A

1.5-2%

220
Q

what is the amniotic fluid and what cells does it contain?

A

fluid filled sac that surround the developing fetus and facilitates symmetric growth and lung development

cells shed from fetal skin, fetal urine and fetal respiratory tract = more representative of true fetal genotype

221
Q

how is AF taken?

A

transabdominally using syringe
10-20mls

222
Q

what is the miscarriage risk for AF?

A

0.5-1% RCOG

223
Q

what gestation can AF be taken?

A

2nd and 3rd trimester
from ~15 weeks, early AF has an increased miscarriage risk and low test success as not enough fetal cells. late gestation AFs are also more dirty and prone to failure

224
Q

when can fetal blood be sampled?

A

from ~ 18-20 weeks when veins are sufficiently developed

225
Q

how is fetal blood sampled?

A

blood is taken by needle from umbilical cord or fetal blood vessels

226
Q

what is the miscarriage risk for fetal blood sampling?

A

2%

227
Q

for what reasons may fetal blood be sampled?

A

most commonly for:
- suspected fetal blood disorders e.g. fetal anemia
- inconsistent CVS and AF result follow-up
- fetocide/MTOP
- immunologic tests

228
Q

what other prenatal samples may be taken?

A

pleural effusion when excess fluid removed
POC from miscarriage or TOP

229
Q

what is the main MCC risk in CVS and AF?

A

CVS- need to carefully dissect to remove the maternal decidua

AF- bloodstaining may be maternal (or fetal) also risk of tissue plug
- discarding the first draw of AF can reduce the risk of MCC

230
Q

what is the impact of culturing on MCC for CVS and AF?

A

culturing conditions for AF favors the growth of the amniocytes to reduce or eliminate mat blood cells

In CVS and POC the culturing process does not reduce the risk of MCC as the maternal and fetal tissue will both be grown

231
Q

what confluency of cultures is required for karyotype?

A

75% confluent monolayer- can be less for a targeted test of a few cells

232
Q

According tot CMGS MCC BPG when should MCC be suspected?

A
  • normal female in bloodstained sample
  • XX/XY mixed genotype
  • presence of mixed genotype on PCR
  • mosaicism in a female
  • uncertainty about the ID of a tissue sample in culture
  • slow growth, especially when originating from a single piece of tissue or a small number of colonies.
233
Q

when should MCC testing be performed?

A

if MCC suspected

for all monogenic tests- need to test mate sample against prenatal. lab use QF-PCR whihc has sensitivity to ~10% therefore can only state that significant MCC has been excluded
BP for the testing lab to do the MCC and needs to be on the DNA aliquot that was tested.

sensitivity of MCC assay may be lower than of the test so confirmation of result that are the same genotype as the mother may require confirmation y another method e.g. linkage following positive PCR result

should not issue report until MCC has been completed

234
Q

when should MCC testing be performed?

A

if MCC suspected

for all monogenic tests- need to test materal sample against prenatal. lab use QF-PCR which has sensitivity to ~10% therefore can only state that significant MCC has been excluded
BP for the testing lab to do the MCC and needs to be on the DNA aliquot that was tested.

sensitivity of MCC assay may be lower than of the test so confirmation of result that are the same genotype as the mother may require confirmation y another method e.g. linkage following positive PCR result

should not issue report until MCC has been completed

235
Q

How common in mosaicism in AF?

A

0.1-0.3% (lower than CVS)

236
Q

what is the clinical relevance of CPM?

A
  • observation of aneuploidy in CVS but not in AF = risk of UPD by trisomy rescue (chr 6, 7, 11, 14, 15)

most pregnancies with CPM continue to term with no abn but in some cases the abn in the placenta can lead to placental insufficiency and IUGR or other abn

237
Q

what factors play a role on the impact of CPM?

A

 Origin of error – somatic errors are associated with less severe consequences.
 Level of mosaicism – there is a correlation between a high number of aneuploid cells and poor pregnancy progress.
 Specific chromosomes – the influence of CPM on fetal growth is chromosome specific. Certain chromosomes carry imprinted genes involved in growth or placental function, which may contribute to impaired pregnancy progress when CPM is detected. CPM involving sex chromosomes usually has no adverse effect on foetal development.
 Type of chromosome abnormality – marker chromosomes are more often confirmed in the fetus than trisomies.

238
Q

How can CPM risk be minimised in prenatal testing?

A
  • test multiple villi and chop/digest to ensure mesenchyme and trophoblast are sampled
  • follow-up mosaic CVS with long term cult as mesenchymal core culture results are more likely to reflect a true fetal mosaicism than direct preparation
  • follow-up mosaic with AF and detailed USS

decision regarding pregnancy should not be made on the ????

239
Q

what is the difference between meiotic and mitotic CPM and what are the associated aneuploidies?

A

 Mitotic CPM – Mitotic non-disjunction can occur in a trophoblast cell or a non-fetal cell from the inner cell mass creating a trisomic cell line in the tissue which is destined to become the placental mesoderm. Trisomy 2, 3, 7 and 8 are derived mitotically.

 Meiotic CPM –Due to trisomy rescue of a trisomic conceptions Trisomy 16 and 22 are derived meiotically.

240
Q

what factors influence the pattern of normal and abnormal cells in the developing embryo?

A
  • reduced or improved replicative rates of the normal and abnormal cell lines
  • abnormal cells may fail to differentiate or function properly
  • may be no selection against abnormal cells but there presence could compromise the pregancy e.g. placedntal insufficiency, IUGR
241
Q

what are the 3 types of CPM and there prevalence?

A

 Type I, the abnormal cell line is confined to the cytotrophoblast (40%)
 Type II, it affects only the mesenchymal cells of the stromal villous core (40%)
 Type III, it involves both tissues. (7%)

Type I and II CPMs are usually the consequence of nondisjunction events, and type III is the result of “trisomy rescue”

type III carries a risk of CPM in the rescued diploid fetus

242
Q

which mosaics are more likely to involve the fetus and hence considered high risk?

A

trisomies 8, 9, 12, 13, 15, 18, 20, and 21

243
Q

what is the impact of CPM on arrayCGH

A

Karampetsou et al. (2014) report a case of CPM involving a deletion of exons 7-10 of the STS gene detected by array-CGH using DNA extracted from uncultured CVS.

therefore suggested that reports on uncultured CVS should include the rider that CPM has not been ruled out and ideally confirmation should be on cultured CVS or AF

FRC Path 2023 (15 decks)

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