Prenatal Cytogenetics Flashcards
Describe the features associated with mosaic trisomy 16
IUGR, IUD, preeclampsia, preterm delivery, neonatal death, CHD
Most common trisomy seen in spontaneous abortions
What are the 2 types of Triploidy?
Diandry - Type 1 - Double paternal contribution
Digyny - Type 2 - Double maternal contribution
What is the most common form of Triploidy?
Type 1 - Diandry - Double paternal contribution
What is the placental presentation of Type 1 Triploidy?
Double paternal contribution - Cystic villi that have trophoblastic hyperplasia –> Partial hydatidiform mole
Define Partial Hydatidiform Mole
Subtype of Hydatiform mole forming in triploid pregnancy with double paternal contribution - Cystic villi that have trophoblastic hyperplasia
Give 3 scan findings of a Diandric triploidy
Symmetrical IUGR,
Structural abnormalities including neural tube defects,
Large, cystic placenta,
Oligohydramnios
What is the risk to the mother in a Diandric triploidy?
0.5% risk of a malignant invasive mole
Give 2 mechanisms by which a diandric triploidy may arise
Fertilisation by 2 sperm (dispermy),
Normal egg fertilised by a diploid sperm
What is the placental presentation of Type 2 Triploidy?
Non-hydropic villi, no evidence of molar placenta, generally small
Give 3 scan findings of a Dygynic triploidy
IUGR - often asymmetrical,
Large head,
Oligohydramnios,
Holoprosencephaly
Give 2 mechanisms by which a dygynic triploidy may arise
Fertilisation of a diploid egg by a haploid sperm,
Retention of a polar body in a fertilised egg,
Fertilisation of an ovulated primary oocyte,
Fusion of 2 eggs (dieggy) and fertilisation by a haploid sperm
What may explain the different clinical presentations of type 1 and type 2 triploidies?
Different imprinted states
Give 3 clinical features of triploidy
Face to chest fusion, Limb growth/development retardation, Growth disorganisation, Midface dysplasia, Syndactyly, Heart defects, Renal defects
What is the most common form of gestational trophoblastic disease?
Hydatiform mole
Under WHO classification - give 2 examples of pre-malignant gestational trophoblastic diseases
Complete hydatiform mole,
Partial hydatiform mole
Under WHO classification - give 2 examples of Malignant gestational trophoblastic diseases
Invasive mole,
Choriocarcinoma,
Placental site trophoblastic tumour
What is the unique biochemical marker in gestational trophoblastic disease?
Elevated hCG (human chorionic gonadotrophin) - useful for early detection, diagnosis and follow-up
What causes a Complete Hydatidiform Mole?
Diploid androgenetic pregnancy - both chromosome sets paternally derived
Give 3 clinical presentations of complete hydatidiform moles
No fetal development,
Extensive hydrops,
Swollen villi and marked widespread hyperplasia of trophoblast,
Maternal hypertension, oedema and vaginal bleeding
What is the recurrence risk following a complete hydatidiform mole?
1 in 100 - rising to 1 in 4 following 2 consecutive CHMs
What is the genetic difference between sporadic complete hydatidiform moles and familial recurrent hydatidiform moles?
Pregnancies are genetically biparental in origin
Describe Familial recurrent hydatidiform moles
Maternal-effect AR condition - very rare,
75% pregnancies develop as CHM
NLRP7 (19q13.42) and KHDC3L (6q13) both thought to have role in maintaining maternal imprinting within the ovum
What is the risk of developing an invasive mole from a complete hydatidiform mole?
1-15%
Describe Gestational Choriocarcinoma
Bleeding - local disease,
Metasteses - causing symptoms at site
Highly malignant tumour of uterine wall - 3% risk following CHM and 0.1% risk following partial mole,
Increased levels of hCG
Describe Placental Site Trophoblastic Tumour
Least common form of GTD - commonly follow normal pregnancy,
Presentation varies from slow growing disease limited to uterus to a more rapidly growing metastatic disease similar to choriocarcinoma
PSTT is diploid and arises from the non-villous trophoblast
What treatment and monitoring is offered following gestational trophoblastic disease?
Suction evacuation for partial and complete moles,
Monitor hCG levels for up to 2 years following CHM and 6 months following partial mole,
Pregnancy should be avoided during monitoring
Describe the formation of Oogonia
During embryological development, diploid primordial germ cells migrate to embryonic ovary - undergo rapid mitosis –>approx 7 million oogonia
What happens to oogonia following 7th month of gestation?
Majority die.
Rest enter meiosis 1 –> primary oocytes progress through first meiotic prophase until diplotene - arrest until puberty
What phase are primary oocytes arrested in until puberty?
Diplotene of M1
What happens to primary oocytes at puberty?
Groups periodically resume meiosis - by ovulation oocytes are in Metaphase 2 of Meiosis 2
When is meiosis complete during oogenesis?
After fertilisation
During telophase of oogenesis - describe the 2 daughter cells produced
Small daughter cell with virtually no cytoplasm - 1st polar body,
Larger cell with nearly entire volume of cell contents - secondary oocyte
Describe the secondary oocyte
Cytoplasm contains large number of mitochondria, ribosomes, DNA/RNA polymersases, protective chemicals and morphogenic factors.
Plasma membrane surrounded by thick extra cellular matrix - Zona Pellucida - also surrounded by a layer of foccile cells - cumulus cells - nurture the oocyte before and just after ovulation
Define fertilisation
Process by which an egg and sperm fuse to become a new individual - usually occurs in ampulla of fallopian tube
What is the cortical reaction in fertilisation?
Cortical granules inside oocyte fuse with plasma membrane of the cell - enzymes inside these granules are expelled by exocytosis to the ZP - causes glycoproteins in the ZP to cross linke –>matrix become impenetrable
Why does the zona pellucida degrade in the zygote?
Allows implantation of the blastocyst following migration to the uterus
Define cleavage
Zygote divides repeatedly to form smaller cells (blastomeres) - cell divsions are slow and asynchronous
Define rotational cleavage
1st division is in the vertical plane, in the second round one cleaves vertically and one horizontally
Define compaction
Loosely associated blastomeres of 8 cell embryo flatten against each other to form the Morula - tight packaging seals off sphere and outer cells secrete fluid to form hollow ball of cells, inner cells form gap junctions enabling small molecules and ions to pass between them
What is the fluid filled cavity in the morula called?
Blastocele
Describe the blastocyst
At 16 cell stage can discriminate cells - outer layer (trophoblast) –>CHORION
Inner non-polar cells congregate at one end of blastocele –> INNER CELL MASS
Describe the Inner Cell Mass
Goes on to form all cells of the organism plus 3 other extraembryonic membranes; the yolk sac, amnion and allantois
What happens if splitting occurs at the blastocyst stage?
Monozygotic twins
When does implantation occur during embryogenesis?
Day 5 - Day 6
How does implantation occur during embryogenesis?
Day 5: Enzyme released that bores hole through ZP, causes it to partly degrade and releases blastocyst.
Day 6: Blastocyst attaches tightly to uterine epithelium (implantation).
Trophoblast cells rapidly proliferate and differentiate
Following implantation what do the trophoblast cells differentiate in to?
Inner layer - cytotrophoblast
Multinucleated outer cells layer - Syncytiotrophoblast - invades connective of uterus
Describe the differentiation of the Inner Cell Mass prior to implantation into 2 distinct layers
External layer (epiblast or primitive ectoderm), --> Ectoderm, endoderm and mesoderm + 3 extra embryonic membranes Internal layer (hypoblast or primitive endoderm), --> Extraembryonic mesoderm
What are the fluid filled cavities either side of the bilmainar disc?
Amniotic cavity and yolk sac
Describe gastrulation
The orientation of the body is established and the trilaminar disc is formed - occurs in week three, rapid cell movement
Describe the trilaminar disc
Endoderm, Ectoderm and Mesoderm
What tissues does the Ectoderm form?
Skin (and derivatives)
Nervous System
What tissues does the Mesoderm form?
Blood,
Vessels,
Muscular tissue,
Connective tissue
What tissues does the Endoderm form?
Lung, Liver, Pancreas, Thymus, Endocrine glands
What is cell-free fetal DNA
cffDNA first described by Dennis Lo et al in 1997 is made up of fragments (approx 200-300bp) in the maternal plasma derived from the placenta
When can cffDNA be detected in the maternal plasma?
From 4-5 weeks, reliably from 7, concentration raises with gestation and is rapidly cleared from the maternal blood stream following delivery
Describe 2 challenges associated with distinguishing/isolating cffDNA
1) Concentration of cfDNA is low
2) cffDNA only makes up 10-20% of cfDNA
3) Half of fetal genetic material is maternally derived - indistinguishable from cfmDNA
Give 3 clinical applications of NIPD
1) Fetal sex determination where there is F/H X-linked disease
2) Diagnosis of certain single gene disorders - particularly paternally inherited mutns
3) Detection of fetal aneuploidy
4) Fetal blood typing in RhD-ve mothers
Give 3 disorders where sex determination of the fetus by NIPD may be clinically useful
1) CAH
2) DMD
3) Adrenoleukodystrophy
How is NIPD for fetal sex determination carried out?
Blood spun to isolate plasma - circulating nucleic acids isolated and RT-PCR then used to identify SRY - quantitative test
How can you avoid false negative results in fetal sex determination by NIPD?
Having minimum cutoff for cffDNA present,
Testing for universal fetal markers (paternally derived SNPs or CCR5),
Carry out replicate testing
What restricts the identification of large scale abnormalities in NIPT?
The fragment lengths of the cffDNA - approx 200-300bp
Give 2 examples of placentally expressed genes useful for quantifying fetal fractions in NIPT
SERPINB5 - hypomethylated in placenta, hypermethylated in maternal cells,
RASSF1 - hypermethylated in fetal tissue, maternally hypomethylated
What is the main limitation of using a hypomethlyated marker in fetal fraction determination in NIPT?
Bilufite conversion massively depletes input DNA - use of hypermethylated fetal cells as hypomethylated maternal cells can be removed
How can cffDNA be enriched within a sample?
Targeting short fragments - maternal fragments tend to be longer
Give 2 methods of fetal aneuploidy determination in NIPT
1) Targeting fetal specific RNA/DNA
2) Direct measurement of chromosome dosage
Give 3 benefits of NIPT/D
1) Non-invasive testing much safer - reduce in miscarriage
2) Cheaper for FMUs - less expertise to take blood than an invasive sampling test
3) Potential for earlier diagnosis
4) Less trauma for parents
5) Potential to improve quality of care
Give 3 limitations of NIPT/D
1) In multiple pregnancies cannot distinguish fetuses
2) Proportion of cffDNA in women with high BMI is reduced - higher false negatives and inconclusives
3) Source of cffDNA is placenta - CPM etc
4) False negatives - vanishing twins, triploidy, low fetal fraction
5) Can be complicated by maternal abnormalities
What is DANSR?
Digital Analysis of Selected Regions
Describe DANSR
Enables targeted sequencing of selected loci (SNPs) from specific chrms.
Locus specific DANSR oligos anneal to cfDNA, their terminals form 2 nicks, ligation of these nicks results in product capable of amplification
How does DANSR enable estimation of fetal fraction in NIPT?
Primers for non-polymorphic loci on target chrms give counts of chrms and primers for polymorphic loci on chrms 1-12 facilitate differentation between maternal and fetal DNA
Give 2 types of result to always be reported in prenatal microarray context
Any variant that will potentially inform management of pregnancy, high penetrance neuro-suscept loci with severe pheno, Neuro-suscept loci with associated abnormalities that could be seen on scan, unsolicited pathogenic findings
Give 3 egs of CNVs not to be reported in prenatal microarray context
15q13.1q13.3 Dups, 15q11 BP1-BP2 dels/dups, Xp22.31 (STS) dups, 16p13 dups, heterozygous dels of recessive genes that cannot be linked to phenotype
What 2 considerations should be given when writing prenatal microarray reports of uncertain results?
Do not include patient support leaflets, clinically actionable unsolicited pathogenic CNVs should include clear comment that they are unrelated to presenting phenotype
