Session 5 - Prenatal testing Flashcards
What methods of prenatal sampling are there? At what gestation can they be sampled?
cffDNA - 7+weeks (9+ ideal) CVS - 11-13wks AF - 15-17wks FBS - 20+ wks POC Foetal skin/biopsy
What type of mosaicism are found in prenatal samples?
MCC
CPM
Pseudomosaicism
What three levels of mosaicism are found in prenatal testing? How are they defined?
level 1 - one cell (artefact)
level 2 - multiple cells, once culture (pseudomosaicism)
level 3 - multiple cells, multiple cultures - may represent CPM or true foetal mosaicism.
Why does MCC occur less in AF than in CVS/POC?
Because culture conditions select for amniocyte growth.
According to CMGS BPG, when should MCC be suspected?
- When there is a mix of XX/XY cells
- When a male foetus is XX
- When tissue sample is of unknown origin
- Normal cells in an otherwise abnormal result
- Slow cell growth in culture.
List some BPG recommendations for MCC testing
Performed by testing lab
Should be more sensitive than diagnostic test
“no evidence of significant MCC” or “MCC cannot be ruled out” on report.
Confirm results on cultured cells, rather than uncultured
2 informative markers required
MCC test should be able to detect MCC at levels of 10%+
At which stages can CPM occur?
Mitotic - occurs after non-disjunction in trophoblast or non-foetal tissues
Meiotic - trisomy rescue. (trisomy 16 and 22)
How can CPM affect foetal development?
Reduced replication rates of abnormal cells
Abnormal differentiation of abnormal cells
Cause abnormal placentation
There are three types of CPM. Name them.
Type 1: Affects trophoblast cells only
Type 2: Affects mesenchyme cells only
Type 3: Affects trophoblast and mesenchyme cells
What factors contribute to the likely effects of CPM?
Type of mutation
Origin of mosaicism (somatic, meiotic)
Proportion of WT/M cells
Specific chromosome
What procedures can be put in place to minimise CPM in prenatal cultures?
Mesenchyme core
Take more than one frond from different regions of the CVS biopsy
Enzymatic digestion of the biopsy
Never terminate based on mosaic results in a CVS.
Describe the stages of oogenesis.
- Primordial germ cells migrate to the embryonic ovary and proliferate (mitosis) to form oogonia
- Oogonia enter Meiosis I and progress is halted at diplotene to form primary oocytes
- Primary oocytes resume meiosis during puberty, expel PB1 and are halted at metaphase of meiosis II until fertilisation. This forms the secondary oocytes.
- Ovulation and subsequent fertilisation trigger completion of MII and the second PB is produced.
Describe the stages of Spermatogenesis
- primordial germ cellsmigrate to the embryonic testis and begin mitosis to generate spermatogonia
- Some spermatogonia differentiate to form primary spermatocytes.
- spermatocytes undergo two rounds of division (MI and MII) to generate the spermatids
- Spermatids mature under the control of the sertoli cells to form mature sperm.
- Mature sperm migrates from seminiferous tubules to the epididymis.
Describe the process of fertilisation:
Sperm attaches to follicular cells of oocyte
Sperm reaches ZP
Acrosome reaction allows sperm to penetrate ZP and head is released into the oocyte cytoplasm
Cortical reaction prevents further sperm fertilising egg
Oocyte undergoes second meiotic division for release second PB
Sperm and egg nuclei become the PN
Sperm and egg membranes fuse to complete the first mitosis and generate a diploid cell zygote
List the stages of embryogenesis
Cleavage Compaction Blastocyst and inner cell mass formation Implantation Gastrulation
Which three layers of cells form during gastrulation? Which organs do they go on to form?
Endoderm - Lung, Liver, Pancreas, endocrine glands
Mesoderm - Blood, muscles, connective tissue
Ectoderm - skin, nervous system
What is a Diandric triploid?
Two paternal sets of chromosomes, one maternal set.
What else is diandric triploidy known by?
Partial hydatidiform mole
What are the features of a diandric triploid?
Symmetrical IUGR, normal head size, cystic placenta, high maternal hCG (80% of cases)
How do diandric triploids arise?
dispermy (one egg, two sperm)
fertilisation of a normal eg with diploid sperm
What is a Digynic triploid?
Two maternal sets of chromosomes, one paternal set
What are the features associated with a digynic triploid?
small placenta, macrocephaly/small body, IUGR and body asymmetry, holosprosencephaly
How do Digynic triploids arise?
Fertilisation of a diploid egg by one sperm
Retention of first polar body
fertilisation of an ovulated primary oocyte (still in MI)
fusion of 2 eggs then fertilisation by one sperm.
What is the recurrence risk associated with triploidy?
mostly sporadic
partial moles 1-1.5%
some recurrence of digynic triploidy in a few families.
What is a hydatidiform mole?
The most common gestational trophoblastic disease
What types of gestational trophoblastic disease are there?
Non-malignant complete and partial moles
Malignant: invasive moles, choriocarcinoma, PSTT
What biochemical marker can be used to detect gestational trophoblastic disease?
Raised hCG
What is a complete mole? What is the incidence?
46,XX (90%) or 46,XY (10%) conceptus with all chromosomes originating from a single parent.
1/1000
What are the features associated with a complete molar pregnancy?
No foetal development
Large molar placenta
hydrops
hypertension, oedema and bleeding in the mother
What are the causes of complete molar pregnancies?
20% dispermic fertilisation of a nullisomic egg
80% monospermic fertilisation with the male PN duplicating to form a diploid nucleus.
How do mothers with molar pregnancies present?
Vaginal bleeding, hypertension, pre-eclampsia, hyperemesis, very high levels of hCG
What is the risk of a complete mole in a future pregnancy?
1/100
1/4 if a patient has had two previous molar pregnancies.
Describe Familial Recurrent Hydatidiform Moles.
Very rare
AR maternal effect
Mutations in KHDC3L and NLRP7 cause problems maintaining the maternal imprint. Mothers will never be able to conceive a healthy child.
Conceptuses 46,XX or 46,XY with biparental contributions.
List some malignant forms of Gestational Trophoblastic Disease
Invasive moles - arises from a complete mole
Choriocarcinoma - 3% risk following molar preg
Placental Site Trophoblastic tumours - can occur after normal or molar pregnancies - 3.4yrs post-pregnancy.
All show increased hCG
How can gestational trophoblastic diseases be treated?
monitor hCG - 6mo-2yrs - avoid pregnancy until returned to normal
low toxicity chemo (methotrexate)
Suction evacuation
Which pathway is involved in familial twinning? how does it work?
TGF9 signalling pathway. Promotes ovulation of >1 oocyte at a time
What is the most common gender balance of dizigotic twins?
Male-Female in 50% of cases
What increases the chances of a dizygotic pregnancy?
Increasing maternal age, up to age ~37
How do monozygotic twins arise?
One zygote splits to form two embryos. The point at which this happenes determines the sort of MZ twin:
DC/DA - 1/3 of MZ twins - separation occurs before the morula stage
MC/DA 2/3 of twins - occurs at the blastocyst stage
MC/MA - very rare - forms post amnion development. Occasionally conjoined.
List some problems associated with twinning
Early delivery, increase in perinatal mortality, IUGR, increased pressure on mother during pregnancy.
Prenatal diagnosis is complicated - need to determine if twins are DC or DA as this will affect sampling
Conjoined twins
Parasitic twins
Twin reversed arterial perfusion
TTTS
What causes TTTS? How can TTTS be treated?
Placental joining of arterial flow from one twin to vein of another.
Treat by amnioreduction
Laser ablation of vessel connection
When should you be particularly aware of TTTS?
When testing for BWS
How can Vanishing twin complicate prenatal diagnoses?
cffDNA can remain in the bloodstream for up to 8wks post demise.
Cause misdiagnoses. the vanished twin often vanished because of chromosome abn
All cffDNA tests for NIPT should be accompanied by an early USS
Females diagnosed as male
What is zygosity testing used for?
To determine the degree of identity in the genome of twins.
Why is zygosity testing requested?
when one twin develops symptoms of a disease - used to determine recurrence risk in other twin
HLA-matching for transplant patient
How is zygosity tested for?
Use microsatellite markers from parents and both twins. calculate the probability of inheriting all the same markers by chance.
How many PCR cycles are used for QF-PCR?
24
How many markers should be tested per chromosome in QF-PCR?
4
How large (repeats) are the majority of markers?
4bp - reduces stutter
How large must QF-PCR primers be?
> 22bp
Which sex chromosome markers are often used?
AMEL - present on X and Y, but 4bp difference
X22 - on Xq (PAR2)
DXYS218 - on Xp (PAR1)
HPRT
SRY - non-polymorphic, detec males
TAF9 - present on chr3 and on X - 2bp different in lnegth - used to determine relative copy number of the X chromsome
What are the normal allele ratios for 1:1? When is this not true?
0.8-1.4
This is extended to 1.5 if the two markers are >24bp apart to account for preferential amplification of the smaller allele
How are allele ratios calculated?
by dividing the size of the smaller allele peak by the larger allele peak.
How many informative markers are needed to report a normal result?
- 1 can be used but must be clarified on report
How many informative markers are required to report a trisomic result?
- All other markers must be uninformative
Why is it useful to observe 3 1:1:1 peaks in a trisomic foetus, rather than 2 2:1 peaks?
1:1:1 ratio is indicative of non-disjunction at meiosis, and means that CPM is very unlikely.
How can inconclusive results be investigated and reported?
Use additional markers for single chromosomes.
Cannot report if all ratios are inconclusive, or if there are normal ratios for any of the markers.
If an abnormal result is identified, how should this be confirmed?
Repeat test, confirm on cultured cells (rule out MCC in AF), can be confirmed by FISH. Shouldn’t be reported until result confirmed.
How is MCC evident in a QF-PCR result?
Skewed allele ratios. Additional peaks (the two smaller peaks should add up to the large peak)
How does best practice state MCC should be dealt with?
Run maternal sample.
- If low level MCC report result
- if single foetal genotype report result
- if inconclusive - don’t report.
How does mosaicism show on QF-PCR?
skewed peaks/extra alleles for a particular chromosome. e.g. mosaic Trisomy 21 or mosaic 45,X
Diploid/triploid mosaics will look like MCC
What can cause normal and abnormal patterns on a single chromosome?
Somatic microsatellite mutation - increase of 1-2repeats
Polymorphic submicroscopic duplications - abnormal single marker flanked by normal markers
Partial chromosome imbalance - normal and abnormal results on one chromosome. If telomeric may suggest translocation. Need two consecutive markers to report
CNVs - abnormal markers flanked by normal markers - may be benign inherited, may require further follow-up
Primer binding site polymorphism - reduce annealing temp
Homozygosity - all markers uninformative
What technical factors might cause interpretation problems?
Stutter peaks - taq slippage
Spikes - genetic analyser artefact
Bleedthrough - dye blobs.
What advantages does QF-PCR have over FISH?
High throughput Better resolution Can detect MCC and UPD (possibly) Less volume of AF required Easier Cheaper Can detect some unbalanced translocations
What statements should be used when reporting QF-PCR results?
Results assume that the tissue analysed was foetal in origin
The results ARE CONSISTENT WITH…. This is CONSISTENT with a diagnosis of….
For cases with trisomy detected and all 2:1 markers - CPM cannot be excluded. May recommend waiting for karyotype results, especially in the absence of USS abnormalities.
Regarding prenatal array analysis. What did Huang and Crolla (2010) conclude about the use of higher density arrays?
That increasing the resolution did not result in higher detection rate of pathogenic findings, but did result in an increased detected rate of VUSs and benign findings.
List the advantages of microarray analysis for prenatal diagnosis.
Higher resolution that karyotyping, dividing cells/cultured cells not required, doesn’t identify carriers of balanced familial translocations.
List the disadvantages of microarray analysis for prenatal diagnosis
High quality DNA required
Increased detection of incidental findings
Increased detection of VUSs due to higher resolution
No balanced rearrangement detection
Lower level mosaicism may be missed - this may be picked up on karyo/FISH screen
Triploidy missed
Difficult to meet TAT if follow-up studies required
Increased costs if follow-up required
What cautionary measures can be taken when introducing an array service for PND?
Use the same array for PND and post-natal diagnosis to utilise in-house expertise.
Minimise false negatives by using a high res array
Why can a CNV inherited from an unaffected parent not be automatically classed as benign?
- Imprinting
- Unmasking recessive allele in proband
- Variable penetrance
What where the eligibility criteria for the EACH study?
1 or more structural abnormality on the 11-14wk or 18-20 week scan
isolated NT >3.5mm at the 11-14wk scan
Which two arrays were used in the EACH study?
Nimblegen 12*135K array
ISCA 8*60K oligo array
What results were reported from the EACH study?
Only de-novo variants consistent with a diagnosis of a known microdeletion/duplication syndrome
Any other significant imbalances identified suggestive of an unbalanced translocation.
What is the required turnaround time for a prenatal array?
14days
Which variants does the BSGM recommend are never reported back to patients?
15q13.1q13.3 duplications
15q11 BP1-BP2 duplications or deletions
Xp22.31 (STS) duplications
16p13 duplications
heterozygous deletion of recessive genes that cannot be linked to the presenting phenotype.
For foetuses with multiple abnormalities, what is the expected mutation rate using CGH?
What is the expected rate for foetuses with abnormalities of a single organ system?
~9%
3-7%
List the advantages of QF-PCR over interphase FISH
high throughput multiple markers/chromosome cheaper (£5 compared to £40 per test) lower failure rate (0.09% vs 0.7-3%) Detection of MCC Quality not affected by gestational age
What factors can be used to determine risk of T21 to a pregnancy?
MSS markers
USS scan
Maternal age
NT measurement
What is the risk of a DS baby at 20yr old and 40 yr old?
1/1500
1/85
According the the UK NSC, what should all pregnant women be offered?
Information to help them decide if they want screening
A screening test for DS that meets national standards
An USS at 18-24weeks to check for physical abnormalities
What is the UK cutoff for high/low risk pregnancies after the combined test?
1:150
What proportion of women with a DS foetus choose to terminate the pregnancy?
91-93%
How is the NT measurement used for screening?
Measured at 11-14 weeks.
>3.5mm indicative of chromosome abn, heart problem
Can also be measured at 18-20weeks; should be <6mm
How is MSS used for screening?
Use MoM (calculated per marker) - divide mother’s values by median of concentration of unaffected pregnancies at same gestational age.
What maternal factors should be taken into account when calculating risk from MSS?
Maternal age, weight, ethnicity, smoking, gestation, diabetes, twinning (PAPP-A is lower in MC twins than in DC twins)
Which is the recommended test for Fetal Anomaly Screening? What markers are screened and when does the testing take place?
Combined test.
Performed at 10-14 weeks
NT, PAPP-A and b-hCG
Which test outperforms the Combined test? Which markers does this screen? Why is this not the recommended test?
1st Trimester: NT, bhCG, PAPP-A
2nd Trimester: integrin A, uE3, AFP
25% of patients don’t turn up to their second test.
Which additional disease did UK NSC recommend are included in the test for DS in 2014?
T13 and T18
At what gestation is the foetal anomaly scan performed?
18-21 weeks
Which normal variant markers are considered “soft” USS markers and should not be reported?
Choroid plexus cysts, mega cisterna magna, 2-vessel cord, echogenic foci in heart
Which normal variant markers are considered worth following up?
Ventriculomegaly Echogenic Bowel Increased nuchal fold (>6mm at 18-21 wks) Renal pelvic dilatation IUGR - small for gestational age
What is the future for prenatal screening programmes in the UK?
NIPS likely to replace MSS as it is more sensitive and specific - decrease the number of invasive tests required.
Proteomics of the maternal serum - profiling the proteins present in the maternal serum to detect markers indicative of foetal trisomy. Use SELDI-TOF, MALDI-TOF and SDS-PAGE
What are the sources of fetal tissue in the maternal circulation?
Intact fetal cells
cffDNA
cffmRNA
Why are intact fetal cells unsuitable for NIPT currently?
They are present in too smaller numbers
They persist for years post-delivery
Lack distinct markers for easy enrichment
What proportion of cfDNA in the maternal plasma is fetal in origin?
5-20%
When does cffDNA appear in the maternal plasma? When is it at high enough concentration for testing?
Week 4-5.
Week7+
Where does cffDNA originate?
From placental cells
What size range are cffDNA fragments
200-300bp
Why is placental-derived cffmRNA unusual?
It is present in the plasma in a stable form. IT is bound to trophoblast-derived microparticles that protect it from nuclease degradation
What is different between cffmRNA and cffDNA?
cffmRNA doesn’t increase with gestation
What are the technical challenges associated with cffDNA?
The relatively low concentration in maternal plasma
The mixture of maternal and foetal fragments
Half of the alleles seen in the foetus will also be present in the mother
Name some clinical applications for NIPD
Foetal sexing before further testing for X-linked or sex-limited disorders.
Diagnosis of single gene disorders
RHDO
Trisomy testing
Foetal blood type - RhD testing
How is foetal sexing performed. What is the information used for?
How accurate are the results?
Detect presence of SRY in maternal plasma - absence of SRY indicative of female foetus.
False neg results may be a results of too little cffDNA - run repeats, quantify cffDNA using foetal specific markers (CCR5 or paternal SNPs)
Use prior to DMD testing, CAH testing (dexamethosone treatment from week 6+ if female) etc.
99.5%
Which single gene disorders are currently able to be tested by cffDNA?
Achondroplasia Deafness Aperts HD TD
What are the problems encountered during NIPT for single gene disorders?
Can only detect paternally-inherited alleles and de-novo alleles.
Large deletions (>300bp) cannot be detected
How can NIPT be used to screen for recessive disorders?
IF paternal allele is absent, and low new mutation rate, then foetus can be identified as a likely carrier. This reduces the invasive procedure numbers by 50%
Exclusion tests have been used by Lo et al to diagnose B-thal; if the foestus has inherited the normal paternal allele they are unlikely to be affected.
Relative mutation dosage in cases of both parents carrying the same mutation - compares mutant vs normal alleles
Name foetal specific markers be used to confirm present of cffDNA
Paternal SNPs cannot always be used - may be absent or homozygous.
Epigenetic markers:
SERPINB5 (Maspin) is on chr 18 and is hypomethylated in placental tissue. As Maspin is hypermethylated in the maternal blood this can be detected by b-s modification and ms-PCR
RASSF1 is hypermethylated in the placenta and hypomethylated in the maternal tissues - use methylation sensitive enzymes to digest maternal fraction.
What are the limitations of of the epigenetic marker approach to identifying foetal cffDNA presence in maternal plasma?
Bisulphite modification of DNA causes it to degrade (up to 95% loss of sequence).
What methods of cffDNA enrichment are available?
Enrich by size
Detect hypermethylated markers, only - digest maternal fraction
What methods can be used to identify aneuploidy by NIPT?
Target foetal-specific nucleic acids - calculate the RNA:SNP allele ratio (need polymorphic markers in these genes)
Direct measurement of chromosome dosage (dPCR, NGS)
What markers are available on which chromosomes to quantify chromosome number using NIPT?
PLAC4, not expressed in mother, present on Chr21
HLCS, on chromosome 21
SERPINB2 cffmRNA on chromosome 18
SERPINB5 hypomethylated in placenta on chromosome 18
What is the principle of measuring chromosome dosage?
What methods can be used to detect direct measurement of chromosome dosage?
Comparing the number of reads/copies of a sequence on chromosome A to chromosome B. If more reads are generated from chromosome 21, trisomy can be detected
dPCR
NGS
What is the reported sensitivity and specificity of NGS to detect aneuploidies by chromosome dosage?
For T21 and T18 it is close to 100%
Which large scale study has proven that NGS can be used to detect foetal aneuploidy, accurately?
Chitty, et al (2015) used NGS to identify mutations in FGFR2 associated with Achondroplasia and thanatophoric dysplasia - RAPID
What is RHDO?
Relative haplotype dosage. It is used to track foetal inheritance in a region in LD with the mutated allele
What is required for RHDO?
Parental samples
Robust statistical analysis
Money! It’s very expensive.
What are some future possibilities for NIPD?
WGS of a foestus from maternal blood
Widespread roll out of testing in the NHS - though it is now recommended by UK NSC
Single cell screening - move back to intact foetal cells?
Which current projects are involved with/working on the study of cffDNA?
PHG foundation SAFE RAPID NIPSIGEN PROOF
List some benefits of NIPT
Less invasive
Reduced pregnancy loss
More acceptable -> more woemn take up screen
Earlier diagnosis
Les risk to mother (infection etc.)
Specialist operatives not need to sample CVS/AF
List some limitations of NIPT
Expensive
Won’t work for multiple pregnancies
Not good in fat women
invasive test may still be required to confirm an abnormal result
CPM may be a problem as the cffDNA is derived from the placenta
False positives and false negatives are still a risk
Name some ethical issues to consider concerning NIPT
Use of the technology - could be applied to sex-selection, minor abnormalities, paternity testing
Increased secondary findings in WGS, equity of access, appropriate consent.
What can be used to enable structural variant detection by NGS in PND?
Paired-end reads
Which diseases were investigated by Lench et al (2013) to identify mutations prenatally using NGS?
PKD, Fraser syndrome, Skeletal dysplasia
Describe the 2015 study by Drury et al on exome sequencing for foetuses with USS abnormalities
Test 24 cytogenetically normal pregnancies with abscan
Used excess CVS or FBS
Group split into 2 cohorts: 1st cohort (n=14) - proband only; 2nd cohort trios (n=10)
Used a combination of clinical expertise and filtering to determine likely pathogenic variants in both cohorts
Results Sanger confirmed
Definitive diagnosis in 5/24
Likely diagnosis in 1/24
2/24 had results suggestive of AR inheritance
2/24 had incidental findings
What is the PAGE project and what does it seek to do?
1000 exome/genome of foetuses with abscan.
Trios
DNA from CVS/AF/POC
It seeks to identify the relative contribution of different mutation types to USS findings
To introduce testing into the NHS as a diagnostic service
To develop a cost-effective test/assay for improved prenatal diagnosis of abscans
What is the EACH study and what does it seek to do?
Evaluation of the use of aCGH in prenatal diagnosis for foetuses with structural abnormalities and normal QF-PCR.
Aims to detect additional pathogenic abnormalities by aCGH, determine the best array platform for prenatal arrays, determine whether cost/TAT are appropriate for prenatal testing and find out about patient attitudes to testing
What is the RAPID study and what does it seek to do?
Aims to improve quality of care to patients undergoing prenatal testing in the NHS by reducing the number of invasive procedures performed.
Objectives:
Develop lab standards
Establish sensitivity and specificity of testing for DS
Economic evaluation
Develop educational material for people undergoing testing
Analysis of healthcare needs to support commissioning
What NIPT tests are now available after RAPID?
Achondroplasia (FGFR3)
Apert syndrome (FGFR2)
Sexing
Paternal exclusion of CF mutation
What is the screening ‘high-risk’ cut-off for patients entering RAPID?
1:1000
What two categories of preimplantation testing are there?
PGD
PGS
Why is PGS not available on the NHS?
Studies have shown it has little effect on pregnancy outcome, and can worsen outcomes in some cases.
What is PGD used to detect?
Single gene disorders
Structural abnormalties
Cancer susceptibility - tenuous
What criteria do couples wanting to under PGD have to meet for NHS treatment?
No live, unaffected children Not being treated solely for infertility Genetic disorder listed on UKGTN and licensed by HFEA Risk of unborn child >10% Non-smokers Maternal BMI between 19 and 30 Couple at risk of having child with serious genetic condition Referred by Clinical Genetics Received genetic counselling Female <40yrs old
How many disorders is PGD now licensed for? Name the common ones
> 100
DM1, CF, SMA, DMD, HD, FRAX, HBB disorders
What policy exclusions to PGD treatment are there?
Infertility only
Social sex selection
PGS
ICSI is used over IVF in PGD testing - why?
To reduce the likelihood of paternal contamination during testing.
At what stages can cells be biopsied?
Oocyte - biopsy of 1st and 2nd PB - only screens maternal genes, no information about post-mieotic aneuploidy
Cleavage stage - day 3, take 1/2 cells - upto 60% are mosaic at this stage and correct by day 5
Blastocyst stage - day 5 - take multiple cells, need to replace embryos by day 6
Blastocoele biopsy/blastocentesis - investigated currently
What testing methods are available for PGD?
Single cell whole genome amplification FISH PCR aCGH SNP array
Give an example of a testing for a monogenic disorder using PCR/PGD.
What limitations are there of using this technique
Test for CF where both parents are carriers.
Use allele specific primers to amplify CF alleles
SRY testing for embryo sexing.
Very prone to contamination, allele drop out in dominant disorders
Why is mitochondrial disease testing easier with PGD? What are the limitations?
There are multiple copies per cell, so the technique is less likely to fail.
Factors such as mutation type and plasmy can make interpretation of results difficult.
What is Pre-implantation Genome Haplotyping? (PGH)
Uses WGA or a single cell and involves haplotyping embryos across multiple loci to determine if embryo has inherited a high risk haplotype.
What are the advantages of PGH?
Can screen for multiple mutations in a single gene (not mutation-specific)
Less likely to see allelic dropout as other markers can compensate
Can identify structural rearrangements
Less likely to fail due to uninformative markers
Less intensive PCR workup
Can identify unaffected males
What are the disadvantages of PGH?
Need to haplotype multiple family members
Recombination between haplotypes to knock markers out of linkage
What technique is used for PGH?
Multiples Displacement Amplification - this generates large amounts of DNA from small samples
What are the limitations of PGD?
Misdiagnosis
Wrong embryo transferred
Maternal/paternal contamination
Epigenetic abnormalities associated with ART - normal BWS 1/16,000; ART BWS 1/4000 - waves of denovo methylation occur post-fertilisation/pre-implantation by the actions of DNMT3A/B
What are the moral/ethical concerns surrounding PGD?
Choice of disorders to test for. Availability Death of embryo vs death of foetus Designer babies Testing for late-onset disorders Parents wishing to have affected children (Achondroplasia, Deafness).
What proportion of liveborn children have chromosome abnormalities?
<1%
What proportion of conceptuses have chromosome abnormalities?
~50% - most abort before pregnancy is recognised
What are the most common trisomies observed in the first trimester?
What proportion of foetuses are abnormal in first trimester?
16 22 21 18 13 2 45,X
10%
What is the rate of miscarriage in the second trimester? What proportion of foetuses are cytogenetically abnormal?
2-5%
20%
What loss rates are associated with the following chromosome abnormalities?
+16, +21, +13, +18, triploidy, tetraploidy, 45,X, Balanced structural, Unbalanced Structural, Normal karyotype
\+16 - 100% \+21 - 80% \+13 - 95% \+18 - 95% triploidy - ~100% tetraploidy - ~100% 45,X - 99% Balanced structural - 15% Unbalanced Structural - 95% Normal karyotype - 8%
Describe the investigation of recurrent miscarriage.
Karyotype 3rd and subsequent miscarriages (RCOG) - If abnormality found, test parents.
Why did the RCOG guidelines change from karyotyping parents?
Studies found that parents with balanced rearrangements were likely to have a normal or balanced child.
Unbalanced rearrangements are likely to abort - low risk of affected child.
Testing POC will give a reason for this miscarriage, even if not inherited.
An abnormal karyotype lowers the risk in the next pregnancy.
What problems can be encountered in analysis and culture of POC?
Maternal contamination
Dead tissue
Increased risk of infection.
If cultures fail, what other approaches can be used to determine cause of miscarriage?
QF-PCR for common trisomies MLPA Sub-telomere MLPA Array CGH WES
What is the point of the first trimester scan?
dating check viability identify multiple pregnancies placental localisation fetal anomalies
Which major markers are screened in the first trimester scan?
NT
Nasal bone
fetal heart
what abnormalities can be detected at the 1st trimester scan? which syndromes are they associated with?
Holosprosencephaly T13 (T18 and 7q36 del)
Exomphalos T18, T13 and BWS
Megacystis T13 and T18
Limb anomalies - T21, T18, triploidy and TS
What prenatal USS findings are associated with DS?
Short femur Patent ductus ateriosis AVSD Echogenic bowel brachycephaly duodenal atresia 5th finger clinodactyly mild ventriculomegaly Sandal gap Nasal bone hypoplasia increased NT
What prenatal USS findings are associated with T18
Strawberry-shaped head Ventriculomegaly Renal anomalies mega cisterna magna choroid plexus cysts absent corpus collosum Micrognathia Heart defects Diaphragmatic hernia Single vessel cord rocker bottom feet exomphalos
What prenatal USS findings are associated with T13
Holosprosencephaly cleft lip/palate cardiac anomalies echogenic kidneys post-axial polydactyly
What prenatal USS findings are associated with Diandric triploidy?
Small foetus
large cystic placenta
hydropic
What prenatal USS findings are associated with Digynic triploidy?
Small foetus Macrocephaly small placenta oligohydramnios hitch-hiker toe
What prenatal USS findings are associated with 45,X?
Increased NT (cystic hygroma) oedema horseshoe kidney cardiac anomalies ascites
List some prenatal features of the following syndromes: Mosaic trisomy 7 Pallister-Killian WHS CDC Miller-Dieker Williams syndrome
Mosaic trisomy 7 - low set ears, rocker-bottom feet, cliteromegaly, renal agenisis
Pallister-Killian - diagphragmatic hernia, short limbs, abnormal hands and feet
WHS - IUGR, microcephaly, cleft lip/palate
CDC - cleft lip/palate, heart defects
Miller-Dieker - lissencephaly
Williams syndrome - suprevalvular aortic stenosis, IUGR
List the prenatal features of achondroplasia
difficult to spot until 3rd trimester
short long bones
Trident hand
finger separation
frontal bossing
What proportion of CVS and AF samples is mosaicism detected in?
CVS - 1-2%
AF - 0.1-0.3%
How can mosaicism be excluded?
By scoring cells in 1 or more additional cultures or colonies
Describe a basic work-up for mosaicism in suspension culture
Screen 20 cells from two independent cultures
Describe a moderate work-up for mosaicism in suspension culture
Screen 20 cells from at least 1 other culture not known to have an abnormality
Describe an extensive work-up for mosaicism in suspension culture
Screen 20 cells in each of two independent cultures not known to have the abnormality
What are the indications for a basic prenatal mosaicism work-up?
SINGLE cell with: 45,X unbalanced structural rearrangement balanced structural rearrangement centromeric break
What are the indications for a moderate prenatal mosaicism work-up?
Extra sex chromosome (multi-cell or single cell)
Trisomy involving: 1,3,4,6,7,10,11,17 or 19
45,X multiple cells
monosomy in >1 cells
Marker chromosome in a single cell
balanced rearrangement in >1 cell
What are the indications for an extensive prenatal mosaicism work-up?
Trisomy of: 2,5,8,9,12,13,14,15,16,18,20,21,22 in single or multiple cells
unbalanced rearrangement in >1 cell
Marker chromosome in >1 cell
What level of mosaicism should be reported?
Level 3 - likely to represent true foetal mosaicism.
Level 2 mosaicism should be reported with care and a follow-up AF should be advised and high resolution USS
Give and example mosaic karyotype for a Turner’s mosaic.
45,X [5]/46,XX[15]
What methods are available to investigate marker chromosomes identified during PND?
FISH - 15, 13/21, 14/22, XIST
C-banding / DAPI-DA staining
NOR staining
aCGH
How many pregnancies are ‘affected’ by marker chromosomes?
1/1000
What series of tests recommended by the 2009 best practice guidelines for prenatal diagnosis to identify marker chromosomes?
C-banding to determine number of centromeres, and if the marker is heterochromatin or euchromatin.
NOR for satellites
Take parental bloods to investigate inheritance
FISH for centromeres of 15, 13/21 and 14/22
FISH for X and Y
FISH for chromosomes associated with UPD
Whole chromosome paints
Which chromosome markers require further investigation?
15 - check for inclusion of PWAS region
22 - check for inclusion of cat-eye region
i12p, i18p, i18q - confirm by FISH
What are the limitations of using CGH for the detection of marker chromosomes?
Low level mosaicism may not be detected
Poor coverage of centromeric/heterochromatic regions
Lack of positional information
What is the clinical significance of marker chromosomes?
Duplication of disease-associated genes can produce a phenotype:
PWAS region in idic(15)
i22q - cateye syndrome region
chr14-derived markers - UPD risk
X-derived markers with XIST are low risk; those lacking XIST are high risk
After one trisomic pregnancy, what are the recurrence risks of being affected with any subsequent trisomy in the future?
Which paper was this data published in?
T13, 18, 21 - ~1% (+ any maternal age effect)
45,X - no increased risk
Warburton et al, 2004
If a common trisomy or monosomy X is identified at prenatal diagnosis, when should parental follow-up be offered?
Only after recurrence (2 or more trisomic pregnancies).
The risk is low, so this isn’t routinely offered at the diagnosis of the first trisomic pregnancy.
