1
Q
where does cffDNA originate from?
A
placenta - shed highly fragmented DNA into the maternal circulation during normal apoptosis
2
Q
what is the fetal fraction and what amount is there in maternal blood? from what stage can it be detected? how do we know it is specific to the current pregnancy? how is fetal DNA distinguished from mums DNA?
A
- the total cell free DNA in maternal plasma that comes from the placenta (up to 20%)
- reliably detected from 7 weeks
- increases with increasing gestation
- rapidly cleared from circulation within an hour after delivery
- fetal DNA is shorter -approximately 200bp for fetal fragments and larger for maternal fragments. in silico size selection in data analysis enriches for fetal DNA
3
Q
what are the technical challenges of isolating cffDNA?
A
- low abundance - outnumbered by cell free mat DNA
- need specialist STRECK tube to stabilise maternal blood and prevent release of maternal DNA
- DNA is indistinguishable from mat DNA (apart from size)
4
Q
what advantage do intact fetal cells in maternal circulation have over cff-DNA?
A
stays in blood for 27 years whereas fetal DNA cleared rapidly after delivery
5
Q
how does NIPD work?
A
- uses informative SNPS where mum homozygous to quantify level of paternal SNP present
- can also look at methylation in fetal epigenetic markers but some methods such as bisulphite conversion degrade DNA
6
Q
why is an invasive procedure needed for NIPT but not NIPD?
A
- Confined placental mosaicism has not been reported for single gene diseases. USS recommended to rule out vanishing twin however that might interfere with genotyping
7
Q
Describe uses of NIPD for Fetal sex determination. How does the testing work? why might there be a false negative result (no Y material in a male?) how can this be mitigated? what is the accuracy?
A
- sex determination to rule out X linked diseases eg. DMD/CAH. DMD -If Y chromosome material detected > do invasive sampling or stop if female. CAH - administer dexamethasone in female fetus to prevent virilisation (abnormal genitalia). not required for males. RT-PCR quantified amount of SRY on Y chromosome present.
- undetectable levels of cff-DNA - overcome by having cut-off limit, have control locus eg. CCR5 to check for presence of fetal DNA, replicate testing of each sample
- accuracy = 95.5%
- RAPID project still recommends scan to confirm fetal sex
8
Q
Describe uses of NIPD for AD/de novo mutation detection?
A
- cffDNA will contain altered alleles that are not present in the high background of maternal cfDNA
- may use targeted NGS panel eg. FGFR3 or bespoke RT-PCR assay for relevant variant
- can only detect SVs (expansion, insertion or duplication ) <300 bp due to shorter fetal DNA length
- workup from affected proband or carrier parent required beforehand
9
Q
how does NIPD work for AR or XL disorders? paternal exclusion testing and RMD/RHDO
A
PATERNAL EXCLUSION TESTING
- test fetal DNA for paternal variant, if absent the fetus is at most a carrier
- invasive testing offered if paternal variant identified
- eg. cystic fibrosis offered by targeted NGS at GOSH
relative mutation dosage (RMD)
- difficult to test for maternally-inherited variants due to background of maternal cfDNA
- In relative mutation dosage (RMD) the relative amounts of mutant and normal alleles in the maternal plasma are determined.
- • Equal amounts of wild type and mutant alleles would be expected in a female carrier with a carrier fetus.
- more mutant than expected = fetus homozygous for mutation
- more wildtype than expected = normal fetus
- need high enough fetal fraction for accurate calculation
advantage = doesnt require proband or dad
disadvantage = technically challenging , targeted to one mutation
Relative haplotype dosage
- multiple SNPs around region of interest are sequenced with targeted NGS
- o Haplotyping (linkage analysis) is used to determine whether the fetus is likely to have inherited a high risk or low risk haplotype
- o In diagnostic use for SMA, DMD and CF
advantages: statistically robust as uses several SNPs, can be used for mutation types or genes that are not amenable to NGS - disadvantages: risk of recombination, requires proband and paternal sample, expensive, consaguinity may be an issue with SNPS
10
Q
what might the future hold for NIPD?
A
whole fetal genome present in mat blood so may be able to do WGS
non-invasive exome screening in USS abnormalities for paternally inherited alleles and de novo pathogenic variants
many more disorders added for RHDO and ‘proband free’ direct parental haplotyping from cell-free DNA
11
Q
what was the RAPID project?
A
Project - 5 year UK national programme funded by NIHR. Aims: (1) to improve the quality of NHS prenatal diagnostic services by evaluating early non-invasive options based on cffDNA and RNA in plasma, (2) to develop standards and recommendations for use of NIPD/NIPT in NHS practice
12
Q
what are the benefits of NIPD?
A
- no invasive sampling & therefore no miscarriage risk
- less expertise required for blood sample
- earlier testing to allow for decisions
- better targeting of anti-D therapy, which is currently given to all RhD negative women without testing to prevent a potentially fatal maternal immune response against a RhD positive fetus)
13
Q
what are the limitations of NIPD?
A
- • In multiple pregnancies it is not possible to differentiate between the foetuses
- • The fetal fraction cffDNA in women with a high BMI is lower
- • The potential for incorrect results: false negatives can be the result of failure to extract or detect sufficient material, due to the low amount of total cell-free DNA and the small proportion of fetal versus maternal cell-free DNA - mitigated by fetal fraction quantification. False positives - result of either technical issues, such as contamination, or clinical abnormalities such as the presence of a non-identical vanishing twin
- wider issues include consent, equity of access, false negatives and positives, paternity testing, sex selection, , available DTC - should this be regulated?
14
Q
what is aneuploidy?
A
a cell, having one or more chromosomes extra or missing from the normal full euploid set
15
Q
how does QF-PCR work? what are the advantages?
A
amplification of STRs on chromosomes of interest used to determine copy number
● STRs (short tandem repeats or microsatellites) are a pattern of 2 - 6 bp that are repeated directly adjacent to each other.
● STRs are known highly polymorphic markers; a patient is therefore likely to have different numbers of repeat units on each allele.
- 4 markers for each chromosome of interest and sex chromosome markers if referral indicates sex chromosome abnormality eg. AMEL, SRY and DXYS218(Xp) and X22
- quantitative pcr - extracted DNA added to fluorescent primer multiplex and pcr. The reaction must be quantitative to detect copy number, therefore the PCR is stopped while in exponential phase to detect copy number -“ During the exponential phase of the reaction, the amount of product is directly proportional to amount of template “
- rapid, cheap, small quantities of DNA needed
16
Q
what does a normal QF-PCR result ratio look like? what is the ratio range? how many informative markers are needed on one chromosome to interpret as normal?
A
1:1 if heterozygous or 1:0 if homozygous (uninformative as can be monosomic)
ratio is 0.8-1.4
need at least 2 informative markers per chromosome to interpret as normal
17
Q
what does a trisomic QF-PCR result ratio look like? what is the ratio range? how many informative markers are needed on one chromosome to interpret as trisomy?
A
2:1 (0.45 &0.65 and 1.8 &2.4.) or 1:1:1 (0.8-1.4)
2 abnormal markers required for trisomy
if all alleles have same sized marker it is uninformative
● Best Practice: Cannot report a sample as trisomic if any ratios are inconclusive (between normal and abnormal) or if any normal ratios for an otherwise trisomic chromosome are obtained.
18
Q
1:1:1 or 2:1
which is associated with M1 event? what might the other possibility be associated with and what could this mean if seen in a CVS sample?
A
1:1:1
M2 or mitotic non-disjunction
Where this pattern is seen in CVS the risk of CPM is increased
Best practice: The villi are now chopped/dissociated together and tested to give better sample representation (inner mesenchyme core and the outer cytotrophoblast layer
19
Q
what is an inconclusive QF-PCR result? how can this be resolved?
A
result between normal and abnormal
● May be resolved by using single chromosome kits.
● Inconclusive ratios may be the result of preferential amplification of the smaller allele. This is more likely to occur if the distance between the alleles is increased,
20
Q
do you need to confirm a positive QF-PCR result? do you need to confirm a negative QF-PCR result? In what circumstance should it be confirmed and how?
A
no - but sample identity must be confirmed prior to reporting (N.B. by a repeat test of the original sample or genotype comparison with a maternal blood sample).
- Normal results do not need repeating (N.B. there is a requirement for rigorous checking of sample transfers), unless the result is only based on a single marker result – this should be stated in the report and these results should be confirmed by a second method, e.g. karyotype or FISH.
21
Q
what level of MCC can QF-PCR detect? how can this be identified from the trace? what should be done if MCC is suspected? when can a result be reported if there is MCC? If there is MCC, what alternative testing should be done?
A
10% - May show skewed allele pattern for all chromosomes.
if foetus is normal will have 3 peaks and the sum of the two smaller will equal the larger peak
run mums sample to compare genotypes
– if a low level of maternal genotype present but majority is foetal & has no inconclusive allele ratios
- QF-PCR on cultured cells or karyotype
22
Q
how is mosaicism identified on QF-PCR?
A
skewed allele ratios and extra peaks
in diploid/triploid mosaic - all markers will be affected
23
Q
what might normal and abnormal allele patterns obtained for a single chromosome with QF-PCR mean?
A
- somatic microsatellite mutation. usually increase or decrease of 1-2bp. sum of mutation and original peaks should give normal ratio when compared to unaffected allele
- Polymorphic Submicroscopic Duplications/Submicroscopic microsatellite duplications: a single marker consistent with trisomy and all others are normal . usually parents have same pattern for that marker.
- CNV: abnormal markers flanked by normal markers
- primer binding site polymorphism - primer binding less efficient and results in skewed ratios. lower annealing temperature.
24
Q
what advantages does QF-PCR have over FISH?
A
● Less sample volume required (0.5-1ml amniotic fluid for PCR vs 3-4ml for FISH).
● Greater range of gestation (12-34wks PCR vs 15-21wks FISH).
● Less intense labour and higher through-put possible.
● Cheaper cost.
● Can detect MCC in all samples rather than just male conceptions.
● Can be used to infer UPD.
● Can detect certain unbalanced rearrangements (distal 13q, 18q, 21q).
25
what is the UK screen-positive cut-off rate for Down Syndrome?
>1:150
26
what NT thickness is associated with increased aneuploidy risk?
>3.5mm
Increased NT reflects foetal heart failure and is strongly associated with a chromosomal abnormality
(+21,18,13, 45,X and triploidy) also structural anomolies including cardiac if normal karyotype
27
which screening test only includes down syndrome and when does this occur?
quadruple
2nd trimester
Trisomies 13 and 18 can be picked up on the ultrasound scan from 18 weeks
28
when is the more detailed USS performed? what % of malformations does this detect?
from 18 weeks
identified 40% of malformation
29
what is the miscarriage rate for CVS 12 weeks and amnio 16 weeks and fetal blood >18 weeks (usually following abnormal USS?
- <2%
- <1%
<2.5%
30
what is the sensitivity of cff-DNA for Trisomy 21? what happens when a positive result is found?
sensitivity of 99% and false positive rate of <0.1%. It is not as reliable for other aneuploidies such as trisomy 18 and 13 but can detect both. PPV for turners is only 40%
• All positive NIPT results need to be confirmed using an invasive diagnostic test such as CVS or amniocentesis.
31
what was the result of the RAPID study?
UKNSC recommended that NIPT should be implemented into the NHS as part of the Fetal Anomaly Screening Programme.
32
when is cff-DNA testing offered for NIPT in the uk?
screening risk >1:150
33
what does the combined test take into account?
maternal age, biochemical markers – free beta human chorionic gonadotropin (bhCG) and pregnancy associated plasma protein-A (PAPP-A) ultrasound measurements – nuchal translucency (NT) and crown rump length
34
what does the quadruple test take into account?
AFP, β-hCG, uE3+ Inhibin A
screens for T21 only, when NT measurement cannot be obtained
35
can FISH detect mosaicism?
Yes and No (depends on if 18/X/Y used and if fetus is male)
36
can FISH detect triploidy
can qf-pcr detect triploidy
both yes
37
when should a prenatal array result be reported
where there is clinical significance i.e. the finding explains the observed (ultrasound) abnormality, or a clinically actionable result has been obtained.
It is important that the laboratory informs the clinician when a pathogenic CNV is not consistent with the ultrasound findings or when the origin of the CNV may have a contributing effect.
The BSGM has reported a list of incidental findings not to be reported.
38
when is prenatal array most likely to be used?
to pregnancies with abnormal ultrasound scan (USS) results and a normal QF-PCR result for the common aneuploidies
39
can arrays be performed on uncultured samples?
yes
if DNA extraction on uncultured material is too poor for a quality microarray result, cultured cells can be grown for repeat DNA extraction
40
what are the Advantages of Microarrays in prenatal diagnosis
- don't need to culture cells > faster TAT and no artefacts
- high resolution and pick-up rate - Increases detection of chromosome abnormalities by up to 6% compared to karyotype for USS findings
- carrier status for balanced translocations not revealed
41
what are the disadvantages of Microarrays in prenatal diagnosis
- difficult to interpret & limited TAT
- VUS - whether to report requires MDT
- incidental findings
- requires high quality and quantity of DNA
- doesn't detect balanced rearrangements (may have phenotypic consequence if gene disrupted)
- misses low level mosaicism <10%
- doesn't detect triploidy
- may need to culture for follow-up FISH
- cost for array + follow-up more expensive than karyotype
- balancing coverage and optimal resolution
- counselling for variable penetrance/phenotype
42
why might a pathogenic CNV be inherited from a normal parent?
- may have SNV in AR gene
- may be imprinted region and only show effect when inherited from certain gender
- incomplete penetrance
43
what was the conclusion from the EACH study?
CMA is a robust, acceptable, and probably cost-effective diagnostic test and should replace karyotyping in care pathways when the indication for fetal testing is one or more structural anomalies or an isolated NT of ≥ 3.5 mm on an ultrasound scan after a normal QF-PCR result.
44
what are the disadvantages of karyotyping for prenatal analysis?
- cell-culture failure
- limited resolution 5Mb
- clonal selection
45
what is the increased diagnostic yield % of array CGH for prenatal testing over karyotyping?
6%
46
All pregnant women in the UK are offered a minimum of 2 ultrasound scans during their pregnancy in the first and 2nd trimester. what are the 3 groups of abnormality that USS detects?
• neural tube defects
• chromosome abnormalities
• congenital heart defects
47
what T21 screening markers are visible in the first trimester and 2nd trimester?
1st trimester
NT - detects 60% of cases
serum markers PAPP-A and free beta
absent nasal bone
doppler evaluation for regurgitated blood flow
limb abnormalities - short long bones
2nd trimester
cardiac defects
duodenal atresia closure in the first part of their small intestine
soft:
echogenic bowel
sandal gap
48
what might echogenic bowel be associated with?
- cystic fibrosis (up to 80%)
- Down syndrome
- Trisomy 18
- sometimes Trisomy 13
- uncertain significance
- normal variant
49
what might prenatal IUGR be associated with?
_ trisomies 13, 18, 21, turner, triploidy, russel-silver, di george, williams syndrome, transient neonatal diabetes
50
what is linkage analysis?
FAMILY BASED
linking heritable traits to their chromosomal location
- suited to mendelian high penetrance disorders
- requires large families
- poor resolution
- LOD score given
- used for PGD and prenatal testing
- requires informative markers (no homs and enough variation)
tendency for genes and highly polymorphic genetic markers to be inherited together - can be used to track a gene not yet identified
51
what is linkage disequilibrium?
non-random association of alleles at two or more loci with a frequency greater than expected by chance
- due to chance of natural selection
- association found between alleles and disease if disease in LD with marker allele
- affected by recombination, selection, new mutation, genetic drift and population history
52
what is an association study?
- compares cases:controls to identify association between allele and genetic disease
53
what is GWAS? why might there be an association if it is not true causative variant? what are disadvantages? are the variants detected common or rare and what is their effect size?
POPULATION BASED
studies genetic variation across the whole genome ‘hypothesis free’ to find association between a genetic marker and a trait- often uses SNP arrays
- association may be due to chance, linkage disequilibrium between marker and true variant, population stratification bias
- cannot test causality, confounding effects, expensive, statistical significance hard to reach, need large studies to have sufficient power, missing heritability - SNPs confer small effects
GWAS have a high power to detect common variants of high or moderate effect
54
what are the stages of a linkage analysis?
- identify families who carry the disease
- genotype genetic markers across whole genome (microsatellites, SNPs- polymorphic and mendelian
- identify regions with linkage to disease (LOD scores)
- Fine mapping of region with more densely-packed markers, look for candidate genes within region
55
what is parametric linkage? what parameters are there?
(also known as standard LOD score analysis)
used for simple Mendelian disorders
a series of parameters need to be specified before analysis can begin
- MOI
- gene frequencies
- large meioses
- penetrance
•Not suitable for common and complex disease such as diabetes or schizophrenia (no idea of gene frequencies or penetrance of any susceptibility alleles or sometimes mode of inheritance).
56
what is the recombination fraction in linkage?
A genetic marker will segregate with the disease more often the closer it is to the disease gene. The further apart two loci are on a chromosome, the more likely it is that a crossover will separate them.
recombination fraction is designated θ. The value of θ will never exceed 0.5: two independent markers on homologous chromosomes will segregate independently so that on average 50% of offspring will receive one allele and 50% the other allele at that locus
• A recombination fraction of 0.01 in a pedigree corresponds to 1% recombination which equals a genetic distance of 1centiMorgan
• Double crossovers can involve 2 or more chromatids, but if the region crossing over is close to the gene, a second event in the immediate vicinity is unlikely
57
what is the LOD score in linkage? if there are no recombinants what is the LOD score? what is the LOD Z score threshold for linkage?
odds ratio of linkage (linked: non-linked)
log (odds ratio) = Z
lod scored can be added across families
• If there are no recombinants, the highest lod score will be where θ = 0
• Z= 3.0 is the threshold of significance for accepting linkage
• Linkage can be rejected at values of Z<-2 and values between -2 and +3 are inconclusive
negative LOD scores tell us where the gene is not = exclusion mapping
multipoint mapping = data from more than two loci are analysed
58
what is autozygosity mapping?
used in consanguineous families
• Autozygosity occurs when individuals are homozygous at a particular locus because the alleles are identical by descent (IBD)- inherited from a common ancestor
- shared blocks of homozygous markers that segregate with the disease of interest can be analysed to determine the location of the disease gene
- affected sib pairs used for Shared segment analysis
59
what is affected sib pair analysis?
• Model free method used to identify alleles shared by affected siblings (regions identical by descent)
• If they inherit an allele more or less than would be expected by chance this indicates that the allele or its locus may be involved with the disease.
60
how is linkage analysis used in a diagnostic lab?
give an example of why this is used in SMA and HD?
- applied to families where the causative pathogenic variant has not been identified
- assumes the locus responsible for the disorder in a family is known, therefore the clinical diagnosis must be clearly defined.
- several informative microsatellite markers flanking the locus of interest to identify the high-risk haplotype
- analysis of key family members to assign phase and the high-risk haplotype and informativity of markers
eg. SMA - proband has homozygous deletion but parent has two copies of SMN1 on one allele - can help to clarify the carrier risk for other family members that have two copies of SMN1
eg. HD – Linkage exclusion method mostly used for prenatal testing to identify the grandparental high risk haplotype, where the linking parent does not want to know his/her HD status. If the grandparental high risk haplotype is present in the fetus, the risk to the fetus is increased to approximately 50%, this test should be set up at the laboratory prior to the invasive test to ensure that there are informative markers within the family and has important ethical considerations
largely surpassed by WGS to identify causative genes in families and GWAS in populations
61
what % of CVS samples have CPM?
up to 2%
less for amnio as more fetuses lost by then and more mosiacism in placenta than fetus
62
how is MCC reduced for CVS samples?
dissection - remove maternal deciduas
63
what is the advantage of using CVS over amnio?
12 weeks vs 16
64
what are the advantages of amniocentesis over CVS
lower risk of miscarriage and more accurate representation of fetal genotype. Amniocentesis can also be used to test for neural tube defects while CVS cannot (although these are often picked up by USS).
65
where is FBS taken from? when is it taken? what is a drawback?
blood vessels of the umbilical cord or fetal blood vessel
>18 weeks
• FBS is a reliable indicator of fetal karyotype but risk of miscarriage higher 2%
• It is used when ambiguous or inconclusive results are found with other methods (e.g. mosaic cases where a definitive result is required quickly).
May also be used during termination
66
how does MCC affect fetal sampling? how is it detected?
- CVS - maternal decidua. more likely
- amnio = bloodstaining. less likely. even less likely if cultured as the culture conditions favour growth of the amniocytes and eliminate maternal cells.
- microsatellite markers - presence of XY or mixed genotypes, abnormal + normal cell lines together,
- backup cultures should be set up
- mat sample required and state that significant MCC is excluded (min two informative markers)
- • Prenatal reports should not be issued before MCC testing is complete
67
what is Confined placental mosaicism?
which trisomies are more likely to be real?
the presence of abnormal cells restricted to the extraembryonic tissues.
• Most often, when CPM is found it represents a trisomic cell line in the placenta and a normal diploid chromosome complement in the fetus (80% of time)
• Three types of CPM have been described and are based on which placental cells are affected.
Type I, the abnormal cell line is confined to the cytotrophoblast (40%) - non-disjunction
Type II, it affects only the mesenchymal cells of the stromal villous core (40%) - non-disjunction
Type III, it involves both tissues. (7%)- trisomy rescue (risk of UPD)
• Trisomies involving chromosomes 13, 18 and 21 are more commonly seen in fetal rather than placental tissue
- can lead to false positives in prenatal array
68
where can CPM originate from? (2 ways)
Mitotic CPM – Mitotic non-disjunction can occur in a trophoblast cell creating a trisomic cell line in the tissue
Meiotic CPM – Alternatively, CPM can occur through the mechanism of trisomic rescue. If a trisomic conception undergoes trisomic rescue in certain cells, including those that are destined to become the fetus, then the remaining trisomy cells may be confined to the placenta
69
what is mosaicism risk in amniotic fluid?
what are levels I, II and III mosaicism? which is most likely to be true?
<0.3%
• Level I mosaicism involves the observation of a single abnormal cell- artefact =pseudomosaicism.
• Level II mosaicism occurs when two or more cells with the same chromosome abnormality are seen in a culture from a single flask
• Level III mosaicism is defined as the presence of two or more cells with the same chromosome abnormality that is distributed over two or more independent cultures - true mosaicism
70
how can you minimise CPM in prenatal testing?
- culture rather than direct testing
- use more than one frond and from different areas of the biopsy
- follow up amnio if CPM suspected
• Under no circumstances should a decision to terminate a pregnancy be based entirely on a CVS mosaic result.
71
what is the clinical relevance of CPM?
- aneuploidy in CVS + normal AF karyotype = UPD risk
- may have IUGR
- somatic errors are less severe
- higher mosaicism = worse pregnancy progress
- imprinted chromosomes
72
at which stage do the primary oocytes become arrested until puberty?
what stage are the secondary oocytes in at ovulation?
meiosis 1 prophase
meiosis 2 metaphase - don't continue until fertilisation when meiosis 2 is completed and 2nd polar body released
73
when does Spermatogenesis begin?
puberty until death (quality decreases)
74
when does Epigenetic re-programming occur?
In the fetal germline, all DNA methylation patterns are erased (gray line), and then paternal (blue) and maternal (red) methylation imprints are established during gametogenesis.
The two germline genomes that are combined at fertilization undergo parent-specific genome reprogramming in the early embryo, during which most germline patterns are erased again and somatic patterns (green) are established. Only imprinted genes maintain their germline patterns during development of the new organism.
75
what are the two types of twins and how are they formed? are they di or mono amniotic and chorionic? can they be inherited?
1. monozygotic = identical. formed from same egg which splits and forms two embryos. may be dichorionic/diamniotic, monochorionic/diamniotic or - monochorionic/monoamniotic (may be conjoined). 100% genetically similar. epigenetic and environmental effects cause differences. rarely familial.
2. dizygotic = 2 eggs fertilised by different sperm. more common than MZ. may be familial - genes in GDF9. Incidence increases with increasing mat age. always dichorionic/diamniotic
76
what are problems with twinning?
six fold increase in perinatal mortality in twins - more likely premature, IUGR and congenital anomolies.
- MC/MA - Greatest perinatal mortality rate mainly due to cord entanglement
- • It is important to determine chorionicity in management of twin pregnancies due to increased complications of monochorionic placentations. This is also important to clarify when taking PND samples as the origin of the sample has to be clearly identifiable (i.e. twin1/twin 2 or upper sac/lower sac).
- may have Intrauterine death of one twin and severe fetal anaemia in the other due to fetal blood transfer
- Twin reversed arterial perfusion (TRAP) risk in monochorionic twins - Poorly oxygenated arterial blood passes to the acardiac twin & returns even more poorly oxygenated to the normal twin
- Twin to twin transfusion syndrome (TTTS)-Affects 4-35% of MC/DA pregnancies. connecting the artery from one twin with the vein of the other allowing unidirectional blood flow from a donor twin to a recipient twin, resulting in asymmetrical fetal growth and fetal mortality in 80%
- vanishing twin
77
what is vanishing twin? why can this result in false positive NIPT? what can be done alongside NIPS to reduce risk?
- one twin suffers early fetal death - occur in dichorionic & monochorionic pregnancies
- causes false positive NIPT. cffDNA from the demised twin can be detected 8 weeks or longer after demise
- all cffDNA testing for fetal sex should be accompanied by an ultrasound scan, which could be done early in pregnancy, as loss of the twin usually occurs in the first seven weeks
78
when might zygosity testing be used in twin testing? how is zygosity testing performed?
- one twin has developed a clinical phenotype which is thought to be genetic in origin. Determining whether the twins are monozygotic can help to establish recurrence risk in the other twin
- transplantation: HLA identical twin and is selected as a potential donor. If they are identical then there is likely to be less of a graft versus leukaemic effect in the transplant.
- likelihood of monozygosity can be determined by amplifying polymorphic microsatellite markers from both parents and both twins and calculating the likelihood of the same alleles being inherited by chance
- can use bayes theorm: MZ risk is 1/3 and DZ is 2/3 or 1/2 and 1/2 if the sex is known to be the same
79
what are the 3 stages that PGT can be performed? what are advantages & disadvantages of each stage?
- 1st polar body prior to conception or 2nd polar body after fertilisation - BUT lacks paternal contribution & needs analysis of every polar body as some wont be fertilised. Have longer time for testing before freezing
- Blastomere -day 3 cleavage stage 5–8 cell embryonic stage - high mosaicism risk. well established methods. Most common stage
- blastocyst biopsy is performed on day 5–6 (best as more cells can be biopsied) fewer embryos at this stage and may not represent ICM but more likely to be implanted and more material. can quantify mosaicism.
80
what testing is used for PGT?
- arrayCGH or SNP array (usually for structural rearrangements), NGS (also detects CNVs from familial rearrangements), FISH, PCR+sanger , RT-PCR , ARMs assay, restriction enzyme digest or indirectly by linkage. can also use mitochondrial testing by quantifying mutation load in heteroplasmy.
non-invasive preimplantation genetic testing under investigation - Based on discovery of DNA within the blastocoele fluid of blastocysts. day 5 blastocyst - may have contamination.
81
what is preimplantation tissue typing used for?
– embryo testing to ensure an exact tissue match to an older sibling with a life-limiting blood disorder where a close relative isn’t available
82
what is PG-A, PG-SR, PG-M
PG-A = aneuploidy - used to be preimplantation genetic screening
PGT-M Preimplantation genetic testing for monogenic/single gene defects
PGT-SR Preimplantation genetic testing for chromosomal structural rearrangements
PGT-M and PGT-SR previously called PGD1
•PGT-A has not demonstrated clinical benefit in a number of studies and is not supported by the NHS
83
under what circumstances does the NHS fund up to 3 cycles of PGD?
• Couple should be at risk of having a child with a serious genetic condition
• Referred by an NHS Clinical Genetics Service and have been counselled by a clinical geneticist or genetic counsellor
• Risk of a pregnancy affected by the condition should be 10% or more
• Female partner should be under 40 years of age at the time of treatment (c.f. 43y in France)
• No living unaffected child from the current relationship
• HFEA must have licensed the indication for PGT https://www.hfea.gov.uk/treatments/embryo-testing-and-treatments-for-disease/approved-pgt-m-and-ptt-conditions/
• Couple should not be seeking PGT primarily because they are infertile or for any other reason be unable to have children on their own
84
what is the method for PGT? when is ICSI used?
- ovarian stimulation and oocyte retrieval, IVF or ICSI and embryo culture
- ICSI used for male fertility problems or where egg difficult to penetrate. intracytoplasmic morphologically selected sperm injection may be better as selects better sperm based on morphology
- Biopsy of 1st and 2nd polar bodies, blastomere (day 3) or blastocyst (day 5-6)
85
what are advantages and disadvantages of haplotype linkage analysis for PGT?
disadvantage - not possible to determine haplotype for de novo, may not have family members available. low quantity of DNA-amplification failure, DNA contamination or allele drop-out BUT can also amplify nearby markers to determine allele drop-out, contamination and recombination
advantages - same assay can be used for multiple families, can be used for large expansions difficult to amplify by PCR, can be used if causative variant unknown, exclusion testing for HD so parental status unknown
86
what are ethical considerations of PGT?
- higher risk of imprinting disorders
- designer babies
- later onset conditions eg. BRCA,
- deaf couples wanting deaf child
- maintaining heterozygous sickle cell carriers
- sex selection for family balancing - legal in US
87
what % of liveborns have unbalanced chromosome abnormality?
<1%
88
when is a miscarriage?
when is a stillbirth?
what % of 1st trimester pregnancy ends in miscarriage
what % of 2nd trimester pregnancy ends in miscarriage
<24 weeks gestation
after 24 weeks gestation
15%
5%
89
what is the most common cause of trisomy?
maternal meiosis I non-disjunction
• Exceptions: Trisomy 18 which is mostly due to an error at maternal meiosis II
• Aneuploidy is significantly associated with maternal age
90
what are the different types of triploidy?
2/3 =diandry two paternal chromosome sets usually from dispermy. present as partial hydatidiform mole - growth retardation
1/3 = digyny - two maternal sets due to diploid egg either non-disjunction or failure to exclude polar body. Nonmolar and mostly abort or present with severe growth retardation.
91
what is a complete mole? what condition does it have?
• Diandric diploidy (complete mole) two sperm enter an ‘empty’ ovum. No embryo - uniparental diploidy
• Dygynic diploidy (ovarian teratoma) 46 chromosomes maternal in origin due to abnormal development of primary oocyte. Fatal. May contain fully differentiated tissue e.g. hair, nail.
92
what % of women experience recurrent miscarriage? what % of recurrent miscarriage couples have balanced structural chromosomal rearrangement?
1%
recurrent miscarriage = three or more miscarriages before 24 weeks post-menstruation
• In ~2-5% of recurrent miscarriage couples, one of the partners carries a balanced structural chromosome rearrangement.
93
what types of referrals are accepted for miscarriage genetic testing?
• Pregnancy loss or termination with significant fetal malformations (irrespective of gestation)
• Pregnancy loss >24 weeks
• Miscarriages (<24 weeks) for 3rd and subsequent miscarriages (in line with RCOG 2011 guidelines)
94
what testing is carried out for recurrent miscarriage referrals?
Cytogenetic analysis should be performed on products of conception of the third and subsequent consecutive miscarriage(s). Parental peripheral blood karyotyping of both partners should be performed in couples with recurrent miscarriage where testing of products of conception reports an unbalanced structural chromosomal abnormality.”
Testing of products of conception will lead to detection of unbalanced rearrangements due to parental rearrangements but also sporadic chromosome aneuploidy and polyploidy that is associated with miscarriage……..Knowledge of the karyotype of the products of conception allows an informed prognosis for a future pregnancy outcome to be given. If the karyotype of the miscarried pregnancy is abnormal, there is a better prognosis for the next pregnancy
95
what are issues of karyotyping POC for recurrent miscarriage?
MCC
• Culture failure common due to lack of viable fetal cells
- infection risk
- mosaicism: biological (eg: vanishing twin, chimerism, confined placental mosaicism, true constitutional mosaicism) and technical (cultural artefact, maternal contamination). Particular difficulties may be encountered if a normal cell line overgrows that of an abnormal line, or the abnormality (especially an extra marker chromosome) is lost in vitro.
96
apart from karyotype, what other methods can be used for POC recurrent miscarriage testing?
1. QF-PCR - FRONTLINE test: whole chromosome gains & losses for chromosomes 13, 15, 18, 21, 22, X & Y
2. Array-CGH – detects imbalances throughout the genome to a high resolution
3. Conventional karyotyping – not routine but available when required eg familial cytogenetic rearrangement, to investigate low level cytogenetic mosaicism
4. Cell culture – to facilitate biochemical assays or for long term storage
97
what two cell populations for the trophoblast?
ICM (forms embryo) and trophoblast (forms placenta) which is more rapidly dividing and more likely to have mosaicism.
In CVS sample, mosaicism may be confined to placenta.
trophoblast develops into mesoderm
ICM develops into ectoderm and endoderm
98
When an abnormal cell is detected during a routine analysis what should be done to investigate mosaicism?
follow Hsu and Benn guidelines (1999)
basic workup - examination of a total of 20 cells from two independent cultures, one of which may contain the abnormal metaphase eg. single cell with Monosomy X or structural rearrangements
moderate workup - 20 cells from additional separate culture(s) without the initial observation should be examined eg. multiple cell with 45, X or extra sex chromosome
extensive - the examination of 20 cells from each of two further separate cultures excluding the culture with the initial observation eg. trisomy 16, 13, 18, 21
99
how do you Exclude mosaicism in fetal blood?
mosaicism screen of a minimum of 30 cells
100
what would you do if level III mosaicism identified in CVS?
follow up amniocentesis or fetal blood analysis along with detailed ultrasound assessment of fetal morphology
the report must include a statement to the effect that the level detected at analysis will not necessarily reflect the proportion or the tissue distribution in the fetus
Testing parents and referral to clinical genetics may also be appropriate.
101
why is Pallister-Killian syndrome – iso12p tissue specific mosaicism not present in blood?
high turnover - additional abnormal chromosome is lost
Abnormal cells are found at significant levels in fibroblast, AF, CVS and BM samples
102
what was the EACH study? what was the conclusion?
- o Test whether array CGH should replace karyotyping in PND of fetal anomalies detected by routine ultrasound screening
- o Evaluate whether NIPD can replace conventional invasive testing for diagnosis of Downs + other major trisomies
Conclusions: CMA is a robust, acceptable and probably cost-effective method to detect more clinically significant chromosomal imbalances in the anomalous fetus. The results suggest that CMA should replace karyotyping in these care pathways.
. Increases detection of chromosome abnormalities by up to 6% compared to karyotype for USS findings
103
what was the RAPID study? what was the conclusion?
• 5 year UK research programme to improve quality of NHS prenatal diagnostic services by evaluating early NIPT diagnosis based on cff DNA + RNA in maternal plasma
investigated the use of NIPT to detect fetal aneuploidy as part of the NHS Down syndrome (DS) screening
pathway
NIPT detected T21 pregnancies in 100% of cases (95% CI: 88% - 100%). No false negative results have been
identified
104
what was the PAGE study? what was the conclusion?
Prenatal assessment of genomes and exomes.
Whole exome sequencing improves prenatal diagnosis in euploid fetuses with abnormal ultrasound scans.
diagnostic variant identified in 8.5% of cases
diagbistic variants identified in 15% of fetuses with more than one structural anomaly and 15% of fetuses with skeletal anomolies
only 3% in isolated NT cases
105
how would you manage identification of marker in prenatal setting?
what is risk of abnormality if a marker is identified prenatally?
1. test parents for inheritance - request bloods. inherited less likely to be pathogenic or if same level of mosaicism
2. check chromosomal origin, size and gene content - karyotype to examine structure and mosaicism, agNOR can tell acrocentrics, FISH including for UPD chromosomes and if 46,X +mar: X and Y (SRY) probes - gonadoblastoma. If XIST lacking it is associated with more severe phenotype.
If 47 XX, + mar
If same size as chr20: small chr 15 without euchromatin often do not contain PWAS critical region. Large acrocentric chr 15 might do. identify (i12p)=Pallister killian syndrome
if smaller than chr 20: FISH analysis using centromeric probes for all acrocentric chromosomes 15, 13/21, and 14/22
can then do arrayCGH if not identified. only detects unbalanced euchromatic material. may not identify mosaicism.
outcome is dependent upon the size, origin, content, tissue distribution and inheritance
overall risk of an abnormality associated with the presence of a marker chromosome at prenatal diagnosis is 13%
FRCPath genomics 2022
flashcards
Decks in class (10)
# Cards
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Session 1: DNA and chromosome structure, function and gene discovery222
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session 6: cancer genetics & treatment170
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Session 2: Laboratory techniques and bioinformatics194
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mixed SAQs93
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Session 3: Modes of inheritance and constitutional genetics181
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session 7: lab management42
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session 6: prenatal105
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session 5 population genetics63
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Hot topics12
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essays1
