Cyto

Question 1

what is the purpose of the new CNV (2019) guidelines?

Answer 1

the guidelines introduce a semi-quantitative, evidence-based framework with a 5-tier classification system with separate schemes for gains and losses. The aim was to provide consistent, evidence-based clinical classification across labs.Pathogenic 0.99 or higherLikely pathogenic 0.90 to 0.98Uncertain significance -0.89 to 0.89Likely benign -0.90 to -0.98Benign -0.99 or fewer

Question 2

what is involved in CNV assessment?

Answer 2

CNV genomic content: size,overlap with established triplosensitive (TS), haploinsufficient (HI) or benign genes/genomic regions; gene number and content population frequency, evaluation of literature, public databases, internal lab data; inheritance pattern/family history for patient being studied

Question 3

give examples of recurrent CNV syndromes caused by low-copy repeats which may misalign during recombination at meiosis

Answer 3

Prader-Willi / Angelman syndromes 15q11q13DiGeorge/velocardiofacial syndrome 22q11.21 deletion including TBX1Williams syndrome 7q11.23 deletion including ELNSmith-Magenis syndrome 17p11.2 deletion including RAI1Miller-Dieker syndrome 17p13.3 deletion including PAFA1B1 and YHWAE

Question 4

why should number of protein-coding genes in a CNV be assessed?

Answer 4

• CNVs containing no protein-coding genes or other known functionally important elements are more likely to be benign.• a small CNV in a gene-rich region could have a larger impact than a much larger CNV in a gene-poor region. ACMG guidelines suggest adding evidence if ≥25 genes for copy number loss and ≥35 genes for copy number gains. Consideration should be given for clusters of genes where a relatively large number of genes may be present with little phenotype impact e.g. olfactory receptor gene clusters

Question 5

why might Pathogenic CNVs may also be present at low frequency in control populations ?

Answer 5

recessive inheritance, incomplete penetrance, parent of origin effect, late onset phenotypes and affected individuals in poorly phenotyped control cohorts

Question 6

name sources for population frequency of CNVs

Answer 6

DGV - The DGV Gold is a curated set of variants from selected high resolution and high quality studies in DGV, clustering variants and removing low-resolution studies with high false-positive rates GnoMad SVo In-house laboratory database of normal control samples can be useful to identify common variants in a local population and/or technical artefacts that may be platform specific.

Question 7

what kinds of evidence might aid CNV interpretation?

Answer 7

• multiple unrelated patients with a similar CNV and a consistent phenotype (more likely pathogenic)- de novo supports pathogenicity. Additional weight is given if the phenotype is highly specific and relatively unique to the gene/genomic region.- Segregation amongst similarly affected family members supports pathogenicity‚Ä¢ Non-segregations EXCEPT incomplete penetrance, phenocopies (variation caused by environment that resembles a genetic variation), accurate phenotyping of family members, age of onset and inheritance pattern - for a CNV identified in recessive gene - look for SNV on other allele

Question 8

what is haploinsufficiency? how can it be found for a gene?

Answer 8

loss of one allele resulting in a single normal allele at a particular locus is inadequate for normal function resulting in a phenotype. Regions of copy number loss containing established HI genes are more likely to be pathogenic. HI info available on ClinGen, decipher, knockout mouse models, literature, ClinVar, HGMD, OMIMpLI available on GnoMad

Question 9

what are intergenic deletions and could they be pathogenic?

Answer 9

do not affect the coding sequence of a gene. may be pathogenic if disrupt regulation of expression of nearby genes An intergenic deletion in close proximity to a known OMIM morbid gene with a relevant phenotype may warrant further investigation.intragenic dels affecting the 5’ or 3’UTR, or are entirely within an intron, is less clear, but it is possible that such deletions could affect RNA transcription, splicing, stability, or translation.

Question 10

what is Triplosensitivity (TS)? how is it assessed?

Answer 10

where an additional copy of a gene/genomic region results in a phenotype. Fewer genes appear to exhibit TS than HI and therefore copy number gains are more likely to result in a milder phenotype or to be benign than the reciprocal deletion. clingen, literature and database searching show TS.

Question 11

why might 5’ or 3’ duplications be pathogenic?

Answer 11

may disrupt regulatory elements

Question 12

why is it important to determine the source of a homozygous dup?

Answer 12

Homozygous duplications manifest as a copy number of 4, which could also represent a heterozygous triplication. Parental studies are therefore necessary to determine whether the imbalance is monoallelic or biallelic. A biallelic gene-disrupting duplication can be causative of a recessive disorder, whereas a monoallelic triplication cannot be associated with a recessive disorder but may cause disease through a different mechanism.

Question 13

What are the three types of cell culture systems available?

Answer 13

Direct preparation - no culturing performedSuspension - cells in media in tubes/flasksIn-situ - cells cultured on a coverslip and colonies form

Question 14

What supplements can be added to the culture medium to promote growth?

Answer 14

L-glutamineFoetal calf serumAntibioticsAntifungals

Question 15

What mitogens are available to stimulate cell growth? Which cell types do they stimulate?

Answer 15

PHA - T cell stimulationPWM - B cell and T-cell stimulationLPS - B-cell stimulation (used for patients with chronic lymphoproliferative disordersTPA - B cell stimulation

Question 16

How long are Bone Marrow cultures routinely cultured for?

Answer 16

12/48hours

Question 17

How long are AF/CVS samples cultured for?

Answer 17

5-7 days, undisturbed

Question 18

What additional step needs to be performed prior to BM culture?

Answer 18

White Cell Count

Question 19

Name three Chromosome Breakage syndromes. State which gene is mutated in each.

Answer 19

Fanconi Anaemia - 15 genesAtaxia Telangiectasia - ATMBloom syndrome - BLMXeroderma Pigmentosa - multigenic

Question 20

What chemicals/treatments can be used to highlight chromosome breakage in culture?

Answer 20

MMC or DOB - they crosslink the DNA and enhance the rate of breaks. This can be used to diagnose FA or AT.UV-light - induces increased SCE in Bloom syndrome. Detected by adding BrdU to culture and G-banding.

Question 21

How can the identification of a balanced translocation lead to new gene discovery?

Answer 21

Gene interruption (DMD, SOTOS)Result in submicroscopic deletions/duplications (CHARGE syndrome)Gene removed from cis-acting elements by balanced translocation (PAX6 in Aniridia)Gene translocated next to an enhancer (BCR-ABL1, t(9;22)(q34;q11))

Question 22

How can recurrent deletions/duplications leads to gene identification?

Answer 22

Microdeletions can narrow down a region linked to disease. Study genes in these regions to identify possible cause. - Miller Dieker (17p13.3), LIS1

Question 23

why is parental testing important for CNV analysis?

Answer 23

. If a variant detected in a patient is found to be inherited from an unaffected parent, it is unlikely to be pathogenic through a fully-penetrant, autosomal dominant inheritance pattern – but incomplete penetrance, recessive inheritance, parent-of-origin dependent effects etc are not excluded. De novo origin of a variant supports its pathogenicity but is not by itself sufficient to class a variant as pathogenic, particularly if relationships have not been confirmed. X-linked variants inherited from carrier mothers can be tested for in male relatives to help determine clinical significance.

Question 24

should CNV carrier status be reported?

Answer 24

Reporting of carrier status for recessive conditions (i.e. unrelated to the reason for testing) is at the discretion of the laboratory. However, carrier status for X-linked recessive conditions in females should be reported due to her own future reproductive risk, the implications for other family members, as well as the possibility that the patient may be a manifesting carrier.

Question 25

how is a reciprocal unbalanced translocation identified by array and what follow up is required?

Answer 25

terminal deletion of one chromosome and a terminal duplication of a different chromosome is suggestive of an unbalanced product of a parental balanced translocation. This can be confirmed by G-band analysis in the proband, or FISH if the translocated segment is too small to be reliably detected by G-band.

Question 26

what is an isochromosome?

Answer 26

Isochromosomes are recognizable by copy number gain of one chromosome arm. For example, Pallister Killian syndrome is caused by mosaic isochromosome of 12p, resulting in mosaic tetrasomy of 12p.

Question 27

when should a CNV be reported?

Answer 27

o account for patient’s phenotype.o have other implications for the patiento have implications for the patient’s familya VUS should be reported if it may be pathogenic and further investigations may clarify pathogenicity

Question 28

when should a PRENATAL CNV be reported if not related to referral reason?

Answer 28

o Report high penetrance neuro-susceptibility loci that are associated with a risk of a severe phenotype o Report neuro-susceptibility loci associated with an increased incidence of anomalies detectable on scan, as reporting these may help direct further scanningo Report deletions of cancer susceptibility genes.o Report a DMD deletion in a female fetus.o Do not report low penetrance neuro-susceptibility loci eg 15q11.2 deletions.o Do not report heterozygous deletions associated with recessive conditions unrelated to the fetal anomalies.o Do not report unsolicited pathogenic variants for which there is no available intervention.o Do not report variants of uncertain significance that cannot be linked to a potential phenotype on the basis of genes involved.

Question 29

What are the cytogenetic and molecular techniques for CN detection

Answer 29

G-bandingFISHMLPAQF-PCRNGSARRAY

Question 30

What is G-banding?

Answer 30

Manipulation of the cell to obtain metashase chromosomes. Cells are harvested and then banded using giemsa staining so the banding pattern can be visualised. - requires actively dividing cells- to get the highest yield of metaphases cells are synchronize by being stopped in S phase by BrDU-cells are then released and allowed to passage to Metphase before being harvested ans stained- Treatment with a hypotonic salt solution just prior toharvest permits swelling of the nuclei. Incubation in adilute KCl or sodium citrate solution for 10–30 mingenerally achieves good spreading. Insufficient hypotonic treatment results in chromosome spreads that are tightly knotted; individual chromosomes are difficult to virtuallyimpossible to visualize. - last step is to preserve the cells with fix (at this point they contain no risk and can be handled without gloves or safety cabinet). Fixation with Carnoy’s solution, amixture of methanol and glacial acetic acid, arrests theprocess of hypotonic swelling and all metabolic processes- slide making is by dropping the fixed solution onto slides. the humidity and height the solution is dropped from affects the chromosome spreading and is optimised for max spread between chromosome so they can be analysed without broken cells- cells are aged in an oven and can then be stained.

Question 31

what is G-banding used for?

Answer 31

NAME?

Question 32

What are the benefits and limitations of G-banding?

Answer 32

• low resolution- requires actively multiplying cells- labour intensive, slow TAT- can’t detect UPD- risk of cultural artefact (PND)- some abnormalities not seen in cultured cells e.g. some mosaic aneuploidies- aneuploidy detection and can determine recurrence risk- provides structural information- can detect balanced rearrangements- whole genome screen- can detect mosaicism- sensitivity increases with more cells counted- relatively robust and inexpensive

Question 33

describe the use of FISH for CNV detection

Answer 33

uses flourescently labelled ssDNA probes targeted to the region of interest. In the presence of a deletion there will be no signal so also need to include a control probe ensure that hybridisation has occurred. For a duplication there will be extra signal either in the same location for tandem dups which may appear as one brighter signal or elsewhere if the duplication is present on another chromosome.

Question 34

What are the uses of FISH

Answer 34

mainly used in cancer on FFPE or BM to ID recurrent genomic rearangements for prognostic or treatment stratification. Aneuploidy detection and microdels/dups (arrayCGH has superseded this for most referrals except urgent family testing)origin or marker from g-banding.If G-banding fails e.g. AML screengene amp in cancer e.g. HER-2 for breast cancerpost transplant chimerism monitoringPGD

Question 35

what are the benefits of FISH

Answer 35

• can be done on interphase (don’t need to be dividing) or metaphase cells (positionl information)- can scan a lot of interphase cells to detect mosaicism- rapid FISH can provide a fast TAT (same day if lab aware of sample and ready to set-up)- can analyse single cells- higher resolution than G-banding- probes can be deisgned for alomost any genomic region- can detect subtelomeric and cryptic rearrangements missed by G-banding

Question 36

what are the disadvantages of FISH

Answer 36

• can get artefacts fromt he co-localiastion of 2 probes so they appear as one signal-non-specific binding– targeted test so need to know what you are looking forcannot detect UPD-cannot detect MCC in female pregnancies- limited number of probes can be used at a time due to a limited number of flourophores- interphase FISH doers not provdie positional information-micro duplications can be missed due to low result ion on metaphase spreads.

Question 37

Describe QF-PCR for copy number detection

Answer 37

quantification of polymorphic repeat regions using flourescently labelled primers. Quantification is relative to the other markers in the multiplex, not absolute. PCR reactions is stopped whilst still in the exponential phase where the amount of input DNA is proportional to the amount of PCR products- it is most commonly used to detect anueploidy in prenatal samples and MCC- can also be used for UPD (requiresd samples from both parents), post transplant chimerism monitoring and sexing

Question 38

what are the advantages of QF-PCR

Answer 38

• detects mosaicism to ~10%- cheap, quick, fast technique- can be high throughput- requires very little starting material- does not require confirmation by another method

Question 39

what are the disadvantages of QF-PCR?

Answer 39

NAME?

Question 40

What is real time QF-PCR?

Answer 40

PCR where the amplification is measured during each PCR cycle using fourescent DNA binding dyes- SYBR green (non-specific)- taqman (seq specific)measure during the exponential phase where the amount of product amplified is proportional to the amount of starting materialThe ‘threshold’ number of cycles requires for the fluorescent signal to be detected is called the Ct However, the efficiency of amplification is often variable among primers and templates. Therefore, the efficiency of a primer-template combination is assessed in a titration experiment with serial dilutions of DNA template to create a standard curve of the change in (Ct) with each dilution. To quantify gene expression, the (Ct) for an RNA or DNA from the gene of interest is subtracted from the (Ct) of RNA/DNA from a housekeeping gene (usually ABL1) in the same sample to normalize for variation in the amount and quality of RNA between different samples. This normalization procedure is commonly called the ŒîCt-method[14] and permits comparison of expression of a gene of interest among different samples.

Question 41

what are the considerations of prenatal culturing?

Answer 41

Precious sample, usually limited material available and irreplaceable- therefore long term cultures are established and are grown in situ. This is in case additional DNA is required for testing or FISH or karyotype analysis is needed for follow-up testing - to increase the success rate and reduce the risk of contamination or culture failure 2-3 cultures should be set-up using 2 different media batches and stored in 2 separate incubators. only 1 culture should be harvested at a time and cultures should not be discarded until the final report has been issued

Question 42

what are the considerations of CVS culture?

Answer 42

cytotrophoblast are most distantly related to the fetus than the mesoderm. Short term incubations of CVS (for approximately 24hours) for cytogenetic analysis reflect “spontaneous”trophoblast cell divisions. In contrast, longer-term cultures of CVS consist of the mesenchymal cells and thus represent the ICM - can follow up abn result on uncutlured CVS with cultured CVS to determine cell lineage and how likely it is to represent the fetus-uncultured CVS if chpeed/digested will contain both cell lineages

Question 43

How is CVS processed for culturing?

Answer 43

needs to be carefully dissected to remove any maternal decidua (microscrope and needles/forceps). Morphology and weight of CVS is estimated and cultures are set-up- any remaining cleaned villi is kept in the fridge until the report has been authorised. CVS is spread over the gowing curface and gorwn in chage media at 37oC- change contains the additives- fetal bovine serum, glutamine for growth, antibiotics to prevent infection and bicarbonate to buffer and control the pH

Question 44

How is AF processed for culturing?

Answer 44

centrifuged, pelleted and supernatant removed. pellet is resuspended and used for DNA extraction or resuspended in amniomax for culturing (same additives as chang)

Question 45

When are cultures assessed for CVS and AF?

Answer 45

cultures are assessed after 6-7 days, if colonies are present the media is replaced. - media is chaged to ensure optimum conditions for growth- If 5+ active colonies present the culture should have enough metaphases for harvesting- to get methaphases you need actively dividing cells and overgrowth a a culture can limit the no. of cells (contact inhibition) - ‘full’/confluent cultures can be split and supplementary cultures set-up to provide space and ensure the cultures remain active until the case has been reported

Question 46

How are CVS and AFs harvested?

Answer 46

should be carried out in a class 2 hood as uses the mutagen BrDU- BrDU (longer chromosomes) and colcemid (inhibits the mitotic spindle) are added to the cultures the afternoon before harvesting-following morning the media is decanted and the cells are resuspened in trypsin -centrifuge to pellet cells and supernatant is removed- resuspend cells in hypotonic solution so cell swell for optimal chromosome spreadingadd fix (dropwise at first to prevent cell clumps forming)cells are now biologically inert and can be handled without gloves and slides can be made. Temp and humidity is controlled to produce consistent high quality metaphse spreads- left to dry, aged in oven and checked for metapahses under a phase microscope before staining

Question 47

Why is a coulter counter used in cancer cutlure

Answer 47

used to count cells and allow for appropriate seeding of the culture so the media is not exhausted by overseeding but that there are enough cells cultured for analysis

Question 48

what are the considerations of cancer cell culture?

Answer 48

• usually use BM- due to the aberrant increased growth of acute leukemia’s most do not require PHA for stimulation, chronic leukemia’s with slower growth may do. neoplastic cells are already dividing but in a normal individual white blood cells are non-dividing and need a mitogen to cause them to de-differentiate back to an immature stateoften a variety of cultures are set-up to try and capture the mutant - 24hour, stimulated/unstimulated, long termno synchronisation

Question 49

what are the steps in cell culture?

Answer 49

1. obtain your sample e.g.PB2. add to culture vessel (tube) with growth media (RPM1640) and stimulants as required3. synchronise cell cycle to obtain maximum number of metaphase chromosomes4. arrest in metaphase- add colcemid5. add a hypotinic solution (KCL) to swell cells so chromosomes are spread and not tangled and tight6. fix cell suspension (carnoy’s fix- add dropwise to start to stop cells clumping) 7. make slides8. satin for analysis e.g. giemsa staining for standard g-banding

Question 50

what is included in growth media?

Answer 50

amnio acid, balanced salt solution, Hepes buffer, energy source, hormones, pepetides, fatty acidsL-glutamine, antiobiotics fetal calf serum and PHA often addedPHA

Question 51

why is PHA added to cell culture

Answer 51

T stimulate mitosis. It is a mitogen that de-differentiates leucocytes back to an immature state where they can undergo divisionnot routinely added to acute leukemias because stimulation if division may interfere with analysis of spontaneously dividing malignant cells.

Question 52

How are cells synchronised?

Answer 52

synchronised to obtain maximum metaphases unless a rapid result is required. - 72 hour culture processthymiding is added to block DNA synthesis so cells are paused in s-phase. Thymiding inhibtis the uptake of DOC (deoxycitidine) causing DNA synthesis to slow down- Excess DOC is added to remove block, all cells then leave S-phase together and are synchronised

Question 53

How are cells harvested in metaphase?

Answer 53

After releasing the S pahse block cells progress through the cell cycle to metaphase ~4.5 hourscolcemid is then added which blocks the cells in S-pahse and prevents them progressing to anaphase. The longer the culture is in colcemid the more metaphases will be harvested but they will be shorted so need to balance the two requirements.

Question 54

why is fix added?

Answer 54

Fix removes water from cells, kills and preserves them (biologically inert and can be handled without gloves) and harden the membranes and chromatin- preserves the cells and chromosomes

Question 55

what satin is used in G-banding

Answer 55

Trypsin is used to digest chromosomes- need to be careful not to over digest as chromosomes become fuzzy with less clear banding.stained with leishmanns stain

euchromatin= active, gene and GC rich, early replicating, stains paleheterochromatin= inactive, AT rich, late replicating, stains dark