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Flashcards in Arrays Deck (81):
1

What is a DNA microarray?

- Gene chip, DNA chip or biochip array is a collection of DNA probes attached to a solid surface.
- Allows to simultaneously examine copy number changes within a DNA sample.
- Probe-target hybridisation is usually detected and quantified by detection of fluorochrome labelled targets.

2

What is the most common type of array technique used in diagnostics? What is the principle behind the technique?

- Array CGH is the most common type of array technology in clinical practice.
- It relies on the concept of competitive hybridisation of differently labelled reference and test DNA.

1). DNA extraction and digestion
2). Test DNA labelled red with Cy5, ref DNA labelled green with Cy3.
3). Test and ref DNA combined in 1:1 ratio and left to hybridise to the DNA spotted chip.
4). Where there is a lack of a DNA region in the test DNA only the ref DNA will bind and it will appear green on scanning. Where there is a gain more test than ref DNA will bind and it will appear red. Yellow indicates no difference between the test and ref DNA (i.e. no losses or gains) in that location.

3

Very simple, how does a microarray scanner work and what is done with the data?

- It measures the intensity of fluorescence at each of the oligonucleotide spots simultaneously.
- Software allows normalisation, visualisation, breakpoint analysis and comparative analysis of the data. This allows for the accurate detection of CNVs.

4

What different types of arrays are there?

- Comparative genomic hybridisation: assessing genome content in different cells or closely related organisms.

- Gene expression arrays: assessing gene activity

- Chromatin immunoprecipitation on Chip: combines chromatin immunoprecipitation (ChiP) with DNA microarray.

- SNP array: Identifying SNPs

- Exon array: designed to detect different spicing isoforms

- Tiling array: consits of overlapping probes designed to densely represent a genomic area

- Fusion gene microarray: detects fusion transcripts

5

What are the basic types of arrays used for assessing copy number variants in cytogenetics?

- BAC arrays: amplified BAC DNA spotted onto glass slides, effectively the same as multiple FISH probes. Resolution about 0.5 to 1 Mb.
- Targeted arrays are designed for specific regions of the genome for the purpose of evaluating targeted segments.
- Oligo arrays: Custom oligos spotted onto glass slides. Higher resolution. Resolution for cytogenetics is about 100-200kb. With increased coverage the resolution is improved.
- SNP array: Detects SNPs. Allele specific oligonucleotide probes are used to detect polymorphisms.
- At present platforms are combining both aCGH and SNP genotyping to optimise diagnostics.

6

Describe BAC arrays.

BAC arrays: amplified BAC DNA spotted onto glass slides, effectively the same as multiple FISH probes. Resolution about 0.5 to 1 Mb.

7

Describe targeted arrays.

Targeted arrays are designed for specific regions of the genome for the purpose of evaluating targeted segments.

8

Describe oligo arrays.

Oligo arrays. Custom oligos spotted onto glass slides. Higher resolution. Resolution for cytogenetics is about 100-200kb. With increased coverage the resolution is improved.

9

Describe SNP arrays.

SNP array: Detects SNPs. Allele specific oligonucleotide probes are used to detect polymorphisms.

10

In what areas of cytogenetis can array technologies be applied?

- Constitutional cytogenetics
- Prenatal diagnosis
- Solid tumours
- Products of conception
- Malignancies

11

Briefly describe the history of arrays.

- In 2004 across the human genome genomic imbalances were defined by several studies. Approximately half of these regions were found to overlap with clones and coincide with segmental duplications. These phenotypic variations show a more dynamic human genome structure.

- The 1st gen CNV map of the human genome was a study of 270 individuals from 4 populations with ancestry from Europe, Africa and Asia. This is the HapMap collection. DNA from these individuals were screened for CNVs using 2 complimentary technologies, SNP based arrays and clone based CGH. A total of 1,447 copy number variable regions (12% of the genome) were defined in these populations. These CNVs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably the CNVs encompassed more nucleotides content per genome than SNPs thus underscoring the importance of CNVs in general diversity and evolution. The utility of CNVs for genetic disease studies was also demonstrated.

- Another study found that routine karyotype analysis is not sensitive enough to detect subtle chromosome rearrangements less than 5Mbs. The presence of subtle DNA copy number changes was investigated by aCGH in 50 patients with LD and dysmorphism using a DNA microarray constructed from large insert clones spaced at approximately 1Mb intervals across the genome. 12 copy number variations were defined in 12 patients - 7 deletions (6 de novo and 1 inherited from phenotypically normal parent) and 5 duplications (1 de novo and 4 inherited from a phenotypically normal parent). These CNVs ranged in size from those involved in a single clone to regions as large as 14Mb. On the basis of these results it was anticipated that aCGH would become a routine method of genome wide screening for unbalanced rearrangements in children with learning disabilities.

12

What kinds of things can be performed using aCGH?

- Rapid and accurate screening for subtle DNA copy number changes along the whole genome which cannot be detected by karyotype.
- Precise delineation of deleted or duplicated segments in order to investigate the link between the presence of one unbalanced rearrangement and the observed phenotype.
- Deletion/duplication breakpoint mapping and sequencing. A new way to investigate if some rearrangements could be driven by specific DNA sequences/motifs/conformations.

13

List some of the referral criteria for aCGH testing.

- Prenatal onset abnormal growth pattern
- Global developmental delay and learning difficulties
- Behavioural problems
- One or more congenital malformations
- Cranio-facial dysmorphism
- Abnormal dermatoglyphic or trichoglyphic patterns
- Family history of multiple miscarriages, learning difficulties, or congenital malformations.

14

Describe how and why aCGH has replaced karyotyping.

- aCGH at least doubles the detection of chromosome imbalances compared to karyotype in patients with neurodevelopmental disorders.
- Identifies both the location and gene content of pathogenic imbalances.
-aCGH investigations of IMR/Dysmorphic patients is revealing new deletion and duplication syndromes.
- Arrays have also revealed unexpected levels of complexity and imbalance in cytogenetically balanced rearrangements. Approximately 40% of patients with apparently balanced de novo rearrangements and abnormal clinical phenotypes will have imbalances detected by arrays.
- aCGH will detect imbalances which are the unbalanced products of balanced parental rearrangements.

15

What are the limitations of aCGH?

- Cannot detect 'balanced' chromosome rearrangements such as reciprocal translocations and inversions. Particularly the order and orientation of the rearranged segments involved cannot be determined.
- May not detect mosaicism where the proportion of abnormal cells is less than 30%.

16

Describe Copy Number Variations (CNVs).

- A CNV is a segment of DNA that differs in copy number compared with a reference genome.
- CNVs are found in 12% of the human genome and up to 40% overlap with known genes.
- Contribute up to 7% of genome variability within humans.
- Copy number variation has also been associated with autistic spectrum disorders and idiopathic learning disability.
- Some CNVs have been associated with susceptibility or resistance to disease.
- Some CNVs are common whilst others are rare.
- Many CNVs are benign. Over 29,000 CNVs are catalogued in the database of Genomic Variants.
- Some CNVs are known to be pathogenic.
- Array interpretation has to distinguish innocuous variation from that associated with disease.

17

What % of the human genome are CNVs found in?

CNVs are found in 12% of the human genome and up to 40% overlap with known genes.

18

What % of human genome variability is due to CNVs?

Contribute up to 7% of genome variability within humans.

19

Describe the considerations for clinical interpretation of CNVs.

1). Comparison of CNV with internal and external databases.
2). Association of CNV with well established syndromes.
3). Genomic content.
4). CNV size.
5). Follow up studies.
6). Inheritance of CNV.

20

How would you go about the comparison of a CNV with internal and external databases when investigating the clinical significance of a CNV?

- Look at the frequency of the CNV in internal database.
- Comparison with the Database of Genomic Variants (DGV).
- Is the CNV associated with known syndromes (see Decipher, PubMed, OMIM, ISCA, ECARUCA).

21

List some useful resources for array interpretation.

- UK Best Practice Guidelines - very general and their usefulness is limited.
- American College of Medical Genetics gives more detailed standards and guidelines for interpretation and reporting of postnatal constitutional CNVs.
- Another useful resource is 'Practical guidelines for interpreting copy number gains detected by high resolution array in routine diagnostics'. This was a paper published in European Journal of Human Genetics in 2012.
- Also useful is 'Diagnostic Interpretation of Array Data Using Public Databases and Internet Sources' published in Human Mutation.

22

What considerations should be applied when assessing the genomic content of CNVs to aid interpretation of clinical significance?

Consideration of Genomic content in CNV:
- Are there genes relevant to the phenotype?
- Are the genes dosage sensitive? Review Decipher, OMIM and relevant publications.
- If there is a loss, is a phenotype associated with haploinsufficiency? This may not have an effect when their is gain.
- CNVs involving genes with dominant mutations may not have relevance or result in a different phenotype.
- Single copy deletions of a recessive gene may only suggest carrier status, depending on a mutation on the other homologue.

23

How important is CNV size? Explain your answer.

CNV size is not always important:
- Not all large CNVs are pathogenic!
- There are even large euchromatin chromosomal variants that are known to be benign.
- Very small CNVs can be pathogenic.
- The threshold for detecting imbalances should be based on array performance balancing specificity and sensitivity.
- Potential pitfalls of using arbitrary size cut-offs.
- Careful consideration of gene content of unbalanced regions is required when interpreting aCGH data.

24

Describe what follow up studies may be conducted after aCGH.

Follow up studies for an abnormal CNV:
- In the earlier stages of the aCGH service, all reportable CNVs were confirmed in proband either with FISH or with MLPA/PCR to assess artefacts.
- Confirmation is not required in high coverage arrays and in platforms which combine aCGH and SNP arrays.
- Around 25% of referrals have imbalances which would require parental bloods to assist interpretation (unknown significance CNVs).
- 15-20% of there imbalances turn out to be de novo and depending on gene content may be causative.
- Parental bloods are tested to ascertain their recurrence risk (parents may carry a balanced rearrangement or can be asymptomatic in cases of pathogenic CNVs with reduced penetrance).
- FISH is preferable - e.g. the position of signals on chromosomes in a balanced parent can unravel submicroscopic balanced rearrangements (translocations, inversions, insertions) which would confer high recurrence risk.

25

Are familial CNVs clinically relevant?

- Although in clinical practice it is considered that if a CNV is inherited from a phenotypically normal parent then it is likely to be benign, this is not always correct however. This depends on the penetrance of the CNV.
- Syndromic CNVs have reduced penetrance and variable expressivity.
- De novo CNVs are more likely to be causal but this is by no means always the case.
- Inherited copy number gains could also be causal as may often be inherited from an asymptomatic parent.
- Phenotype may depend on the co-occurrence of other CNVs and mutations.

26

What are the essential steps in classifying a CNV as 'benign'?

1). Comparison with the Database of Genomic Variants (DGV).
2). >1% frequency and 100% coverage with same gene content and similar dosage (gain or loss) in at least one publication - happy it is benign and no need to report it.
3). Sex for X-linked CNVs.
4). Comparison with Internal Database. >3% and preferable 100% coverage. May represent a local polymorphism or be platform related.

27

How do we decide that a CNV is clearly pathogenic?

- Clinical significance is established in peer reviewed publications.
- Includes large CNV which overlap a smaller interval with clearly established significance.
- Smaller CNVs which include critical causative genes of known clinical syndromes.

28

Give some examples of pathogenic CNVs.

All known cytogenetic syndromes: DiGeorge, Prader Willi, Williams, Turner, Potocki-Shaffer and Kleefstra syndromes, MECP2 duplications, ring chromosomes and imbalances derived from balanced rearrangements. Some examples:

- A de novo loss of 3.75Mb as 22q13.31-q13.33 which overlaps the 22q13.3 syndrome (patients show some degree of mental retardation, and absence of expressive speech).

- De novo amplification of a 0.79Mb at Xp11.22. This includes HUWE1 gene which is linked to non syndromic X-linked learning difficulties.

- Maternally inherited loss of 750kb of 22q11 within the DiGeorge critical region.

29

What phenotype will a de novo loss of 3.75Mb as 22q13.31-q13.33 which overlaps the 22q13.3 syndrome region produce?

A de novo loss of 3.75Mb as 22q13.31-q13.33 which overlaps the 22q13.3 syndrome (patients show some degree of mental retardation, and absence of expressive speech).

30

What phenotype will de novo amplification of a 0.79Mb at Xp11.22 produce?

- De novo amplification of a 0.79Mb at Xp11.22. This includes HUWE1 gene which is linked to non syndromic X-linked learning difficulties.

31

Give some examples of CNVs associated with neurodevelopmental disorders of reduced penetrance.

These include:
- Loss of 1q21.1 (proximal and distal)
- Loss of 16p11.2 (proximal and distal)
- Loss of 16p13.11
- Loss of 16p12.1
- Loss of 17q12
- Loss of 15q13.2
Gains in some of these regions are also associated with milder and different phenotypes.

There are also emerging syndromes such as the 2q11.1-2 deletion and 15q11.2 deletion which has only 10% penetrance and is not universally accepted as syndromic.

The presentation of these syndromes is usually variable and can be inherited from an asymptomatic parent. Inheritance studies would establish the recurrence risk for parents.

The outcome of future pregnancies which carry these CNVs is difficult to predict which makes prenatal diagnosis questionable.

32

What is the phenotype related to loss of 1q21.1 (proximal and distal)?

Neurodevelopmental disorder of reduced penetrance.

33

What is the phenotype related to loss of 16p11.2 (proximal and distal)?

Neurodevelopmental disorder of reduced penetrance.

34

What is the phenotype related to loss of 16p13.11?

Neurodevelopmental disorder of reduced penetrance.

35

What is the phenotype related to loss of 16p12.1?

Neurodevelopmental disorder of reduced penetrance.

36

What is the phenotype related to loss of 17q12?

Neurodevelopmental disorder of reduced penetrance.

37

What is the phenotype related to loss of 15q13.2?

Neurodevelopmental disorder of reduced penetrance.

38

When might a CNV remain classified as a CNV of unknown significance?

When the further work required to classify it is not able to be carried out. For example, parental samples may not be available for testing.

39

What does the CNV classification of 'likely pathogenic' mean? What evidence might qualify a CNV to be classed as 'likely pathogenic'?

- 'Likely pathogenic' means that there is still some uncertainty but there is some evidence to suggest that this CNV is probably pathogenic.
- Gene(s) within the interval with function that is relevant to the reason of referral.
- CNV has been described in a case report with phenotype relevant to patient.
- Large CNV has been described with several genes but their combined function is difficult to determine and it is suspected to contribute to the phenotype.
- Inherited from a parent with a similar phenotype.
- It is a de novo rearrangement and the genes may be indirectly linked to the phenotype.

40

Read the following example of a likely pathogenic CNV:

- A child was referred with sever developmental delay, prominent forehead, midface hypoplasia, hypoxic ischaemic encephalopathy.
- Paternally inherited loss of a 217,974bp section of 11q13.3.
- This deletion includes part of one HGNC gene - SHANK2.
- Deletions of this part of the SHANK2 gene have been described in patients with autistic spectrum disorder and mild to moderate mental retardation.
- The inheritance pattern of ASD is complex as it possibly involves interaction of several genes.

41

Read the following example of a likely pathogenic CNV:

- A child is referred with learning difficulties, Marfanoid phenotype, tall stature and ligament laxity.
- A 27.8Mb amplification of a region of 4q25-2q28.3 was found which included 50 OMIM genes and several OMIM diseases.
- This gain has been confirmed by G band examination.
- Parental bloods have not been received as yet.
- Given the size of the imbalance and the number of genes involved it is likely to be associated with the patient's clinical problems.

42

When may a CNV be classified to be of uncertain clinical significance - likely benign?

- A CNV is likely to be classified as likely benign when there are no genes in the interval unless it is located in close proximity to a well characterised gene with clinical relevance to the reason of referral. If this happens then the CNV may have disturbed the regulatory elements of that gene.
- Inherited from a phenotypically normal parent.
- Genes which do not appear to have clinical relevance to the phenotype.

43

Read the following example of a likely benign CNV:

- A child was referred for developmental delay, short stature and dislocations.
- A 2.8Mb loss of a region of Chromosome 1 was detected between 1q32.1 and 1q32.2.
- Parents were deceased.
- This region contains several refseq genes and 5 OMIM genes.
- This call does not appear to contain any genes which can be directly linked to the phenotype and the clinical significance. Therefore it is likely benign.

44

Why might a CNV be classified as having 'Uncertain Clinical Significance'?

- There is insufficient evidence available to determine the clinical significance of the CNV.
- CNV is described in contradictory publications.
- CNV contains genes but it is not known whether the genes in the interval are dosage sensitive.
- Parents are not available and it cannot be established if the CNV is de novo or inherited.

45

Read the following example of a CNV of uncertain significance:

- A child is referred with learning difficulties, microcephaly, behaviour problems, dysmorphic features and deafness.
- A maternally inherited 199kb loss of a region of chromosome 15 at band 15q15.3 was detected.
- This was confirmed by FISH.
-This region contains the autosomal recessive STRC OMIM gene, mutations of which are associated with deafness
- If this deletion has an unmasked mutation in the other copy this may account for the mutation but not for the rest of the phenotype.
- Other molecular testing may clarify this finding.

46

What should be considered when deciding whether or not CNVs of questionable importance should be reported?

1). Intronic deletions in a gene relevant to the phenotype:
- Often thought that intronic deletions in genes relevant to the phenotype should be reported.
- e.g. There are highly conserved intronic sequences upstream of exon 7 containing binding sites in NRXN1 which also has incomplete penetrance.
- Disruptions of these sequences have been associated with expression of the gene which has been linked to schizophrenia and intellectual disability.

2). Loss of a region which includes a recessive gene which could cause the clinical phenotype if there is a mutation on the other allele:
- e.g. loss of MERTK gene of which heterozygous mutations or homozygous deletions have been associated with Retinitis Pigmentosa (Rod-Cone Dystrophy) which was associated with the reason of referral. Repeated sequencing confirmed a mutation.

47

Should intronic CNVs of questionable importance be reported?

- Often thought that intronic deletions in genes relevant to the phenotype should be reported.
- e.g. There are highly conserved intronic sequences upstream of exon 7 containing binding sites in NRXN1 which also has incomplete penetrance.
- Disruptions of these sequences have been associated with expression of the gene which has been linked to schizophrenia and intellectual disability.

48

Should CNVs of questionable importance be reported when there is a loss of a region which includes a recessive gene?

- Loss of a region which includes a recessive gene could cause the clinical phenotype if there is a mutation on the other allele:
- e.g. loss of MERTK gene of which heterozygous mutations or homozygous deletions have been associated with Retinitis Pigmentosa (Rod-Cone Dystrophy) which was associated with the reason of referral. Repeated sequencing confirmed a mutation.

49

What incidental findings might we discover on aCGH testing?

Incidental findings are those that do not relate to the clinical problems for which the patient was referred. These include:

- Late onset conditions

- Cancer-suceptibility genes

Most labs will report genes that are strongly associated with malignancy such as BRCA1.

50

Describe the efficacy of chromosomal microarray analysis in prenatal analysis.

- For prenatal diagnosis the fetal DNA derives from amniocytes or chorionic cilli samples.
- Arrays can detect aneuploidy and copy number changes.
- SNP arrays can also identify triploidy and complete moles.
- aCGH identifies additional clinically significant abnormalities in approximately 6% of fetuses with USS abnormalities and a normal conventional karyotype.
- aCGH detected an abnormality is 1.7% of fetuses with a normal karyotype.
- Difficulty in interpreting unknown significance variants. Samples from both parents may be required to help understand the significance of these results.

51

Where is DNA derived from for prenatal aCGH?

For prenatal diagnosis the fetal DNA derives from amniocytes or chorionic cilli samples.

52

What prenatal abnormalities can be detected by array?

- Arrays can detect aneuploidy and copy number changes.
- Cannot always reliably detect triploidies etc.

53

What prenatal abnormalities can be detected by SNP array?

SNP arrays can also identify triploidy and complete moles.

54

Are arrays better than karyotyping in a prenatal context? What may arrays detect?

- Chromosomal microarray analysis identifies almost all of the abnormalities that are identified by fetal karyotyping and may identify additional specific genetic disease.
- Arrays will not detect balanced rearrangements whereas karyotyping will.
- Copy number variants of reduced penetrance may be identified. Their impact cannot be predicted even if they are inherited.
- Diseases with variable expressivity may be identified (clinical presentation may vary greatly and range from mild to sever).
- The test may identify consanguinity or non-paternity.
- Unknown significance variants are difficult to interpret in a prenatal diagnostic setting. Samples from both parents may be required to help understand the significance of these results.
- Test results may identify adult onset diseases. This may also indicate that one of the parents has the same adult onset disease but has not yet developed symptoms.

55

What will arrays detect in a prenatal context?

- Chromosomal microarray analysis identifies almost all of the abnormalities that are identified by fetal karyotyping and may identify additional specific genetic disease.
- Arrays will not detect balanced rearrangements whereas karyotyping will.
- Copy number variants of reduced penetrance may be identified. Their impact cannot be predicted even if they are inherited.
- Diseases with variable expressivity may be identified (clinical presentation may vary greatly and range from mild to sever).
- The test may identify consanguinity or non-paternity.
- Unknown significance variants are difficult to interpret in a prenatal diagnostic setting. Samples from both parents may be required to help understand the significance of these results.
- Test results may identify adult onset diseases. This may also indicate that one of the parents has the same adult onset disease but has not yet developed symptoms.

56

What considerations should be taken into account when using arrays in the prenatal setting?

- The pretnatal setting is different from postnatal and different considerations are applicable.
- Recommendations are required for the use of chromosome microarray in pregnancy.
- Patient counselling about the benefits, limitations and results of testing should be given to enable patients to make informed decisions.

57

What is the minimum resolution that the BSHG recommends throughout the genome for prenatal array testing?

A microarray platform capable of detecting a minimum resolution of approximately 400kb throughout the genomes as a balance of analytical and clinical sensitivity.

58

Describe the BSHG recommendations for prenatal array testing.

- To link clinical and molecular data for consistent national diagnostic interpretation of results.
- To obtain parental samples to assess the significance of novel duplications and deletions.
- To use a microarray platform capable of detecting a minimum resolution of approximately 400kb throughout the genome as a balance of analytical and clinical sensitivity.
- Designs with added targeted coverage in known disease associated genes and regions should explicitly state the specific design and mean minimum detection threshold for targeted regions.
- To state the sensitivity of detection of mosaic findings.

59

What are the BSHG recommendations for indications of prenatal array testing?

- One or more structural anomalies detected on USS.
- An isolated nuchal translucency NT=3.5mm when crown-rump length measures from 45mm to 84mm (at approximately 11 weeks 0 days to 13 weeks 6 days).
- Fetuses with a sex chromosome aneuploidy that is unlikely to explain the ultrasound findings.
- Further updating as more evidence becomes available on the diagnostic use of microarrays in pregnancy.

60

What variants should be reported in the context of a prenatal setting?

- Pathogenic variants relevant to the referral indication.
- Neuro-susceptibility loci associated with an increased incidence of anomalies detectable on scan.
- High penetrance neuro-susceptability loci that are associated with a risk of a severe phenotype.
- Deletion of a known cancer predisposition gene such as BRCA1.
- Deletion of the dystrophin gene in a female fetus, again to allow the mother to be tested for carrier status.

61

What incidental aCGH findings should be reported in a prenatal setting?

- Deletion of a known cancer predisposition gene such as BRCA1.
- Deletion of the dystrophin gene in a female fetus, again to allow the mother to be tested for carrier status.

62

Should neuro-susceptibility loci associated with an increased incidence of anomalies detectable on scan be reported in a prenatal setting?

Yes.

63

Should high penetrance neuro-susceptability loci that are associated with a risk of a severe phenotype.be reported in a prenatal setting?

Yes.

64

What incidental findings should not be reported in a prenatal setting?

- Any finding that is not linked to potential phenotypes for the pregnancy or has no clinically actionable consequences for that child or family in the future.
- Variants of uncertain significance that cannot be linked to a potential phenotype on the basis of the genes involved.
- Low penetrance neuro-susceptibility loci and unsolicited variants (such as 5q13.1q13.3 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.

65

Should low penetrance neuro-susceptibility loci and unsolicited variants be reported in a prenatal setting?

No.

66

Should 5q13.1q13.3 duplications be reported in a prenatal setting?

No. Low penetrance neuro-susceptibility loci and unsolicited variants should not be reported in a prenatal setting.

67

Should 15q11 BP1-BP2 duplications or deletions be reported in a prenatal setting?

No. Low penetrance neuro-susceptibility loci and unsolicited variants should not be reported in a prenatal setting.

68

Should Xp22.31 (STS) duplications be reported in a prenatal setting?

No. Low penetrance neuro-susceptibility loci and unsolicited variants should not be reported in a prenatal setting.

69

Should 16p13 duplications be reported in a prenatal setting?

No. Low penetrance neuro-susceptibility loci and unsolicited variants should not be reported in a prenatal setting.

70

Should heterozygous deletion of recessive genes that cannot be linked to the presenting phenotype be reported in a prenatal setting?

No.

71

Should variants of uncertain significance that cannot be linked to a potential phenotype on the basis of the genes involved be reported in a prenatal setting?

No.

72

Should any finding that is not linked to potential phenotypes for the pregnancy or has no clinically actionable consequences for that child or family in the future be reported in a prenatal setting?

No.

73

When may array testing of Products of Conception (POC) be carried out?

- To investigate the aetiology of the miscarriage.
- To counsel for the proper assisted reproductive technology (ART) to improve the reproductive outcome in a future pregnancy.
- Indicated in women suffering a first trimester fetal loss.
- Is particularly relevant in couples with recurrent miscarriages and couples with a miscarriage after an ART cycle.

74

What are the advantages of analysing POC by arrays?

- Tissue culture is not required unlike in G band analysis.
- Successful results in more than 98% of the analysed samples.
- Analysis to rule out maternal cell contaminations.
- SNP arrays can detect triploidy and complete mole.
- Can detect unbalanced rearrangements which derive from a balanced parental rearrangement. This would have a recurrence risk for the parents and may also have a risk for an unbalanced offspring.

75

Describe some of the common findings in POCs analysed by arrays.

- Aneuploidy. Aneuploidy of all chromosomes can be found in POCs.
- Recurrent aneuploidy contributes to recurrent miscarriage in a small number of patients - it is believed that this is caused by mutations of genes that control various steps in meiosis.
- Triploidy and complete moles can be detected as well as derivatives from balanced rearrangements (translocation/inversion/insertion).
- Duplications/deletions are often inherited from a normal parent.

76

Describe the applications of arrays in haematological malignancies.

- Several studies identified a wide spectrum of copy number changes (gains/losses) in AML-ALL-MM patients.
- Copy number neutral loss of heterozygosity (LOH) are identified with SNP arrays and this is not a common finding in haematological malignancies.
- Chromothripsis could be identified.
- Genetic alterations allow us to define subgroups with worse prognosis and rapidly stratify treatment.
- New expressed biomarkers are used for development of target leukaemia therapy.
- Individualised, personalised leukaemia therapy becomes reality.

77

Describe the applications of aCGH in the clinical management of tumours.

- Reveal copy number markers for cancer prediction and choice of treatment.
- Monitor cancer progression and can distinguish between mild and metastatic lesions.
- Measure the DNA copy number or oncogenes and tumour suppressor genes.
- Identify and understand the genes that are involved in cancer and help to design therapeutic drugs.

78

What can expression arrays be used for?

- Expression arrays can be used in the classification of tumours.
- Gene expression profiling helps molecular classifications.
- Identification of subclones and tumour cell heterogeneity.
- Phenotype is often associated with different genetic profiles which helps classification of tumour.
- Identification of clonal heterogeneity before and after treatment, clonal selections under treatment and Minimal Residual Disease (MRD).
- Maximizing cure rates by personalising therapy is one of the major aims of modern therapy.

79

What type of array might you use for identification of subclones and tumour cell heterogeneity?

An expression array.

80

What can expression arrays identify before, during and after tumour treatment?

Expression arrays can be utilised in the identification of clonal heterogeneity before and after treatment, clonal selections under treatment and Minimal Residual Disease (MRD).

81

What does the future hold for microarrays? Is NGS likely to replace microarrays any time soon?

- Microarrays have transformed and revolutionised clinical cytogenetics.
- Microarrays are generally considered easier than NGS to use and analyse data from.
- More economical than NGS and yield higher throughput.
- The transition away from microarrays to NGS is a long and varied one.