21. Use of microarray for detection of idiopathic learning difficulties

Question 1

What types of referral are received for microarray?

Answer 1

1. Postnatal blood for dev delay, congenital abnormalities
2. Tissues from TOP or pregnancy loss (after QF-PCR pre-screen)
3. Amnio/CVS due to abnormal USS (after QF-PCR pre-screen)

Question 2

What are LCRs?

Answer 2

Low copy repeats aka segmental duplications

Highly homologous sequences of >95% homology, >1kb in size

Mediate NAHR if in close proximity leading to dels/dups

Question 3

How does a SNP array work?

Answer 3

Solid surface containing 1000s of unique probe that hybridise adjacent to SNP

Fragmented DNA hybridises to probe - single base extension to determine genotype of SNP based on colour of fluorescence

Question 4

What is the resolution of a SNP array?

Answer 4

Varies across genome

Illumina 850k array has 850k probes across the genome - enriched for >3000 dosage sensitive genes, with lower resolution backbone

Average spacing 1kb in targeted genes, 5kb in backbone

Question 5

What are the two metrics used in SNP array analysis?

Answer 5

1. Log ratio - total intensity of both fluorescent signals combined
2. B allele frequency - colour of fluorescence emitted at each SNP

Question 6

Why are there 2 measures of copy number in SNP array analysis?

Answer 6

Independent measures used to corroborate one another

Question 7

What is a cluster file?

Answer 7

Reference file

Generated from cohort of normal patient samples, used to compare test sample against

Captures normal variation in population, operators, sample types

Question 8

What formula is used to determine the BAF?

Answer 8

B / (A+ B)

Question 9

What BAF pattern is seen for a normal copy number?

Answer 9

3 clusters at 1, 0.5 and 0

Question 10

What BAF pattern is seen for a het deletion?

Answer 10

2 clusters at 1 and 0

No heterozygous SNPs so no cluster at 0.5

Question 11

How does a hemi/hom deletion appear on SNP array?

Answer 11

Copy number 0 on log track

Random allocation of SNPs on BAF track

Question 12

What BAF pattern is seen for a het duplication?

Answer 12

4 clusters at 0, 0.33, 0.66 and 1

Question 13

How does mosaic CNV appear on SNP array?

Answer 13

Small loss/gain on log ratio

Duplication pattern on BAF track - het ratios differ from 0.5 depending on level of mosaicism

Question 14

How does triploidy appear on SNP array?

Answer 14

Duplication pattern for all of every chromosome

Not visible on log ratio as normalised for entire chromsome

Question 15

What indicates consanguinity?

Answer 15

Multiple regions of LOH affecting many chromosomes across the whole genome

Total autosomal LOH of >1.5% = consanguinity

Question 16

How does UPD appear on SNP array?

Answer 16

LOH on a single chromosome (deletion pattern)

Log ratio normal

Question 17

What techniques are used to follow up array and what are their advantages & disadvantages?

Answer 17

FISH to confirm - provides positional info but CNV may be below resolution

Targeted array if below resolution but no positional info

MLPA if kit available

Karyotyping if balanced rearrangement suspected

Question 18

What are the advantages of SNP array?

Answer 18

High resolution than karyotyping - higher pick up rate

Amenable to automation and high throughput

BAF track can detect consanguinity therefore increased risk of AR conditions and UPD therefore additional diagnoses

Question 19

What type of UPD can SNP array detect?

Answer 19

Isodisomy only

Heterodisomy if parents are also tested

Question 20

What are the disadvantages of SNP array?

Answer 20

1. Can’t detect balanced rearrangements
2. Can’t give positional info
3. Increased detection of VUSs –> parental anxiety, counselling
4. Risk of incidental findings (BRAC2 deletion, DMD CNVs)

Question 21

What is the purpose of testing for dev delay/LD?

Answer 21

Provides prognostic info, direct clinical care & educational needs

Allow for future prenatal diagnosis

Question 22

What evidence is used to determine the pathogenicity of a CNV?

Answer 22

Number of genes involved

Known haploinsufficient or triplosensitive genes involved?

Previous de novo cases, taking into account phenotypic overlap

Segregation

Case-control data