12 - Molecular Cytogenetics 1

Question 1

Structural Variation (SV)

Answer 1

• Rearrangements of the DNA in a genome resulting in novel breakpoint junctional events
• Copy number neutral (Inversion or balanced translocation)
• Copy number variants (Deletion, duplication, triplication)

Question 2

Copy number variants

Answer 2

• Major source of genetic diversity
• Common
• Mostly benign polymorphic structural
  variation (SV) with no phenotypic effect
• Can have a role in human disease (e.g. obesity and cancer predisposition

Question 3

Role of genomic architecture on human genetic disease

Answer 3

Structural features of the human genome or the genomic architecture, can result in region-specific susceptibility to rearrangements and thus genomic instability which can result in human genetic disease.

Question 4

Genomic disorders

Answer 4

Loss or gain of:
- Whole chromosome (aneuploidy)
- Several adjacent genes in a contiguous gene syndrome
(microdeletion/microduplication syndrome)
- Single gene
- Exons (part of a gene)

Question 5

Molecular mechanisms through which phenotypes of genomic disorder can arise

Answer 5

• Gene dosage (altering copy number of a dosage sensitive gene, e.g haploinsufficiency)
• Gene interruption (exon deletion)
• Gene fusion
• Position effect
• Deletion unmasking recessive variant on the other allele

Question 6

Methods of detecting CNVs

Answer 6

• Karyotype
• FISH (metaphase & interphase)
• QF-PCR
• DNA microarray
• MLPA
• NGS

Question 7

QF-PCR

Answer 7

• Prenatal diagnosis of abnormal chromosome copy number (aneuploidies)
• Only a few aneuploidies are compatible with life (13, 18, 21, X and Y)
• At risk pregnant woman undergoes invasive sampling of amniotic fluid or CVS (foetal DNA is obtained)
• Performed using multiplex of fluorescent, polymorphic STR (short tandem repeat) markers from chromosomes 13, 18, 21, X and Y
• Copy number is determined from the relative quantification of STR markers (normal if peak 1:1, trisomy = 1:1:1)

Question 8

Advantages of QF-PCR

Answer 8

• Accurate
• Low failure rate
• Rapid
• Cost effective
• Suitable to automation

Question 9

What does QF PCR stand for

Answer 9

Quantitative Fluorescent PCR

Question 10

DNA microarray

Answer 10

• DNA chips
• Whole genome analysis
• A collection of DNA spots attached to a solid surface
• Each DNA spot contains a specific DNA sequence, known as a probe (oligonucleotide)
• Probes are used as hybridisation targets
• Probe-target hybridization is usually detected and quantified by detection of fluorophore

Question 11

Advantages of microarray compared to karyotyping

Answer 11

• Much higher resolution and therefore diagnostic yield
• Cheaper
• Robust
• Tissue culture not needed

Question 12

Disadvantages of microarray

Answer 12

• Unable to detect balanced rearrangements
• No positional information for a duplication
• Can be time consuming to analyse and report
• Many CNVs are novel with unknown clinical significance
• Can detect incidental findings (eg deletion of cancer suppressor genes)

Question 13

Microarray uses

Answer 13

• Prenatal diagnosis
• Pregnancy loss
• Cancer

Question 14

Types of chromosome microarrays

Answer 14

• Array CGH (Genomic gains and losses)
• SNP array (genomic gains and losses plus copy neutral aberrations)

Question 15

Array CGH

Answer 15

• Patient genomic DNA compared to normal reference control DNA
• Fragment DNA, fluorescently labelled in different colours and then competitively hybridised on to the array and read with a fluorescence scanner.

Question 16

SNP arrays

Answer 16

• Do not use control reference DNA
• Fragmented patient DNA is hybridised to the array
• SNP allele frequency and absolute fluorescence levels are compared to a standard consisting of averaged results for multiple normal samples

Question 17

Detection of CNVs

Answer 17

Log R Ratio and B Allele Frequency

Question 18

Log R Ratio (LRR)

Answer 18

• Log R Ratio (LRR) is a normalised measure of the total signal intensity for the SNP
• Any deviations from zero for LRR is evidence for copy number change.

Question 19

B Allele Frequency (BAF)

Answer 19

Measure of the allelic intensity ratio

Question 20

Diagnostic implications regions of homozygosity (ROH)

Answer 20

• Uniparental disomy (single large ROH on same chromosome)
• Identity by descent (multiple ROH across different chromosomes)

Question 21

5 tiers to classification of CNV

Answer 21

• Pathogenic
• Likely pathogenic
• Uncertain
• Likely benign
• Benign

Question 22

Reporting pathogenic / likely pathogenic microarray results

Answer 22

• Testing family members may be appropriate.
• Test parents for recurrence risk.
• Rule out balanced rearrangement
• Genetic counselling recommended

Question 23

Reporting CNV uncertain microarray results

Answer 23

• Testing parents may be helpful
• Genetic counselling may be appropriate

Question 24

Multiplex Ligation dependent Probe Amplification (MLPA)

Answer 24

• Targeted to specific regions of interest NOT whole genome analysis
• Have probes that target a specific genome sequence
• MLPA probe consists of two parts (left and right probe oligonucleotide - LPO, RPO)

Question 25

LPO and RPO

Answer 25

• Contain PCR primer and DNA hybridisation sequences
• Stuffer sequence gives prove a unqiue length

Question 26

Steps of MLPA

Answer 26

1. Sample denaturation and probe hybridisation
2. Probe ligation
3. Probe amplification
4. Fragment separation by capillary electrophoresis
5. Data analysis

Question 27

Problem with detection of SVs by NGS

Answer 27

• Structural variants usually occur is regions with complex genomic architecture: flanked by repetitive sequence elements (LCRs).
• Difficult to sequence