Principles of mutation screening

Question 1

What are the three types of genomic changes?

Answer 1

Gross chromosomal abnormalities, copy number alterations, gene mutations (including insertions and deletions)

Question 2

What are the methods used to investigate genomic changes?

Answer 2

Karyotyping, FISH, SNP arrays, Sanger sequencing, NGS, RFLP and size change detection

Question 3

What are the two types of balanced chromosomal rearrangements. Give an example for each.

Answer 3

1. Formation of a chimeric fusion gene - This leads to the production of new fusion mRNAs which produce abnormal proteins e.g BCR-ABL
2. Deregulated expression of structurally normal genes - generally following translocation, brings an oncogene under the control of an active promoter e.g BCL2 and MYC

Question 4

What are the two types of chromosomal imbalance mechanisms?

Answer 4

1. Genomic gain - could be complete or partial trisomy.
2. Genomic loss - Could lead to monosomy (single Chr loss) or a large scale deletion where not all of the chromosome is lost

Question 5

What is Array CGH?

Answer 5

1. Patient and control DNA are labelled with fluorescent dyes and applied to the micro array (the micro-array contains individual glass slides/solid supports that contain probes of varying size - oligos to represent areas of interest or broad genomic clones such as bacterial artificial chromosomes).
2. Patient and control DNA compete to attach or hybridise to the micro-array
3. The micro-array scanner measures the fluorescent signal.
4. Computer software generates a signal to be read. Equal hybridization leads to a mix in the fluorescent signal of control Vs patient DNA. Patient DNA loss means the signal will be stronger from the control DNA. Patient DNA gain will increase the signal in favor of the patient fluorophore.

Question 6

What is an SNP?

Answer 6

A variant nucleotide at a specific position in the genome found in more than 1% of the population

Question 7

How many SNPs are used per array?

Answer 7

more than 6,000,000 probes across the entire genome

Question 8

What is the purpose of SNP arrays?

Answer 8

Allow for genotyping of hundreds and thousands of selected SNPs across all chromosomes
provide information on copy number (amplification, deletion) by intesnsity of the total signal
Provide information on paternal or maternal origin of genomic region by changes in heterozygosity (copy number neutral LOH)

Question 9

What is the use of a SNP array in AML?

Answer 9

Provide information on copy number and look at copy number neutral LOH. If this happens recurrently in a region of the genome it tells us that something changing is advantageous to the cancer.

Question 10

How does uniparental disomy occur? (copy number neutral LOH).

Answer 10

During mitosis, mitotic recombination takes place which induces recombination at a particular locus, making one cell homozygous for the paternal allele and the other homozygous for the maternal allele.

Question 11

How is UPD detected by SNP arrays?

Answer 11

The total copy number doesn’t change in the region. However, heterozygous SNP call shows LOH. Looking at allele specific copy number shows that one allele has been enrighed compared to the other. This region has experienced copy number neutral LOH.

Question 12

What is an example of UPD in AML?

Answer 12

One copy of FLT3 is mutated (activating mutation). At relapse or disease progression cells found to be homozygous for the mutant allele due to copy number neutral LOH.

Question 13

Which chromosome experiences the highest frequency of deletions in AML?

Answer 13

Chromosome 7

Question 14

Which chromosomes often experience trisomy in AML?

Answer 14

Chr8 and 21

Question 15

What is uniparental disomy?

Answer 15

Copy number neutral LOH by mitotic recombination. If this gives an survival benefit to a tumour it is selected for.

Question 16

When is NGS not a good method for investigating changes in the genome>

Answer 16

If there have been insertions as it assumes its from somewhere else in the genome.

Question 17

Whats the benefit of using a 52 NGS gene panel in AML?

Answer 17

More affordable, higher average depth of coverage following many reads, simpler IT requirements, fewer variants to interpret, shorter turn-around time in a diagnostic setting.

Question 18

What internal tandem duplication is seen in AML?

Answer 18

FLT3 - an in frame repeated sequence (can be of 3 to more than 300 nucleotides in length) - produces a constituitively active FLT3 protein.

Question 19

How is the FLT3 ITD diagnosed?

Answer 19

Use primers that extend over the region of FLT3 where the ITD takes place. Amplify this and run it on a gel. Compare to WT band, there are two bands in patients with FLT3 ITDs.

Question 20

What is the other FLT3 mutation found in AML (not ITD) How are they detected?

Answer 20

Tyrosine kinase domain point mutations detected by RFLP analysis.

Question 21

How is RFLP analysis used to diagnose FLT3 point mutations?

Answer 21

The region in which mutations are commonly found is an EcoRV restriction enzyme recognition motif. PCR the region of FLT3, add EcoRV and compare uncut DNA, WT DNA and the sample.
Uncut = 1 band
WT = 2 bands
Mutant = majority uncut

Question 22

What are the causes of non-synonymous consequences of point mutations/indels

Answer 22

AA substitution (missense)
Introduction of a STOP codon (nonsense)
Frameshift typically causes a premature stop.

Question 23

What are the causes of synonymous or non-coding consequences of point mutations/indels

Answer 23

Alter the splice site

Affect regulatory region

Question 24

What intergenic region has been found to be proto-oncogenic in T-ALL?

Answer 24

Intergenic region upstream of TAL1 (an oncogene). Mutation in the non-coding region creates a novel binding site for a transcription activator complex (MYB). MYB binds and drives the production if TAL1, drives T-ALL

Question 25

What are the common chromosomal translocations found in AML?

Answer 25

Translocations that alter genes responsible for regulation of transcription.

Question 26

In patients that have reciprocal translocations, what is significant about the timing of the mutation?

Answer 26

Thought to be the initiating lesion in those that have it. Found in all tumour cells and persist at relapse.

Question 27

How do reciprocal translocation fusion proteins change the normal process of haematopoiesis?

Answer 27

Fusion proteins are typically regulatory TFs. The DNA binding domain of the WT TF remains unchanged in the fusion protein. The fusion partner is able to recruit repressors of gene expression (HDACs, corepressors, Dnmt’s). Gene for normal haematopoiesis becomes deacetylated/methylated, turning gene expression off leading to a block in normal differentiation of blood cells.

Question 28

What genes experience point mutations and deletions in AML?

Answer 28

Genes encoding transcriptional regulators (RUNX1, CEBPA, GATA1). Mutations cause a loss in their ability to bind DNA to turn on genes for differentiation etc.

Question 29

What is suggested by finding mutually exclusive mutation events.

Answer 29

They work in the same signalling pathway/ offer the same benefit to the tumour.

Question 30

What is the result in mutations in IDH1/2?

Answer 30

It causes a change in their enzymatic function. Instead of catalyzing Isocitrate to alpha-Ketoglutarate + NADPH. Catalyses a new reaction - alpha-ketoglutarate to 2-hydroxyglutarate + NADP+. 2HG inhibits alpha KG mediated reactions. In AML, alpha KG is prevented from acting as a cofactor for the hydroxylation of methylated cytosine residues. This alters the epigenetic state of the cell, affecting cell differentiation.

Question 31

What are thought to be the requirements for the development of AML?

Answer 31

mutually exclusive genetic changes affecting cell signalling, hydroxymethylation, transcription factors and epigenetic modifiers.