18.01.16 Origin of structural abnormalities

Question 1

What mechanisms can lead to genomic rearrangement? Give examples.

Answer 1

1. Non-replicative non-homologous repair mechanisms e.g. NHEJ, MMEJ, Breakage-fusion-bridge cycle
2. Replicative non-homologous repair mechanisms e.g. FoSTeS, MMBIR, chromothripsis

Question 2

What are the potential underlying mechanisms of reciprocal translations?

Answer 2

Could be potentially formed via NHEJ, MMEJ, FoSTeS or MMBIR.

Question 3

What is NHEJ?

Answer 3

Example of non-replicated non-homologous repair mechanism to repair double-strand breaks (DSB).

It requires no homology and often leads to small (1-4bp) deletions or insertions of free DNA at the breakpoints as a result of editing at breakpoints to make them compatible for ligation.

NHEJ is considered to be the major mechanism generating chromosomal translocations in cancer.

Question 4

What is MMEJ?

Answer 4

This mechanism requires short regions of homology (5-25bp) at either side of a double strand break allowing repair to occur through annealing and ligation.

This mechanism often leads to deletion of sequences located between the regions of homology. This mechanism appears to be the one associated with chromosomal fusions formed between critically shortened telomeres

Question 5

What is the breakage-fusion-bridge cycle?

Answer 5

Chromosome or sister-chromatid fusion due to telomere erosion can create dicentric chromosomes; during anaphase, these centromeres can be pulled to separate nuclei resulting in breakage of the chromosome.

The broken ends of this chromosome are then prone to further rounds of fusion and subsequent breakage.

This cycle will stop when the chromosome acquires a telomere.

This repeated cycle of breakage and fusion is believed to play an important role in the generation of chromosome instability in cancer.

Question 6

Which mechanisms is thought to be a large contributor to translocations seen in leukaemia?

Answer 6

Incorrect repair by NHEJ and MMEJ in the presence of multiple double strand breaks is thought to be the major contributor to translocations seen in leukaemia.

Sequence motifs (e.g. TTTAAA) and repetitive sequences (such as LTRs, LINE, Alu) that promote secondary structure formation in DNA such as hairpin and cruciform are prone to DBSs and this may explain why such regions are often found in proximity of translocation breakpoints.

Question 7

When do replicative non-homologous repair mechanisms act?

Answer 7

during DNA replication

Question 8

What is FoSTeS?

Answer 8

During replication fork stalling, the 3’ end of the lagging DNA strand can disengage from the original template and anneal, by virtue of microhomology, to a single-stranded section of DNA in a nearby replication fork, where synthesis restarts. “Nearby” refers to spatial closeness and not necessarily adjacent in primary sequence.

Question 9

How does FoSTeS lead to genomic rearrangements?

Answer 9

This mechanism could cause deletions, duplications, inversions or translocations depending on the relative position of the two replication forks.

Switching to a downstream fork would result in deletion, while switching to an upstream from would result in duplication. FoSTeS can occur multiple times in series leading to more complex rearrangements.

Like other mechanisms leading to rearrangements, FoSTeS is likely to be influenced by local genomic architecture (e.g. palindromes or cruciform), with fork stalling caused by secondary structure formation, lesions in the template strand or a shortage of dNTPs.

Question 10

What is MMBIR?

Answer 10

This mechanism is often associated with restart of a collapsed replication fork, it is initiated by a single-single stranded region at a double-strand break, the 3’ end anneals to any single-stranded template with which it shares micro-homology and is present in close proximity. The break is repaired through DNA replication of this end, using the invaded region as a template.

Question 11

How does MMBIR lead to genomic rearrangements?

Answer 11

Annealing of the invading end within a sister chromatid and depending upon the position and orientation of invasion can generate deletions, duplications and inversions, while annealing within a different chromosome can lead to translocation. Repeated MMBIR cycles can lead to complex chromosome rearrangements. MMBIR represents one model for the molecular mechanism of FoSTeS.

Question 12

What is chromothripsis?

Answer 12

Chromothripsis: Joining, possibly via NHEJ, of chromosome portions that have been shattered into hundreds of pieces. This mechanism can explain highly complex derivative chromosomes seen in some cancers and in very few constitutional rearrangements. See review by Forment et al., (2012).

Question 13

Give two examples of a recurrent translocation.

Answer 13

t(11;22)(q23.3;q11.2)

t(4;8)(p16;p23)

t(X;Y)(p22.3;p11)

Question 14

Describe the formation of the recurrent t(11;22) translocation.

Answer 14

the most common recurrent translocation in humans is mediated by similar palindromic AT rich repeats (PATRR). A 450bp PATRR present on chr 11 and a 590bp PATRR on chr 22 (within LCR22) lead to intra-strand pairing and the formation of hairpin or cruciform structures. The centre of the palindrome is susceptible to DNA breakage that could lead to NHEJ between the two regions.

Question 15

Describe the formation of the recurrent t(4;8) translocation.

Answer 15

t(4;8)(p16;p23): 8p23 contains two repeats of oldfactory receptor (OR) gene clusters, the distal (REPD) and the proximal (REPP)

4p16 also contains repeats of OR gene clusters with high degree of homology to the 8p23 repeats. It is thought that the similar REPs on chr 4 and 8 mediate the t(4;8)(p16;p23) translocation, however a specific mechanism does not seem to have been proposed.

Question 16

What other rearrangements are associated with the OR gene clusters at 8p23?

Answer 16

inv dup (8p), +der(8)(p23pter), del(8)(p23p23) and a benign submicroscopic inversion within the region.

This inversion is present at a heterozygous status at a high frequency (25%) in the population and it is thought to convey structural susceptibility to the other rearrangements.

Question 17

What is the proposed mechanism for the formation of the recurrent t(X;Y)?

Answer 17

t(X;Y)(p22.3;p11): It has been suggested that an inversion on Yp might predispose for this translocation leading to XX males and XY females.

Question 18

Which factors influence partner choice in reciprocal translocations?

Answer 18

Spatial proximity

In cancer - IGH and MYC loci

the choice of partner in reciprocal translocations is governed by multiple factors including spatial proximity within the nucleus and the frequency and likelihood of a locus harbouring a double strand break.

Question 19

What are the five proposed mechanisms of Robertsonian chromosome formation?

Answer 19

1) centric fusion of two acrocentric chromosomes (monocentric)
2) break in one short and one long arm (whole-arm translocation, monocentric),
3) break in both short arms and formation of a dicentric chromosome
4) misdivision of centromere (Homologous Robertsonians only)
5) U-type exchange (break in both chromatids that then loop around to join each other) and formation of an isochromosome at the next cell division (Homologous Robertsonians only).

Question 20

What are the two most common Robertsonian translocations? What is their method of formation?

Answer 20

Centric fusion is the most common mechanism of formation of the two most common Robertsonians, rob(13;14) and rob(14;21).

The breakpoints are consistent and cluster between two satellite -III sequences on 14p and a satellite -I and rDNA sequence on 13p and 21p. It has been suggested that similar sequences are orientated in an opposite direction on chromosome 14 compared to 13 and 21, promoting paring and recombination of these chromosomes.

The rest of the Robertsonian translocations have variable breakpoints suggesting other mechanism of formation.

Question 21

How are terminal deletions thought to arise?

Answer 21

Terminal deletions can be caused by DSBs that are repaired and stabilised by:

1) the synthesis of a new telomere (telomere healing)
2) obtaining a telomere sequence from another chromosome (telomere capture)
3) chromosomal circularization, leading to a ring chromosome.

The first two mechanisms have been identified in del(22)(q13.3) (Phelan-McDermid syndrome).

The presence of repetitive elements (Alu, LINE, SINE, LTR) and simple repeats may play a role in generating and stabilizing terminal deletions, although not always.

Question 22

How are ring chromosomes formed?

Answer 22

Ring chromosomes usually result from two terminal breaks in both chromosome arms, followed by fusion of the broken ends, or from the union of one broken chromosome end with the opposite telomere region, leading to the loss of genetic material.

Alternatively, they can be formed by fusion of subtelomeric sequences or telomere-telomere fusion with no deletion, resulting in complete ring chromosomes. NHEJ or MMBIR have the proposed mechanisms of repair in ring 22 chromosomes.

Question 23

How are idic(15q) formed?

Answer 23

The breakpoints of small idic(15q) chromosomes seem to involve BP1 and BP2 of the Prader-Willi/Angelman syndrome region.

The breakpoints of larger idic(15) chromosomes involve BP3, BP4 and other more distal loci.

The majority of idic(15q) are thought to occur via a U-type exchange between LCRs on homologs. However non-NAHR/LCR based mechanisms have also been proposed.

Question 24

How are idic(22(q11.2) formed?

Answer 24

Cat-eye syndrome (CES). LCR22-A and LCR22-D have been shown to be responsible for the idic(22)(q11.2) formation. As with idic(15q),

The majority of idic(22q) chromosomes are thought to occur via a U-type exchange between LCRs on homologs, but non-NAHR/LCR based mechanisms have also been proposed.

Question 25

How are idic(X) formed?

Answer 25

The proximal Xp region is highly enriched for large and highly homologous palindromic segmental duplications.

NAHR between the palindromes catalyses the formation of idic(X) chromosomes with recurrent breakpoints.

FoSTeS or MMBIR are more likely to catalyse the formation of non-recurrent-breakpoint idic(Xq), although NHEJ induced by cruciform structure and breakage cannot be excluded.

Question 26

What are the theoretical mechanisms of isochromosome formation?

Answer 26

1) misdivision at the centromere (centric fission)

2) U-type exchange

Question 27

How are i(Xq) and i(Xp) thought to arise?

Answer 27

i(Xq): Monocentric i(Xp) are thought to mainly derive by NAHR between inverted alpha satellite repeats within the centromeric heterochromatin (centromeric misdivision).

Most i(Xp) chromosomes are however dicentric. The ones with recurrent breakpoints can arise by NAHR between inverted LCRs, but misdivision of the centromere or replication based mechanisms cannot be excluded.

A U-type exchange mechanism between sister chromatids or homolgous X chromosomes has also been proposed. i(Xq)s with unique breakpoints could arise via NHEJ or FoSTeS.